Association induction hypothesis

The association induction hypothesis (AIH) is a scientific theory of the cell created by Gilbert Ling, a cell physiologist and biochemist. Introduced in 1962, it attempts to explain all biologic life phenomena on the basis of the properties and activities of microscopic assemblies of molecules, atoms, ions, and electrons of the smallest unit of life called nano-protoplasm. The AIH theory is considered to be a bulk phase theory that is an alternative and controversial hypothesis to the generally accepted Cell membrane theory and Membrane pump theories.

Overview of the theory

Ling claims that

• Water and ions in cells exist in a different physical state than in a dilute water solution.
• Water in cells is dynamically structured into polarized-oriented multilayers
• Structured water is in a higher (negative) energy and lower entropy state than in free water and can differentially exclude solutes according to the “size rule” with lower solubility for larger solutes.
• Most cell cations (the positively charged ions) are associated with fixed anions (negatively charged ions) on cell proteins.
• Most intracellular ions are adsorbed onto charged sites on proteins; the preferential adsorption of potassium ion being due to the inductive effect of the neighboring adsorbed detachable molecular entity called electron-withdrawing cardinal adsorbent or EWC reducing the electron density of the carboxyl groups on proteins. The most important EWC are the end product of energy metabolism called ATP
• Once the EWC like ATP and potassium ions are adsorbed in place on the cell protein, they cause the protein to unfold. A force of attraction offered by the exposed dipolar charged groups on the unfolded protein-backbone-chain causes water molecules to line up in multiple layers. This means the water molecules are no longer free or random but exhibit an orderly arrangement, which in principle resembles that of ice crystals although cellular structured water is vastly less solid than ice.
• Various protein EWC systems are interwoven in a latticework that extends throughout the cell to form a skeleton-like structure resembling a sponge. The EWC laden skeleton controls ion concentrations by choosing potassium in preference to other available monovalent ions like sodium and by structuring water.
• Although much extracellular sodium ion and water molecules diffuse in and out of the cell, no energy is required for the exchange. In theory, the cell could hold these asymmetrical concentrations forever without using energy. Only when cells are engaged in reversible life activities is the metabolic activities called upon to replace the momentarily depleted ATP.

Beginnings of the theory

In 1947, as a graduate student at the Department of Physiology of the University of Chicago, Gilbert Ling in preparation for a talk on "The Sodium Pump" could not find in the libraries any genuine experimental evidence to support the theory which had been accepted as scientific fact. Despite warnings from two highly respected professors to not become a martyr and to leave the sodium pump alone, he went on to carry out a number of experiments to test the validity of the membrane (pump) theory.

The microscope offered a new view of life and in 1849 with the publication of Theodor Schwann's Cell theory presented in his book in German 'Mikroscopische Untersuchungen über die Übereinstimmung in der Struktur and dem Wachstum der Thiere und Pflanzen' and its English translation Schwann, T. (1847) Microsopical Researches into the Accordance in the Structure and Growth of Animals and Plants. The living cell was erroneously thought to be a puddle of watery solution enclosed in a membrane in which some microscopic devices (later to be called pumps) regulated the chemical contents of the fluid inside and outside the cell. The mistake of thinking of a cell as a watery sac as opposed to a solid was rectified in 1923 by cell anotomists but the cellular free water and membrane pump concept of cell physiologists remained, possibly due to the “unquestioning acquiescence of the German textbooks” toward what were introduced in Schwann’s Magnum Opus.

Ling, did not leave the sodium pump alone “because above all, I thought it was a very serious question that needed to be resolved.” and in the ensuing years produced a number of independent experimental sets of evidence against the membrane theory. He also wrote a number of papers and 5 books to support his alternative view of the cell called the Association Induction Theory or AIH for short.

Key concepts

Ling’s fixed charge hypothesis

First presented in 1952. Ling pointed out why fixation in space of an ion enhances its association with its oppositely-charged counter-ion. The intensity of mutual attraction between the counter-ion and the fixed ion varies directly with the product of their charges and inversely with the square of the distance between them. Although a naked Na⁺ is smaller than a naked K⁺, a hydrated K⁺ is smaller than a hydrated Na⁺. Accordingly, a hydrated K⁺ can stay closer to the singly charged oxygen atom of an oxyacid group. As a result, preferential electrostatic adsorption of K⁺ over Na⁺ follows. It was pointed out parenthetically that in muscle cells, the contractile protein, myosin alone carries enough fixed anionic sites to adsorb all the cell's K⁺.

Polarized orientated multilayer theory

First published in 1965 and revised and extended in (2003–2006) Ling maintained that virtually all cell water assumes a dynamic structure different from that of normal liquid water and all or nearly all the water molecules in a living cell are adsorbed as polarized-oriented multilayers on the fully extended protein chains. The carbonyl (CO) and imino (NH) groups of these fully extended proteins offer properly spaced, alternating negatively and positively charged sites. Together, these charged sites adsorb a layer of (oppositely oriented) water molecules or dipoles.

The water molecules thus oriented and polarized in turn polarize and orient a second layers of water molecules and this continued until virtually all cell water is oriented and polarized. Initially Ling in 1965 based his polarized multilayer (PM) theory on the earlier theories of deBoer and Zwikker (1929) and Bradley (1936).

In 1993, Ling had presented the theory of solute distribution and supportive evidence on how the physicochemical properties of water assuming the dynamic structure of polarized multilayers would become different from those of normal liquid water. Foremost among these altered properties is the theoretically predicted and experimentally confirmed reduced solvency for hydrated Na⁺ and other large solutes in polarized-oriented water in living cells and model systems.

In 2003 Ling introduced a new theoretical foundation for the long-range polarization and orientation of water.

More recent experiments have confirmed the molecular layering near charged surfaces, in particular through the work of Gerald Pollack.(2001, 2013) and Martin Chaplin on structured water.

Nano protoplasm

The AIH attempts to introduce microscopic physiology in which molecules, atoms, ions and electrons replace and give new meanings to the macroscopic concepts of membranes, pumps, rigid pores, semi-permeability etc. Central to this revolutionary leap is the introduction of the concept of the (unit of) microscopic protoplasm or nano-protoplasm as the smallest unit of life.

