Antioxidants within this class comprise non-enzymatic molecules that are capable of rapidly inactivating radicals and oxidants [ 95 ]. Based on the source of non-enzymatic vitreous antioxidants, they can be classified into metabolic and nutrient non-enzymatic antioxidants. Metabolic antioxidants are endogenous antioxidants produced by the body and include glutathione, metal-chelating proteins, uric acid, and transferrin. Nutrient antioxidants include the class of non-enzymatic antioxidants that are exogenously sourced through foods and supplements, for example, vitamin C, vitamin B2, and trace metals (zinc and selenium) [ 96 ].

ii. Vitamin B: Riboflavin has been detected in both human and animal vitreous, with 0.8 µg/100 mL and 8.0 µg/L average concentrations detected in the ox and bovine vitreous, respectively [ 104 105 ]. Riboflavin plays an essential role in the glutathione redox cycle and guards against lipid peroxidation [ 106 ]. Riboflavin acts as the precursor for two coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are involved in energy metabolism. FAD is essential for the activity of glutathione reductase, which converts oxidized glutathione (GSSH) into reduced glutathione (GSH) (see discussion of GSH below) [ 107 ]. Also, riboflavin functions as an antioxidant through the oxidation of dihydroriboflavin, the reduced form of riboflavin, to produce reducing equivalents for the deactivation of hydroperoxides [ 108 ]. Dihydroriboflavin also protects against reperfusion oxidative damage by reducing oxidized iron in hemeproteins [ 109 110 ]. Riboflavin directly scavenges for free radicals produced by mutagens, thereby inhibiting their mutagenicity [ 111 ]. For a review of the antioxidant ability of riboflavin, see [ 106 107 ]. On the other hand, riboflavin is a photosensitizer which can mediate a riboflavin-sensitized photochemical reaction and result in age-related liquefaction of vitreous [ 57 ]. Thus, therapeutic use of riboflavin for eye diseases may be a two-edged sword that needs to be wielded carefully to achieve a salubrious outcome.

i. Vitamin C: Also referred to as ascorbic acid (AA), Vitamin C is a water-soluble molecule present in most tissues in its anionic state [ 17 ]. Humans cannot synthesize AA de novo and source this molecule exogenously [ 97 ]. The vitreous gel receives its supply of AA from plasma by active transport from the ciliary process of the ciliary body [ 98 ]. AA concentration within the vitreous body approximates to 2 mmol/L, about 33 times higher than plasma concentration [ 99 ]. Also, AA within intact gel vitreous is higher than in liquefied vitreous and in vitreous of proliferative diabetic retinopathy (PDR) patients [ 27 100 ]. As an antioxidant, AA is oxidized in order to convert superoxide anions and lipid hydroperoxidases into stable forms, thereby preventing lipid peroxidation, the oxidative damage of lipids. AA consumes oxygen released at the vitreo–retinal interface, in an ascorbate-dependent fashion, and guards against intraocular oxidative stress and nuclear cataract development [ 27 ]. AA also functions as an intrinsic modulator of hyalocyte proliferation and of extracellular matrix production by hyalocytes [ 101 102 ]. AA serves as an enzyme co-factor to a number of enzymes, especially hydroxylases, which are involved in collagen synthesis [ 103 ].

4.1.2. Proteins and Free Amino Acids

Proteomic analysis of human vitreous has revealed proteins and several amino acid constituents that play important roles in ocular development as well as function as antioxidants [ 112 113 ]. The majority of vitreous antioxidant proteins are located within the central vitreous, among them are glutathione, taurine, crystallin, cysteine, uric acid, tyrosine, human serum albumin, transferrin, and pigment epithelium-derived factor [ 114 ].

