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33
k Russian Journal / of Biomechanics
www.biomech.ac.ru
THE RESEARCH IN VIVO OF PEPSIN PROTEOLYTIC ACTIVITY CHANGE UNDER THE INFLUENCE OF VORTICAL MAGNETIC FIELDS WITH D-
AND L-FORM OF CHIRALITY
A.P. Vatorinov*, D.A. Kuznetsov*, T.I. Subbotina*, A.A. Yashin**
* Tula State University, 126, Boldin Street, 300600, Tula, Russia
** Scientific Research Institute of New Medical Technologies, 126, Lenin avenue, 300026, Tula, Russia; e-mail: [email protected]
Abstract. The experimental research of influence of low-frequency vortical magnetic field on Wistar rat organism has been carried out. The influence of the field with right and left rotations of vortex on proteolytic activity of pepsin was investigated. The results showed that the influence of the right rotatable field increases pepsin activity, and the influence of the left rotatable field oppresses pepsin activity. The possible mechanisms of dynamics of pepsin activity have been considered.
Key words: vortical magnetic field, proteolytic activity, pepsin, chirality, gastroenterology, gastrin, pepsinogen
The present article is the continuation of a cycle of works [1, 2] devoted to research of influence of low-frequency vortical magnetic fields (VMF) on alive organism, carried out in scientific cooperation by a team of researchers from Russia (Tula), Ukraine (Dnepropetrovsk) and Austria (Vienna). The object of researches is the study of change of the proteolytic activity of pepsin under the influence of the right and left (D- and Z-form of chirality) directions of rotation of a whirlwind. The given subject of research is of great importance both in the theoretic-biological plan, and for the clinical practice - from the positions of creating new methods of non-medicamental treatment of very widespread gastroenterological diseases.
In work [1] the influence of VMF on proteolytic activity of pepsin in vitro was investigated (the field influenced gastric juice taken at patients suffering from ulcer of the stomach and duodenum, and activity of pepsin was estimated on the final product of digestion of fibers up to aminoacid). The result obtained by the authors [1] is declared as the scientific discovery the essence of which is as follows: the influence of VMF on gastric juice changes proteolytic activity of pepsin, moreover at the right rotation of the field this activity grows, at the left - it is reduced.
However the more objective parameters for clinical practice are obtained from research of VMF influence on the entire organism that is a subject considered in the present article.
Introduction
Basic mechanisms of regulation of pepsin proteolytic activity under the influence of a
vortical magnetic field
It is shown in [3] that chirality of the whole alive world (in particular - of biomolecules, making it) allows to believe that efficiency of electromagnetic fields (EMF) influence on alive organism is determined in many respects by their own polarization (chirality). From the results of research [1] is appears that the D-form (right) spatial symmetry is characteristic for molecular structure of pepsin. Let us briefly consider the basic mechanisms of pepsin activity regulation [4] taking into account that the process of VMF influence on the entire organism is informational, phenomenological [5] in its essence.
The inactive proenzyme (pepsinogen) is synthesized in the main cells of a mucous stomach and is secreted into the stomach cavity. For our investigation it is essential that pepsinogen is also contained in other biological liquids (blood, wetting, sperm, spinal liquid) and it represents a "heavy" biomolecule - with molecular weight 42500 and stability at pH = 6...7.
The terminal inactive part of pepsinogen is formed by six peptid with M = 3100, and the unblocking of the active centre is possible only into strong-acid environment. The inhibitor of the active centre dissociates at pH <5.4 and is digested by active pepsin at pH =3.5...4.0; activation of pepsinogen acquires an auto-catalytic character.
The secretion stimulators of pepsinogen are choline-ergic fibres, the wandering nerve, sympathetic fibres terminating on in fS-adrenoreceptor, gastrin, histamine, secretin, cholecystoclenin (the last two enzymes directly stimulate synthesis of pepsinogen). So the research of VMF influence on the entire organism, instead of in vitro, as shown in [1], allows to "involve" the whole complicated system of pepsinogen secretion.
