CC BY
73
© Ye. O. Posokhov, A. S. Tkachenko*, Ye. M. Korniyenko**
UDC 547. 787. 2 + 535. 33/. 34
Ye. O. Posokhov, A. S. Tkachenko*, Ye. M. Korniyenko**
Influence of Carrageenan (E 407) on the Membrane of Enterocytes Investigated by Fluorescent Probes Research Institute of Chemistry (Kharkiv)
*Kharkiv National Medical University (Kharkiv) **V. N. Karazin Kharkiv National University (Kharkiv)
The results presented in this article were obtained in following research «Investigation of long-term effects of regular consumption of foods containing genetically modified organisms in damaged epithelial barrier of the gastrointestinal tract» (state registration number 0110 U000653).
Introduction. Food additive E 407 (carrageenan) is widely used in the modern food industry. It is sulfated polysaccharide, which is produced by extraction from red seaweed. Carrageenan serves as natural thickener in dairy, confectionery and meat products. Moreover, in experimental medicine carrageenan is widely used to model the inflammatory processes. It has been used in the pathophysiology for modeling of peritonitis, pleurisy, arthritis and carrageenan-induced edema of the limbs in rats [6, 13]. Carrageenan-induced inflammation, originally described by Winter, is an acute, non-immune and easily reproducible. Edema and erythema, hyperalgesia develop immediately after subcutaneous injection of carrageenan and they are the result of pro-inflammatory agents such as bradykinin, histamine, tachykinins, components of the complement system, reactive oxygen and nitrogen species [6]. There is an evidence of oncogenic transformation of cells under the influence of carrageenan, the positive correlation between the use of products with this additive in the diet and an increased risk of breast carcinoma has been found [17]. Also the model of ulcero-necrotizing carrageenan-induced gastroenterocolitis has been reported [18]. In this connection, the question about the safety of carrageenan as a food additive arises. In modern literature, there are no reliable data about the effect of systematic consumption of carrageenan neither by an adult nor by child or by pregnant women. A model of moderate chronic carrageenan gastroenterocolitis without necrotizing process was elaborated at Kharkiv National Medical University on the basis of the model of necrotizing carrageenan-induced gastroenterocolitis.
At the same time, the mechanism of carrageenan action on enterocyte biomembranes during continious consumption remains unexplored. It is very urgent task, because of the spread of food products containing carrageenan in the diet of the consumers. The investigation of the mechanisms of biomembranes damage will help to develop regulation rules of carrageenan use in food.
The purpose of the research was to study the influence of carrageenan (E 407) on the lipid membrane of enterocytes in rats with fluorescent probes (ortho-hydroxy derivatives of oxazole), which non-covalently bind to cell membranes of experimental animals and have a quick response to changes in their microenvironment [3-5].
Subjects and methods. The female Wistar rats were used for the experiment. Chronic carrageenan-induced gastroenterocolitis was reproduced by the free access of animals to 1 % solution of carrageenan in drinking water. Laboratory animals were divided into 2 groups. Group № 1 consisted of intact animals, group № 2 consisted of experimental animals with chronic carrageenan-induced gastroenterocolitis. A month after the start of the experiment animals were taken from the experiment by decapitation. Manipulations with animals were carried out in accordance with the provisions of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, 1986). The intestine was removed and placed on the cold immediately after decapitation of rats. Intestinal perfusion was performed using cooled physiological solution. Epithelial cells were detached by scraping the inner surface of the intestine by anatomical knife. A suspension of epithelial cells was made in Tris-HCl buffer (pH 7. 4). Fluorescent probes were dissolved in acetonitrile to the initial concentration of 2x10-4 mol / l. 10 |il of each corresponding probe was added to the suspension of enterocytes. The final concentration of each probe in the suspensions of investigated membranes is 1x10-6 mol/l, thus, the molar ratio of lipid / probe was 1000:1. Then, we measured the fluorescence probes O1O (2 - (2 -OH-phenyl)-5-phenyl-1,3-oxazole) and PH7 (2 - (2 -OH-phenyl)-phenanthrene [10,11] -1,3-oxa-zole) in physiological solutions containing enterocytes of rats with chronic carrageenan-induced gastroen-terocolitis. Measurement of the fluorescence was performed by spectrofluorometer «Hitachi F4010» after 1 hour after the addition of probes to a solution of cells. The fluorescence spectra of probes were measured in the diapason of 340-600 nm with monochromator slit width of excitation and fluorescence 5 and 5 nm, respectively, and the excitation wavelength of 330 nm. The enterocytes of intact healthy animals were used as
Fig. 1. Scheme of excited state proton transfer (ESIPT) reaction in 2 - (2-OH-phenyl)-5-phenyl-1,3-oxazole (probe O1O). Upwards arrow shows the electronic excitation and downwards arrow represents the light emission (fluorescence). Corresponding maxima of fluorescence are measured in nanometers.
