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Д1АГНОСТИКА ТА Л1КУВАННЯ: ХВОРОБА В1ЛЬСОНА Труфанов е. О.
Хвороба Вшьсона - хрошчне спадкове, аутосомно-рецесивне захворювання, пов'язане з порушенням обмiну мiдi. Незважаючи на характерну клiнiчну картину, дiагностика захворювання ускладнена. Несвоечасно поставлений дiагноз призводить до того, що при вщсутност лiкування швидко прогресують невролопчш розлади та патологiчнi змiни в печшщ, якi протягом кiлькох роюв призводять до смертi хворого. Адекватне, безперервне лкування (дiета, пенiциламiн), розпочате на початкових стадiях захворювання зменшуе клiнiчнi прояви та значно покращуе прогноз хвороби Вiльсона.
Ключовi слова: хвороба Вшьсона, клшчш прояви, дiагностика, лiкування.
Стаття надiйшла 10.05.18 р.
DIAGNOSIS AND TREATMENT: WILSON'S DISEASE Trufanov Ye.O.
Wilson's Disease is a chronic hereditary, autosomal-recessive disorder associated with excessive deposition of copper in the liver, brain, and other tissues. Despite of a characteristic clinical picture, diagnostics of Wilson's Disease is complicated. Diagnosis delay and untimely initiation of treatment lead to a rapid progression of neurological symptoms, pathological changes in the liver and patient's death for several years. Adequate, continuous treatment (diet, penicillamine) initiated in the early stages of the disease greatly improves clinical symptoms and the prognosis of Wilson's Disease.
Keywords: Wilson's Disease, clinical features, diagnostic, treatment.
Рецензент Катеринчук I.П.
doi 10.26724/2079-8334-2018-4-66-120-124 udc 616.697-02:616.69-007.5
PROOXIDANT/ANTIOXIDANT BALANCE IN SPERM CELLS OF INFERTILE MEN
E-mail: [email protected]
Hyperproduction of reactive oxygen species and disturbances of prooxidant/antioxidant balance can damage sperm cells and are considered as possible mechanisms of male infertility. The aim of present paper was to study the prooxidant/antioxidant balance in sperm cells of infertile men with different forms of pathospermia. In this study, we found the levels of intracellular TBARS in spermatozoa to be elevated and activity of glutathione antioxidant protection system (GPx, GR, GsT) and GSH content to be decreased in infertile individuals with different forms of pathospermia compared with the healthy man with normozoospermia. The index of antioxidant status was calculated by dividing the sum of the activities of antioxidant enzymes (GPx, GR, GsT) and GSH content to the sum of the TBARS and GSSG level. The extent of decrease of indexes of antioxidant status of spermatozoa of infertile men revealed a disorder-associated trend: oligozoospermia > oligoasthenozoospermia = asthenozoospermia >> leucocytospermia. Unidirectional change in the index of antioxidant status in different forms of pathospermia allows to use it as biotest for intensity free radicals processes and resistance to free radicals in spermatozoa.
Keywords: antioxidant enzymes, lipid peroxidation, ^ male infertility, pathospermia..............
The present study is a fragment of the research projects "Investigation of systemic and paracrine regulatory mechanisms in providing homeostasis of functional and metabolic parameters of an organism in conditions of adaptation to the extreme factors of different nature " (state registration No 0116U004510) and "Application of mathematical methods for the study ofphysical and chemical processes in biotechnical systems " (state registration No 0117U001078).
Oxidative stress is a potential prerequisite for the development of pathologies of different genesis [7]. It is considered as the main cause of male infertility Oxidative stress is defined as an imbalance of free radicals processes and antioxidant defense mechanisms, which is associated with sperm abnormalities, resulting in infertility [8]. In physiological norm antioxidant defense system which includes both enzymatic and non enzymatic scavenges free radical species, mainly reactive Oxygen species, and protect from overexposure to oxidative stress. The omnipresent nature of both enzymatic and non enzymatic components of the antioxidant machinery underlies the necessity of ROS detoxification for cell survival [9].