It is possible to assume as the first approximation that the influence of VMF activates the following mechanisms of pepsinogen secretion amplification:
- increase of Ca 2+ transfer into cells and stimulation of Na +/K AdenosinoThreePhosphatase;
- stimulation of intracellular movement of zymogen granules;
- amplification of activity membrane phospholytase and output of zymogen granules from cells;
- activation of system cyclicGuaninoMonoPhosphate and cyclicAdenosinoMonoPhosphate.
While analyzing the processes of activation it is necessary to take into account various characteristics (M and pHAc ~ conditions of the greatest activation) of seven known pepsinogen fractions - pepsin [4]: pepsin-1 (pi) (M = 43800; pHAc =1.9); p2 (M = 39950, is auto-catalyticly degraded pepsin); p3 (M = 37150; pHAc =2.4 ... 2.8); p4 (pHAC < 3.5); p5 (M = 34600; pHAC =2.8...3.4); p6 (is equivalent p4); p7 (pHAC =3.7...3.9). The named fractions are in complex secretional interrelationship with each other, and VMF influences on the secretional system as a whole.
In the natural process pepsinogen turns into active pepsin after separating (from its polypeptidical chain) of a fragment consisting of 44 amino-acid rests which shield its active centre. Unblocking of the latter is possible only in the strong-acid environment at pH=2 [6] (it is a phenomenological fact), therefore for optimization of auto-catalytic process of activation the sufficient contents of hydrochloric acid in gastric juice is necessary; accordingly, ions H+ are secreted by parietal cells of a mucous stomach. In the result of transmembrane transmission of H+/K+, -grad H+ is present, and hydrolysis of H+/K+-AdenosinoThreePhosphatase serves as a source of energy for it.
The ions of chlorine act in the stomach cavity coming from blood (that allows to assume the effect of VMF influence on transport systems of a microcirculational channel of blood [1]) in exchange for bicarbonic ions, which are formed in the cells of gastric
epithelium: C02 + H20 <-> H2C03 <-» H + HCO3" The exchange is initiated with the help of anion-transport protein. Thus the hydrochloric acid is formed in the stomach cavity.
One of the basic stimulators of parietal cells is gastrin. In reply to gastrin stimulation a lot of gastric juice with high acidity is secreted. Actually gastrin (enzyme) is developed in a mucous stomach and represents polypeptid in two forms (consisting of 34 and 17 aminoacids, respectively). It is small gastrin that most effectively stimulates parietal cells: the speed of HC1 secretion grows as much as 8 times. From positions of biochemical specificity pepsin hydrolyses only peptidal bonds and it is also specific for optical configuration of the aminoacid rests on both sides of the hydrolysed bond:
O
H2N-CH-C
R1
O
•NH-CH-C
R
PEPSIN
0
11
[NH - CH - C] „ — -
1
R"
(1)
Efficiency of pepsin is the highest in relation to dicarbonic acids and aromatic aminoasids. It is considered [4] that presence or absence of the certain charged groups in the certain position in relation to hydrolysed bond serves as the crucial factor of pepsin activity. However it is necessary to take into account that pepsin is active and it concerns the aromatic not charged and not polar groupings, for example tyrosine and phenylalanine.
Thus the considered (in the context of a theme of the article) mechanisms of regulation of pepsin activity give sufficient numbers of degrees of freedom when estimating the effect of VMF influence. The experimental research considered below enables to specify these mechanisms.
Experimental researches: materials and methods
The experiments were carried out in the period from November, 2000 till February, 2001 on pubescent rats - males of a line Wistar at the age of 6 ... 8 months. To create VMF the installations constructed by the author were used.
For 48 hours prior to the beginning of the experiment the rats were operated: gastrostomy was executed to create the conditions of direct collection of gastric juice. The operation was carried out under intramuscular cetaminic narcosis at the rate of O.lml/lOg of weight of an animal. A catheter for intervenal injection such as "butterfly" in author's updated modification has been used to serve as gastrostomic tube. To exclude an opportunity of independent removal of catheter by rat its proximal end was hypodermicly removed onto the back surface of a neck, that simultaneously is the optimum for collecting gastric juice.
