Radio-isotopic determination of hydrogen peroxide in aqueous humor and urine

doi:10.1016/0014-4835(91)90167-D

Experimental Eye Research

Volume 53, Issue 4, October 1991, Pages 503-506

https://doi.org/10.1016/0014-4835(91)90167-D Get rights and content

Abstract

The concentrations of hydrogen peroxide in the aqueous humor and urine of several animal species and humans have been determined. The determinations are based on peroxide-dependent decarboxylation of I-[¹⁴C]-α-ketoglutaric acid and measurement of the resulting ¹⁴CO₂ by quantitating the radioactive disintegration. The levels of H₂O₂ in most animals varied between 5·0 and 41 μm for aqueous, and 115 and 187 μm for urine. The levels of peroxide in the urine of steer, cat and baboon were lower and fell out of the above range. In the aqueous of humans with cataracts, the levels ranged from 33 to 324 μm, the overall average being 189 ± 88 μm. The source of such high levels in the aqueous of cataract patients is currently being studied.

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High production of ROS during aging or a significant decrease in the ROS scavenging ability of the lens, causes oxidative stress and leads to cataracts [44–48]. H2O2 concentration in the aqueous humor is ∼30–70 μM; higher concentrations (>600 μM) are seen in certain cataract patients [47–49]. Maintaining a balance of H2O2 is critical for lens transparency since H2O2 in excess causes oxidative damage to lens proteins, lipids, and DNA that results in opacity and cataract [50].
High levels of reactive oxygen species such as hydrogen peroxide (H₂O₂) cause oxidative stress in the lens and lead to cataractogenesis. The present investigation was undertaken to find out whether the mammalian lens aquaporins (AQPs) 0, 1, and 5 perform H₂O₂ transport across the plasma membrane to reduce oxidative stress. Our in vitro cell culture and ex vivo lens experiments demonstrated that in addition to the established water transport role, mouse AQP0, AQP1 and AQP5 facilitate transmembrane H₂O₂ transport and function as peroxiporins. Human lens epithelial cells expressing AQP1, AQP5 and AQP8, when treated with 50 μM HgCl₂ water channel inhibitor showed a significant reduction in H₂O₂ transport. Data obtained from the experiments involving H₂O₂-degrading enzyme glutathione peroxidase 1 (GPX1) knockout lenses showed H₂O₂ accumulation, suggesting H₂O₂ transport level by AQPs in the lens is regulated by GPX1. Under hyperglycemic conditions, there was an increased loss of transparency, and enhanced production and retention of H₂O₂ in AQP5^−/− lenses compared to similarly-treated WT lenses. Overall, the results show that lens AQPs function as peroxiporins and cooperate with GPX1 to maintain lens H₂O₂ homeostasis to prevent oxidative stress, highlighting AQPs and GPX1 as promising therapeutic drug targets to delay/treat/prevent age-related lens cataracts.
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While GSH appears to the predominant form released under isosmotic conditions, in the presence of the oxidative stressor, H2O2, we revealed that GSSG was the predominant form released (Figs. 5C and 6C). While the concentration of H2O2 in the rat vitreous humour is unknown, studies have demonstrated that the rat aqueous humour contains ∼10 μM H2O2 (Giblin et al., 1984; Ramachandran et al., 1991). This indicates that release of GSSG by the lens in response to 10 μM H2O2 may be a more physiologically relevant reflection of what occurs in vivo.
In this study, we have investigated whether the lens was capable of exporting the antioxidant glutathione. Pairs of rat lenses were cultured in isosmotic artificial aqueous humour for one, two, three, or six hours in low oxygen conditions (90% N₂, 5% CO₂, 5% O₂), and reduced glutathione (GSH) and oxidised glutathione (GSSG) levels measured in the media and lenses. We show that the rat lens is capable of releasing ∼5 nmol GSH for each time point suggesting that GSH release is regulated since it does not appreciably increase over time. We also demonstrated that the predominant form of glutathione released was the reduced form. We next cultured lenses in the absence or presence of acivicin, a γ-glutamyl transpeptidase (GGT) inhibitor, and found that GSH levels were significantly increased (p < 0.001) in the presence of this inhibitor, which indicated that GSH released by the lens undergoes degradation into its constituent amino acids. GSH release was significantly decreased (p < 0.001) in the presence of 100 μM MK571, a multidrug resistance-associated protein (Mrp) inhibitor, suggesting that Mrp transporters mediate GSH efflux from the lens. Culturing lenses in low (10 μM) or high (70 μM) concentrations of H₂O₂ for one hour significantly increased total glutathione levels (p < 0.05) relative to controls, due to the increased release of GSSG. Our results show that in response to oxidative stress, the rat lens is able to release GSH or GSSG, thereby serving to maintain lens redox state or potentially influence the redox state of nearby tissues.