One of the most important enzymatic antioxidant defence mechanism in spermatozoa consist of the glutathione system which includes glutathione peroxidase (GPx, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2) and glutathione S-transferase (GsT, EC 2.5.1.18). The GPxs are a family of enzymes that contain the unique amino acid selenocysteine. They use low-molecular-weight thiols, such as GSH, to reduce H2O2 and lipid peroxides to their corresponding alcohols GSH reductase (GR) converts GSSG to GSH, a co-substrate for the GPxs. GSTs, another antioxidant enzyme family, inactivate secondary metabolites, such as unsaturated aldehydes, epoxides, and hydroperoxides [10]. GSH (low molecular weight thiol tripeptide y-glutamyl-cysteinyl-glycine) is the major soluble non-enzyme antioxidant which is highly abundant in all cell types. Depletion of glutathione system leads to cytotoxic and destructive lesions [11].
© R.V. Fafula, O.K. Onufrovych, 2018
On the other hand it is known that products of lipid peroxidation can be used as damage biomarkers, since the intensity of free radical processes can be estimated by their content. One of the most sensitive markers of lipid peroxidation and oxidative stress are thiobarbituric acid reactive substances (TBARS). In our previous studies the intensity of lipid peroxidation processes [6] and activities of enzymes of glutathione antioxidant protection system [12] in spermatozoa in sperm cells of infertile men with different forms of pathospermia were studied.
The aim of present paper was to study the prooxidant/antioxidant balance in sperm cells of infertile men with different forms of pathospermia.
Materials and methods. Patients. This study involved 72 infertile men with different forms of pathospermia. They were recruited between January 2014 and April 2016. A detailed medical history was performed for all studied cases. Exclusion criteria: subjects currently on any medication or antioxidant supplementation were not included. In addition, subjects with infertility over 10 years, azoospermia, genital infection, chronic illness and serious systemic diseases, smokers and alcoholic men were excluded from the study because of their well-known high seminal reactive oxygen species (ROS) levels and decreased antioxidant activity [4,13].
According to semen analysis all infertile men were divided into 4 groups: oligozoospermia (16.7 %), asthenozoospermia (23.6 %), oligoasthenozoospermia (13.9 %) and leukocytospermia (45.8 %). The control group consisted of 20 healthy men with somatic fertility, normozoospermia and confirmed parenthood (married for 3-10 years and have healthy 1-3 children). All patients and healthy donors were matched by age. Semen samples were obtained by masturbation and collected into sterile containers, following 3 - 5 days' abstinence from sexual activity. After liquefaction at 37 C with 5% CO2 in air, semen samples were examined for volume, sperm concentration, pH, morphology and motility according to the World Health Organization guidelines [14]. Before turning to the study, all men were aware of patient information leaflets and gave informed consent to participate in research. Terms of sample selection meet the requirements of the principles of the Helsinki Declaration on protection of human rights, Convention of Europe Council on human rights and biomedicine and the provisions of laws of Ukraine. Approval for the study was taken from the ethics committe of Danylo Halytsky Lviv National Medical University (Ethical Committee Approval, protocol No 2 from February 16, 2015).
Cell preparation. Sperm cells were washed from semen plasma by 3 times centrifugation at 3000 xg for 10 min in media which contained (mM): 120 NaCl, 30 KCl, 30 Hepes (pH 7.4). The content of total protein in the samples was determined by Lowry method using a kit to determine its concentration ("Simko Ltd"). Determination of oxidative stress parameters was carried out in permeabilized spermatozoa. The detergent saponin in a final concentration of 0.5% was added to sperm suspension for permeabilization of sperm membranes [12].
