CC BY
38
ОЗЫК МАКАЛА / ОРИГИНАЛЬНАЯ СТАТЬЯ / ORIGINAL ARTICLE
Материал поступил в редакцию: 13-01-2014 Материал принят к печати: 03-03-2014 УДК 616.3;61:575
Effect of GPX1 Gene Polymorphism Distribution on the Indices of Cellular Adhesion and Functional Condition of the Endothelium in Patients with Chronic Diffuse Liver Diseases
The aim. To study peculiarities of the indices of the cellular adhesion and endothelial functional state in
CO
q: uj ад
Uj §
о
CO I
ic
N
ic u_ О
UJ
Chympoy K.A., Pashkovska N.V. ^
Bukovinian State Medical University, Chernovtsi, Ukraine
UJ
S
patients with CDLD (chronic diffuse liver diseases) depending on Pro197Leu polymorphism GPX1 gene. о
Methods. 28 patients with CDLD aged were examined. We have determined Pro197Leu polymorphism о GPX1 gene, the level of soluble intercellular adhesion molecule - 1 type (ICAM-1), desquamated endothelial cells and the content of nitrogen monoxide (NO) metabolites in the blood serum.
Results. In patients with CDLD there is a relation between the expression of Pro197Leu polymorphism of GPX1 gene and indices of cellular adhesion, which is revealed by a reliably higher content of ICAM-1 in the blood serum in homozygotic carriers of Leu-allele. Changes of the endothelial functional state in patients with chronic diffuse liver diseases are associated with Pro197Leu polymorphism of GPX1 gene, which is proved by a reliably higher index of desquamated endotheliocytes amount and lower level of NO metabolites in the carriers of LeuLeu-genotype.
Conclusions. It was еstablish dependence of level cellular adhesion and endothelial function in patients with chronic diffuse liver disease from gene polymorphism distribution Pro197Leu GPX1.
Key words: chronic diffuse liver disease, polymorphism, intercellular adhesion molecule, endothelium
J Clin Med Kaz 2014;1(31):33-37
Автор для корреспонденции: Чимпой Кристина Андреевна, Буковинский государственный медицинский университет, г. Черновцы, ул. Красноармейская 77Б/14, г.Черновцы, Украина. 58000 Е-mail: [email protected]
БАУЫРДЬЩ СОЗЫЛМАЛЫ ДИФФУЗДЫ АУРУЫ БАР НАУКАСТАРДА СРХ1 ГЕН1 ПОЛИМОРФИЗМ1 ДИСТРИБУЦИЯСЫНЫЦ ЭНДОТЕЛИЙДЩ КЫЗМЕТ ЖАГДАЙЫ МЕН ЖАСУШАЛЫЩ АДГЕЗИЯ К0РСЕТК1ШТЕРШЕ ЭСЕР1 Чимпой К.А., Пашковская Н.В.
Буковинский мемлекептж медициналык университет^ Черновцы к., Украина
Зерттеудщ чаксаты. Бауырдыц созылмалы диффузды ауруы бар наукастарда ОРХ1 геш полиморфизм! дистрибуциясы кезщде эндотелийдщ кызмет жагдайы мен жасушалык адгезия керсетюштерщ зерттеу.
Эдастерь Бауырдыц созылмалы диффузды ауруы бар 28 наукас тексершдь ОРХ1 генщщ полиморфизмщщ Рго197Ьеи, десквамацияланган эндотелиоциттердщ б1рщш1 типт жасушааралык адгезияныц молекулалык децгей жэне кектамыр кан сарысуындагы нитроген монооксидщщ метаболиттер1 к^рамы зерттелдг
Нэтижесь Бауырдыц созылмалы диффузды ауруы бар наукастарда ОРХ1 генщщ полиморфизмщщ Рго197Ьеи экспрессиясы мен жасуша адгезиясыныц керсетюштер1 арасындагы байланыс аныкталды. Ол Ьеи-аллеляныц гомозиготты тасымалдаушыларыныц кан сарысуында 1-шi типт жасушааралык адгезиясыныц жогары децгешмен керщк тапты. Сонымен катар, бауырдыц созылмалы диффузды ауруы бар наукастарда эндотелийдщ кызмет жагдайыныц бузылуы ОРХ1 генщщ Рго197Ьеи полиморфизм1мен байланысы байкалды. Б^л ЬеиЬеи-генотипт тасымал-даушыларда азот оксидщщ метаболиттерщщ темендт мен десквамацияланган эндотелиоциттердщ саныныц жогарылауымен керщк тапты.