The three most abundant components of living cells and their constituents are water, protein and potassium ion (K⁺). The individual units of each of these components in the form of single molecules and single ions are all directly or indirectly in contact or associated with one another. Through electronic polarization or induction, each entire nano-protoplasmic unit or their bigger aggregate can function coherently.

Resting and active living state

left, active living state;right, resting living state”

A nano-protoplasmic unit is an electronic machine. As such, it can exist in one of two alternative states: the resting living state and the active living state (See Figure).

In the resting living state, nano-protoplasm (or its larger aggregate) is at equilibrium and therefore does not require continual energy expenditure for its maintenance. Instead, for a nano-protoplasm unit to assume and sustain its resting living state, a special region (or site) of the protein component must be occupied and acted upon by a specific molecular agent called ATP. A product of the cell's energy metabolism, ATP serves its functions by its steady adsorption as such and exerts a far-reaching and powerful electronic impact on the protein (and its associated molecular and ionic partners.)

Cardinal absorbents

In the AIH, both internally produced agents like ATP and external applied drugs and hormones belong to what are collectively called cardinal adsorbents. Cardinal adsorbents fall into two categories: electron-withdrawing cardinal adsorbents or EWC and electron-donating cardinal adsorbents or EDC. (A third kind, called electron-indifferent cardinal adsorbents or EIC is of rare theoretical significance and can be ignored). ATP and calcium ion (Ca⁺⁺), for example, are highly important EWC’s.

Importance of proteins and other chemicals

Among the three major components of living cells, protein alone is made only by living cells of one kind or another and thus unique to life. As a rule, the proteins in different kinds of nano-protoplasm are different. As a long-chain polymer, the building blocks of a protein are some 20 different small molecules called amino acids. Each amino acid can be represented by the general formula, NH₂CHR₁COOH, where R₁ differs among different amino acids but the remainders are almost always the same. The symbol R₁ for the amino acid, glycine, is a simple neutral H atom, that for alanine is a neutral methyl group, that for aspartic acid is COOH(CH)₂ with a negatively charged, anionic β-carboxyl group hanging on its end. For the amino acid, glutamic acid, it is COOH(CH2)2 with an anionic γ-carboxyl group at its end. For the amino acid lysine, it is a NH₂C(H)₂(CH₂)₃ with a terminal positively charged cationic ε-amino group. For arginine, it is (NH₂)₂H(CH₂)₃ with a terminal cationic guanidyl group. Equation 1 below shows how a miniature protein or short polypeptide or tripeptide containing two peptide linkages (CONH) is formed by joining three free amino acids with the loss of four hydrogen atoms and two oxygen atoms in the form of two water molecules.

NH₂CHR₁COOH + NH₂CHR₂COOH + NH₂CHR₃COOH → NH₂CHR₁CONHCHR₂CONHCHR₃COOH + 2 H₂O                         (1)

As a part of a protein molecule, what remains in the polypeptide or protein of each amino acid is called an amino acid residue. The symbol R₁ that each amino acid carries is called a side chain. In a neutral medium, the β- and γ-carboxyl groups are fully ionized and thus each endows the protein with a single negative electric charge of a fixed mono-valent anion. Each ε-amno group and guanidyl group, on the other hand, endows the protein with a positive charge in the form of a fixed mono-valent cation. β- and γ-carboxyl group may function as adsorption sites for K⁺, Na⁺ or fixed cations. Pairing of a fixed cation and a fixed anion forms a salt linkage. The peptide linkage, CONH, may bind onto similar peptide group on the same protein chain fourth removed in either direction, forming an α-helix structure or alternatively bind multilayers of water molecules. The choice between the two is determined by the electron density of the sites involved, a subject which the section farther below will discuss. Above all, the alternative structure of the peptide linkage undergoes extremely rapid transition or resonance, which makes the polypeptide chain highly polarizable and thus suitable for long-distance information and energy transfer. In addition, resonance also endows short-range negative electric charge to the carbonyl oxygen atom and positive charge to the imino NH group.

Details of the theory

The association induction hypothesis explains the four cell physiology subjects:

1. Solute Distribution
2. Cell Volume Control
3. Membrane Permeability
4. Resting Potential

Association

The association aspect of the AIH refers to the association between water molecules and the carbonyl oxygen atom and imino hydrogen atom of amino acid residues in polypeptide chains. It also refers to the association of potassium ions with beta and gamma carboxyl (COOH) groups on the protein chains as well. But above all, association indicates the state of direct and indirect physical and electronic contact among all the major components of the living matter (protoplasm) including water, protein, potassium ion and ATP when the protoplasm is in its resting living state

Induction

The inductive aspect of the AIH refers to the ability of adsorbed molecular entities to influence the electron distribution of the functional groups of the nano-protoplasm units and larger living structures.

Solute distribution

Living cells contain a large amount of water, making up some 80% of the cell's weight, though it could be as low as 50% and as high as 90%. The rest of the cell consists mostly of giant protein molecules (and in much smaller amounts, the nucleic acids, DNA or RNA, and carbohydrates like glycogen). The cell also contains an assortment of small molecules and ions. Some of these small molecules and ions like adenosine triphosphate (ATP) are vital to life.

When a salt dissolves in water, it splits into two oppositely charged particles or ions, the positively charged ion is called a cation and the negatively charged ion is called an anion.

Most living cells spend their lives in a salt-water environment. When common salt, or sodium chloride, dissolves in water, the ionically-bonded molecule splits into two charged particles, or ions — positively charged sodium ions (Na⁺) and negatively charged chloride ions (Cl⁻).

In the process of dissolution, some of these ions take up a more or less permanent coat of strongly bound water molecules and are then referred to as hydrated sodium ions, for example.

The sodium-ion concentration in most living cells is low, equal to about one tenth to one third of that in the fluid outside the cell. In contrast, another univalent positively charged cation, the potassium ion, though chemically very similar to the sodium ion, distributes itself in such a way that its concentration inside the cells is some forty times higher than in the surrounding medium (interstitial fluid).

The asymmetries in the distribution of the sodium ions (3 to 7 times greater outside concentration) and the potassium ions (40X greater inside concentration) are found in virtually all living cells. How does the cell physiologist explain this unusual pattern of distribution of the potassium and sodium ions?

