Memory of water

Template:TOC-right The memory of water is a phrase mostly associated with homeopathy and the controversial research of Jacques Benveniste and his colleagues. This team reported that particular solutes subjected to sequential physical processing and dilution appeared to have some biological effects that were different from the "control" effects of the water used for the dilutions. The work resulted in considerable controversy, and some other labs were later unable to reproduce the reported effects. However, the memory of water controversy has extended beyond these initial studies, and some scientists assert that it is based on sound scientific discoveries. An overview of the issues surrounding the memory of water and its relationship to homeopathic medicine was the subject of a special issue of the leading academic journal on homeopathy.

Water is not simply a collection of molecules of H₂O, it contains several molecular species including ortho and para water molecules, and water molecules with different isotopic compositions such as HDO and H₂¹⁸O. These water molecules as part of weakly-bound but partially-covalently linked molecular clusters containing one, two, three or four hydrogen bonds, and hydrogen ion and hydroxide ion species. In addition, there are always adventitious solutes in liquid water. Even double-distilled and deionized water always contains significant and variable trace amounts of contaminating ions, and different samples will differ in the contaminants that they contain.

There is some support for the notion that water can have properties that depend on how it has previously been processed (that is, water has, in some sense, a kind of "memory"). The experimental evidence indicates that the "memory" are due primarily to solute and surface changes occurring during this processing. In particular, water, as a result of repeated vigorous shaking, might include Redox molecules produced from water, dissolved atmospheric gases and airborne contaminants, silicates (i.e., tiny glass "chips"), nanobubbles and their material surfaces, dissolved ions, including from the glassware. It may be contaminated by material that adheres to the surfaces of glassware, for example by bacterial material. There might also be some effects of successive shaking on water structure that causes "clustering" of water molecules.

These mechanisms are not mechanisms of memory in any cognitive or electronic computing sense; the term memory here is used as a metaphor, implying only that the past history has a discernible influence on the present properties. History may be more accurate, as the content of a biological or electronic memory varies as a function of time, typically losing information. If there is a "memory of water", degradation over time could explain the counterargument against homeopathy that water should retain the memory of all of its encounters.

The Benveniste studies

Human basophils are a rare granulocyte cell type accounting for 0.1–1% of white blood cells; these cells contain large numbers of "granules" which store inflammatory mediators, including in particular histamine. These cells can be cultured readily and studied in vitro. In these cells, exposure to anti-human-IgE antibodies triggers a "degranulation" process in which the granules fuse with the plasma membrane to release their contents, including histamine, into the extracellular fluid. At high concentrations (>10⁻⁶ M) histamine binds to H2 receptors on the surface of the basophils, and regulates the basophil degranulation by feedback inhibition.

Basophil activation can be measured in several different ways. First, degranulated cells can be stained and then counted; this is a subjective measurement and is prone to variable outcomes depending on the observer. Second, histamine release into the culture medium can be measured using fluorimetric assays. Third, the fusion of cytoplasmatic granules leads to the expression of the marker CD63 on the surface of the basophils; the percentage of basophils that express CD63 can be determined with flow-cytometry, and correlates well with histamine release.

In 1988, Jacques Benveniste (1935-2004) a distinguished French immunologist published a controversial paper in Nature reporting on the action of very high dilutions of anti-immunoglobulin E on the degranulation of human basophils.. At the high dilutions used, the solutions should have contained only molecules of water, and no molecules of (anti-IgE) at all. Benveniste concluded that the configuration of molecules in water was biologically active, and a journalist coined the term water memory.

Nature published the article with two unprecedented conditions: first, that the results must first be confirmed by other laboratories; second, that a team selected by Nature be allowed to investigate his laboratory following publication. Benveniste accepted these conditions; the results were replicated in Milan, Italy; in Toronto, Canada; in Tel-Aviv, Israel and in Marseille, France, and the article was accompanied by an editorial titled "When to believe the unbelievable."

