New Frontiers in
Exploration of Energy and Biological Systems
March 2005
Biophotonics Group
oxygen-dependent processes in water and aqueous solutions of different amino acids where conditions for low level but continuous production of reactive oxygen species are arranged.
oxygen-dependent processes in taken out human and animal blood. We study blood basing on the assumption that it exemplifies an integrated and coherent living tissue behaving according the Principle of Stable Non-Equilibrium of Erwin Bauer (see above), rather than a liquid conglomerate of diverse cellular and molecular elements.
oxidative processes in water (including tap chlorinated water) and water solutions of inorganic salts and gases such as air, nitrogen, argon and helium followed with PE from these aqueous systems dependent presumably on quasi-polymer structure of water.
Part 1: Model Photo-Chemical and Photo-Biochemical Reactions
“Gurwitsch Reaction” – Slow oxidation of an amino acid in aqueous solutions triggered by brief irradiation with ultra-weak source of UV-photons or addition of hydrogen peroxide.
This reaction was discovered by A. and L. Gurwitsch at the end of 1930-ies [6,7]. They found that after a brief irradiation of glycin or some other non-aromatic amino acid solution with a chemical or biological source of MGR or with strongly attenuated physical source of UV-photons (mercury lamp) the solution becomes itself a long-lasting source of MGR provided that it is kept in diffuse daylight and contacts with air. Basing on the results of “Mitogenetic spectral analysis” (a unique analytical method developed by Gurwitsch and his associates) Gurwitsch claimed that UV irradiation induces an autocatalytic reaction of oxidative deamination of an amino acid. A polypeptide-like substance emerging in the solution after its irradiation and staying in it at a very low stationary level performs the catalysis. Thus, the process induced in this system is extremely unusual. In an apparent contradiction to the 2-d law of thermodynamics, free energy of the system after initiation of the process seems to increase from its initial level; the system could keep going in a highly non-equilibrium state for very long periods of time. However, this discovery “passed without comment by other workers in this field” [8], as it sharply contrasted with the theoretical concepts that had been dominating at this time and are still dominating now.
In our own studies of this reaction and its modifications using modern physical and chemical analytical approaches we confirmed most of Gurwitsch’s conclusions and provided evidence in favor of degenerative branching chain reaction mechanism (chain reactions with delayed branching) of this process. We also suggested that this particular type of physical-chemical processes taking place in aqueous systems provides for the building up of stable non-equilibrium conditions in these reaction systems. Such a state is maintained due to continuous inflow of energy of electron excitation coming from the emergence and annihilation of reactive oxygen species taking place in the presence of molecular oxygen.
The results of these works and conclusions made on their basis are published both in Russian and in English [9-15]. Briefly, he major results include the following:
The analysis of macro-kinetic regularities of slow oxidative processes with reactive oxygen species (ROS) participation initiated and proceeding in aqueous amino acid solutions and accompanied with spontaneous or luminofore-amplified luminescence revealed that critical conditions, in particular, thresholds of reagents concentrations, of surface to volume ratio, and some others should be overpassed for its exponential development. The development of the process is preceded with the latent period; the duration of it strongly depends upon minor admixtures of pro- and/or antioxidants. All these features are characteristic for the processes developing according to the mechanism of a chain reaction with delayed branching.
For the first time a strong hysteresis was observed in the dependence of the reaction rate upon cyclic temperature changes indicating that a high level of cooperativity between elementary chemical transformations (single reaction events) exist in these reaction systems.
Using modern methods of analytical chemistry and biochemistry it was shown that a brief irradiation of glycin solution with an ultra-weak source of UV-photons induces a process characterized with the appearance of a polymer substance in the solution that catalyses oxidative deamination of an amino acid. Supposedly, the energy released in the course of oxidation is used to support the catalytic activity of a polymer, which rapidly loses its activity after isolation from the system.
For the first time in was demonstrated that the intensity of the chemical process of oxidative deamination in amino acid aqueous solutions depends upon the optical properties of these solutions, in particular, upon the presence and concentrations of fluorophores of different chemical nature, but capable to be excited with UV- and visible photons.
Maillard reaction (amino-carbonyl reaction).