Корытынды. Бауырдыц созылмалы диффузды ауруы бар наукастарда ОРХ1 генщщ полиморфизмщщ Рго197Ьеи экспрессиясы мен жасуша адгезиясыныц керсетюштер1 арасындагы байланыс аныкталды.
Мацызды свздер: Бауырдыц созылмалы диффузды аурулары, полиморфизм, жасушааралык адгезия молекуласы, эндотелий
■ ВЛИЯНИЕ ДИСТРИБУЦИИ ПОЛИМОРФИЗМА ГЕНА СРХ1 НА ПОКАЗАТЕЛИ КЛЕТОЧНОЙ АДГЕЗИИ И ФУНКЦИОНАЛЬНОГО СОСТОЯНИЯ ЭНДОТЕЛИЯ У БОЛЬНЫХ ХРОНИЧЕСКИМИ ДИФФУЗНЫМИ ЗАБОЛЕВАНИЯМИ ПЕЧЕНИ Чимпой К.А., Пашковская Н.В.
Буковинский государственный медицинский университет, г. Черновцы, Украина
О С
Д] §
O 41
о
2
S m о о
m
0
■п ^
2 N 2 X
1
СО
2
N3 О
О
с S m
DD
m a
3
Цель исследования. Изучить особенности клеточной адгезии и функционального состояния эндотелия у больных хроническими диффузными заболеваниями печени в зависимости от Рго197Ьеи полиморфизма гена ОРХ1.
Методы. Обследовано 28 больных хроническими диффузными заболеваниями печени. Определен Рго197Ьеи полиморфизма гена ОРХ1, уровень молекулы межклеточной адгезии 1-го типа, десквамированных эндотелиоцитов и содержание метаболитов монооксида нитрогена в сыворотке венозной крови.
Результаты. У больных хроническими диффузными заболеваниями печени установлена связь между экспрессией Рго197Ьеи полиморфизма гена ОРХ1 и показателями клеточной адгезии, которая проявляется достоверно высоким содержанием молекулы межклеточной адгезии 1-го типа в сыворотке крови у гомозиготных носителей Ьеи-аллеля. Изменения функционального состояния эндотелия у больных хроническими диффузными заболеваниями печени ассоциируются с Рго197Ьеи полиморфизмом гена ОРХ1, о чем свидетельствует достоверно выше показатель количества десквамированных эндотелиоцитов и низкий уровень метаболитов оксида азота у носителей ЬеиЬеи-генотипа.
Выводы. Установлено зависимость показателей клеточной адгезии и функционального состояния эндотелия у больных хроническими диффузные заболевания печени от дистрибуции Рго197Ьеи полиморфизма гена ОРХ1.
Ключевые слова: хронические диффузные заболевания печени, полиморфизм, молекула межклеточной адгезии, эндотелий.
INTRODUCTION
Genetic polymorphism is the basis of a phenotype difference of peculiarities, and it can stipulate congenital susceptibility to various diseases. The study of this question draws much attention to the gene coding factors involved in the development of variable pathology [2,12,15].
