Резюме
Сердюк В.Н. Исследование уровня продуктов окислительного повреждения белков и липидов в слезной жидкости и камерной влаге у больных ПОУГ.
Цель настоящей работы заключалась в исследовании уровня продуктов окислительного повреждения белков и липидов в слезной жидкости и камерной влаге у больных ПОУГ. При глаукоматозном процессе выявлены значительные повышения скорости оксидативного повреждения белков и липидов, что особенно выражено в слезной жидкости больных (содержание малонового диальдегида повысилось на 138,5%, диеновых конъюгатов — на 83,3%, карбонильных групп — на 68,2% по сравнению с нормой).
Ключевые слова: глаукома, белки, липиды, нейропатия, перекисное окисление.
Резюме
Сердюк В.Н. Дослідження рівня продуктів окисного окислювального пошкодження білків і ліпідів в слізній рідині і камерній волозі у хворих на ПВКГ.
Мета цієї роботи полягала в дослідженні рівня продуктів окисного пошкодження білків і ліпідів у слізній рідині та камерній волозі у хворих на ПВКГ. При глаукоматозному процесі виявлені значні підвищення швидкості оксидативного пошкодження білків і ліпідів, що особливо виражено в слізній рідині хворих (вміст малонового діальдегіду підвищився на 138,5%, дієнових кон’югатів – на 83,3%, карбонільних груп – на 68,2% порівняно з нормою.
Ключові слова: глаукома, білки, ліпіди, нейропатія, перекисне окислення.
Summary
Serdyuk V.N. Investigation of the level of products of proteins’ and lipids’ oxidative damage in the tear fluid and chamber moisture in POAG patients.
Studing the level of products of oxidative damage to proteins and lipids in the tear fluid and moisture chamber in patients with POAG. In glaucomatous process revealed significant increase rate of oxidative damage of proteins and lipids, which is especially pronounced in the tear fluid of patients (malondialdehyde content increased to 138.5% of diene conjugates – 83.3% carbonyl groups – 68.2% compared the norm).
Key words: glaucoma, proteins, lipids, neuropathy, lipоperoxidation.

Рецензент: д.мед.н., проф. А.М. Петруня

УДК 617.7-007.681:577.19-07

Областная клиническая офтальмологическая больница (Днепропетровск)

49000, г. Днепропетровск. Октябрьская площадь 14.

Regional Clinical Ophthalmological Hospital, Dnipropetrovsk

49000, Dnepropetrovsk. 14 October Square.

kzdokol@ukr.net

Introduction. Glaucoma is a widespread disease, in particular, in developed countries which often leads to fatal blindness and disability, and therefore represents an urgent medical and social problem of the state and society as a whole [3].

Glaucoma represents a topical and complex problem since, on the one hand, the modern ophthalmology has at its disposal the wide range of medicinal preparations, methods of non-surgical and surgical treatment, and, on the other hand, these remedial measures are not always effective. It is explained by the complexity of pathogenic mechanisms of development of the disease and symptomatic, instead of pathogenic, approach to its treatment and prevention [4,15,17,23].

In the light of the current views, glaucoma is considered a neurodegenerative disease of the organ of vision, i. e. the glaucomatous optic neuropathy [19].

In the POAG development, microstructural changes at various levels are directly observed because of violation of a number of processes: involution, biomechanical processes, mechanisms of blood circulation and vascular autoregulation, acceleration of apoptosis of the nerve cells and reduction of the level of natural neuroprotection. Immune changes, elasto-tonic properties of sclera, age, ethnicity, vascular disregulation, arterial sclerosis and so on may also play an important role in the pathogenesis of the disease [13,16].

At present time, the importance of free-radical processes and, in particular, the active forms of oxygen in the pathogenesis of neurodegenerative diseases is shown [5,6,14].

According to the metabolic concept of pathogenesis of the glaucomatous optic neuropathy which exists now, oxidative stress and exito-toxic damage to the third retinal nerve play the specific role in glaucoma. The prominent role is also given to the pathologic processes with the involvement of activated neuroglia which in the conditions of hypoxia produces the excessive amount of toxic metabolites having the damaging effect on the neurons of retina and axons of the optic nerve [22,24,25].