Maillard reaction is the reaction between carbonyl- and amino-containing compounds, e.g., between reducing monosaccarides and simple amino acids, leading to a spontaneous synthesis of a variety of low- and high molecular weight compounds; many of them were shown to have remarkable biological activities. This reaction is accompanied with low-level PE resulting from slow “background” oxidative processes due to the emergence of ROS in its course. However, the authors who had observed chemiluminescence accompanying Maillard reaction practically always performed the process at very high temperatures, and they did not ascribe any significant role to the emergence of electron excited species in its course.
We found that brief heating of glucose (or ribose)/glycin basic aqueous solution is just the initiating condition for the development of the process of PE from the reaction system that can proceed for dozens of hours at room temperature [16, 17]. This process is similar to Gurwitsch reaction and it displayed many features characteristic of chain reactions with delayed branching. Furthermore, it turned out that under certain conditions pronounced oscillations in PE intensity in the course of this process might be observed, and under these conditions several new phenomena of significant importance were discovered [18-20].
In simple solutions of certain carbonyl compounds (e.g., methylglyoxal) and amino compounds (e.g., glycin, ethanolamine, putrescine) under moderate basic conditions there develop non-linear oscillations of chemiluminescence and of red/ox potential with the amplitudes reaching 50-500% of the respective mean values with typical periods from several to tens of minutes and lasting for tens of hours. Development of oscillations critically depends on concentration of reagents, in particular of an amino compound, on the reaction volume, or more precisely on the ratio of air/water interface surface area to the reaction volume, on the temperature and some other factors. All these phenomena indicate that cooperative phenomena may emerge in the reaction systems and that the genuine process of self-organization (orderliness) occurs there under the specific conditions.
Oxygen plays a dual role in the overall process: it is absolutely necessary for the initiation of the process and its initial development, however at the advanced stages oxygen paradoxically inhibits oxidative processes and PE from the reaction system. It has been shown for the first time that when at these stages dissolved oxygen is substituted for the inert gases the process “flames up” again. Anaerobic stage of PE augmentation that follows afterwards lasts for many hours. In its course the products appear in the solution that give rise to an extremely intensive oxidative burst on contact with air.
Slight mechanical and electromagnetic impulses induce fast and pronounced increase in luminescence intensity fading to the initial level according to the hyperbolic law. In entirety the results allow to suggest that the reaction systems studied may reside very far from the equilibrium for a long time due to the continuous presence of electronically excited constituents despite the fact that the reaction systems are in a thermal equilibrium with the surroundings.
Conclusions
Slow amino acid oxidation in water induced either by hydrogen peroxide or by carbonyl compounds proceeds according to the mechanism of chain reaction with delayed branching. Such reactions are essentially non-linear and critically dependent upon initial and boundary conditions. Both temporal and spatial self-organization may emerge in these reaction systems. Complexity of the system including structural complexity of chemical species participating in the processes increases as the process develops. Starting from initially most simple compounds there appear heterocyclic and aromatic chemical species, which may serve as precursors of nucleic acid bases, new amino acids, and of polymers of different structure. Reaction systems studied may serve a model of processes that may take place in nature and give rise to complex bio-organic molecules without participation of any living organisms. One may even speculate, that these organized processes can be looked upon as proto-biochemical: “building blocks” for living cells could emerge in the course of these processes at the pre-cellular stage of the evolution. On the other hand, as such processes undoubtedly take place in the organisms, most probably, in the intercellular matrix one may expect that some biologically active substances or their precursors may be regularly synthesized in course of them, that is without enzyme participation.
It should be stressed that these curious processes markedly differ from usual chemical and biochemical reactions by regular appearance in their course of reactive oxygen species, and by generation of electron excited states due to free radical reactions. Data obtained in our laboratory strongly favors the idea that this particular feature of such reactions provides for their ability to withstand chaotization and to develop temporal, spatial, and structural orderliness which arise in their course.
Part 2: Living blood outside an organism: Patterns of photon emission and erythrocytes sedimentation argue for its dynamic cooperative and coherent properties maintained due to oxygen-dependent oxidative reactions.
