Libmonster ID: BY-1588
Author(s) of the publication: Alexander KONOVALOV

by Acad. Alexander KONOVALOV, Arbuzov Institute of Organic and Physical Chemistry, RAS Kazan Science Center, Tatarstan, Russia

As shown by Russian scientists, the biological effects of highly diluted water solutions are due to tiny, nanomolecular assemblies (nanoassociates) formed under the action of two effectors: the dissolved substance and the outer electromagnetic field. This phenomenon confronts physicists, biologists and chemists with difficult problems and calls for further research.

"Facts inexplicable by extant theories are particularly dear to science, and... they hold the key to its progress in the near future." This idea, as formulated by Alexander Butlerov, an eminent organic chemist, back in 1879, is still valid today, in the 21st century. Unfortunately many scientists will not abide by this truth. Sure that our present knowledge is all-embracive and sufficient for an understanding of natural phenomena, we tend to negate the very facts rather than search for their root causes. Such causes, however, may be hidden in hitherto unknown phenomena. This has a close connection with our subject-matter.

Today we know of thousands (!) of works at different laboratories of the world dealing with all levels of biological organization of matter (biomacromolecules → cells → organs → organisms → populations) and demonstrating: water solutions of biologically active substances (BAS) can touch off biological effects at different concentrations in a regular response of a particular bio-

стр. 4

logical system. The range is from "ordinary" dilution levels (10-3-10-7 M,--no objections in this regard) to high-level dilutions, an object of questions and doubts. The "dead zone" lies in between--that's where bioeffects are absent. Since solutions are diluted sequentially, we speak of "highly diluted solutions" versus low-level solutions. In this country such studies have been on over the past 30 years in the laboratory of Dr. Yelena Burlakova (Emanuel Institute of Biochemical Physics).

Now why the queries about the effects of high dilutions? Such doubts are not groundless either. According to orthodox views, these solutions should have no effects--"outlawed", so to speak! Indeed, the correlation between the number of molecules of dissolved matter and water is one to billion at 10-8 M, while if the concentration is down to 10-18 M, the ratio will be one to a billion of billions. Given conditions like that, any bioeffect is out of the question: such solutions should take on a solvent's characteristics, water in this case. The concept of infinitely diluted solutions rests on that: dilutions diminish the concentration of the basic substance, and thus the diluted solution tends--and rather fast--to adopt the solvent's characteristics. For this reason most experimentalists blame bioeffects in the solution/bioobject system on the object. It is thought to react to very low concentrations or individual BAS molecules somehow. But how? One cannot tell! It's in the realm of guesswork.

But supposing solutions are responsible after all? What if they give rise to molecular assemblies at different concentrations (dilutions) of a solution, and bioobjects respond namely to such conditions? How can we explain the "dead zone" phenomenon?

Perhaps we know but too little about the true cause? The results of our work done jointly at the Arbuzov Institute of Organic and Physical Chemistry together with Dr. Irina Ryzhkina and M.S. Liasan Murtazina and M.S. Yulia Kiseleva have shown that this is really so. We have made a broad and comprehensive study of water solutions of different chemical composition in a wide range of concentrations using a set of physical and chemical methods.

Thus far we have studied about 100 compounds in a 10-2 to 10-20 M concentrations diluted sequentially

See: V. Pchelyakova, "Ultra Small Doses: Are They Good or Bad?", Science in Russia, No. 1. 1995.--Ed.

(proceeding from the initial sample (suspended-sediment) concentration). Our list included antioxidants, plant growth regulators, neuromediators, vitamins, tranquilizers, hormones, a variety of drugs as well substances unknown to us in their biological characteristics. Chemically this list included compounds of different structure: from simple molecules (like glycine, which is the simplest amino acid) to complex macrocyclic compounds like porhyrins* or calyxarenes**.