The analysis of genetic associations plays an important role in the examination of the role of genetic factors involved in the development of polymorphic diseases, and chronic diffuse liver disease (CDLD) in particular. The difference of marker allele frequency in patients with certain pathology and healthy individuals gives the evidence to draw a conclusion about the link between a particular allele and corresponding pathology [12,16]. The information available concerning the links of CDLD pathogenesis
MATERIALS AND METHODS
28 patients with CDLD aged from 25 to 74 (an average age - 52,3±6,09) were included into the study. There were 19 men (67,9%) and 9 women (32,1%), an average duration of the disease was 5,9±1,30 years. The control group included 20 practically healthy individuals (an average age - 52,2±12,15), 13 men (65,0%) and 7 women (35%) among them.
The diagnosis of chronic hepatitis (CH) was made in 13 individuals (46,4%) with an average age of 49,6±8,59. There were 7 men (53,8%) and 6 women (46,2%) among them, an average duration of the disease was 6,0±2,10 years. A mild form of CH was found in 8 patients (28,6%) and moderate form - in 5 patients (17,8%).
Liver cirrhosis (LC) was diagnosed in 15 patients (53,6%) with an average age of 55,0±7,43. Men constituted 11 patients (73,3%), women - 4 (26,7%), an average duration of the disease was 5,7±1,80 years. A mild form of LC was found in 9 patients (32,2%) and moderate form - in 6 (21,4%).
The study was conducted on the basis of the Department of Gastroenterology, Chernivtsi Regional Clinical Hospital.
The diagnoses of CH and LC were made according to the Classification of the World Congress of Gastroenterolo-gists (Los Angeles, 1994, with additions of V Desmet et al, 1995) and specifications of the International Classification of Diseases (ICD) of the 10th revision [4].
allows detecting the range of genes-candidates which potential relation with this pathology needs further investigation.
Due to recent scientific research both of Ukrainian and foreign scientists the concept of relations between indices of cytokine regulation, endothelium functional state and expression of various genes is beyond any doubt [15]. Although dependence of the above indices upon GPX1 Pro-197Leu gene polymorphism in patients with CDLD remains above the attention of researchers.
The aim: Peculiarities of the indices of the cellular adhesion and endothelial functional state in patients with CDLD depending on Pro197Leu polymorphism in GPX1 gene.
CH and LC were verified on the basis of complaints, anamnesis, objective status, common laboratory methods of examination (general clinical blood and urine analyses, biochemical blood test - general bilirubin and its fractions, sublimate and thymol tests, ionogram, proteinogram, co-agulogram). The activity of the following blood enzymes was examined: alaninaminotranspherase (AlAT), aspar-tateaminotranspherase (AsAT), gammaglutamyltransph-erase (GGT), alkali phosphatase (AP). The levels of urea, creatinine were detected in the blood as well as serum markers of hepatitis B and C viruses. Instrumental examinations were conducted (USD of the abdominal organs, esophago-gastroduodenofibroscopy (EGDFS)).
The degree of activity of CH and LC was found on the basis of clinical manifestations and biochemical signs - AlAT, AcAT activity, thymol test, bilirubin level in the blood [5].
The degree of LC compensation was estimated by the criteria of CG. Child and J.G. Turcotte (1964) in the modification of K.N.H. Pugh (1973). The levels of bilirubin, albumins, prothrombin were detected in the blood serum, the presence of ascites and encephalopathy was found [13].
The degree of portal hypertension was determined on the basis of varix dilatation of the lower esophageal portion, subcutaneous veins of the anterior abdominal wall, umbilical veins, splenomegaly, ascites and hepatic encephalopathy [7].
Inclusion criteria were: the age from 25 to 76, diagnosed CH and LC (of a mild and moderate activity) verified by means of clinical, laboratory and instrumental examinations, informed written concern of the patient to participate in the study.