While performing gastrostomy the over-middle laporotomy was used as an access. In the area of stomach bottom, on its front surface there was imposed a purse-string serose-muscular suture, with the cut in the centre being carried out, through the end of which the gastrostomic tube was introduced into the cavity of the stomach. Purse-string suture was tightened and in addition - with the purpose of fixing the tube - peritonitical string serose-muscular suture was imposed.
Two series of experiments (each consisting of five tests) were carried out. In the first series D - VMF (right-adjustable) influenced on rat, in the second - Z-VMF (left-adjustable). In all these cases the digesting activity of pepsin was investigated on an empty stomach. For the control a portion of gastric juice taken at experimental rat directly before the beginning of the experiment was used. Then the rat was placed into the working chamber of the installation
to be subjected to the influence of VMF for 15 minutes. Directly after this session a portion of gastric juice was collected.
The research of enzyme-generative function was carried out in parallel by two methods: by the unified Tugolukov's method, based on estimation of proteolytic activity of pepsin by the amount of the split protein of plasma, and unified method of Anison-Mirskoy in Chernikov modification, based on pepsin ability to split molecular haemoglobin [4].
Numerical estimation of experimental results
The control research of proteolytic activity of gastric juice in the first series of experiment has allowed to establish that digesting activity of pepsin in relation to proteins of blood plasma makes up from 0.027 mg up to 0.04 mg, the average activity - 0.032. After influence of D-VMF the digesting activity has increased and reached 0.04...0.08 mg, the average - 0.053 mg. The correlation of average parameters of protelytic activity of pepsin in relation to proteins of plasma in the control stage is shown in Fig. la.
The definition of protelytic activity of pepsin in relation to standard haemoglobin has also revealed the increase of enzyme activity under the influence of D-VMF; the activity of pepsin in the control has made up 0.6... 1.2 mcg/1, the mean value - 0.66 mcg/1; in the experiment the activity of pepsin has increased up to 0.9... 1.9 mcg/1, the mean value - 1.29 mcg/1 (Fig. lb). In Table 1 the parameters of proteolytic activity of pepsin, received in the samples of gastric juice in the first series of experiment (D-VMF) are submitted.
In the second series of experiments Z-VMF was used; the digesting activity of pepsin in relation to plasma in the control has made up 0.045...0.06 mg, the mean value - 0.05 mg; in the experiment the proteolytic activity decreases up to 0.04...0.05 mg, the mean value -0.044 mg (Fig. lc) was observed.
0.053
D-VMF a)
mcg/1-i .5
D-VMF b)
0.044
mcg/1
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 -I 0
0.75
0.63
□ Control
□ Experience
L-VMF
L-VMF
c) d)
Fig. 1. The correlation of average parameters of protelytic activity of pepsin in experiment and control.
78
Table 1. Parameters of proteolytic activity of pepsin (Z)-VMF).
№ of test Method of Tugolukov Method of Anison-Mirskoy in Chernikov modification
Control Experiment Single test Control Experiment
1 0.045 0.05 0.2 0.8 0.9
2 0.027 0.04 0.2 0.6 0.85
3 0.04 0.08 0.2 0.1 1.3
4 0.03 0.04 0.25 0.6 1.5
5 0.03 0.05 0.2 1.2 1.9
Mean value 0.032 0.053 0.21 0.66 1.29
Note: spread in values in some cases being rather significant ( îowever there is an expressed
tendency); it is explained by the change of the position of an experimental animal and natural variation of physiological norms of organism.
Table 2. Parameters of proteolytic activity of pepsin (Z-VMF).
№ of test Method of Tugolukov Method of Anison-Mirskoy in Chernikov modification
Control Experiment Single test Control Experiment
1 0.06 0.05 0.2 0.8 0.7
2 0.045 0.04 0.2 0.85 0.5
3 0.045 0.04 0.18 0.58 0.52
4 0.05 0.045 0.2 0.75 0.7
5 0.05 0.045 0.21 0.8 0.75
Mean value 0.05 0.044 0.19 0.75 0.63
Defining proteolytic activity of pepsin in relation to standard haemoglobin the similar dynamics of digesting activity of enzyme has been revealed. In control portions of gastric juice the digesting activity of pepsin has made up 0.58...0.8 mcg/1, the mean value - 0.75 mcg/1. In the experiment the decrease of activity has made up 0.52...0.75 mcg/1, the mean value - 0.63 mcg/1 (Fig. Id).