Now what about our methods and instruments? They were standard. True, we used most up-to-date gadgets. But no minidata, and all beyond experimental errors. Our results had to be reproduced, that was our condition. What was most unusual was the object of our research: we took extra low concentrations. We were looking into such things as specific electric conductivity (x), surface tension (a), pH and, in some cases, dielectric permeability and optical activity. All that belongs to

* Natural pigments, such as hemoglobins, chlorophylls, cytochromes and the like.--Ed.

** Compounds derived from phenols regarded as substances with nearly unlimited possibilities.

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Dependence of proteinkinase activation on phenozane (potassium salt) concentration, with phenozane added to a culture of smooth muscle cells of rat (a); inhibition of proteinkinase C activity depending on a-tocopherol concentration (b).

the characteristics of solutions. The DLS (Dynamic Light Scattering) methods were quite helpful: they enabled us to assay for the presence of nanoobjects in vitro. Otherwise our results would have trembled in the balance. We must also note that the Zetasizer Nano ZS device of Malvern Instruments (Britain) that we used for measuring the mean hydrodynamic diameter (D) of a nanoobject also makes it possible to determine the zeta potential characterizing the interaction of a moving object with the medium. And so we had two essential things at our disposal: the parameters of solutions, and the parameters of nanoobjects.

We carried out our experiments both in laboratory and in "hypoelectromagnetic" (induced) conditions, that is, on being ready the solutions were allowed to stand for 24 h within a three-layer permalloy (iron/ nickel) container shielding the enclosed matter from external electromagnetic fields. Thereby the geomagnetic field induction was brought down to a thousandth fraction, which is a good indication for screening.

We found the concept of infinitely diluted solutions to be not universal--it applies in some cases, but does not in others. We named solutions where this theory holds (25 percent of compounds studied) "classical", and the other category (75 percent)--"nonclassical".

The "classical" pattern means that a solution attains rather fast to a solvent's characteristics under conditions of sequential diluting, with no further changes there. As we demonstrated, both the surface tension and the specific electric conductivity values at 10-6 to 10-7 M concentrations were down to the water values and stayed stable in subsequent dilutings (the surface tension and the specific electroconductivity values of twice distilled water were equal to 71-72 m/H/m and 1.5-2.0 µCm/cm, respectively). These indicators held in about 25 percent solutions investigated. But how about the rest 75 percent?

Here we were in for a bit of surprise. We saw the properties of our solutions to be changing in further dilutions, and not linearly at that. For instance, it was shown for solutions of potassium phenozane (an antioxidant synthesized at the Semenov Institute of Chemical Physics) that the surface tension in sequential dilutings (namely at concentrations 10-6 to 10-7 M) falls unexpectedly by 10-20 mH/m. Specific electric conductivity is up to 40 µCm/cm and keeps changing. All that beyond experimental error. A comparative study of changes in bioeffects and physicochemical characteristics of diluted solutions suggested this conclusion: there is a correlation between the two phenomena: both are a result of "nonclassical behavior". Both have common causes. But what in particular?

We found a clue to this phenomenon: in high-dilution water solutions of "nonclassical" compounds there appear nanosize molecules which we called nanoassociates. Their dimensions that may be down to 400 nm change in subsequent dilutings--not in the linear and not in the monotonic fashion, but rather jumpwise. The picture thus obtained looks like a serrated saw. Again, potassium phenozane solutions are a graphic example here, and changes brought about thereby are of definite significance. We determined zeta potentials as well,

стр. 6

Concentration dependences of surface tension (1) and specific electrical conductivity (2) in diluted solutions of potassium phenozane, 25 °C.

changing nonlinearly, too, and having negative values in high solutions.

A comparison of their parameters, i.e. the dimensions and zeta potentials of nanoassociates and the characteristics of consequential dilutions are correlated too. Such dynamics is of regular, not stochastic, random nature. This means that nanoassociated "rule" in diluted solutions exhibiting "nonclassical" properties.

But nanoassociates do not arise in diluted "classical" solutions. So, we do not have "classical" nanoassociates, but deal with "nonclassical" only.