a control sample. The fluorescent probes, successfully used in the past for studies of biological membranes [3-5]: 2 - (2 -OH-phenyl)-5-phenyl-1,3-oxazole (probe O1O) and 2 - (2 -OH-phenyl)-phenanthrene [10,11] -1,3-oxazole (probe PH7), were used for investigation of carrageenan influence on the membrane of en-terocytes. The choice of fluorescent probes O1O, PH7 (ortho-hydroxy 2,5-diaryl-1,3-oxazole) for investigation of carrageenan influence on physico-chemical properties of biological membranes was due to the fact that the fluorescent characteristics of the probes depend on the physico-chemical properties of their microenvironment: hydrogen bonding ability (i. e., the ability to form hydrogen bonds), the polarity and viscosity of the microenvironment [2-5, 8-10].
Results and discussion. It has been known that in the excited state the ortho-hydroxy 2,5-diaryl-1,3-ox-azole is characterized by excited state proton transfer (ESIPT) reaction (Fig. 1): hydroxyl group in the orthoposition of the lateral benzene ring acts as protonodonor and the nitrogen atom of oxazole ring acts as proton acceptor [2, 8-10]. The result of the ESIPT reaction is the formation of phototautomeric form (T*), fluorescent in significantly longer wavelengths in comparison with the initial form (N*) [2, 8-10].
The presence of two-band fluorescence allows to perform ratiometric measurement, i. e. to use the ratio of phototautomeric form and the initial form fluorescence intensities (IT./IN.) as a parameter for evaluation
Fig. 2. Expected location and orientation of fluorescent probes O1O and PH7 based on their fluorescence properties in lipid membranes [3-5] and on the basis of their structural similarity with fluorescent probes with known localization in lipid membranes [15]. Two molecules of phosphatidylcholine from the outer monolayer are showed to denote the localization of the probes.
of the physical and chemical properties of the microenvironment. The use of ratiometric fluorescent probes allows to exclude the measurement error associated with the deviation of the fluorescent probe concentration (e. g., uneven distribution of the fluorescent probe in various membranes) and the measurement error associated with a deviation of fluorescence technique settings (deviation of the intensity of the exciting source, a change in focus, changes in the sensitivity of the photodetector, etc.) [10].
Compounds that differ in their lipophilicity [3-5] were selected for the present study. It is expected that the regions of localization of selected probes in the membrane are different and correspond to the lipophi-licity of the probes (Fig. 2) [1, 3-5]. Expected location and orientation of O1O and PH7 in lipid membranes is based on their fluorescence properties in lipid membranes [3-5] and on the basis of their structural similarity with fluorescent probes with known localization in lipid membranes [1]. The location of the probes in lipid membrabes: probe O1O is located in the region of glycerol residues of phospholipids and in the region of carbonyl groups of phospholipids; probe PH7 is located in the region of methylene chains of phospholipids and in the center of the bilayer (Fig. 2).
A noticeable increase in the intensity and hypso-chromic (i. e. short-wavelength) shift of the lonwav-elength fluorescence band of probe O1O tautomeric
Table
Fluorescence intensity of probes O1O and PH7 in enterocyte membranes of animals with
carrageenan-induced gastroenterocolitis
Sample Fluorescence intensitv.a. u.
Probe 010 Probe PH7
375 nm 470 nm 425 nm 485 nm
Control 5.0 23.7 4.7 67.4 124.1 1.8
Gastroenterocolitis 5.3 79.4 15.0 64.6 117.9 1.8
350 400 450 500 550 600 650
Wavelength, nm
Fig. 3. The fluorescence spectra of the probe O1O in solutions containing enterocytes: (a) control (solid line), (b) the animals with carrageenan-induced gastroentero-colitis (dashed line).
form (IT*) was observed as a result of carrageenan action on enterocytes, at the same time, the intensity of the fluorescence band of the probe O1O normal form (IN*) was almost unchanged (Table, Fig. 3). Thus, the intensity ratio of the tautomeric and the initial forms IT*/ IN* of probe O1O increases under the influence of car-rageenan (E 407) (Fig. 4).