In Table 2 the parameters of proteolytic activity of pepsin obtained in tests of gastric juice in the second series of experiment (Z-VMF) are submitted.
Comparing dynamics of pepsin activity change under the influence on an experimental animal of D - VMF and L - VMF it is possible to note that in the second case (at the obviously expressed tendency) these parameters are lower that coincides with results in vitro in [1]. It can be explained logically: any vectorized process (biological, physical, etc.) under the influence of the external factor has expressed dynamics at positive, that is coinciding with a vector of process, gradient of external influence and it has got reduced dynamics at a negative gradient. In the present situation the process of activization of pepsin is connected with the right-hand symmetry of molecules of that enzyme, therefore grad D-VMF > grad L-VMF can be observed in experiment. The other subjective moment is as follows: the influence of Z-VMF in experiment was carried out by the generator with smaller intensity of a field (see the photo in above mentioned article).
Analysis of results of research
Revealed in [1] in vitro and in the present work in vivo biological effects formed as a consequence of influence of VMF with D- and ¿-forms of chirality on alive organism testify to decisive significance of the form of chirality in formation of the feed-back physiological or pathological reaction. The change of proteolytic activity of enzymes of gastric juice confirms the given data [1, 2] about the opportunity of direct influence of VMF on subcellular, including molecular, structure. The dependence of pepsin activity from the form of chirality gives evidence about occurrence of feed-back reactions not only on organismal or fabric levels, but also at a molecular regulation level of metabolism processes, as catalytic activity of enzymes is involved.
In the present research the authors did not set the goal of solving the task concerning subtle mechanisms of catalytic activity change. Nevertheless the following explanations of mechanisms of running processes probably are not deprived of the certain degree of correctness.
Considering the increase of pepsin activity in experiment it is possible to assert about displacement of pH-balance for this type:
HC1<->H+ + C1
That is in this variant the increase of H+ concentration initially occurs; it serves a sublimator for HC1. The increase of FT can be connected with change of an orientation of hydrogen bonds under the action of grad Z)-VMF. The durability of hydrogen bonds is maximal in case when all atoms involved in it are located along one straight line:
—(h>-(H>...-(H>-
Water of intercellular matrix of organism is most likely to be the source of H+ in this case; it is characterized by the least expressed electrical dipole moment.
In a molecule of water the atom of oxygen gets a partially negative charge and atoms of hydrogen - partially positive charge. In norm each molecule of H2O is connected with four other molecules as the atom of oxygen participates in formation of two hydrogen bonds, and the atom of hydrogen - in one. Other molecules can serve a source of H+, in which H+ is covalently attached to electronegative atoms. At last both extracellular and intracellular matrix water can be a source of H+.
There is sense in such a reason: active, not dependent from concentration of H+ and correspondingly from the meaning of pH, the transformation of pepsinogen into pepsin under the action of VMF can be a consequence of breaking in a molecular circuit (1):
H2N - CH - CO -NH----NH - CH - COOH (2)
! ??!
R (break of chain) R1
The breaking of bonds in (2) from the positions of physicochemical sciences can be associated with even larger right-hand curling of biomolecules.
As it concerns the variant of stimulation of gastrin synthesis in the chain: gastrin -» pepsinogen —» pepsin, such mechanism is less probable, as to start and realize this process, in
view of its biochemical inertia, we need quite a long period of an exposition, anyway much more than 15 minutes of the present experiment. Besides this mechanism is impossible in experiment in vitro, that is excluded by results of research in [1].