It should be emphasized that initial damage to all ganglion cells or majority thereof in glaucoma starts from the damage of their axons in the domain of the head of the optic nerve disk (OND) and that individual rate of ganglion cells’ rate would be to a considerable degree determined by the metabolic state of cells themselves and their dendrites. Therefore, axons of dying ganglion cells in glaucoma are in the more advanced stage of dying than the bodies of their cells. It is comparable to the death of ganglion cells in the retinal ischemia, where the bodies of ganglion cells are more severely damaged than their axons. It is necessary to state that the processes leading to death of ganglion cells in glaucoma occur very slowly, with the variable rate, depending on the degree of hypoxic damage in the domain of the optic nerve head. Damage occurs gradually, continuously or periodically over many years [15,20,21].

The most of researchers are of opinion that at the bottom of the process of cell membrane damage there is the process of LPO (lipid peroxidation) involved in the regulation of cell division, biosynthesis of prostaglandins, nucleic acids, leukotrienes, reactions of oxidative phosphorylation, and regulation of membrane permeability [1,18].

 Activation of the peroxidation process leads to decrease in unsaturated fatty acids, which promotes the growth of viscosity of the lipid bio-layer of cell membranes. Products of the lipid peroxidation (aldehydes, ketones, free radicals, peroxides) inactivate thiolic groups – ferments, suppress the oxidative phosphorylation, and cause destruction of elastic fibers and de-polymerization of muco-polysaccharides. This being the case, protein-lipid and lipid-lipid bonds are disrupted, and activity of membrane-bound enzymes decreases. All of that causes damage to bio-membranes and disrupts the transport processes and selective permeability for ions. Consequently, the ions of calcium and sodium accumulate in cells, which leads to cell death. Nevertheless, the facts for considering the lipid peroxidation as the initial trigger mechanism of glaucomatous process development are not enough to date [2].

In the course of previous investigations we revealed that in the conditions of glaucomatous process modeling the dramatic increase in products of lipid peroxidation in the retina and optic nerve was registered. For example, concentration of malondialdehyde during the final period of observation increased by 110,7%, and that of diene conjugates — by 54,9% [11].

Furthermore, it was shown that in modeling of the experimental glaucoma intensity of processes of quenching of free-radical oxygen form – superoxide in tissues of the retina and optic nerve decreased considerably: indices of activity of superoxide dismutase were 79,9% и 69,9% during the second and the third periods of the stable hypertension.

Reduction of the level of thiolic groups of proteins of the retina and optic nerve in modeling of the glaucomatous process was revealed during the final period of observation (by 25%). The same period featured considerable growth of disulfide groups’ concentration (by 45%) [9].

In the conditions of development of the glaucomatous process in the retina and optic nerve of experimental animals, progressive decline in the level of reduced and oxidized glutathione —  42% and 17%, accordingly, was observed at the end of the 3^rd observation period. Besides, the level of glutathione peroxidase and glutathione-S-transferase reduced accordingly by 18,7% and 23,7%  [10].

Therefore, experimental study of this pathology because of which severe impairment of visual functions occurs and complications develop, determines the direction of searching for the ways of pathogenic therapy and prevention of the process progressing, providing for reduction of complications of this disease and conducting clinical and biochemical studies in POAG patients.

Objective of this work consisted in the investigation of the level of products of the oxidative damage to proteins and lipids in the tear fluid and chamber moisture in POAG patients.

Materials and Methods.

Investigations on studying patho-chemical indices in the chamber moisture were carried out on 44 patients.

24 patients represented the control group, and 20 patients – glaucoma patient group.

Investigations on biochemical studying of the tear fluid were carried out on 90 patients.

24 patients represented the control group, and 66 patients – glaucoma patient group.

In the tear fluid and chamber moisture of patients, the investigators determined carbonyl groups of proteins, as well as the content of malondialdehyde and diene conjugates using spectrophotometric methods.

Principle of the method of determination of malondialdehyde content is the following. At temperature of 100°С in the acid medium malondialdehyde reacts with 2-thiobarbituric acid forming the colored trimethine complex with the maximum of absorption at wave length of 532 nm.