Blood provides vital functions of an organism as a whole. A lot of knowledge about particular functions of blood components was gained due to the relative ease of their isolation from whole blood. However, this approach does not allow to get information about interactions of blood components in whole blood, though their interactions are undoubtedly very significant for efficient blood functioning.
It is rather difficult to study blood in its integrity in an intact organism and even in model systems, where its circulation is provided. However, it is reasonable to expect, that interactions between components of blood are not completely lost after it has been taken out from an organism. We suppose that taken out blood retains some properties of a living system. Thus it should obey the Principle of Stable Non-Equilibrium of Erwin Bauer. To realize this principle a living system should possess energy and material resources and also a specific structure (form). Taken out blood has enough resources for long-lasting metabolic activity. Whether it has a specific structure may be revealed only experimentally or at least deduced from the observations of its behavior.
Biological term “behavior” means patterns of reactions of a living system upon external and internal irritants. Its observation presumes continuous monitoring of certain parameters of a system under study. To accomplish this goal we developed methods allowing to evaluate the holistic properties of blood through continuous registration of two different parameters, which presumably reflect integral properties of blood – luminescence of whole blood and the so called erythrocyte sedimentation reaction.
Studies of luminescent properties of whole non-diluted blood.
It has been shown as far as at the beginning of 1930-ies by A.G. Gurwitsch and then by other authors that animal and human blood as well as nervous tissue unlike other animal tissues spontaneously emit mitogenetic photons [21,22]. Spontaneous MGR of blood was strongly dependent upon oxidative reactions taking place in it, and especially upon oxidative glycolysis, in the course of which ROS should emerge by analogy with the Maillard reaction. Several decades later it was discovered that neutrophils - the most abundant type of blood leukocytes may be induced by multiple factors (in particular by bacteria) to produce the whole array of ROS [23]. This reaction of immune cells is known as “oxidative burst”, and it is accompanied with PE from a cell suspension. Intensity of ROS-dependent PE may be enhanced by many-ten-fold with fluorescent substances such as lucigenin and luminol added to a cell suspension. It is commonly accepted that lucigenin-dependent luminescence (LGL) indicates of O2· ¾generation in a system, while luminol-dependent luminescence (LML) is a probe for the generation of predominantly other reactive oxygen species (H2O2, OH·, OCl¾, NO·). A great number of publications is devoted to the studies of chemiluminescence accompanying neutrophil and other white blood cells production of ROS. However, practically nobody studied luminescence from non-diluted whole blood presumably because the very opportunity of such an opaque liquid to emit photons is counterintuitive.
We found that that although undiluted blood is a highly opaque substance it may be a sufficiently intense source of PE [24]. Free radical reactions in which reactive oxygen species participate are the most plausible source for electron excited states generation that eventually expresses itself in PE. We found that LGL is relatively intense in fresh undiluted blood healthy donors’ blood, even if blood is completely isolated from ambient air, indicating that continuous oxygen reduction takes place in it, and oxygen is supplied by erythrocytes. Blood dilution with physiological saline or with platelet-free plasma immediately attenuates LGL, indicating that close contact between oxygen reducing cells – leukocytes, and oxygen-donating cells – erythrocytes is needed. Blood strongly reacts upon subtraction of some part of it by enhancement of PE, figuratively speaking, upon cutting it into parts. In healthy donors’ blood LML is usually much lower than LGL, but addition of substances provoking non-specific immune reactions – respiratory burst (RB) in neutrophils, results in rapid elevation of LML intensity. PE from even small portions of blood (0,2 ml) may last for many hours indicating of its vast reserves. Photons may be emitted in oscillatory manner even if blood is not bothered. When blood is irritated with periodic slight mechanical push, well pronounced low-frequency waves of PE are often observed.
Blood of patients with cardiovascular diseases was characterized by higher amplitudes of oscillations and in general by much higher spontaneous levels of LM-CL than healthy donors’ blood. Such a pattern of behavior of sick persons’ blood was used for monitoring of therapeutic intravenous low-intensity red laser irradiation of blood of angina pectoris patients [25].
Studies of PE from whole blood helped to reveal some new fundamental properties of this tissue.