Comparisons with membrane-pump theory

The mechanisms and explanations offered by the membrane-pump theory and the association induction hypothesis are profoundly different.

In the Membrane pump theory, a living cell represents essentially a bag-full of water, an aqueous solution of proteins, a lot of potassium ions, lesser sodium ions, and other dissolved substances in an aqueous solution. With the membrane-pump theory, the water inside the cell shows no major difference from normal liquid water bathing the cells. Nor are the small and large molecules and ions inside the cell markedly different from similar substances dissolved in normal liquid water.

With the membrane-pump theory, cell proteins dissolved in this normal liquid cell water are themselves in their so-called native state (a misnomer) that is, a stable, and reproducible state, which a protein assumes reproducibility in vitro when purified by certain standard technical procedures and dissolved in water. However, this so-called native state is not the normal, natural state of proteins found in living cells, particularly cells in the resting living state.

In the membrane-pump theory, an all-important but very thin membrane, called the cell membrane or plasma membrane encloses this bag of watery solution. In the membrane pump theory, it is this very thin membrane which determines the chemical makeup and ionic distribution (potassium more in the inside, sodium more on the outside) of the cell.

The cell membrane accomplishes these tasks by virtue of postulated critical diameters of rigid membrane pores (or channels), which admit small molecules and ions but bar larger ones and by the ceaseless inward or outward transportation of ions by a postulated energy-consuming sodium-potassium pump located in the cell membrane.

Then there are also pumps for the different sugars, for the many different (free) amino acids and the many different positively charged as well as negatively charged ions etc.

The importance of structured water in the AIH theory

In Ling's theory a distinction is made between normal water as you might find in a wet sponge and water that is bound to for example raw hamburger meat. In the former it is possible to squeeze out all the water but in the latter it is impossible even after spinning the meat in a centrifuge at 1,000 times the force of gravity for 4 minutes, water still remains in chopped-up muscle cells. Cell water cannot be normal liquid water. Were the cell water truly normal liquid water, it would have been extracted by squeezing or even more so by centrifugation. What should remain in squeezed hamburger or centrifuged muscle fragments should be nothing more than dried proteins like a fully squeezed-out sponge. But that does not happen while the cells are still alive or close to being alive as in the fresh hamburger.

Our next question is to find out how water (making up some 80% of the weight of the fresh muscle (as well as other cells) can be held so tenaciously inside the cell, resisting centrifugation at 1,000-times gravity. Since the cell is primarily water and proteins, one naturally seeks an explanation in terms of the interaction between the more mobile water molecules and the more fixed proteins. Theoretically speaking, all proteins have the potential of reacting with a large amount of water. In reality, only some proteins interact with a large amount of water "permanently."

One familiar water-retaining protein is gelatin, the major ingredient of the powdered material that comes in Jell-O packets. Jell-O is almost all water and yet in Jell-O, water can "stand up" as no normal pure liquid water ever can. This ability of the water in Jell-O to stand up against gravity, which ice can also, indicates that the water-to-water interaction in the Jell-O water has been altered by the only other component present, gelatin. Why and how is this possible? First, what is gelatin? Gelatin is a product of the "cooked" animal skin, hoof, horn, etc. The main source material of gelatin from these animal parts is the protein known as collagen, the major protein component of our tendons and skin. That gelatin is an unusual protein has been known for a long time. Thus the term colloid is its namesake.

Ling's association induction hypothesis attempts to offer an explanation for the uniqueness of gelatin (as well as colloids) and the "living substance" or protoplasm.

Proteins are long chain molecules. However, unlike ordinary chains where each link is just like another link, the proteins are chains of some twenty different kinds of links, called amino-acid residues which are amino acids in a "joint" form. Viewed at close distance, each amino acid component of the protein chain (a long string of amino acid residues) offers a pair of electrically charged or polar groups: a negatively charged carbonyl oxygen atom carrying a “lone pair” of (negatively charged) electrons; and an imino H atom, which is lacking in one electron and thus positively charged.

In most isolated proteins, each backbone carbonyl oxygen atom is joined (or hydrogen-bonded, or H-bonded) to the H atom of the imino group belonging to the fourth amino acid residue down the chain. In this way, the protein chain assumes what is known as the alpha-helix structure. Both the polar imino group and the carbonyl groups also have affinity for water molecules. The O end of the H₂O water molecules can adsorb onto the protein backbone’s positively charged imino site; the H end of the H₂O water molecules adsorb onto the negatively-charged carbonyl oxygen atom of the protein backbone.

However, in most isolated proteins in their so-called native state, backbone NH and CO groups are joined together intra-molecularly via H-bonds just mentioned. Thus joined, they are unable to interact with water. However, as first pointed out by Ling in 1978, a large portion of the gelatin chain cannot fold into the alpha-helical folds because 54% of the amino acid residues making up gelatin are either unable (proline, hydroxyproline) or disinclined (glycine) to assume the alpha-helical structure. Accordingly, a large portion of the gelatin protein molecules remains permanently in the fully extended conformation just like the proteins in a resting living cell. In this fully extended conformation, the backbone CO and NH groups are exactly properly spaced and directly exposed to and they are free to interact with not just one layer, but multiple layers of water molecules. Water thus polarized endows gelatin with many of its unusual properties, which it shares with living cells.

This is then the essence of what has been known as the polarized multilayer theory of cell water first introduced by Ling in 1965.

Parenthetically, by multiple layers, of water this means no more than a few layers (5, 6, or 7 layers of stacked-up water molecules) on each protein chain (and there are a vast number of such protein chains in a typical cell). Stacking 5 to 7 layers of water molecules on top of one another would be quite adequate to account for all of the intercellular water existing in the dynamic structure of polarized multilayers as proposed by the AIH.

In 1993, Ling wth Niu and Ochsenfeld published a paper entitled "Predictions of polarized multilayer theory of solute distribution confirmed from a study of the equilibrium distribution in frog muscle of twenty-one nonelectrolytes including five cryoprotectants" that provided strong experimental support for the Polarized orientated multilayer theory together with his quantitative theory of solute distribution.