The increase of the intensity of the tautomeric form fluorescence band (IT*), hypsochromic (i. e. short-wave-length) longwavelength shift of the tautomeric form fluorescence band and increased ratio of IT./IN. for O1O probe indicates a decrease in polarity and hydrogen-bonding ability of the microenvironment of probe O1O in the enterocyte membranes of rats with gastroenteroco-litis. Such decrease in polarity and hydrogen-bonding ability of the microenvironment of probe O1O indicates
Fig. 4. The ratio of the tautomeric and the initial form intensities IT./IN. for O1O probe in the membranes of enterocytes.
a decrease in hydration of the probe microenvironment in the erythrocyte membranes of the experimental group.
Taking into consideration the emulsifying properties of carrageenan [14, 16], the discussed reduction of hydration might be caused by the possible accumulation of carrageenan (E 407) in the regions of probe O1O localization may increase the microviscosity of the lipid bilayer and, therefore, may lead to increase of orderliness of the membrane phospholipid molecules (i. e. may lead to the increase in membrane lipid order), which in turn, may contribute in dehydration of the lipid bilayer [7, 11, 12]. According to literature [7, 12], the similar dehydration of the lipid membrane due to the increase in orderliness of the phospholipid molecules (i. e. due to the increase in membrane lipid order) is observed in case of cholesterol and its esters accumulation in the lipid bilayer. At the same time, in case of the probe pH 7 no significant change was found in its fluorescence parameters under the influence of carrageenan (E 407) on enterocytes (Table). The absence of changes in the regions of the probe pH7 localization (i. e., in the rather hydrophobic regions of the lipid bilayer) may be explained by the fact that, carrageenan, the structure of which have polar hydroxyl and charged sulfonyl groups, is localized in more polar regions of the lipid bilayer.
Thus, it was shown that fluorescent probe O1O (2 -(2 -OH-phenyl)-5-phenyl-1,3-oxazole) might be used as an indicator for estimation of the nature and types of changes in the structure of enterocyte membranes of rats under the influence of carrageenan (E 407). It was found that the changes in the membranes of enterocytes under the influence of carrageenan occurred in the regions of probe O1O localization, i. e. in quite polar regions of the membrane: presumably, in the region of glycerol residues of phospholipids and in the region of the carbonyl groups of phospholipids. The influence of carrageenan (E 407) didn’t lead to changes in the regions of probe pH 7 membrane localization, i. e. in a more hydrophobic region of the enterocyte membranes: presumably, in the in the region of methylene chains of the phospholipids and in the bilayer center.
It has been known that the absorption of essential nutritional factors such as amino acids, glucose, vitamins takes place in the small intestine. Disturbance of enterocyte membrane, which occurs under the influence of carrageenan, reduces the absorption of nutrients in the gastrointestinal tract, which may significantly aggravate the course of gastroenterocolitis. In addition, carrageenan-induced disorder of enterocyte transport function may be explained by its possible effect on the structure of protein transporters localized at the apical surface of enterocytes, which indicates the impact of a multi-vector influence of carrageenan on the morpho-functional state of gastrointestinal tarct in experimental animals.
Conclusions.
1. The consumption of carrageenan leads to the following structural changes in the enterocyte membranes of experimental animals: to increase in microviscosity of the lipid bilayer, to increase in the orderliness of
PROBE O1O, ENTHEROCYTES + E407
phospholipid molecules in the membrane (i. e. due to the increase in membrane lipid order) and to dehydration of membranes.
2. The use of fluorescent probes (ortho-hydroxy oxazole derivatives) allows to estimate the type and nature of the damaging effect of carrageenan on the structural components of membranes. Thus, the mentioned method, using ortho-hydroxy oxazole derivatives
as fluorescent probes, may be suggested for application in the practical medicine for the study of the negative effects on the human body similar to carrageenan food supplements.
Prospects for further research. Our data substantiate the importance of research aimed at understanding the mechanisms of action of food additives and to evaluate their safety for human health.