Patients with decompensated LC (III degree of hepatic-cellular failure, hypoalbuminemia less than 30%, III-IV degree of hepatic encephalopathy, resistant ascites, systemic hypotension), chronic hepatitis of a viral etiology, Wilson's disease, congenital Œj-antitripsin insufficiency (Œj-inhibitor of pro-teinases), idiopathic (genetic) hemochromatosis, autoimmune hepatitis, diabetes mellitus, III-IV degree of chronic heart failure with ejection fraction of the left ventricle less than 45%, acute disorders of the cerebral circulation and acute coronary syndrome, psychic disorders, residents of the III-IV zones of radiation contamination, individuals during pregnancy or lactation period or those receiving oral contraceptives, with any acute inflammatory processes, other concomitant decompen-sated diseases or acute conditions able to affect the results of the study, were excluded from the investigation.
Depending on GPX1 gene Pro197Leu polymorphism there were 12 homozygotes by Pro-allele, 8 - by Leu-allele and 8 ProLeu-heterozygotes.
The diagnosis of CDLD was made on the basis of anamnesis, generally accepted complex of clinical-laboratory and instrumental investigation methods, USD of the abdominal organs. Patients with chronic hepatitis and cirrhosis of a viral etiology, Wilson-Konovalov disease, congenital insufficiency of a-antitripsin (a-inhibitor of proteinase), idiopathic (genetic) hemochromatosis, autoimmune hepatitis were excluded from the study.
Alleles of Pro197Leu regions in GPX1 gene were studied by means of excretion of genome DNA from leukocytes of the peripheral blood with further amplification of a polymorphic region by means of polymerase chain reaction (PCR) on the programmed amplificatory "Amply-4L" ("Biocom", Moscow) with individual temperature program for the parameters of every gene. Table 1 presents succession of oligonucleotides in primers and their calculation positions on chromosomes.
Table 1. Succession of oligonucleotides in primers used for polymerase chain reaction (PCR) to identify Pro197Leu polymorphism of GPX1 gene
Gene name Gene localization on chromosome Primer Succession of oligonucleotides in primers
GPX1 3p21 Direct 5'-TCGAAGCCCTGCTGTCTCA-3'
Reverse 5'-CGAGACAGCAGCACTGCAA-3'
CO
q: uj
0Q
Uj §
o
CO I
sc
N
sc u_ O
UJ
o 8
s
o u_
0
1
o: o
DNA extraction was conducted by means of "DNAsorb-B" reagents, variant 100 (Russian) according to the instruction. Purified DNA was kept under the temperature of 20±20C. Samples for PCR were prepared by means of "AmplySense - 200 - 1" set (Russian).
The content of soluble intercellular adhesion molecule - 1 type (ICAM-1) in the blood serum was detected by im-munoenzymatic method with the use of commercial test system "BenderMedSystems" (Austria).
Functional endothelial state was estimated by the content of NO metabolites and the amount of desquamated endothelial cells in the blood. NO content in the blood serum was estimated by the concentration of its final stable metabolite - NO2 and the content of total final metabolites NO (nitrates+nitrites). The method to detect NO2 content in the venous blood plasma is based on the photocolorimetric detection of optic density of NO2 stained complex by Griess test [11]. The amount of desquamated endothelial cells (EC) in the blood was estimated by J.Hladovec method in N.Petrishchev et al. modification [6].
The protocol of examination of the patients was approved during the proceedings on Biomedical Ethics at Bukovinian State Medical University. The document is compiled according to the requirements stipulated by the 6th chapter of CH GCP (1996). While compiling the protocol, the main principles of the Helsinki Declaration on Biomedical Research (1974) adapted during the 41st International
Assembly in Gong Kong (September, 1989) were followed. The protocol corresponds to the basic principles of proper medical practical work such as respect of a personality, awareness of the patient, estimation of the risk of harm and benefit [17,18,19].
Primary findings of patients' examinations were included into the data base in the system of MicrosoftExcel. Formalization, standardizing of the results and statistical analysis were included into the further processing of our findings.
Before checking statistical hypotheses the analysis of regular distribution of the values in randomized surveys by means of detection of asymmetry and excess coefficients with the help of Khan-Shapiro-Wilcky criterion was conducted.