If photons emitted by blood during the RB were reflected back to the sample, they influenced metabolic processes in blood. In particular, back reflected photons accelerate slowly developing RB at the stage of its development, and at the stage of RB decay they prolong the process. We suppose that photons excite haemoglobin (Hb) molecules and energy of excitation may be used for enhanced oxygen release from multiple HbO2 molecules tightly packed in erythrocytes [26]. Increased oxygen availability eases reactive oxygen species generation by white blood cells, therefore intensity of PE from blood is increased.
We also found that carbon monoxide (CO) sharply intensified LC-CL in undiluted blood. Presumably, high energy released in the reaction of CO binding to free Hb molecules induces accelerated oxygen release from multiple HbO2 molecules resulting in a similar effect upon CL as that produced by blood self-irradiation. It is known, that CO is produced in the organism, especially in brain.
Studies of temperature dependence of PE from blood in which oxidative burst was induced in the presence of luminol revealed in principle the same type of hysteresis as it was observed in Gurwitsch reaction (see above). When blood temperature was cyclically changed in the range of 35-380C, a profound hysteresis in PE intensity was observed. In particular, at the stage of oxidative burst development temperature lowering down was hardly followed with the decrease of PE, while at the stage of oxidative burst decline increase of the temperature was followed with the intensity increase after a significant lag. PE responded in a highly paradoxical way to temperature elevation over the physiologically tolerable level. When it increased to 410C a sharp decline in PE was observed. This collapse could not be explained by some irreversible processes in blood because as soon as the temperature started to decrease PE decline was halted and its intensity began to increase irrespective of blood cooling down.
Very sensitive single photon counters with low dark count rate allow observing specific patterns of PE from non-diluted blood even if no PE enhancers such as luminol and lucigenin are added to it. These patterns are revealed using sophisticated mathematical approaches for analysis of long time series. In particular non-linear complex oscillatory patterns were revealed in blood. These oscillatory patterns differ in their configuration, period length, length of their exhibition. Complex oscillatory patterns of blood behaviour strongly depend on blood state and on the physiological state of a donor.
All these results definitely demonstrate that blood behaves as a cooperative living system whose parts unceasingly interact in time and space in order to provide maximal efficiency in exerting its physiological functions.
From our results it follows also, that oxygen delivery -- one of the most important functions of blood –is actively used by other blood constituents for electron excited states (EES) generation. It can be suggested that this high density energy may be used by a feedback mechanism for discharge-like oxygen release from erythrocytes – the necessary property of oxygen transport function of blood that has been generally neglected. Besides further easing of oxygen release, EES as we suggested elsewhere [27], are used in living systems for triggering biochemical processes, for continuous “pumping” of the non-equilibrium state of inter- and intracellular structural components, while the structural patterns of ROS reactions determine biochemical and physiological rhythmic modes. Thus, chemiluminescent properties of undiluted blood reflect its behaviour as of a non-linear cooperative system, whose elements interact to provide the highest efficiency of performance of this tissue as a whole.
Cooperative processes in blood as revealed with the new technique: ESR-grapy
If the idea that whole blood represents a living tissue behaving according to the Principle of Stable Non-Equilibrium is right one may expect to observe characteristic features of this behavior using different methods. However, currently such methods are practically lacking. Most hematological diagnostic methods are based on blood fractionation and studies of particular properties of its constituents under the conditions when interactions of these constituents with others and with blood as a whole are lost. There is only one established diagnostic method -- Erythrocyte Sedimentation Rate (ESR) measurement in which holistic properties of blood may be evaluated, though up to now this advantage of the method was missed. The method seems very simple: blood with an anticoagulant is taken into a pipette which is vertically installed and after a certain period of time (usually, 1 hr) the distance passed by the boundary between sedimenting erythrocytes and clear plasma is measured. Generally the process of red cell mass sedimentation is regarded in terms of more or less complex hydrodynamic theories, though there are a lot of indications, that specific biological, in particular, bioenergetic properties of blood may play a major role in this process [28].