After publication, the follow-up investigation was conducted by a team including the editor of Nature, Dr John Maddox, American scientific fraud investigator and chemist Walter Stewart, and "professional pseudoscience debunker" James Randi. With the cooperation of Benveniste's team, under double-blind conditions, they failed to replicate the results. Benveniste refused to withdraw his claims, and the team published in the July 1988 a detailed critique of Benveniste’s study. They claimed that the experiments were badly controlled statistically, that measurements that conflicted with the claim had been excluded, that there was insufficient"avoidance of contamination, and that there were questions of undisclosed conflict of interest, as the salaries of two coauthors of the published article were paid for under a contract with the French company Boiron et Cie.

In the same issue of Nature (and subsequently) Benveniste vigorously attacked the Nature team’s "mockery of scientific inquiry."

Subsequent attempts by other labs to reproduce Beneviste's results have failed to reproduce the effects .

However other studies have looked at the effects of very low concentrations of histamine on degranulation induced by anti-immunoglobulin E (IgE) antibodies, and again reported effects at very low concentrations .. As degranulation itself produces relatively high concentrations of histamine in the medium, one would only expect an effect with very high concentrations of added histamine - and indeed the most recent study reported significant effects only at 10-2M histamine. These experiments generally involved dilutions of histamine to concentrations of as low as 10-38M, and the dilutions were performed conventionally not according to the protocols used in homeopathy.

So how is it possible that adding vanishingly low concentrations of histamine to a preparation that is already secreting high concentrations might have any effect? What could possibly explain the extraordinary results reported by Beneviste and others?

One difficulty with the basophil preparation is that, in these cells, degranulation can be triggered by many different stimuli, including slight mechanical disturbances and environmental variations in temperature, and is sensitive to small differences in incubation time, making adequate controls very difficult. For example, in the experiments of Giggisber et al, the authors found no significant effects of low dilutions of histamine, but did find significant effects for row numbers of the microtiter plates - i.e. there was a significant effect simply of the order in which the samples were assayed.They concluded that seemingly, trivial differences in the experimental set up can lead to significant differences of the results.

Water in living organisms

Water is essential for functional macromolecular folding, stabilization and activity, transport, membrane formation and protein insertion into membranes; it the intracellular matrix in which biological molecules interact. Understanding exactly how water diffuses when confined in proximity to complex macromolecules inside a cell is therefore an important challenge. The diffusion coefficient of water in biological tissues has been measured using nuclear magnetic resonance, and these have shown that, within a cell, water diffuses much more slowly than pure water in aqueous media. This is at least partly explained by tortuosity effects, macromolecular crowding and confinement effects, but some (but not all) have suggested that the interaction with macromolecules might cause "clustering" of water molecules -that it might change the structure of the intracellular water.

In contrast with this post-hoc analysis of water behavior in cells (proteins may cause water to arrange in clusters - or not), Patterson proposes, in an invited paper for Chaplin's Water structure and science resource, "the cluster-domain model (which) depicts the cytoplasmic space as a densely packed world: not as a fluid solution, and not as protein molecules in empty space". Further, it postulates a "protein-water network (...) an energized gel whose units, protein domains and water clusters, manipulate pressure and tension to perform mechanical steps." This model is intended "to explain (...) the mechanical and energetic events of the cell (because) the traditional view (statistical mechanical principles) cannot explain (how these events) proceed without failure (...)". According to this model, the interaction of water with macromolecules does not cause "clustering" of water molecules, it is a cluster: a “pressure pixel”.

The cell can be seen, from a somewhat extreme structuralist point of view, as organized water. There is an incipient order in liquid water, which is given long-range coherence and permanence by the protein framework. In the words of A. Szent-Gyorgyi, “Life is water dancing to the tune of solids”.

Enzyme function: random events or coherent action? (2004) JG Watterson in Pr. Chaplin's Water structure and science online resource.

As we have seen, the materials science of liquid water, in a similar fashion, is abandoning the random network model of liquid water. The physics and the biophysics of water could be united in a synthesis that may accomodate homeopathy.

Water structure as a basis for homeopathy

"Based on this evidence we would be ready to accept that homoeopathy can be efficacious, if only the mechanism of action were more plausible". Kleijnen J et al.(1991). Clinical trials of homeopathy. British Medical Journal, 302:316–23.

