Influence of low intensity laser radiation on different biological systems [Elektronische Ressource] / vorgelegt von Olga Tsivunchyk

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Influence of low intensity laser radiation on different biological systems Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Chemie der Philipps-Universität Marburg vorgelegt von Olga Tsivunchyk aus Grodno Marburg/Lahn 2003 Vom Fachbereich Chemie der Philipps-Universität Marburg als Dissertation am 15.1.2004 angenommen Erstgutachter: Prof. Dr. H. Bäßler Zweitgutachter: Prof. Dr. M. Hofrichter Tag der Disputation am 16.1.2004 2 Sincere Gratitude My work would be not done without support of these people and here I convey all my cordial gratitude to them. I am truly thankful to Prof. Dr. H. Bäßler for the given possibility to finish my work under his leadership; for used laser equipment and working place; for advising and help in preparing of discussion; for his understanding and taken responsibility at my Ph.D. work. I do consider my work under his supervision as a great honour for me and appreciate it very much to Prof. Dr. M. Hofrichter from Zittau for his agreement to take co-supervision at my work; for his advising concerning experiments and writing of thesis structure; for kind hospitality visiting Zittau to Prof. Dr. D.

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Influence of low intensity laser radiation on
different biological systems




Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)



dem
Fachbereich Chemie
der Philipps-Universität Marburg
vorgelegt von


Olga Tsivunchyk


aus Grodno


Marburg/Lahn 2003
Vom Fachbereich Chemie

der Philipps-Universität Marburg

als Dissertation am 15.1.2004 angenommen




Erstgutachter: Prof. Dr. H. Bäßler
Zweitgutachter: Prof. Dr. M. Hofrichter




Tag der Disputation am 16.1.2004



















2 Sincere Gratitude

My work would be not done without support of these people and here I convey all my cordial
gratitude to them. I am truly thankful

to Prof. Dr. H. Bäßler for the given possibility to finish my work under his leadership; for
used laser equipment and working place; for advising and help in preparing of discussion; for
his understanding and taken responsibility at my Ph.D. work. I do consider my work under his
supervision as a great honour for me and appreciate it very much

to Prof. Dr. M. Hofrichter from Zittau for his agreement to take co-supervision at my work;
for his advising concerning experiments and writing of thesis structure; for kind hospitality
visiting Zittau

to Prof. Dr. D. Gemsa for the given opportunity to work in his working team; for his
leadership during my DAAD study in Philipps-University of Marburg and all help for it

to Prof. Dr. L.-O. Essen for the help and advises in preparing of discussion topic and
presentation of my thesis

to Prof. Dr. P. Galland for the help and consulting in preparation of discussion topic

to Dr. E. von Löw for the great help in laboratory work; for very nice and pleasant
atmosphere in joint co-operation; for his help in literature study and friendly support

to Dipl. Biol. R. Kottke for teaching and help in practical work in microbiological laboratory

to Dr. T. Khomich for time of training in her working group in Institute of biochemistry; for
advices in planning of experiments

to Dr. K. Mandrik for teaching and help in work with plant tissues

3 to Irina Osakovich for her help in biochemical laboratory and work with experimental
animals

to Dmitry Zverev for his help in physical laboratory, with data processing and for our
friendship

to Dr. Y. Romanovsky for his help in work with laser equipment during experiments
performance

to Prof. Dr. A. Rubinov for his help in preparing of discussion topic

to Prof. Dr. R. Frey from Brandenburg Technical University Cottbus, for his interest towards
my work and help in continuation in Marburg

to Claudia and Andreas Karber for their help in cases of technical PC-problems



to my parents and family for possibility to study further more and make researches,
especially to my father, who had shown me the world of science


to my dear husband Andrei and our son Eugen for their great patience and understanding
of my work; for lovely support and home feeling being far away from each other


to my best friend Dr. R. Haas for the given power to finish this work; for his help
overcoming difficulties and problems; for best and worst time that we have spent in fun and
work together.

4 Index
page
Zusammenfassung 8
1 Introduction 9
1.1 Background 9
1.2 Lasers
1.2.1 General description 9
1.2.2 Quantum Properties of Light 11
1.2.3 Stimulated Emission 11
1.2.4 Characteristics of Laser Light 12
1.2.5 Kinds of lasers 13
1.3 Biological antioxidant system 14
1.3.1 General overview 14
1.3.2 system 15

2 Literature review: Physiological and biochemical effects of laser light 16
2.1 Effects of LILI in general 16
2.2 Influence of on cell membrane 17
2.3 The mechanisms of photo- and biological LILI activation 18
2.4 Photoactivation of enzymes 20
2.5 Biostimulation 21
2.6 Biological responses on LILI and their application 24
2.7 Summary 25