To study whole blood behavior we devised also a significantly modified version of ESR method. To register the movement of the boundary between a red cell pillar and clear plasma in blood taken into a vertically installed pipette with high temporal resolution we developed a special opto-electronic apparatus and an appropriate software. We named the velocity-time curves obtained using this device “ESR-grams”, and the method – “ESR-graphy”. It turned out that the boundary between red blood and plasma moves in an oscillatory, rather than in monotonous manner. The latter could be expected for a passive process, while the former indicates that active and non-linear processes are taking place in blood. Patterns of ESR-grams strongly depend on a state of health of an individual, as well as on blood pre-treatment and on additions to it of different substances [29].
As a rule, mean velocities and deviations from the mean (amplitudes of oscillations) in sick persons’ blood are much higher, than in healthy donors’ blood. ESR-grams for healthy donor’s blood obtained on different days when his state of health is stable are very conservative. Changes in their patterns precede other symptoms of the developing disease.
An interesting result obtained using ESR-graphy was objective evaluation of an individual’s sensitivity to geomagnetic storms. Amplitude of oscillations of sedimentation velocities increased during geomagnetic storms in blood of patients of an intensive care ward up to 80-fold over the same parameter on “quiet” days [30]. It is known that the risk of a heart attack, hypertension crises much increase in periods of instability of the geomagnetic fields, and it is interesting to speculate that blood is the major “receptor” of geomagnetic field variations.
Addition of different substances or even of physiological saline in very small quantities to blood may significantly affect its sedimentation behavior. It is notable that patterns of ESR-grams change in multi-phasic manner in response to a monotonous change of an acting parameter (e.g., blood dilution with physiological saline). Effects of saturation of blood with oxygen, addition of small amounts of H2O2 to blood, of inducing RB in it, of blood dilution with own plasma, studies of the microscopic events on red cells plasma boundary were correlated to changes of luminescent properties of the same blood.
Taken together the results suggest that the overall picture of blood sedimentation depends on the extent of initial red cells saturation with oxygen and their ability to release it, on the intensity of metabolic activity of blood, on the degree of its organization and efficiency of cooperation of oxygen donating and oxygen consuming cells, on the state of the route of oxygen transport. Just after blood is taken to a pipette a 3-dimentional structure of erythrocytes associated into 1-dimentional “rouleaux” is formed in it, and depending on all the above mentioned factors this network either gradually “shrinks”, as it happens in healthy donors’ blood, or chaotically collapses as it is seen in blood of sick persons. For example, in blood of very sick individuals initial sedimentation sometimes was very slow, followed with a sudden downfall of a red blood pillar and even its disintegration.
Practical application of ESR-graphy
As ESR-graphy allows to register even slight changes in blood behavior, it served a basis for the new test on substances which may be unsafe for a certain individual, in particular, substances contained in food. It was shown, that extracts of some foodstuffs added to a person’s blood retard initial sedimentation, other extracts significantly accelerate its sedimentation, still other do not influence it. These reactions are highly specific: an extract which affects behavior of blood of a particular donor do not influence ESR in blood of another donor. State of health of hundreds of patients with such different diseases as diabetes, asthma, rheumatoid arthritis, cardiovascular diseases, multiple sclerosis, lupus erythematosus, significantly improved if they avoided foods, extracts from which affected their blood sedimentation [31].
Conclusions.
Studies of dynamic properties of blood outside an organism indicates of its high bio-energetic potential and of its decline and deficiencies in the mechanisms of its employment in patients’ blood. In particular, the process of blood sedimentation reveals blood reactions upon different stressful factors as of a “surviving” tissue, dependent on the volume of its internal resources, on the level of temporal and structural organisation of all its components in their co-ordinate functioning. Studies of blood as of an integrated biological system allows to augment our knowledge of both its particular functions and its special role in unification of all tissues and organs of an organism. Besides, new haematological methods described here may widen opportunities of a practical medicine in diagnostics, prognostics and therapy of ailments.
Part 3. Oxidative processes in water.
Studies of oxidative processes in water (including tap chlorinated water) and water solutions of inorganic salts and gases such as air, nitrogen, argon and helium followed with PE from these aqueous systems started only recently in our laboratory. However, some scientifically interesting and practically important results have been already received.