Assay of oxidative stress parameters. Oxidative stress was evaluated by the measurement of the thiobarbituric acid reactive substances (TBARS). The TBARS reaction evaluates the levels of malondialdehyde, a product of lipid peroxidation [5]. GPx activity was determined by the oxidation of glutathione [3]. GR activity was assayed by measuring the oxidation of NADPH [1]. GsT was determined by the rate of conjugate by reaction with 1-chloro-2,4 dinitrobenzene as described [2]. The content of total glutathione was determined after complete reduction of glutathione through the use of glutathione reductase by means of Ellman's reagent [15]. The level of 5-trinitrobenzoic acid was monitored with a spectrophotometer at 412 nm. To determine the content of oxidized glutathione (GSSG), 2-vinylpyridine was added to the incubation mixture to a final concentration of 2 % 60 min before the determination [16]. The content of reduced glutathione (GSH) was calculated as the difference of contents between total glutathione and its oxidized form. All results concerning antioxidant ratios were expressed in relative units - R.U. [17].
Statistical analysis. Experimental data were processed by methods of variation statistics using software MS Office. Differences were calculated using the t-Student test for independent groups, assuming p < 0.05 as the minimum significance level. The results are presented as the mean ± standard deviation of the mean.
Results of the study and their discussion. TBARS and GSSG level in the sperm cells was found to be significantly higher in the abnormal groups than in fertile group (Fig. 1). The highest TBARS level is observed in patients with leukocytospermia than in other forms of pathospermia (p<0.001 compared to normozoospermic samples). This can be explained by the fact that leucocytes stimulate the ROS formation, induction and development of oxidative stress, thus inhibiting the sperm motility and functional activity [18].
The activity of antioxidant enzymes of glutathione system in ejaculated spermatozoa infertile men in represented on Fig. 2. GPx activity was significantly decreased in patients with all forms of pathospermia compared with healthy men with normozoospermia (p<0.01). The activity of GR also was decreased in all studied groups of infertile men (p<0.05). However, the most expressed decrease in GR activity was observed in leucocytospermic and oligozoospermic samples. Furthermore, GsT activity was significantly decreased in the abnormal groups with the most decrease in leucocytospermic samples (p<0.05). Infertile groups showed significantly decreased values of reduced glutathione in sperm cells vs. fertile men (p<0.01), indicating an alteration of oxidative status (Fig. 3). The most expressed changes in the GSH content were observed in patients with oligozoospermia. However, the GSH level in leucocytospermic patients was lower than in patients with normozoospermia, but these changes were not significant.
Fig. 1. Normalized indices of TBARS and GSSG levels in the sperm cells of infertile men. Hereinafter: 1 represents the value in sperm cells of normozoospermic samples (men with preserved fertility).
Fig. 2. Normalized indices of activities of antioxidant enzymes of glutathione system in the sperm cells of infertile men
Fig. 3. Normalized indices of GSH level as non-enzyme antioxidant in the sperm cells of infertile men
It should be noted that each form of pathospermia can be characterized by a certain level of oxidative stress markers (TBARS) and activity of enzyme (GPx, GR, GsT) and non-enzyme (GSH) antioxidant systems. The decrease in activity (level) of enzyme and non-enzyme antioxidant systems in patients with pathospermia can be explained by the depletion of compensatory mechanisms occurs in sperm cells. The changes in glutathione antioxidant defence system might either result from GSH synthesis or its utilisation in the detoxification process against oxidative stress. Most diseases are characterized by the intensification of free radical processes and the decrease of the antioxidant capacity. Reduction in activity antioxidant enzymes can be related both to the oxidative destruction of the corresponding DNA regions and with the direct damage to enzyme molecules as a result of the reactive oxygen species effects [19]. In addition, studies indicate that OS is also involved in impaired sperm DNA packaging [20].
An important factor that promotes the pathogenesis of many diseases is the ratio of antioxidant system to antioxidant systems, which is known as the antioxidant status of cell. The index of antioxidant status was calculated by dividing the sum of the activities of antioxidant enzymes (GPx, GR, GsT) and GSH content to the sum of TBARS and GSSG level. The activities of antioxidant enzymes, GSH, GSSG and TBARS level in sperm cells of normozoospermic samples (men with
Fig. 4. Indexes of antioxidant status of spermatozoa of fertile and infertile men with different forms of pathospermia
preserved fertility) was assumed as 100% and consequently the index of antioxidant status equalled to 1 (which contributes to the unification of this index) [21]. The calculated index of antioxidant status indicates the resistance of cells to oxygen free radicals.