Roles for the ubiquitin-proteasome pathway in protein quality control and signaling in the retina: Implications in the pathogenesis of age-related macular degeneration
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Most data regarding the degradation of oxidized proteins were collected in lens or cultured lens epithelial cells. H2O2 is a natural oxidant in the eye (Spector and Garner, 1981; Giblin et al., 1984; Ramachandran et al., 1991) and is often used to model oxidative stress in the eye. Exposure of proteins or cells to H2O2 results in generation of protein carbonyls, disulfide and non-disulfide-linked aggregates (Shang et al., 1994, 2001; Dudek et al., 2005; Huang et al., 1995; Jahngen-Hodge et al., 1994; Taylor and Hobbs, 2002).
The accumulation of damaged or postsynthetically modified proteins and dysregulation of inflammatory responses and angiogenesis in the retina/RPE are thought be etiologically related to formation of drusen and choroidal neovascularization (CNV), hallmarks of age-related macular degeneration (AMD). The ubiquitin–proteasome pathway (UPP) plays crucial roles in protein quality control, cell cycle control and signal transduction. Selective degradation of aberrant proteins by the UPP is essential for timely removal of potentially cytotoxic damaged or otherwise abnormal proteins. Proper function of the UPP is thought to be required for cellular function. In contrast, age – or stress induced – impairment the UPP or insufficient UPP capacity may contribute to the accumulation of abnormal proteins, cytotoxicity in the retina, and AMD. Crucial roles for the UPP in eye development, regulation of signal transduction, and antioxidant responses are also established. Insufficient UPP capacity in retina and RPE can result in dysregulation of signal transduction, abnormal inflammatory responses and CNV. There are also interactions between the UPP and lysosomal proteolytic pathways (LPPs). Means that modulate the proteolytic capacity are making their way into new generation of pharmacotherapies for delaying age-related diseases and may augment the benefits of adequate nutrition, with regard to diminishing the burden of AMD.
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The ubiquitin–proteasome pathway (UPP) plays important roles in many cellular functions, such as protein quality control, cell cycle control, and signal transduction. The selective degradation of aberrant proteins by the UPP is essential for the timely removal of potential cytotoxic damaged or otherwise abnormal proteins. Conversely, accumulation of the cytotoxic abnormal proteins in eye tissues is etiologically associated with many age-related eye diseases such as retina degeneration, cataract, and certain types of glaucoma. Age- or stress-induced impairment or overburdening of the UPP appears to contribute to the accumulation of abnormal proteins in eye tissues. Cell cycle and signal transduction are regulated by the conditional UPP-dependent degradation of the regulators of these processes. Impairment or overburdening of the UPP could also result in dysregulation of cell cycle control and signal transduction. The consequences of the improper cell cycle and signal transduction include defects in ocular development, wound healing, angiogenesis, or inflammatory responses. Methods that enhance or preserve UPP function or reduce its burden may be useful strategies for preventing age-related eye diseases.
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Ascorbate and several phenolic compounds readily oxidise in cell culture media to generate hydrogen peroxide. However, addition of α-ketoglutarate, which is known to be released by several cell types, decreased the levels of H₂O₂, and the α-ketoglutarate was depleted and converted to succinate. These observations could account for previous reports of the protective effects of α-ketoglutarate in promoting the growth of cells in culture, and may contribute to explaining some of the variability in the literature in reported rates of H₂O₂ production from autoxidisable compounds in cell culture systems.
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Radio-isotopic determination of hydrogen peroxide in aqueous humor and urine

Abstract

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Exp. Eye Res.

Am. J. Ophthalmol.

Exp. Eye Res.

Exp. Eye Res.

A sensitive method for the estimation of hydrogen peroxide in biological materials

Nature

Role of oxygen in the genesis of reterolental fibroplasia

Br. J. Ophthalmol

The biology of oxygen radicals

Science

Free radical theory of aging. Effect of a free radical reaction inhibitor on the mortality of male mice

J. Gerontol.