In particular, using single photon counting technique we discovered that under certain conditions of argon treatment of aqueous solutions of hydrogen peroxide and sodium chloride the reaction of Cl¾ oxidation to hypochlorite is significantly accelerated. Argon also turned out to catalyze some other chemical and biochemical reactions proceeding in water, and this its action turned out to be specific, differing it from such inert gases as helium and nitrogen [32]. This research is in progress and mechanisms of such an unexpected action of this noble gas are under investigation.
Another research related to water properties is aimed to develop more efficient methods of water purification. Using chemiluminescent analysis we revealed that chlorinated tap water contains long-living free radicals, presumably, high molecular weight radical particles originating from chlorinated organic molecules present in water taken for chlorination. These radicals interact with oxygen and evoke branched chain reactions lasting until most of them disappear. If water containing such species is consumed, they are very likely to induce oxidative bursts in the organism. It is possible that positive correlation between regular consumption of chlorinated water and development of some chronic diseases may to a large extent be explained by the presence of macro-radicals in such water [33].
We developed a device for water purification containing only natural materials – a Karelian fullerene-containing mineral Schungite, dolomite and ceramics. We have shown that this device much more effectively purify water of macro-radicals than common charcoal filters, and it also cleans water from other impurities [34]. Especially intriguing was that water passed through this device had some residual ultra-weak photon emission that did not arise from free radical reaction and had definite bacteriostatic properties.
Development and Commercial Projects
Some applications of our biophotonic research and other research projects has been mentioned above and they are covered by a variety of patents. Quite a few of these projects are currently used on a commercial basis.
The most advanced project is the test for “food immunoantagonists” a new test on the so called delayed or hidden food allergy, allowing to reveal particular foodstuffs which may induce adverse reactions in a particular individual. To perform this test 20 ml of venous blood is taken, food extracts are added to it aliquots, and ESR-graphy is performed. In the case if food extract changes the parameters of erythrocytes sedimentation in comparison with the control the particular food is recommended to be excluded from the diet. Currently 130 foodstuffs are tested. The test is performed in a specially organized clinical laboratory belonging to the Institute for Ecological Rehabilitation of a Human Being, of which the Biophotonic Laboratory is one of the constitutors.
ESR-graphy together with luminescent analysis of whole human blood solves some other practical medical and diagnostic problems, in particular they may be used for in vitro tests for compatibility of medical drugs for particular patients, for monitoring of the process of treatment and for other diagnostic purposes. Common research is performed together with some engineering groups to develop new generations of ESR-graphs for clinics and medical research institutions.
Another project concerns the properties of tap water and methods of its purification. As mentioned above single photon detectors may be used for evaluation of free radical reactions in water, and currently such tests are performed in the Biophotonic laboratory. The test are continuously developing, and common research is performed together with some engineering groups to devise a specialized photon-measuring apparatus for water tests.
This project is closely related to the project of designing water cleaning devices based on “natural technologies”, that is on the natural principles and natural materials for obtaining safe and even “vitalized” drinking water.
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Voeikov V.L., et al. Patent of Russian Federation № 2167101 of May 20, 2001.
(*) Relevant papers on the subject available in the electronic form through E-mail
Interview with prof. Vladimir Voeikov. Russian Biologist Comments On What Distinguishes Living Systems. A leading researcher discusses the breaking frontiers of biophysics presented at the Second International A.G. Gurwitsch Conference, held in Moscow in September 1999. 21st CENTURY Science and Technology, pp. 58-66, Winter 1999–2000.
V.L. Voeikov The Scientific Worldview and Christian Consciousness. In: Bratus’ B.S., Voeikov V.L., Vorobiev S.L., et al. The Beginnings of Christian Psychology. Textbook for Universities. Nauka Publ. Co., Moscow, 1996.
Fedorov M.V., Belousov L.V., Voeikov V.L., Zenchenko T.A., Zenchenko K.I., Pozharski E.V., Konradov A.A., S.E.Shnoll. Structure of Distribution of Amplitude of Photomultiplier Dark Current Fluctuations as Correlated by The Earth Rotation. Astrophysics and Space Science. In Press. 2002.