The obtained indexes of antioxidant status of spermatozoa of fertile and infertile men with different forms of pathospermia are represented on Fig. 4. The index of antioxidant status reflects changes in the system of prooxidant/antioxidant balance. The extent of decrease of indexes of antioxidant status of spermatozoa of infertile men revealed a disorder-associated trend: oligozoospermia > oligoasthenozoospermia ~ asthenozoospermia >> leucocytospermia. The most expressed decrease in index of antioxidant status was observed in spermatozoa of patients with leucocytospermia. Evaluation of the indexes of antioxidant status can be helpful in the diagnosis and the prognosis of the male infertility.
The extent of decrease of indexes of antioxidant status of spermatozoa of infertile men revealed a disorder-associated trend: oligozoospermia > oligoasthenozoospermia ~ asthenozoospermia >> leucocytospermia. The most expressed disturbance of prooxidant/antioxidant balance was in spermatozoa of patients with leucocytospermia. Unidirectional change in the index of antioxidant status in different forms of pathospermia allows to use it as biotest for intensity free radicals processes and resistance to free radicals in spermatozoa.
1. Onufrovych OK, Vorobets DZ, Vorobets ZD. Vplyv preparativ z antyoksydantnymy vlastyvostyamy na stan antyoksydantnoyi systemy spermatozoyidiv pry ekskretorno-toksychniy formi neplidnosti cholovikiv. Visnyk Dnipropetrovskoho universytetu. Biolohiya, medytsyna. 2013;4(2):52-6. [in Ukrainian]
2. Fafula RV, Onufrovych OK, Yefremova UP, Nakonechnyy YA, Vorobets' ZD. Intensyvnist protsesiv lipoperoksydatsiyi u spermatozoyidakh cholovikiv iz porushennyam fertyl'nosti. Visnyk problem biolohiyi i medytsyny. 2017;1(135):199-204. [in Ukrainian]
3. Agarwal AL, Tvrda E, Sharma R. Relationship amongst teratozoospermia, seminal oxidative stress and male infertility. Reproductive Biology and Endocrinology. 2014;12:1-8.
4. Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 2010;48:909-30
5. Birben E, Sahiner MU, Sackesen C, Erzurum S, Kalayci O. Oxidative Stress and Antioxidant Defense. World Allergy Organ J. 2012 Jan;5(1):9-19.
6. Kalinina EV, Chernov NN, Novichkova MD. Role of glutathione, glutathione transferase, and glutaredoxin in regulation of redox-dependent processes. Biochemistry. 2014;79(13):1562-83.
7. Fafula RV, Onufrovych OK, Vorobets DZ, Iefremova UP, Vorobets ZD. Glutatione antioxidant protecyion system in ejaculated spermatozoa of inferrtile men with different eorms of pathospermia. Biol Studii. 2017;11(1): 17-24.
8. Atig F, Raffa M, Habib B-A, Kereni A, Saad A., Mounir A. Impact of seminal trace element and glutathione levels on semen quality of Tunisian infertile men. BMC Urol. - 2012;12(6). Availiable from: URL: doi: 10.1186/1471-2490-12-6
9. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva: World Health Organization Press; 2010. 271p.
10. Hamilton TR, de Castro LS, Delgado JdeC, de Assis PM, Siqueira AF, Mendes CM et al. Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status. Reproduction. 2016 Apr;151(4):379-90.
11. Hammadeh M, Hamad M, Montenarh M, Fisher-Hammadeh C. Protamine contents and P1/P2 ratio in human spermatozoa from smokers and non-smokers. Human Reproduction. 2010;25:2708-20.
12. Bodnar OI, Lukashiv OYa, Vinyarska GB, Grubinko VV. Changes of indicators of the oxidative status of rats in experimental type 2 diabetes and their correction by a selenium-chrome-lipid substances from chlorella Vulgaris beij. Med. and clin. chem. 2017 Nov;19(3):71-81.