Conclusions
Results of research of VMF influence on bio systems submitted in [1 2] and in the present work actually create a new and extremely effective trend in magnetotherapy. The experimental confirmation of pepsin activation at the right rotation of VMF and its oppression at the left rotation in experiments in vitro and in vivo have already now allowed to use this effect for treatment of gastroenterologic diseases such as ulcer of the stomach and ulcer of the duodenum [2]. However the perspectives of application of vortical magnetotherapy are not limited by the given nosological form. Now the authors carry out the experimental research on the influence of VMF on liver, pancreas and other organs functioning.
Prospective mechanisms of pepsin activation under the influence of D-VMF have been briefly considered in this article. They do not give any unequivocal explanation. Therefore the elucidation of physicochemical and biochemical mechanisms of VMF influence on alive organism is also of paramount importance.
References
1. ЖИТНИК H.E., НОВИЦКИ Я.В., ПРИВАЛОВ В Н., РУДЕНКО А.И., СОКОЛОВСКИЙ С.И., ФИЛИППОВ Ю.А., ФИЛИППОВА А.Ю., ЯШИН А.А. Вихревые магнитные поля в медицине и биологии. Вестник новых медицинских технологий, 7(1): 46-57, 2000 (in Russian).
2. ЖИТНИК Н.Е., ПРИВАЛОВ В.Н., РУДЕНКО А.И., СОКОЛОВСКИЙ С.И., ФИЛИППОВ Ю.А., ХОМЕНКО Е.Н., ЯШИН А.А. Вихревые магнитные поля в практике лечения гастроэнтерологических заболеваний. Вестник новых медицинских технологий, 7(3-4): 99-100, 2000 (in Russian).
3. ЯШИН А.А. Четвертое измерение в конструктивной физике живого: эффекты киральности в биологии. Вестник новых медицинских технологий, 7(2): 50-55, 2000 (in Russian).
4. ДИКСОН М., УЭББ Э. Ферменты: Пер. с англ. Москва, Мир, 1982. Тт. 1-3 (in Russian).
5. СУДАКОВ К.М. Информационный феномен жизнедеятельности. Москва, изд-во РМА ПО, 1999 (in Russian).
6. АНДРЕЕВА Н.С. Зачем и почему пепсин стабилен и активен при рН2. Молекулярная биология, 28(6): 1400-1406, 1994 (in Russian).
ИССЛЕДОВАНИЕ ИЗМЕНЕНИЯ ПРОТЕОЛИТИЧЕСКОЙ АКТИВНОСТИ ПЕПСИНА ПРИ ВОЗДЕЙСТВИИ ВИХРЕВЫХ МАГНИТНЫХ ПОЛЕЙ С О- И L- ФОРМАМИ КИРАЛЬНОСТИ IN VIVO
А.П. Ваторинов, Д.А. Кузнецов, Т.И. Субботина, А.А. Яшин (Тула, Россия)
Исследовано воздействие вихревых магнитных полей (ВМП) на живой организм на примере крыс линии Wistar. Объектом исследования является изучение изменения протеолитической активности пепсина при воздействии ВМП с правым и левым (£>- и ¿-формы киральности) направлением вращения вихря. Это имеет важное значение как в плане теоретической биофизики и биомеханики, так и для клинической практики: с точки зрения создания новых методов немедикаментозного лечения (магнитотерапия) столь распространенных гастроэнтерологических заболеваний типа язвенной болезни желудка и двенадцатиперстной кишки.
Проанализированы основные механизмы регуляции протеолитической активности пепсина при воздействии ВМП, исходя из биохимических свойств фермента пепсиногена. Выполнены экспериментальные исследования магнитной установки авторской разработки и гастростомической трубки-катетера в авторской модификации введения.
В результате экспериментов установлено, что после воздействия D-ВМП активность пепсина значительно возрастала, а после воздействия L-BM1I - также заметно снижалась. Численная оценка при обработке проб желудочного сока (по методу Туголукова и по методу Анисона-Мирского в модификации Черникова) показала, что результаты являются достоверными. Дано обоснование полученных результатов с биохимической и биомеханической точек зрения. Библ. 6.
Ключевые слова: вихревое магнитное поле, протеолитическая активность, пепсин, киральность, гастроэнтерология, гастрин, пепсиноген
Received 15 May 2001