In 2003 Dr. Ling published a new theoretical foundation for the polarized-oriented multilayer theory of cell water that shows that under ideal conditions, a checkerboard of positive and negative sites at the right distance apart can polarize and orient multiple layers of water molecules ad infinitum. Underlying this astonishing finding is the recognition that the reach of orientation could be very far while the reach of polarization is invariably confined to a single or at most two layers. Accordingly, the dynamic structuring of cell water is as a rule more likely to be flatter and less tapering than envisioned before.

Since then it has been fully established that not only gelatin, but a large array of acid- and alkali-denatured (isolated) proteins, as well as long chain organic molecules or polymers that have the properly spaced polar groups can exhibit physico-chemical properties, qualitatively if not quantitatively resembling those of living matter or protoplasm.

Water in all these model systems and in the living cell shares the property of maintaining, at a lower concentration, those molecules and hydrated ions found at low levels in most living cells. The most outstanding is the sodium ion (lower on the inside than the outside of the cell by a factor of 3 to 7).

In summary, according to the association induction hypothesis all or virtually all the water in living cells assumes the dynamic structure of polarized multilayers.

Water assuming this dynamic structure endows the living cells with many attributes which had hitherto been assigned to other (incorrect) causes.

Among these solute distribution attributes is that of maintaining a low concentration of large (hydrated) ions like sodium, sugars, and free amino acids. An underlying assumption is that some of the cell proteins exist in the fully extended conformation even though, unlike gelatin, these proteins do so only conditionally (in the resting living state) rather than permanently.

In other words, they do so only when the cells are alive.

The membrane-pump theory has not been able to produce an answer to this simple but basic question yet.

The resting living state

The major ingredients of living cells are proteins, water, small molecules, some large molecules like DNA and ions (sodium, potassium, and chloride).

In the conventional membrane-pump theory, all these ingredients exist as parts of a dilute aqueous solution. In contrast, according to the association induction hypothesis, proteins, water, and much of the small molecules and ions are closely associated or bonded together and maintain themselves in a high-negative energy and a highly ordered or low-entropy state called the resting living state. A cell maintained at its resting living state is alive.

Most individuals know that matter exists in three different states: a gas, a liquid, or a solid. Water, therefore, exists as gaseous water (water vapor) liquid water, or solid water (ice). However, the liquid water state has two different sub-states: less structured (as in normal liquid water) and more structured (as found inside the cell). The multilayered structured water is due to adsorption of the water molecules to the carbonyl (CO) and imino (NH) polypeptide bonds. Thus structured water (inside of a cell) can be considered as a state of water somewhere in between normal liquid water and ice. Water inside cells is somewhat structured. But water in the solid state is more rigidly structured. Now water and ice comprise the same water molecules represented as H₂O. As mentioned before, these molecules exist in different physical states, which we call respectively the liquid state and the solid state. Note that each of these states specifies the relationship between individual H₂O molecules in characteristic space and time coordinates. In ice, water molecules are rigidly fixed in space and move relatively little in time. Water molecules in liquid water are much more mobile and move about more freely with time.

Similarly, the resting living state specifies interactions among the individual components of the living substance of closely associated proteins, water, small molecules, and ions in relatively fixed-space-and-time coordinates. In particular, special emphasis is on their mutual electronic interactions which provide the basis for their existence in what physicists call "cooperative states" in which there are positive near-neighbour interactions among the individual components of the assembly. To maintain the resting living state, interaction with certain key small molecules like adenosine triphosphate (ATP) is vital. When the cell is deprived of its supply of ATP, the cell dies and the protoplasm enters into another state, called the dead state. This is permanent and irreversible.

In the resting living state, cell proteins cause the bulk of cell water to exist in the dynamic structure of polarized multilayers. Water, assuming a dynamic structure shows reduced solvency for and partly excludes large molecules and ions like hydrated sodium and large molecules like sucrose, and raffinose and PEG (Polyethylene glycol) 900.

The cell proteins also offer their singly and negatively charged beta- and gamma-carboxyl groups (COOH) to adsorb preferentially on a one ion-one site basis hydrated potassium ions (over the hydrated sodium ion, for example). Since there is a high concentration of beta- and gamma-carboxyl groups carried on intracellular proteins, the potassium ion concentration in living cells is as a rule much higher (40 times higher) than in the surrounding medium.

However, there is a hugely larger number of negatively-charged beta- and gamma-carboxyl groups (COOH) on the fully extended cell protein molecules than there are adsorbed potassium ions to neutralize them. The rest of the fixed anions are neutralized by fixed cationic ε-amino groups and guanidyl groups.

Here again one finds another profound difference between the AIH and the membrane pump theory, which requires a continuous supply of energy just to keep the ions and molecules where they are and at the concentrations they are found—a requirement that permitted a set of crucial experiments which has unequivocally disproved the membrane-pump theory. The rest of the fixed anions are neutralized by fixed cationic ε-amino groups and guanidyl groups.

Induction, ATP and cardinal absorbents further explained

Thus far we have dealt with the "associative" aspect of the association induction hypothesis. Equally important is the "inductive" aspect, or electrical polarization. Thus in the AIH, the living cell is essentially an electronic machine, where the electronic perturbations are not carried out through long-range ohmic conduction of free electrons along electric wires but by a falling-domino-like propagated short-range interactions.

In the association induction hypothesis, it is this basic electronic mechanism, which not only permits such key components, referred to as cardinal adsorbents, to sustain the protoplasm of closely associated proteins-ion-water system in its normal resting living state.

It also provides the mechanism for cardinal adsorbents to control the reversible shifts between the resting living state and the active living state. The cardinal adsorbent par excellence is the ultimate metabolic product of the combination of the food we eat and the oxygen in the air we breathe, adenosine triphosphate (ATP).

This ubiquitous and crucial small molecule, ATP, was once wrongly believed to carry extra energy in the so-called high-energy-phosphate bonds. However, there is no doubt that ATP is strongly adsorbed on certain key sites (cardinal sites) on cell proteins and the adsorption energy upon these proteins is what gives ATP its energy, not the "high-energy phosphate bonds." Indeed, the adsorption energy of ATP on the muscle protein, myosin, even exceeds what was once(wrongly) assigned as a "high energy phosphate bond" and this high adsorption energy fits like a glove in its central role in polarizing the protein-water-ion system thus maintaining the assembly in the resting living state.

Being in the resting living state specifies what is living. In the resting living state, all the major components exist in their closely associated high (negative) energy and highly ordered, low entropy state.