3 Materials and methods 26
3.1 Lactic dehydrogenase activity determination 26
3.2 Succinate-dehydrogenase (succinic dehydrogenase system) activity detection 27
3.3 Glucose-6-phosphatase activity detection 28
3.4 Biuret – Method 29
3.5 Alpha-amylase activity detection 29
3.6 Determination of superoxide dismutase (SOD) activity 30
3.7 Electrophoresis of proteins 31
3.8 Detection of glutathione peroxidase activity in red cells 32
3.9 Detection of catalase activity 33
3.10 Detection of malonate dialdehyde (MDA) in red cells 34
5 page

2+3.11 Detection of Mg - ATPase activity in red blood cells 35
3.12 Method for the estimation of phosphate 36
2+3.13 Detection of Ca-ATPase activity 37
3.14 Detection of MnP activity 38
3.15 (Triphenyltetrazoliumchloride) TTC-test 39
3.16 Detection of glutathione reductase activity 40
3.17 Method Lowry 40

4 Experiments 41
4.1 Investigations of the influence of low intensive laser irradiation (LILI)
on the antioxidant system of animals 41
4.2 Low intensity laser irradiation (LILI) as a modulator of the antioxidant
system ofanimals 42
2+ 2+4.3 Investigations of changing activity Ca -ATPase and Mg -ATPase of
erythrocytes-membranes after LILI radiation in vitro experiments 43
4.4 Enzymatic response of animals' tissues on LILI in vitro experiments 44
4.5 Investigations of LILI influence of different energy on activity of alpha-
amylase in grains 45
4.6 The possibilities to activate alpha-amylase in germinated grains 45
4.7 Changing of the activity of the exoenzyme Manganese peroxidase after
laser aditon 46
4.8 Investigation of influence of LILI on biochemical properties of yeast 47
4.9 Investigation of influence of LILI on biochemical properties of bacteria 47
4.10 UV-Vis-Spectra of enzymes 48

5 Result 50
5.1 Investigations of the influence of low intensive laser irradiation (LILI)
on the antioxidant system of animals 50
5.2 Low intensity laser irradiation (LILI) as a modulator of animal's
antioxidant system 54
2+ 2+5.3 Investigations of changing activity Ca -ATPase and Mg -ATPase of
erythrocytes-membranes after LILI radiation in vitro experiments 59

6 page

5.4 Enzymatic response of animals' tissues on LILI in vitro experiments 62
5.5 Investigations of LILI influence of different energy on activity of alpha-
amylase in grains 67
5.6 The possibilities to activate alpha-amylase in germinated grains 70
5.7 Changing of the activity of the exoenzyme Manganese peroxidase after
laser aditon 72
5.8 Investigation of influence of LILI on biochemical properties of yeast 75
5.9 Investigation of influence of LILI on biochemical properties of bacteria 76
5.10 Summary of Results 76

6 Discusion 81

7 Conclusions and outlook 87

8 References 88

9 Apendix 102
9.1 List of abbreviations and units 102
9.2 light sources 104
9.3 List of materials and equipment 105
9.4 chemicals 106
9.5 List of biological systems 108
9.6 UV-Vis Spectra 110
7 Zusammenfassung

In der Literatur sind viele Beispiele des Einflusses von Laserbestrahlung mit geringer Energie
(LILI) auf biologische Syteme beschreiben. Allerdings sind die Ergebnisse wiedersprüchlich.
Ziel dieser Arbeit war es, mit verschiedenen Experimenten den Einfluß von LILI auf
verschiedene biologische Systeme und Objekte detailliert zu untersuchen.

Es wurden verschiedene Experimente mit folgenden biologischen Systemen und Objekten
durchgeführt:
* verschiedene Enzyme des Antioxidations-Systems von Tieren (Catalase, Superoxid-
Dismutase, Glutathion-Peroxidase, Glutathion-Reductase)
2+ 2+* Mg - und Ca -ATPase aus Membranen von menschlichen Erythrozyten und Erythrozyten
von Ratten
* Lactat- und Succinat-Dehydrogenase von Ratten aus Leber, Nieren, Gehirn, Muskel, Herz
* alpha-Amylase aus trockenen und gekeimten Gerstekörnern
* Mangan-Peroxidase aus Lignin-abbauenden Pilzen
* Dehydrogenasen von Hefe
* Dehydrogenasen von Bakterien.

Folgende Laser wurden für die Versuche eingesetzt: YAG-Laser (355 nm und 533 nm),
Argon-Laser (458 nm, 488 nm und 520 nm), Helium-Neon-Laser (632 nm) und CO -Laser 2
(10.6 µm).