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ПРООКСИДАНТНО-АНТИОКСИДАНТНА Р1ВНОВАГА В СПЕРМАТОЗО1ДАХ 1НФЕРТИЛЬНИХ ЧОЛОВ1К1В Фафула Р.В., Онуфрович O.K., Ефремова У.П.,
Воробець М.З., Наконечний Й.А., Мельник O.B., Федорович З.Я., Воробець З.Д. Гшерпродукщя активних форм кисню i порушення прооксидантно-антиоксидантно! piBTOBara розглядаються як можливi мехашзми розвитку чоловiчого неплщдя. Метою роботи було оцшити змши прооксидантно-антиоксидантно! piвновaги в сперматозо!дах неплщних чоловшв з piзними формами патоспермш. Виявлено зниження активност ензимiв глутатюново! ланки антиоксидантного захисту, зниження вмюту вщновленого глутатюну та зростання вмюту ТБК-позитивних продукпв у сперматозо!дах шфертильних чоловшв стосовно величин у сперматозо!дах
ПРООКСИДАНТНО-АНТИОКСИДАНТНОЕ РАВНОВЕСИЕ В СПЕРМАТОЗОИДАХ 1НФЕРТИЛЬНЫХ МУЖЧИН Фафула Р.В., Онуфрович Е.К, Ефремова У.П., Воробец М.З., Наконечный И. А., Мельник О.В., Федорович З.Я., Воробец З.Д.
Гиперпродукция активных форм кислорода и нарушения прооксидантно-антиоксидантного равновесия рассматриваются как возможные механизмы развития мужского бесплодия. Целью работы было оценить изменения прооксидантно-антиоксидантного равновесия в сперматозоидах бесплодных мужчин с различными формами патоспермий. Выявлено снижение активности энзимов глутатионового звена антиоксидантной защиты, снижение содержания восстановленного глутатиона и рост содержания ТБК-положительных продуктов в сперматозоидах инфертильних мужчин относительно величин в сперматозоидах мужчин с
чоловшв i3 збереженою фертильнютю. Коефiцieнт антиоксидантного стану розраховували як вщношення суми активностей основних антиоксидантних ензимiв та вщновленого глутатюну до сумарного вмсту продукпв лiпопероксидацii (ТБК-позитивних продукпв i окисненого глутатюну). З'ясовано, що порушення прооксидантно-антиоксидантноi' рiвноваги утворюе наступну патологiчну асоцiйовану (патоспермш) послiдовнiсть (a disorder-associated trend): олiгозооспермiя > олтоастенозооспермш = астенозооспермiя >> лейкоцитоспермш. Однонаправленiсть змiн коефiцieнта антиоксидантного стану за умов патоспермй дозволяе запропонувати його як бютест для оцнки вiльнорадикальних процесiв та опiрностi вшьним радикалам в сперматозо'дах.
Ключовi слова: антиоксидантт ензими, перекисне окислення лтвдв, чоловiче неплщдя, патоспермiя.
Стаття надшшла: 15.03.18 р.
сохраненной фертильностью. Коэффициент антиоксидантного состояния рассчитывали как отношение суммы активностей основных антиоксидантных ферментов и восстановленного глутатиона к суммарного содержания продуктов липопероксидации (ТБК-положительных продуктов и окисленного глутатиона). Установлено, что нарушения прооксидантно-антиоксидантного равновесия образует следующую патологическую ассоциированную (патоспермия) последовательность (a disorder-associated trend): олигозооспермия > олигоастенозооспермия = астенозооспермия >> лейкоцитоспермия. Однонаправленность изменений коэффициента антиоксидантного состояния в условиях патоспермии позволяет предложить его как биотест для оценки свободнорадикальных процессов и сопротивляемости свободным радикалам в сперматозоидах.
Ключевые слова: антиоксидантные ферменты, перекисное окисление липидов, мужское бесплодие, патоспермия.