The transition into the dead state specifies what is dead. In the dead state, water and ions are to a large extent liberated and exist as free water and free ions, with a large entropy gain (more disorganization). In death, the proteins enter an internally neutralized alpha-helix or beta-sheet state.

On first look, there might seem to be a third state, the active living state. In fact the active living state and the dead state are the same state with the difference that the active living state is transient and reversible, while the dead state is irreversible and lasting.

The role of polarized-oriented water in the phenomena of cell volume control, membrane permeability (and the resting potential)

In 1962, Dr. Ling pointed out that the swelling of frog muscle at low and high pH could be due to the respective discharging of fixed cations or fixed anions of the muscle cell proteins. In consequence, the cell-volume-restraining salt linkages formed between the fixed cations and anions are broken up, allowing additional water to enter the cell and expand the cell volume. The critical role of the dynamically structure polarized-oriented water in membrane permeability is just as profound. Since in the AI Hypothesis, all living matter is made of collections of nano-protoplasmic units (NPU) and each NPU contains a large amount of water, there is no escape that cell membranes are primarily water membranes. This fact readily explains why all living cells are as rule far more permeable to water than to solutes dissolved in water. Since in the membrane theory, the continuous phase of the cell membrane is primarily phospholipid bilayers and phospholid bilayers are virtually impermeable to water, this contradiction adds yet another reason to reject the membrane (pump) hypothesis beyond the six already cited above. Dr. Ling has also shown in his quantitative theory of solute distribution that the dynamically structured water is extremely sensitive to small changes in the polarization energy produced by fully extended proteins. This extreme sensitivity gives the cells the means to achieve a high level of versatility. Thus, it allows most cell membranes to have very high permeability to essential nutrients like D-glucose to keep them alive. Yet it has no problem producing membranes of extremely low permeability toward the same essential nutrients as that on the outside surface of frog skin to prevent loss of the valuable nutrients to pond water. Our next subject is the cellular resting potential.

Closest-contact surface adsorption potential

With the perfection of what has been known as the Ling-Gerard capillary electrode, it became possible to insert the tip of the electrode into living cells like frog muscle cells and record reproducibly an inside-negative electric potential difference of approximately 90 millivolts. In the conventional taught view, indeed even in Dr. Ling’s own Ph.D. Thesis, this potential difference recorded is regarded as a membrane potential, arising from the inside high, outside low K⁺ ion gradient with the critical assumption that the cell membrane is absolutely and permanently impermeable to the Na⁺ ion. When radioactive tracer of Na⁺ became available, its deployment provided for the first time in history the means to determine if all cell membranes are completely impermeable to Na⁺ ion. Soon a unanimous conclusion was reached and it is just the opposite of the past assumption. All cell membranes examined are in fact fully permeable to Na⁺. With the assumed difference between the two ions K⁺ and Na⁺ removed, the theory of membrane potential collapsed. In the wake of this critical juncture, Dr. Ling introduced as a part of the association-induction hypothesis, the closest-contact surface adsorption potential for the resting potential of living cells. In this theory, it is the microscopically thin surface of the cell membrane that carries the K⁺ selective beta and gamma carboxyl groups that give rise to the resting potential. He and his coworkers soon verified this theory by demonstrating for example, that a Corning 015 glass electrode sensitive to H⁺ but not to K⁺ became fully sensitive to K⁺ when it is covered with a thin layer of dried nitrocellulose or colloidin, long known for making K⁺ sensitive thimble electrodes.

Membrane permeability

In 1957, Dr. Ling described the result of a theoretical model, in which selectivity for K⁺ and Na⁺ could be reversed, as in the nerve or muscle action potential. The phenomenon was found to be a result from a difference in the electron density (or c-value) of the negatively charged oxygen atom of a mono-valent oxyacid group. At low electron density and a relatively low c-value (and the low (related) c-value analogue of the protein backbone carbonyl groups), K⁺ is preferred (and cell surface water dynamically structured.) Accordingly, K⁺ is adsorbed on the β- and γ-carboxyl groups of cell surface proteins and an inside-negative 90 millivolts resting potential stands.

In contrast, at a relatively high c-value of the same carboxyl groups created by the detachment of the EWC Ca⁺⁺, this ion selectivity is reversed and Na⁺ is preferred over K⁺. At the cell surface, the momentary rise of the c-value of cell surface β- and γ-carboxyl groups (and c-value analogue of cell surface protein carbonyl groups) produces the neutralization of the inside-negative resting potential as well as the polarity-reversing “overshoot” of an action potential.

Criticisms of the theory

Image from a Transmission Electron Microscope of a lipid vesicle. The two dark bands around the edge are the two leaflets of the bilayer. Similar electron micrographs confirmed the bilayer nature of the cell membrane in the 1950s

The standard theory of the cell's plasma membrane has been experimentally confirmed through various methods of lipid bilayer characterization consistently for many years. This contradicts Ling's contention that the plasma membrane consists of structured water and not a 0lipid bilayer.

Osmium tetroxide, a common stain used to visualize cells, darkly stains the cell membrane without permeating the cell. This is because osmium tetroxide binds the phospholipid heads in the membrane. If Ling's hypothesis is correct, the nonpolar osmium tetroxide should permeate the polar structured water of the cell membrane and stain the interior, but it does not.

A number of criticisms have been raised against Ling's theory which according to Ling have been fully rebutted on both theoretical and experimental grounds.

K+ Ion mobility

Sir Bernard Katz, Nobel Laureate wrote in his booklet: "Nerve Muscle and Synapse" (McGraw Hill, 1966): "If cell K+ is selectively adsorbed, its mobility should be slower than in free solution". Experiments of Alan Hodgkin and Richard Keynes from the University of Cambridge shows K+ mobility in isolated squid axon close to that in free solution. Similarly Kushmerick and Podolsky demonstrated K+ mobility in short frog muscle segment close to that in free solution.