Probability of the difference of an average arithmetic and its errors between the groups of the study was calculated by means of double odd Student t-criterion.
For the data corresponding to normal distribution with equality of general dispersions of sampling checked by means of Fisher-criterion a probable difference was with p<0,05 [8].
Mathematical calculation of the results obtained was conducted on IBM PC Pentium III by means of computer program Primer of Biostatistics, Version 4.03 (S.Glantz, USA) and the standard package of statistical programs of Microsoft Office Excel 2007 [1].
RESULTS
The indices of cellular adhesion and functional endothelial state in patients with CDLD did experience reliable changes depending on
polymorphism of GPX1 gene and were statistically different from the group of practically healthy individuals (table 2).
Table 2. Indices of the endothelial function and fibrinolysis in patients with CDLD depending on Pro197Leu polymorphism of GPX1 gene (M±m)
о с
a §
O 41
о
2
s
m g
о
m
о
■n ^
2
N 2 X ï СО
2
N3 О
О
с s m
DO
m a
3
Index Control group n=20 Genotypes of GPX1 gene, n=28
ProPro, n=12 ProLeu, n=8 LeuLeu, n=8
ICAM-1, ng/mL 259,60+10,324 309,23±12,463 P<0,01 351,38±18,274 P<0,001 P2>0,05 387,41±20,108 P<0,001 P2<0,01 P23>0,05
Stable NO metabolites (NO2, NO3, mcmol/L) 18,14±0,684 13,45±1,002 P<0,01 11,45±1,139 P<0,001 P2>0,05 10,24±1,012 P<0,001 P2<0,05 P23>0,05
Endothelial cells, x 104/L 3,04±0,204 4,63±0,320 P<0,001 5,83±0,549 P<0,001 P2>0,05 6,27±0,625 P<0,001 P2<0,05 P23>0,05
Notes: n- numbers of obseravtions;
Pj - probability of changes concerning the P2 - probability of changes concerning the P3 - probability of changes concerning the
control
group of patients with ProPro-genotype group of patients with ProLeu-genotype
Reliable increase of ICAM-1 content in the blood serum of all the groups concerning the control values was found: for the carriers of ProPro-genotype - on 19,1% (P1<0,001), ProLeu-genotype - on 25,4% (P <0,001) and 49,2% (P1<0,001) for the patients with LeuLeu-genotype. LeuLeu-genotype carriers presented the value of this index on 25,3% higher (Pj<0,001) than that of the patients with ProPro-genotype.
Pro-allele homozygotes revealed reliable decrease of stable NO metabolites in the blood in 1,3 (Pj<0,01) in comparison with the control value, Leu-allele ones - in 1,8 times correspondingly (Pj<0,001), ProLeu-heterozygotes - in 1,6 times (P<0,001). Reliably lower level of NO metabolites (on 23,9%, P<0,001) was found in the blood of LeuLeu-genotype carriers as compared with the patients of ProPro-genotype.
While comparing the index of desquamated endotheliocytes amount in the blood of patients with CDLD depending on Pro197Leu of GPX1 gene with the control value its index has been found to increase in 1,5 times (P<0,001) in the group with ProPro-genotype, in 1,9 times (P<0,001) in the group of patients with ProLeu-genotype, and in 2,1 times (P<0,001) in the group with LeuLeu-genotype. The value of the given index in the blood of LeuLeu-genotype carriers was found to be reliably higher than that in patients with ProPro-genotype on 35,4% (P<0,05).
Thus, homozygotic carrier of Leu-allele in patients with CDLD is associated with a reliable higher level of ICAM-1 in the blood serum, index of endotheliocytemia and lower level of NO stable metabolites.