Рецензент: Саричев Л.П.
doi 10.26724/2079-8334-2018-4-66-124-130 удк 616.12-005.8-036.1-06-08-039.76
П1ДХОДИ ДО ОЦ1НКИ РИЗИК1В ТА ФУНКЦЮНАЛЬНИХ РЕЗЕРВ1В СЕРЦЕВО-СУДИННО! СИСТЕМИ У ХВОРИХ НА 1НФАРКТ М1ОКАРДА З КОМОРБ1ДНОЮ ПАТОЛОГ1еЮ, ЯК1 ПЕРЕБУВАЮТЬ у ГОСТРОМУ ПЕР1ОД1 КАРДЮРЕАБ1Л1ТАЦП
E-mail: [email protected]
З метою вивчення можливостей ильмсно! оцiнки та вiрогiдностi впливу коморбщно! патолоп! на перебiг iнфаркту мюкарда, стан функцiональних резервiв у хворих на шфаркт мiокарда та 11 роль в призначеннi адекватно! шдивiдуалiзовано!' програми реабштацп у дано! категорй пацieнтiв, обстежено 97 хворих, яю проходили гострий (стацiонарний) перiод реабштацп у кардiологiчному вiддiленнi. Встановлено, що параметри загальноприйнятих гемодинамiчних та метаболiчних показникiв у хворих на шфаркт мюкарда з коморбщною патологieю не дозволяють використовувати !х в якост диференцiйно-дiагностичних критерi!в оцiнки функцiональних резервiв серцево-судинно! системи. Одночасно виявлено пряму кореляцшну залежнiсть мiж наявнiстю коморбщного стану та вiком пацieнтiв, тривалютю артерiально!' гшертензп, частотою дихання, рiвнем плазмового креатинiну та iндексом коморбщност Чарльсона, а також обернену достовiрну кореляцiю з лшопроте!'дами високо! густини, 8Р02 та тестом 6-хвилинно! ходьби. Отримаш результати стали обгрунтуванням для стратифжацп ризикiв у пацieнтiв, яга включаються в програму реабiлiтацi!' не лише за традицшними маркерами електрично! та гкмодинамiчно!' стабiльностi серцево-судинно! системи, але i за вищенаведеними прихованими ризик-факторами.
Ключовi слова: iнфаркт мюкарда, коморбщна патологiя, кардiореабiлiтацiя, iндекс Чарльсона, лшопроте'ди високо! густини.
Дане дождження е фрагментом НДР «Комплексный nidxid до контролю cuMnmoMie, безпосереднього i вiддаленoгo прогнозу в умовах кoмoрбiднoг патологи в клшщ внутрштх хвороб та практищ Ымейного лжаря», № державног реестраци 0118U000361.
Формування сучасних негативних медико-демограф1чних тенденцш в Укра'т насамперед вщбуваеться за рахунок серцево-судинних захворювань. Саме вони ютотно впливають на основы показники здоров'я: захворюванють, смертнють, швалщнють, тривалють та яюсть життя населення. За останн роки частка померлих вщ хвороб системи кровооб1гу (ХСК) поступово зростала i досягла в 2015 р. 68,0%. Перше мюце в структур! смертност при ХСК традицшно займае шем1чна хвороба серця (1ХС), питома вага яко' збшьшилась вщ 66,6 в 2005 рощ до 68,9% у 2015 рощ, причому показники смертносп вщ 1ХС за цей перюд серед осб працездатного в1ку залишалися стабшьно високими. Гострий шфаркт мюкарда (1М) на сьогодт залишаеться однею з головних причин смертност, а ризик подальших серцево-судинних ускладнень, включаючи рецидив шфаркту мюкарда, раптову серцеву смерть, серцеву недостаттсть та шсульт для тих, хто переживае гострий шфаркт мюкарда, е ютотними нав1ть у епоху оптимального стратеги реперфузй [1].
Виршення питання надання медично' допомоги патентам з 1ХС часто поглиблюеться наявнютю у таких хворих супутньо' та поеднано' патологи, яка передусм зумовлена подовженням тривалост життя в сучаснш цившзацп. Лшування коморбщного патента потребуе
© М.1. Швед, Л.В.Левицька, 2018