In response, Ling and Ochsenfeld showed that similar high K+ mobility in cell cytoplasm cited above can be reproduced at will if frog muscle cells were deliberately killed by prior exposure to metabolic poisons (or otherwise injured as in the region of the muscle cell close to the two cut ends of a short muscle segments used by Kushmerick and Podolsky in their studies). K+ mobility in healthy frog muscle cytoplasm is only 1/8 of that in free solution from 72 sets of experiments Ling and Ochsenfeld performed. for theoretical reason that while adsorption may slow down the rate of diffusion of K+ in frog muscle cytoplasm as Ling and Ochsenfeld has clearly demonstrated and discussed above, adsorption does not necessarily slow down the mobility of K+ or other ions. Thus, experimental observation showed acceleration of the mobility of adsorbed ion as on the surface of glass, for example).

In 1974, former gradual student to Ling, Chris Miller as a part of the reasons he offered for his rejection of the AI Hypothesis, raised a similar criticism. This criticism was fully and completely answered both on theoretical grounds and by experimental testing

In theory, an electric field effect suggested by Kushmerick and by Miller is not anticipated. The Law of Macroscopic Electroneutrality forbids K+ to travel alone in or out of the cut muscle cells in measurable quantity. It can only move in substantial quantities in or out of the cell (1) by exchange with one or more cation(s) traveling in the opposite direction, carrying a virtually identical amount of electric charges, or (2) accompanied by one or more kind of negatively charged ion(s) carrying a virtually identical amount of negative electric charges. For this reason, the overall phenomenon involved in the outward movement of labeled K+ movement in Ling and Ochsenfeld's study is an electrically neutral affair. As such, it is indifferent to the electrical potential field that may well exist across the cut end of the muscle. Experimental results described in the above-cited reference of Ling, 1979 further confirmed our theoretical anticipation. No influence of electrical potential field on the measured intracellular ion mobility could be observed.

Seat of Semipermeability

In 1973 Ling reported experimental evidence affirming that in agreement with the AI Hypothesis, water is polarized in multilayers at the cell surface which gives rise to its observed markedly higher permeability to water than to solutes (a phenomenon named by van't Hoff "semipermeability"). Later McElhaney (ibid 15, 777, 1975) contended as the title of his paper suggests: "Membrane lipid not polarized water responsible for the semipermeability properties in living cells". Biophysical Journal editor at the time, Frederick Dodge, refused Ling to rebut at a length adequate to answer all the many attacks made in that same journal. Ling's rebuttal was published in another journal four years later (Physiol. Chem. Physics, 9: 301, 1977).

Measurement of intracellular K+ ion activity with an intracellular K+ ion-selective microelectrode

Hinke and others inserted a K+-ion-sensitive glass microelectrode into a variety of giant cells and observed K+- ion activity---which one may define as an effective concentration--- close to that in a dilute solution of a free solution containing a similar concentration of K+ ion. However, Ling pointed out that the K+-ion-sensitive microelectrode used cannot monitor the K+ -ion activity in the whole cell---as the experiment was intended to measure---but only that in a microscopic film of water surrounding the ion-sensitive tip of the microelectrode inserted into the cell and that this part of the cytoplasm is inevitably traumatized by the very same intruding electrode.

As work of this type expanded, the K+-ion activity recorded began to show wide fluctuations, ranging from K+-ion activity that is only a small fraction of the average K+ concentration of the cell, to activity which far exceed the average K+ concentration. Such variations are themselves at odds with the basic tenet of the membrane-pump theory, which requires all intracellular K+ activity measured in all living cells to be the same and equal to the K+ activity of that of a free water solution containing the same concentration of K+ as that in cells.

In a detailed analysis of the whole picture, Ling showed that the wide spectrum of data reported can be neatly explained on the basis of the basic tenets of the association-induction hypothesis: (1) water existing in the normal and healthy state of polarized multilayers has reduced solvency for K+ and Na+ ions; (2) cytoplasmic proteins offer adsorption sites for the selective adsorption of K+ ion when the cytoplasm is healthy and uninjured; (3) cytoplasmic proteins lose the ability of selectively adsorbing K+ ion in rough proportion to the extent of damage the cytoplasmic protein suffers; (4) K+ released from injured cytoplasmic proteins may find its way into the free water film in contact with the electrode while the remaining water remains largely uninjured. (5) K+ released from injured cytoplasmic proteins may find its way into the free water in contact with the electrode while the remaining water is also depolarized.(1) and (2) in sturdy cells can explain the lower K+-ion activity observed than that predicted from the measured K+-ion concentration; (5) can explain the earlier reported data where the observed K+- activity equals the K+-ion concentration; (4) can explain the observed K+-ion activity exceeding the K+-ion concentration (for more details, see Ling, "In Search of the Physical Basis of Life"

Synthetic sodium pump show to be an experimental artifact

The supporters of the membrane-pump theory argued that an enzyme (K+-, Na+-activated ATPase) isolated from living cells---which can catalyze the hydrolysis of ATP in the presence of appropriate concentrations of Na+ and K+ ions--- is in fact the postulated sodium pump. In support, Goldin & Tong, and others incorporated isolated ATPase into phospholipid vesicles and showed that more radioactively labeled Na+ ion remained in the vesicles if the (presumed) energy source, ATP, was added to the buffer containing the labeled Na+. The authors attempted to explain the wrong direction of the Na+ "pumped" on the postulation that the membrane vesicle was inside-out, so that instead of pumping Na+ out of the vesicle making its Na+ level lower as it should, the pump was making the intracellular Na+-ion concentration actually higher.

In a detailed analysis of all the published data Ling and Negendank (Persp. Biol. Med., 23:215-239,1980, pp. 224–236) showed that it was highly improbable the observations and claims of Goldin, Hilden and others.

Ling and Negendank pointed out that the controlling step determining the level of Na+ ion in the vesicles could not be the initial loading (and the postulated pumping during that process). Rather, it was the leakage from the vesicles--- which the authors had overlooked---when the vesicles were subsequently passing through (the labeled Na+-free ) buffer solution in the Sephadex column---a step necessary in order to separate the radioactive Na+ ion trapped in the vesicles from the radioactive Na+ in the loading solution in which the vesicles were suspended before being loaded onto the Sephadex column. In other words, what they demonstrated was not how ATP activated the pumping of Na+ into the vesicle during the loading step. Rather it was that the inclusion of ATP in the loading process somehow had slowed down the subsequent leakage of labeled Na+ from the vesicles. That leakage process is a simple physical dissipative process and has nothing to do with the postulated energy-consuming pumping. One should also not forget that the concept of ATP containing a package of "high energy" had been disproved in the fifties and early sixties.