DISCUSSION
Obtained data is consistent with the results of the last year's research and shows that genetic variation in genes that codes enzymes of the glutathione family affect susceptibility to the occurrence of CDLD, one of which is the pathogenesis of endothelial dysfunction [12,20]. Most researchers have found that certain allelic variants of glutathione peroxides gene may increase the likelihood of oxidative stress.
It is well-known that oxidative stress that accompanies the intensification of peroxidation processes and antioxidant imbalance background of activation is the leading mechanism of liver disease [2,10].
Certain researchers [9,15] indicate that in patients with CDLD homozygotic carrying of Leu-allele gene GPX1 is connected with reliably higher level of general bilirubin and its indirect fraction, as well as bigger activity of aminotrans-pherases.
These results can be explained by increasing free radical oxidation processes in Pro197Leu polymorphism of GPX1 gene carriers [14]. Free radicals are able to directly destroy NO [3]. Increased lipid peroxidation of membranes is the result of damaging the structure of the endothelium
and its violation NO-producing ability. Developing an absolute or relative deficiency of NO, is required for normal regulation of vascular tone. The weakening of the NO-dependent vasodilatatory reactions, in its turn, leads to increased vascular tone, increased blood clots, and as a result, tissue hypoxia. In addition, the reduced inhibitory effect of NO on platelet aggregation, leukocyte adhesion to the endothelium and smooth muscle cell proliferation of the vascular wall, creates prerequisites for vascular disorders. Important role in the pathogenesis of endothelial dysfunction plays adhesion cell growth. Of particular importance in cell migration is given intercellular adhesion molecule type 1 - ICAM-1 [12,14]. It is known that cell adhesion is a violation of not only the development but also to the further progression of endothelial damage [16]. Progression endothelial dysfunction, in turn, leads to tissue hypoxia, disruption of metabolism and infiltration of macrophages subendothelial space, induction of endothelial apoptosis [3,15], which is consistent with our results with respect to the growing number desquamated (circulating) endothelial cells in peripheral blood of patients studied.
CONCLUSIONS
1. In patients with CDLD there is a relation between 2. Changes of the endothelial functional state in patients
the expression of Pro197Leu polymorphism of GPX1 gene with chronic diffuse liver diseases are associated with
and indices of cellular adhesion, which is revealed by a Pro197Leu polymorphism of GPX1 gene, which is proved
reliably higher content of intercellular adhesion molecules by a reliably higher index of desquamated endotheliocytes
of the 1 type (ICAM-1) in the blood serum in homozygotic amount and lower level of NO metabolites in the carriers of
carriers of Leu-allele. LeuLeu-genotype.
REFERENCES
CO
q: uj
OQ
1. Гланц С. Медико-биологическая статистика [пер.с англ.] / С. Гланц. - М.: Практика, 1998. - 459 с. щ
2. Ивашкин В.Т. Клиническая гепатология сегодня и завтра/В.Т. Ивашкин, А.О. Буеверов // Рос. журн. §
гастроэнтерологии, гепатологии, колопроктологии.- 2005.- Т.12., № 1 - С. 4-9.
3. Лазебник Л.Б., Дроздов В.Н., Барышников Е.Н. Роль оксида азота (NO) в этиопатогенезе некоторых заболеваний органов пищеварения / Л.Б. Лазебник, В.Н. Дроздов, Е.Н.Барышников // Эксперимент. и клин. S гастроэнтерол.- 2005.- № 2.- С.4-11.
4. Международная статистическая классификация болезней и проблем, связанных со здоровьем. Десятый пересмотр. (МКБ-10). Т.1 (часть 1). - М.: Медицина, 2003. - Т.1 - 698 с. |
5. Павлов Ч.С. Современные представления о патогенезе, диагностике и лечении фиброза печени / Ч.С. Павлов, ^ Ю.О. Шульпекова, В.Б. Золотаревский, В.Т. Ивашкин // Рос. журн. гастроэнтерол., гепатол., колопроктол.- ^ 2005.- Т. 15, № 2.- С.13-20. £
6. Петрищев Н.Н., Беркович О.А. , Т.Д. Власов Т.Д. [и др.]. Диагностическая ценность определения uj десквамированных эндотелиальных клеток крови. 2001; 1: 50 - 52. о
7. Рациональная диагностика и фармакотерапия заболеваний органов пищеварения / под ред. О.Я. Бабака, Н.В. Q Харченко // «Справочник врача:Гастроэнтеролог». - К.: ТОВ «Доктор Меди», 2007. - 308 с.