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УДК 547. 787. 2 + 535. 33I. 34
ДОСЛІДЖЕННЯ ВПЛИВУ КАРАГЕНІНУ (Е 407) НА МЕМБРАНИ ЕНТЕРОЦИТІВ МЕТОДОМ ФЛУОРЕСЦЕНТНИХ ЗОНДІВ
Посохов Є. О., Ткаченко А. С., Корніенко Є. М.
Резюме. За дoпoмoгoю флyopecцeнтниx зoндiв - opтo-гiдpoкcипoxiдниx oкcaзoлy в eкcпepимeнтi на щypax вивчеі-io вплив на cтpyктypи iweiv^paH eнтepoцитiв твapин дoдaнoгo дo їх pa^oHy кappaгeнiнy (Е 407). Bcтaнoвлeнo, i!o кappaгeнiн змінює гiдpaтaцiю iweiv^paH eнтepoцитiв eкcпepимeнтaльниx твapин. Пoкaзaнo, i!o під впливoм кappaгeнiнy (Е 407) вiдбyвaютьcя зміни y пoляpниx oблacтяx бioмeмбpaн, в той же ча^ вплив цієї xapчoвoЇ дoбaвки не пpизвoдить дo змін y гiдpoфoбниx дiлянкax мeмбpaн eнтepoцитiв. Xapaicrep виявлeниx змін дoзвoляє зpoбити виcнoвoк npo збільшення мiкpoв’язкocтi мeмбpaн eнтepoцитiв щypiв з xpoнiчним кappaгeнiн-iндyцipoвaннм гacтpoeнтepoкoлiтoм.
Ключові слова: eнтepoцит, бioмeмбpaнa, кappaгeнiн, флyopecцeнтнi зoнди.
УДК 547. 787. 2 + 535. 33I. 34
ИССЛЕДОВАНИЕ ВЛИЯНИЯ КАРРАГЕНИНА (Е 407) НА МЕМБРАНЫ ЭНТЕРОЦИТОВ МЕТОДОМ ФЛУОРЕСЦЕНТНЫХ ЗОНДОВ
Посохов Е. А., Ткаченко А. С., Корниенко Е. М.
Резюме. Г^и пoмoщи флyopecцeнтныx зoндoв - opтo-гидpoкcипpoизвoдныx oкcaзoлa в экcпepимeн-те на кpыcax изyчeнo влияние на cтpyктypы мeмбpaн энтepoцитoв живoтныx дoбaвлeннoгo в иx paциoн кappaгeнинa (Е 407). Уcтaнoвлeнo, чтo кappaгeнин изменяет гидpaтaцию мeмбpaн энтepoцитoв экcпepи-мeнтaльныx живoтныx. Пoкaзaнo, чтo гюд влиянием кappaгeнинa (Е 407) пpoиcxoдят изменения в пoляp-ныx oблacтяx биoмeмбpaн, в тo же вpeмя, вoздeйcтвиe этoй пищeвoй дoбaвки не пpивoдит к изменениям в гидpoфoбныx yчacткax мeмбpaн энтepoцитoв. Xapaicrep выявленные изменений пoзвoляeт cдeлaть вывoд o6 увєличєнии микpoвязкocти мeмбpaн энтepoцитoв фью c xpoничecким кappaгeнин-индyциpoвaннм гacтpoэнтepoкoлитoм.
Ключевые слова: энтepoциты, биoмeмбpaнa, кappaгeнин, флyopecцeнтныe зoнды.
UDC 547. 787. 2 + 535. 33I. 34
influence of Carrageenan (E 407) on the Membrane of Enterocytes investigated by Fluorescent Probes
Posokhov Ye. O., Tkachenko A. S., Korniyenko Ye. M.
Summary. The influence on enterocytes membrane structure of added to the diet of rats food-additive carrageenan (E 407) was studied using fluorescent probes (derivatives of ortho-hydroxy oxazole). It was found that carrageenan changed hydration membrane of enterocytes in experimental animals. It was shown that carrageenan (E 407) caused changes in the polar regions of biological membranes and at the same time, the consumption of this food-additive didn’t lead to any changes in the hydrophobic parts of the membrane of enterocytes. Nature of detected changes allows to make a conclusion that enterocyte membranes in rats with chronic carrageenan-induced gastroenterocolitis have increased microviscosity.
Key words: enterocytes, biomembrane, carrageenan, fluorescent probes.
Стаття надійшла 15. 01. 2013 р.
Рецензент - проф. Костенко В. О.