Ling and Negendank then pointed out how these observation on the so-called synthetic ion-transporting systems can be better understood in terms of a long-confirmed part of the subsidiary theory of ion permeability in the AI Hypothesis.

In addition, Ling and Negendank mentioned that since healthy Nature-made cytoplasm -freed, cell-membrane sacs fail to pump Na+- or K+-ion (see linked pagelp6a{2}),would it not be somewhat presumptions to claim that vesicles prepared by Golden, Hilden and others cited above--- highly skilled biochemists though they unquestionably are--- did better?

ATP control of K+ concentration

In the AI Hypothesis, ATP, the ultimate metabolic product of living cells, controls the level of K+ ion in living cells by adsorbing onto specific protein sites (cardinal sites) and in so doing maintains the suitable electron density ( or more precisely, the c-value) of beta- and gamma-carboxyl groups on which K+ ion is preferentially adsorbed. Accordingly, there should be a quantitative relationship between the equilibrium level of ATP and of K+ ion in living cells when the level of ATP was made to change by controlled action of metabolic poisons.

Rangachari et al. published K+ vs. ATP data from the study of rat myometrium. They concluded that the predicted linear relationship "did not always hold". A careful examination of their data showed that their data fully confirmed the original prediction except a single experimental data point (of high ATP and low K+ concentration). And that this point was produced by cooling the rat myometrium to zero °C. In answer, Ling pointed out that this departure actually lent additional support for the AI Hypothesis. The predicted quantitative relationships between K+ concentration and ATP concentration in living cells is restricted to observations made at the same temperature. Reduction of the temperature of warm-blooded mammalian tissues to 0 °C, as Rangachari et al. did, brought into operation another aspect of the AI Hypothesis.

That is, the adsorption of K+ is "cooperative" As such the K+ / Na+ distribution in mammalian cells undergoes a temperature transition characteristic of cooperative states between the K+-adsorbing state at high temperature and Na+-adsorbing state at a temperature below the transition temperature ( without perturbing the cell ATP concentration). And this is what Rangachari et al.'s single departing point confirmed.

In summary, Rangachari et al.'s data not only did not refute the prediction of the AI Hypothesis, they confirmed at once two basic aspects of the theory. (For additional discussion on temperature transition, see: Ling "In Search of the Physical Basis of Life" and Ling "A Revolution in the Physiology of the Living Cell" (Krieger, 1992, Chapter 7; pp. 188–196, also pp. 293–294)

NMR relaxation times of water protons in living and killed cells

Former graduate student to Ling, Peggy Neville and her coauthors see also Civan and Shporer showed that frog lens and muscle when killed by heating showed a shortening of the NMR relaxation times of their water protons, in apparent contradiction to an expected lengthening, if the short relaxation times of normal living cells like those studied were due to motional restriction of the bulk-phase water molecules.

It is true that in the Polarized Multilayer Theory of the living cells (as an integral part of the AI Hypothesis), motional restriction of the bulk-phase water is clearly predicted. However, Ling never argued that the observed shortening of NMR relaxation times was exclusively or almost exclusively due to the motional restriction of bulk-phase water. Ling has repeatedly cautioned against this exclusive interpretation even though this interpretation came originally from scientists who had first provided NMR evidence in support of the AIH theory. Ling has further pointed out that other factors including the rapid exchange with a small fraction of tightly bound water on paramagnetic ions and on cell proteins might play significant roles also.

Alternative theories of ion accumulation

Former graduate students of Ling, Palmer and Gulati claimed in 1976 that their findings on the concentrations of cell K+ ion in frog muscle supported the membrane-pump theory. Earlier between 1971 and 1973 Gulati el al had provided supporting evidence for the Association Induction Hypothesis. In a rebuttal, Ling showed that the criticism of the Association Induction Hypothesis came partly from a misunderstanding, and that the general equation for solute distribution of the Association Induction Hypothesis explained quantitatively all of their data.

K+ accumulation and Na+ extrusion from red cell ghosts

In 1973, another former graduate student of Ling, Jeffrey Freedman demonstrated selective uptake of K+ by, and selective extrusion of Na+ from red blood cell "ghosts" (red cells from which a major part of the intracellular proteins, primarily hemoglobin, has been removed by hypotonic lysis). Freedman saw in his finding a refutation of the AI Hypothesis, according to which, both ions distribute asymmetrically as a result of direct or indirect interaction with intracellular proteins which Freedman believed he had removed from the red cell ghosts.

In a series of papers, Ling and coworkers showed that, contrary to Freedman's assertion otherwise, the specific method used by Freedman to remove all or virtually all hemoglobin (and other intracellular proteins proteins) does not do so at all. Rather, it retains different (as much as 25% of the original) amount of hemoglobin in the cell, depending on the individual blood donor. Using Freedman's procedure rigorously, Ling et al. showed that both the amount of K+ regained and Na+ extruded in the subsequent incubation of "resealed" ghosts in the presence of ATP quantitatively depends on the amount of residual proteins (mostly hemoglobin) in the ghosts in full agreement with the prediction of the association-induction hypothesis. With complete or near complete removal of hemoglobin from the ghosts, there was neither demonstrable re-uptake of K+ in, nor extrusion of Na+ from the resealed ghosts also in full support of the AI Hypothesis.

Minimum energy need of postulated Na+ pump

In the Research News Report section of the Science magazine Volume 192 of 1976 in an article entitled "Water Structure and Ion Binding: A Role in Cell Physiology?", science reporter, Gina Kolata says that "Recently, however, some crucial experiments and calculations have been performed that provide strong evidence for the existence of pumps". More specifically Kolata states that investigators Jeffrey Freedman and Chris Miller discovered that "Ling's data are compatible with a much lower rate of sodium efflux from the cell than Ling estimated and that Ling's analysis of his data led him to assume that sodium was being transported out of the muscle cells at least 20 times faster than the rate accepted by muscle physiologists". Many years later when Ling followed up on these accusation it transpired that the so-called crucial experiments and calculations that provided strong evidence for the existence of pumps were never published and were in fact rejected immediately when submitted to the Journal of Membrane Biology. In an letter to Ling dated June 28, 1996, Miller says "We didn't try to publish it after this rejection...But we may have circulated it around to friends, etc. So maybe she heard about it in the grapevine. That's just conjecture...As for citing it, that's impossible: never having passed through the fire of peer-review, it doesn't exist, and so it isn't part of the literature-nothing for you to argue with. I don't have a copy of the paper, having thrown out the manuscript as useless junk over a decade ago". Miller also admitted that he and Freedman were never actually interviewed for the Science article.