8. Реброва О.Ю. Статистический анализ медицинских данных. Применение пакета прикладных программ g STATISTICA / О.Ю. Реброва. - М.: Медиа Сфера, 2002. - 312 с.
9. Чимпой К.А. Змши тирео!дного гомеостазу у хворих на хрошчш дифузш захворювання печшки та патогенетичне обгрунтування !х корекцп: автореф. дис. на здобуття наук. ступеня к. мед. наук: спец. 14.01.02 ^ «Внутршш хвороби» / К.А. Чимпой. - Кшв, 2011. - 17с.
10. Чимпой К.А. Роль про- та антиоксидантно! систем плазми кровi в порушенш тиреовдного гомеостазу у хворих на хрошчш дифузш захворювання печшки. Буковинський медичний вюник. 2010; №1(53): 95 - 97.
11. Чимпой К. А., Пашковська Н.В. Патогенетичне обгрунтування корекцп ендотелiальноi дисфункци у хворих на хрончш дифузш захворювання печшки iз порушенням тирео!дного гомеостазу. Ктшчна та експериментальна патолопя. 2011; №1(35): 177 - 181.
12. Cai H. Hydrogen peroxide regulation of endothelial function: origins, mechanisms, and consequences / H. Cai. // Cardiovasc Res - . 2005 - Vol.68 - P 26-36.
13. Child-Pugh classification dependent alterations in serum leptin levels among cirrhotic patients: a case controlled study // F.F. Bolukbas, C. Bolukbas, M. Horoz [et al.] // Gastroenterol. - 2004. - Vol.23. - Р. 4-23.
14. Cytokine-induced monocyte adhesion to endothelial cells involves platelet-activating factor: Suppression by conjugated linoleic acid / A.A. Sneddon, E. McLeod, K.W. Wahle, J.R. Arthur // Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids. - 2006. - Vol.761. - Р. 793-801.
15. Genotype-activity relationship for Mn-superoxide dismutase, glutathione peroxidase 1 and catatase in humans / M Bastaki, K Huen, P. Manzanillo [et al.] // Pharmacogenet Genomics. - 2006 - Vol.16. - Р. 279-286.
16. Gluathione peroxidase-1 plays a major role in protecting against angiotensin II-induced vascular dysfunction / S. Chrissobolis, S.P. Didion, D.A. Kinzenbaw [et al.] // Hypertension. - 2008. - Vol.51. - Р. 872-877
17. Otte A. Good clinical practice: historical background and key aspects / A. Otte, H. Maier-Lenz, R.A. Dierckx // Nucl. Med. Commun. - 2005. - Vol. 26, №7. - P. 563-574.
18. Portaluppi F. Ethical and methodological standards for laboratory and medical biological rhythm research / F. Portaluppi, Y. Touitou, M.H. Smolensky // Chronobiol. Int. - 2008. - Vol. 25, №6. - P. 999-1016.
19. Rice T.W. The historical, ethical, and legal background of human-subjects research / T.W. Rice // Respir. Care. - 2008. - Vol. 53, №10. - P. 1325-1329.
20. Upregulation of Glutathione Peroxidase Offsets Stretch-Induced Proatherogenic Gene Expression in Human Endothelial Cells Arteriosclerosis, Thrombosis, and Vascular Biology / H. Wagner Andreas, O. Kautz, K. Fricke [et al.] // ATVBAHA. - 2009 - Vol.21. - Р. 316- 325