To form nanoassociates, a dissolved substance has to be structured. But how? We cannot tell so far. Yet such substance is a must. No nanoassociates were formed in "dummy" experiments (without a substance dissolved) as water was diluted with more water in a series of successive tests. No dissolved substance, no effect. So the substance has to be present anyway.

An external (applied) magnetic field may be another effector. We found that with the aid of a permalloy container shielded from within, as we have said, against outer magnetic fields. We proceeded as follows. We divided the solution in two for each particular concentration. One sample was left, as usual, on the laboratory table. The other was placed into the container. And we obtained different results. The data on the laboratory table sample of potassium phenozane solution was down to 10-16 M, while the figure for the sample in the permalloy container was not lower than 10-7 M. But no nanoassociates in highly diluted solutions locked in. So an external electromagnetic field was needed for that. Therefore it was intriguing to compare these data with the characteristics of physicochemical characteristics of the solutions proper. Thus, in the potassium phenozane solutions that were allowed to stand in the permalloy box, there occurred essential changes in electric conductivity, from 10-6 on it corresponds to that of water only. This confirms, on one hand, our prior conclusion on the absence of nanoassociates in high solutions within the container. Yet on the other hand, this is a clear indication that our results were not wrong because of some methodic error. We observed a regular pattern by using different methods to collect data on our research objects, that is on parameters of the nanoobjects in solution, and on the physicochemical properties of the solutions. The consistency of our results points to an interconnection of phenomena under study. That is why it was a "proof-of-concept" experiment to us.

We may just as well say: in solutions within the permalloy container the "nonclassical" pattern of behavior turned into a "classical" one, for no nanoassociates were formed in the absence of an electromagnetic field. No electromagnetic fields--no nanoassociates--no "nonclassical" behavior!

Our study of the body of data on the "laboratory table" and "permalloy-container" experiments for "nonclassical" substances brings us to the conclusion there is a boundary concentration different for particular compounds but stable in the 10-5-10-8 M range. Next follows a region where nanoassociates do not form in "permalloy-container" experiments. But this is where "dubious" bioeffects show up under ordinary conditions. Hence we suggested: if under conditions of electromagnetic field screening there are no nanoassociates, there should be no bioeffects either. A group of research scientists of the Emanuel Institute of Biochemical Physics--Yelena Maltseva, Nadezhda Palmina and Valery Kasparov--checked up on our supposition. They looked into changes in the lipid component's microviscosity in respective membranes under the effect of potassium phenozane solutions in "laboratory-table"

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Concentration dependencies of dimension (D) changes in nanoassociates formed in potassium phenozane (PP) solutions (A), and in specific electroconductivity (B) in diluted PP solutions prepared under normal conditions (curves 1) and allowed to stand in the permalloy container for 24 h (C) (curves 2).

and "permalloy" conditions, and collated the results. In the former case ("laboratory table") the effects are manifest at 10-6, 10-12 and 10-15 M concentrations; in "permalloy" conditions the effect held at 10-6 M, but vanished at 10-12 and 10-15 M.

A comparison of these results with experimental data on the dimensions of nanoobjects in potassium phenozane solutions under "normal" and hypoelectromagnetic conditions points to rather interesting developments. The bioeffect at 10-6 M on the dimensionality scale correlates with a maximum, and with a minimum at 10-12 and 10-15 M. Both disappear under "permalloy-container" conditions. Hence the first conclusion: the character of effects is different at 10-6 M, and at 10-12 M and 10-15 M.

Another conclusion why our experiment was made: nanoassociates are not formed in the absence of an electromagnetic field in highly diluted water solutions and, as a consequence, bioeffects are absent there. This was yet another "proof-of-concept" experiment. And so our conclusion: no electromagnetic field → no nanoassociates → no bioeffects.

