In general, the importance of GSH in eye tissue protection is established . Metabolism of GSH and its transport through cell membranes or between different tissues are essential for the maintenance of redox homeostasis in eye tissues. GSH efflux outside eye tissues through Na+-independent efflux system is established for conjunctive towards “tear film” [19, 20, 21], which is considered as an essential mechanism of H2O2 neutralization in “tear film” . Also, GSH efflux was shown in the liver to have much higher concentrations of GSH than all other tissues  and can maintain redox homeostasis in the multitude of tissues by releasing GSH into the blood flow.
GSH cannot pass into direct cells and must be metabolized to its constituent amino acids, which pass through the membrane and can be substrates for intracellular GSH re-synthesis. Usually, a limiting factor for GSH synthesis is the availability of cysteine , which justifies the use of NAC as a cysteine precursor in the treatment of different pathologies, including ocular. L-cysteine or L-glutamate transport across cell membrane was shown in several eye cell types including human retinal pigment epithelium (RPE), and it was found, that it does increased L-cystine uptake (oxidized form of cysteine), raising intracellular GSH levels. GSH is assumed to be involved in intracellular repair of protein damaged by oxidative stress . Also, nitrosative stress increased uptake of L-cystine and enzyme activity ɣ-glutamylcysteine synthetase (GCS) responsible for GSH synthesis in conjunctival epithelial cell layers .
Positive effects of treatment with GSH or its prodrugs were primarily seen in pathologic eye tissues with decreased GSH levels [8, 12, 25]. In this study, we have surprisingly observed the elevated levels of GSH in patients with recurrent pterygium compared with the control group or primary pterygium, while systemic pretreatment of pterygium with NAC further increased the level of GSH and topical treatment did not affect it compared with group C or P. Last, at least, means that cysteine does not enter directly into pterygium tissue.
An increase in the level of GSH in recurrent pterygium can be in three main ways: decrease of oxidative stress, stimulation of its synthesis, or by efflux (mobilization) of GSH from the nearby tissue with higher concentration, which could be cornea. The latter could recover this loss of GSH from blood flow proceeding from the liver. In this study, we have observed a decrease in eNOS, NO, and 3NT in pterygium tissue, while in the previous research, no significant change in lipoperoxidation (TBARS) was found in pathological tissues . Then it is likely that nitrosative stress slope decreases the inactivation rate of GSH and CAT in recurrent pterygium resulting in their increase. Also a positive correlation of the CAT level with GSH in study groups may mean a reduction in the inactivation rate of this enzyme due to a rise in the level of GSH in pathological tissues.
A high positive correlation between NO and 3NT levels with eNOS activity in our study groups confirms that this enzyme regulates NO production in pterygium tissue, decreasing its level without affecting significantly GSH level in primary pterygium. But the degree of GSH oxidation (GSSG%) decreases from 35% to 25% in this group and remains decreased in other pathological groups, which can be related to the decrease in nitrosative stress. The behavior of NO and especially of 3NT shows a high positive correlation with this parameter of the metabolism of GSH by the groups of the study, confirming a close relationship between these parameters.
As mentioned above, the decrease in the level of NO in tissue stimulates GSH synthesis, and its increase decreases during endotoxemia in the animal model . In our case, reduced levels of NO and 3NT could stimulate GSH synthesis in primary pterygium tissue, which we have not observed. This discrepancy in the stability of GSH with a reduced level of NO in group P makes it possible to assume that the increase in GSH in recurrent pterygium could be due to the mobilization of GSH from nearby tissue, which is cornea.
Comparing the effects of two pretreatments with NAC does not contradict this assumption. The positive impact of the use of NAC were observed in diabetic retinopathy , in protection against alloxan-induced diabetes in mice by increasing the synthesis of GSH in platelets  and in patients with dry eye syndrome, where treatment improved axis-related symptoms . In literature, there are data on GSH efflux from the conjunctiva to tear film [21, 22]. We do not have data on the possible efflux of GSH from cornea to extracellular space despite its millimolar versus micromolar concentration in the conjunctiva. But its efflux from the liver to blood flow is real, and gradient of its level concerning all other tissues is even higher. We also assume that in an organism, there is a precise balance between efflux of GSH of the liver and its concentration in different tissues, and pre-treatment with NAC could stimulate the synthesis of GSH in the liver with consequent redistribution to a multitude of various tissues, including oculars. Topical pre-treatment showed no effect on intracellular levels of GSH in pterygium, which may be by blocking the direct transport of cysteine into pathological tissue.
NO is a radical and has a short life but high capacity to penetrate cells. It is supposed that the reaction between NO and proteins (S-nitrosylation) allows lengthening its lifetime. Under the term S-nitrosylation, we determine the modification of protein thiols by NO . GSH has a high affinity to NO, and S-nitrosoglutathione (GSNO) is the main intermediate in the production of other metabolic regulating nitrosothiols [14, 15]. In this case, nitrosothiols are NO stabilizers and their possible carriers (carrier molecule) instead of their action . Possible NO release reaction of S-nitrosoglutathione is as follows: GSNO = GSSG + 2NO . If it is true, the NO release product of this reaction is GSSG and there was observed the correlation between NO and grade of GSH oxidation.
Evaluating gender differences, we found that women showed a higher level of GSH and CAT in primary pterygium group, lower level of GSH and a higher level of NO in recurrent pterygium. Women showed a positive correlation in research groups between GSSG% and 3NT, while men with NO. As a NO release reaction of S-nitrosoglutathione has as a product GSSG, women likely show more significant role of S-nitrosation compared with men. The nitrosothiols have a different half-life in water: S-nitrosoglutathione (hours), S-nitrosocysteine and S-nitroso-N-acetylcysteine (minutes) and can affect the synthesis of intracellular GSH or its efflux of tissues with a high level of GSH as the liver for all tissues or cornea for eye tissues. In our study high positive correlation between NO and 3NT with percentage of oxidized GSH in total GSH (GSSG%) in pathological tissues indirectly shows the possible effect of S-nitrosation on GSH metabolism and/or its efflux (release) of tissues.
One of the possible sequences of events in pathogenesis in primary pterygium: decreased activity of eNOS, bioaccessibility of NO and S-nitrosation of GSH or other nitrosothiols, possible release of NO resulting in modulation of the intracellular level of GSH through synthesis and/or mobilization of different tissues. If we are right, treatment with prodrugs-NO substances should be investigated regarding pterygium.
In this study, only systemic pre-treatment with NAC significantly increased the level of GSH in pterygium, which we interpreted as a positive effect, but the number of patients is not sufficient to evaluate the frequency of recurrence of the disease.