To the homogenate under study in volume of 0,1 ml, 3 ml of 1 % orthophosphoric acid (рН 2,0), 1 ml of 0,6 % solution of thiobarbituric acid and 0,1 ml of 0,28 % ferrous sulfate solution were added. The tubes were placed in boiling water bath for 60 min. Further, the tubes were cooled in cold water at 0°С - 2°С, with adding of 4 ml of butanol, mixed thoroughly and centrifuged for 10 minutes at 3 thousand rev./min.

The optical density of the upper phase was measured using Specol – 210 spectral colorimeter at wave length of 535 nm against butanol.

Calculation of the content of products reacting with the thiobarbituric acid was made taking into account the coefficient of molar extinction of malondialdehyde – 1,56 · 10⁵ mol^-1·cm^-1 and expressed in mcmol/g of tissue.

Coefficient of variation of the method was equal to 5,2 % [12].

Principle of the method of determination of diene conjugates is the following.  During peroxidation, at the stage of formation of free radicals in the molecules of polyunsaturated higher fatty acids, the system of conjugated double bonds is emerging, which is accompanied by the appearance of new maximum in the absorption spectrum of 233 nm.

To 0,5 of the homogenate under study 4,5 ml of extraction mixture of heptane with isopropyl alcohol was added in the ratio of 1:1 (V:V). After extraction, 0,5 ml of distilled water was added to the mixture, and stratified sample was taken from the upper (heptane) phase of 0,5 ml, and mixed with 2,5 ml of the absolute ethyl alcohol.

Optical density of the solution under test was measured using СФ-26 spectrophotometer at 233 nm against ethyl alcohol.

The content of diene conjugates was calculated taking into account the molar extinction coefficient of 2,2·10⁵ М^-1·cm^-1 and expressed in mcmol/g of tissue [12].

The data obtained was statistically processed using the package SPSS 11.0 [7].

Results and Discussion. Information on the content of products of lipid peroxidation and protein carbonyl groups in the chamber moisture for glaucoma patients is presented in Table 1 and the diagram (Fig. 1).

As it is shown by this data, level of malondialdehyde in the chamber moisture of glaucoma patients increased to (94,3±6,5) mcmol/ml, comprising 187,8% compared with the norm of (50,2±3,3) mcmol/ml.

The content of diene conjugates in the chamber moisture in the group of glaucoma patients increased to (33,4±2,3) mcmol/ml comprising 156,1% with regard to the norm of (21,4±1,5) mcmol/ml.

When studying the data on the content of carbonyl groups, it can be stated that their level in the chamber moisture of glaucoma patients was raised to (77,4±5,2) nmol/g comprising 148% compared with the norm of (52,3±3,2) nmol/g.

Data on the content of lipid peroxidation products and carbonyl groups of proteins in the tear fluid of glaucoma patients is given in Table 2 and the diagram (Fig. 2).

The level of malondialdehyde in the tear fluid in glaucoma patients increased to the value of (12,4±0,8) mcmol/ml, comprising 238,5% with regard to the norm of (5,2±0,4) mcmol/ml.

In the tear fluid of glaucoma patients, the content of diene conjugates increased to (0,88±0,06) mcmol/ml, comprising 183,3% with regard to the norm of (0,48±0,04) mcmol/ml.

When considering the data on carbonyl groups’ content, it can be stated that in the group of glaucoma patients their level in the tear fluid was raised to (143,3±9,5) nmol/g, comprising 168,2% compared with the norm of (85,2±6,4) nmol/g.

Summarizing the results of the studies, we can state that in glaucoma patients the content of malondialdehyde increases by 87,8% in the chamber moisture and by 138,5% in the tear fluid, diene conjugates’ content increases by 56,1% in the chamber moisture and by 83,3% in the tear fluid, level of carbonyl groups is raised by 48% in the chamber moisture, and by 68,2% in the tear fluid.

General review of the results of biochemical studies of the tear fluid and chamber moisture proves that patho-chemical abnormalities we revealed by the experiments in the eye tissues using the modeling of hypertension were confirmed by the changes in the indices of oxidative damage to proteins and lipids in the tear fluid and chamber moisture.

Conclusions:

1. During the glaucomatous process, significant increase in the rate of proteins’ and lipids’ oxidative damage has been revealed, which was especially pronounced in the tear fluid of patients (malondialdehyde content increased by 138.5%, content of diene conjugates – by 83.3%, and that of carbonyl groups – by 68.2% compared with the norm).

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