A similar "proof-of-concept" experiment was carried out with the participation of M.Sc. Dmitry Konovalov (Kazan-based Physicotechnical Institute) involving a solution of cetyltrimethylammonium bromide (BCTA). Under ordinary conditions at a BCTA concentration of 10-9 M there appear 240 nm nanoassociates. This does not take place if there is no electromagnetic field. But if a 7 Hz field is generated within the container, nanoassociates are formed in solution about the same size as under normal conditions.

Thus, nanoassociates formation holds a key to the behavioral pattern of high water solutions. This is of regular occurrence in highly diluted solutions of biologically active substances (BAS) as a result of nanosize molecular assemblies formed under the action of the dissolved substance and electromagnetic fields.

It is pertinent to ask: will it be possible, proceeding

стр. 8

Concentration dependences of dimension (D) changes in nanoassociates formed in PP solutions (A), and in microviscosity of bilipid layer of synaptosomes (B) acted upon by PP diluted solutions prepared under ordinary conditions (curves 1) and kept in the permalloy container (curves 2) for 24 h.

from the results obtained, to predict bioeffects in highly diluted BAS solutions? It may be possible to do that in some cases by relying on prior physicochemical studies of such solutions. If their behavior is "classical", there will be no bioeffects; but if it is "nonclassical", we may point to the probable areas where such effects are expected. We made such predictions in more than ten cases.

But what do we know about nanoassociates at all? Their structure? Our estimates show that the number of molecules of the dissolved substance at the given concentrations is not sufficient to give rise to nanoassociates of the size observed. The sensitivity of the Zetasizer Nano ZS device in determining the size of nanoobjects calls for their presence in sufficient numbers in solution, no less than a thousand per millimeter, for the greater part of nanoassociates in high solutions is under water molecules. In fact, about 7 mln water molecules correspond to a 100 nm object.

What is the nature of nanoassociates? We cannot tell exactly, though there are sundry speculations on this score. What are the forces capable of holding together millions of molecules? Considering the cubic (volumetric) dependence of the number of water molecules in a nanoassociate on its diameter, the correlation will be about as follows: 100 nm ~7 mln; 200 nm ~50 mln; 300 nm ~20 mln; 400 nm ~500 mln water molecules.

And last, what changes take place in matter acted upon by dissolved substances and electromagnetic fields, in highly diluted water solutions at any rate? Questions, questions...

We have thus discovered a fundamental phenomenon not known before: formation of nanomolecular assemblies, the nanoassociates, in strongly diluted solutions

стр. 9

Dimensionality distribution of particles in BCTA diluted solution kept on the lab bench (A) and in the permalloy container (B) furnished with an electromagnetic field generator.

Concentration dependencies of dimension (D) changes in nanoassociates formed in BCTA solutions prepared under ordinary conditions (curves 1) and allowed to stand in the permalloy container (curves 2) for 24 h. 

under the action of dissolved substances and external electromagnetic fields. These assemblies determine a totality of characteristics--physicochemical, biological--of such solutions. But here new problems crop up. For instance: What happens to nanoassociates in sequential diluting and why? What is the mechanics of action of highly diluted solutions on biological objects? What in particular underlies such action: nanoassociates or a structured solution? Are there any nanoassociates in bioobjects? If so, what is their role and mechanism of action? What is an interrelationship between the molecular structure and nanoassociates formation?

Our results just anticipate further research inquiries and open up a wide field for physicists, biologists, biophysicists and chemists, of course.

To conclude, it would be in place to recall what Albert Szent Gyorgyi von Nagyarpolt, a Hungarian-born American biochemist and one of the founders of bioenergetics (Nobel Prize 1937) said more than fifty years ago:

It appears there is a very important dimension absent from our frame of mind, something that makes it impossible to find an approach to these problems (problems of life). Water is not only the mater (mother), it is also the matrix of life, and biology must have not succeeded in the understanding of most obvious functions because it has centered its attention on a substance in the form of particles, while divorcing them from the two matrices--water and electromagnetic field.

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