116,79, i. Glutathione (GSH): Glutathione is a cysteine-containing peptide and a thiol antioxidant with an average concentration of 0.26 mmol/L [ 115 117 ]. The concentration of glutathione within vitreous is relatively low compared to AA [ 116 ]. As an antioxidant, glutathione can directly remove selected oxygen radicals and indirectly assist in the recycling of vitamins C and E [ 118 ]. Also, GSH inhibits the degradation of HA by acting as a scavenger for hydroxyl radicals [ 119 120 ]. GSH is a cofactor for glutathione peroxidase activity of reducing lipid hydroperoxides, producing alcohol and GSSH in the process [ 107 ]. Reduced intravitreal GSH level has been linked with the pathological complications of inflammation and neovascularization in proliferative diabetic retinopathy (PDR) and Eales’ disease [ 117 121 ]. Other reports, on the contrary, indicate an increase in intravitreal GSH in PDR eyes [ 122 ]. This increase may represent a protective response to detoxify the diabetes-associated redox alteration of vitreous. Indeed, profound structural abnormalities have been identified in human vitreous that are independent of the effects of diabetes on the retina [ 76 123 ].

ii. Taurine: Taurine is a free amino acid that abounds in tissues during development [ 124 ]. Taurine has been detected in rat vitreous at a concentration of 1.72 µmol/mL [ 125 ]. Although the exact role of taurine within vitreous is yet to be elucidated, taurine, as an organic osmolyte, has been proposed to be involved in the vitreous-mediated ionic exchanges that occur between the retina and the anterior segment [ 126 ]. In addition, it has been proposed that the retina possibly receives its supply of taurine from vitreous [ 127 128 ]. Taurine provides antioxidative and neuroprotective functions to ocular tissues, although this mechanism has not been fully understood in the human eye [ 129 ]. Depletion or deficiency of taurine leads to loss of photoreceptors and can impede visual function in man and in animal models [ 127 130 ].

iii. Crystallin: Crystallin is a chaperone or stress protein which accumulates within the lens more than all other ocular tissues [ 131 ]. Both α- and β-crystallins have been isolated in rat vitreous [ 132 ]. β-crystallin B2 (molecular weight ~23 kDa) has been recently identified by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) in normal human vitreous [ 133 ]. β-crystallin S, β-crystallin A4, β-crystallin A3, α-crystallin B chain, and γ-crystallin C have also been found in the vitreous body of both PDR patients and controls [ 134 ]. Crystallin levels were significantly lower in vitreous from PDR patients compared with controls. Crystallin performs an anti-apoptotic role by inhibiting the formation of ROS, thereby reducing oxidative stress [ 135 ].

iv. Cysteine: Cysteine, a non-essential amino acid with a highly reactive thiol group, is found in most peptides and proteins. Cysteine acts as the rate limiting precursor for the synthesis of GSH [ 18 ]. As an antioxidant, its reactive thiol group is oxidized to cystine disulphide and aids in maintaining a redox equilibrium within a cell, tissue, or biofluid [ 136 ].

v. Tyrosine: L-tyrosine is a monophenolic amino acid and a byproduct of the pentose phosphate pathway [ 137 ]. The concentration of tyrosine within adult vitreous is 91 µmol/l [ 138 ]. Antioxidant activities of tyrosine, as observed in vitro include anti-lipid peroxidation, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activities [ 137 ].

vi. Human serum albumin (HSA): HSA is an anionic globular protein with a molecular weight of approximately 69 kDa [ 133 139 ]. HSA is sourced by filtration from blood and constitutes about 80% of the average protein concentration within the healthy vitreous body [ 133 140 ]. The molecular structure of HSA confers multiple antioxidant properties on it including an ability to bind potential ROS-generating ligands (for example, the transition metals copper and iron), scavenge hydroxyl radicals through its reduced cysteine residue (Cys34), and scavenge peroxynitrite through its thiol (–SH) group (for a review of the antioxidant properties of HSA, see [ 141 ]).

vii. Transferrin: Transferrin (molecular weight ~80 kDa) is a glycoprotein with two specific high-affinity binding sites for iron [ 142 143 ]. Vitreous contains a mean transferrin concentration of 0.0878 g/L [ 22 ]. As an antioxidant, transferrin is an iron chelator which keeps ionic iron sequestered at physiological PH and minimizes the involvement of iron in iron-dependent radical reactions [ 144 ]. This property helps to reduce intravitreal iron toxicity during vitreous haemorrhage [ 145 ].