In 2003, Molecular and Cellular Biochemistry published a review of the possible molecular mechanisms underlying the action of homeopathic drugs. In it, Khuda-Bukhsh explains that, even in the time of Hahnemann, it was evident that the vehicle or solvent (water or alcohol) was considered the medicine, rather than the molecule.

The research on the plausibility of homeopathy is an attempt to characterize how the behaviour of the molecules of a solvent could differ depending, first, on the solute that was diluted in it and, second, on the kinetic energy imposed on the solvent ("succusions", "dynamisations", "shakings"); it tries to answer the question: are all waters equal? Should we recognize that "water is water, period", or can some non-random, biochemically significant conformational changes happen in the way water molecules behave with each other, when a molecule is "imprinted" in a solution by dilutions and dynamizations?

The structure of liquid water is generally assumed to be a network of H₂O molecules forming short-lived (on the order of 10⁻¹² s) hydrogen bonds. According to this model the existence of long-term structural changes appears unlikely, and if by chance such structures are formed, they will disappear in a few picoseconds.

Materials scientists and physicists studying liquid water challenged the assumption that the unquestionably short life of these bonds determine an equally short life to the structures found in water, at the larger scale of 200 or more H₂O molecules. At an even larger scale, it can be easily observed that a wave keeps existing despite of the constant doing and undoing of hydrogen bonds, and that ice sculptures are also made of H₂O molecules constantly bonding and separating. In the same way, water clusters of a hundred or more molecules are actually structures that have a longer life than the individual bonds composing it.

The 65 reported anomalies of water reflect and heterogeneity in its structure: liquid water is a combination of different phases (a term in materials science to designate 3D arrangements of molecules or patterns) that are not short-lived, although, at the smaller, molecular, scale, the incessant agitation might evoke the impression that no higher order can exist.

Rao, Roy, et al. published preliminary results suggesting that different homeopathic preparations can be distinguished from each other and from controls, using two spectroscopy techniques that the authors judged better suited to analyze the "co-operative nature of structural differences".

Van der Waals bonds determine the interesting properties of fluids. (...)

(under revision:)

It is known that various phenomena can take place in water. Two physicochemical phenomenas have received special attention in the context of homeopathy research: clathrates and solitons.

Clathrate formation and growth

Khuda-Bukhsh reviews the research, consistent with some work in general physical chemistry, which suggests that the process of homeopathic preparation might indeed have an effect on water. Assuming, for example, that clathrates form, there is no current understanding, in molecular pharmacology, of mechanisms by which clathrates would have a metabolic effect. The role of clathrates and other crystalline structures in biochemistry is under study. (...)

Clathrates are complexes of water molecules around low-molecular-weight molecules (e.g., methane) or atoms (e.g., xenon) that can cause the growth of other clathrates devoid of central molecules. The presence of clathrates affects the results of mass spectrometry. This would be one technique for validating this hypothesis, by testing for clathrates in homeopathic preparations.

Silicates

Vigorous shaking of the water in glass bottles can cause small amounts of silica (silicate) fragments or chips to fall into the water. Homeopathic drug manufacturers use a double-distilled water in making their medicine, and whatever medicinal substance is placed in the water might interact with the silicate fragments and may change the structure of the water in unpredictable ways. Despite this experimental fact that these silicate fragments occur in homeopathic water, it is still unclear how continual dilution and succussion (vigorous shaking) increases the therapeutic effects of the medicine and reduces the necessity for frequent dosing (which homeopaths claim is generally the result of using the higher homeopathic potencies).

Nano bubbles/Nano particles

It has been suggested that micro-bubbles and nano-bubbles, caused by vigorous shaking, can "burst" to produce microenvironments of higher temperature and pressure. One group of material scientists have estimated that the vigorous shaking involved with making homeopathic medicines changes the pressure in the water, akin to water being at 10,000 feet in altitude. These scientists suggest that the homeopathic process of using double-distilled water and then diluting and shaking the medicine in a sequential fashion changes the structure of water.