Nach Laserbestrahlung wurden bei verschiendenen Systemen sowohl Aktivitätserhöhung als
auch Aktivitätsminderung beobachtet.
Abhängigkeiten von der Bestrahlungszeit und Intensität der Bestrahlung wurden ebenfalls
ermittelt.
Verschiedene Antworten biologischer Systeme nach Laserbestrahlung hängen von den
spezifischen Eigenschaften dieser Systeme ab.
Unterschiedliche Reaktionen verschiedener biologischer Systeme wurden nach Bestrahlung
mit verschiedenen Lasern detektiert.



8 1 Introduction
1.1 Background
Scientific experiments at the theme of the Dissertation were started since 1999. At the
beginning the theme of Dissertation was formulated as: "Investigations of the influence of low
intensity laser irradiation (LILI) on the Antioxidant system of animals". Those investigations
were done with the purposes to find out the effects of LILI of some kinds of lasers on the
process of lipid peroxidation (LPO) and the activity of antioxidant system in some organs of
animals and, at the first, in erythrocytes and in blood plasma. In literature sources the effects
of LILI on antioxidant system (AOS) of organism with the most important components such
as reduced glutathione (GSH) and the enzymes superoxide dismutase (SOD), catalase and
glutathione peroxidase (GP) mostly have been described for He-Ne-laser irradiation. Since
they are contradictory [1], the function investigations of the transport proteins of the
erythrocytes' membranes which are responsible to keep the native structure of erythrocytes
and ion balance in these cells had caused especial interest. The contradictory obtained results
had made a basis to carry out additional researches concerning laser light influence on other
enzymatic systems of animals, plants, mushrooms, yeast and bacteria.

1.2 Lasers
1.2.1 General description
The light from lasers differs from ordinary light in several important aspects. Ordinary light
from a light bulb travels randomly in all directions (unless the bulb is equipped with an
integral reflector that directs the light). The light is thus incoherent. Even when incoherent
light is directed with a reflector, it still spreads rapidly.
The light from a laser is temporary and spatially coherent. This means that all of the wave-
fronts of light are lined up in time and space. The waves of light go up and down and travel in
the same direction. Coherent light spreads less than other types of light, for example, the
beam of a tightly focused flashlight would spread between 2 degrees and 5 degrees over a 3
meter throw distance.
The sides of a laser beam are almost parallel but the light still spreads slightly. This spread is
divergence and is measured in milliradians (mrad). A simplified explanation of the process of
stimulated emission, for example for He-Ne-laser can be described in following. If a glass
tube is filled with a mixture of helium and neon gas, and an electrical current is applied to the
electrodes, the gas would emit light energy. This glowing gas is referred to as a plasma. Under
normal conditions the electrons in a gas atom orbit are at a fixed distance and pattern around
9 the nucleus; this is the ground state or most stable configuration of the atom. When an
electrical charge travels through the gas in the tube (energy is pumped into the gas), it excites
or stimulates the atoms. Some of the electrons absorb this energy by jumping up to the next
stable orbit. Such configuration is unstable. The electron trys to return to its regular orbit, the
ground state. As the excited (stimulated) atoms in the gas relax back to the ground state, some
of the energy that excited the electron(s) is emitted (released) in the form of random photons.
This is called spontaneous emission. The photons travel rapidly in all directions. They are
visible along the length of the neon tube or radiate outward from the light source.
Nevertheless the spontaneous emission is not enough to cause lasing action.
Lasers are very different from neon tubes in that they amplify the glowing effect via
stimulated emission. Stimulated emission can only occur when there is a "population
inversion" in the energy state of the lasing medium (in this case gas).
Laser tubes are designed in a long narrow configuration with a central bore. There are mirrors
at every end of the bore. These mirrors must be held in precise alignment for the laser to work
properly.
In most He-Ne lasers the mirrors are permanently attached or sealed onto the ends of the tube,
sometimes referred to as hard seal technology. In higher power lasers the mirrors are usually
not mounted on the ends of the tube itself, but on an external resonator that forms part of the
laser frame. It allows to make changing the mirror optics or adding a littrow prism if a
specific output wavelength (colour) is required. The mirrors must be perfectly aligned so that
the emissions from the gas in the tube will be amplified.
Some of the photons of light randomly emitted by the relaxing gas atoms will be travelling
parallel to the bore (centre) of the laser tube. These photons will strike the mirror at the end of
the tube and will be reflected back through the excited gas (plasma). When the photons
travelling parallel to the bore are reflected from the mirrors, they oscillate back and forth
between the mirrors.
If a laser has continuously emitting light, then there must be power to replenish that lost
energy in such a way that the laser action can continue. The power must maintain the
necessary population inversion to keep the laser process going, and that implies a pumping
mechanism to elevate electrons to that metastable state. The using of helium to "pump"
electrons into a metastable state of neon in the He-Ne-laser is an example of such mechanism.
An air-cooled laser tube with cooling fins, the connections for the cathode/filament are visible
on the right.
In their travels through the plasma, some photons strike other atoms that are in the excited
10