Refutation of alleged proof of the Na+-K+ pump

Horowitz (a former postdoctoral student) and Paine injected melted 10-20% gelatin solution into salamander eggs. On cooling, the injected gelatin solidifies into a semisolid gel globule. By analyzing the K+ and Na+ concentration in the gelatin globule, the authors claimed that they have affirmed the existence of Na+-K+ pump in the egg cell membrane.

Their evidence was built on the finding that after radioactively labeled Na+ had attained equilibrium with labeled Na+ in the bathing fluid, the level of labeled Na+ in the water of the gelatin globule was still only 33% of that in the bathing medium. They concluded that there must be a sodium pump in the egg cell membrane.

Ling, in 1984 pointed out that their conclusion is unwarranted because Na+ is not the only cation present in the gelatin globule. Present also in the globule was K+--- at an even higher concentration. Although labeled Na+ has reached diffusion equilibrium between water in the globule and in the bathing medium, K+ was far from having reached diffusion equilibrium. It was shown on thermodynamic grounds that the non-equilibrium, high concentration of K+ kept the Na+ level low by essentially the same mechanism that the presence of impermeant ion in a dialysis sac, also keeps other permeant solutes carrying the same electric charge at equilibrium levels below that in the bathing medium---in the well-known phenomenon called "Donnan Equilibrium". Thus the low Na+ level in the gelatin globule offers no evidence for the existence of the postulated Na+-K+ pump.

Equal distribution of urea and ethylene glycol does not prove normalcy of cell water

In 1930 A.V. Hill demonstrated that urea distributes equally between water in frog leg muscle and water in the surrounding medium. This discovery was confirmed by the demonstration of similar equal distribution of ethylene glycol between water in surrounding medium and in living red blood cells and between water in surrounding medium and water in frog abdominal muscle cells. These confirmatory findings lent support to Hill's claim that water in living cells is simply normal liquid water in agreement with the membrane-pump theory. At the time, opponents to the membrane-pump theory were caught without an adequate answer for this set of observations; the membrane-pump theory gained a decisive victory, strengthening the belief by many in the membrane(-pump) theory.

With the introduction of the AI Hypothesis and its subsidiary Polarized Multilayer Theory of Cell Water (PM theory), the situation changed. Thus according to the PM theory, water in living cells assumes the dynamic structure of polarized multilayers, in consequence of interaction with (various) cell proteins existing in the fully extended conformation--- with their backbone carbonyl oxygen (and imino groups) at suitable distance apart and able to interact with solvent water. As such, cell water exhibits solvency for different solutes according to their molecular size and their surface molecular structure, when compared to their solvency in normal liquid water.

In this PM theory, urea and ethylene glycol distribute equally between cell water and surrounding medium because urea and ethylene glycol are small and because they possess surface structures compatible with the surroudning cell-water structure. However, in the same polarized water, the theory also predicts that larger solutes like sucrose and (hydrated) sodium ions should be found at a much lower level--- as it is well known to be the case in virtually all living cells.

The PM theory also predicts that linear polymers carrying oxygen atoms (with their lone-pair electrons) at suitable distances apart---like the fully extended protein chains in the living cell just mentioned ---should also be able to modify the solvency of bulk-water like that seen in living cells. This prediction has also been fully confirmed. Poly(ethylene glycol), poly(vinylpyrrolidone), gelatin, and urea-denatured proteins all satisfy the theoretical criteria of carrying properly spaced oxygen atoms (with or without additional polar atoms). They are all capable of producing in bulk-phase water reduced solvency for sucrose, sodium ion etc. as seen in living cells, while at the same time, equal solvency for urea and ethylene glycol as Hill and others have demonstrated for living cells.

List of papers independent of Ling submitted to peer reviewed scientific journals

1. (2014) "Membrane Potential Generated by Ion Adsorption" by Hirohisa Tamagawa and Sachi Morita.
2. (2004) "Extension of Colacicco's experiment supporting the adsorption theory" by Tamagawa, H. and Nogata, F. [on the close-contact surface adsorption theory of resting and action potential of living cells.]
3. (2003) "Long-range forces extending from polymer-gel surfaces" by Zheng, J. and Pollack, G. [on the polarized-oriented multilayer theory of cell water]
4. (1988) "Monovalent ions are spatially bound within the sarcomere" by Zglinicki, T. von [on K+ binding in muscle cells]
5. (1981) "Distribution and Function of Water and Ions in Resting and Contracted Muscle" by Tigyi, J., Kallay, N., Tigyi-Sebes, A. and Trombitas, K. [ on selective ion adsorption in cells]
6. (1981) "Electron Microscopic Demonstration of Potassium Binding Sites in Muscle|journal=International Cell Biology" by Edelman, L
7. (1981) "Selective Accumulation of Li +, Na +, K +, Rb +, and Cs ÷ at Protein Sites of Freeze-Dried Embedded Muscle Detected by LAMMA" by Edelmann, L [on localized ion adsorption in cells]
8. (1980) "Cooperative Phenomena in Biology" ed by Karreman, G. [on cooperative adsorption in cells\
9. (1980) "Potassium binding sites in muscle: electron microscopic visualization of K, Rb, and Cs in freeze-dried preparations and autoradiography at liquid nitrogen temperature using 86Rb and 134Cs" by Edelmann, L. [on localized ion adsorption in cells]
10. (1979) "X-ray microanalytical studies on native myofibrils and mitochondria isolated by microdissection from honey-bee flight muscle" by Trombitas, C. and Tigyi-Sebes, A. [on selective ion adsorption in cells]
11. (1979) "X-ray absorption edge fine structure of potassium ions in various environments: application to frog blood cells" by Huang, H. W., Hunter, S.H., Warburton, W.K. and Moss, S.C. [on selective adsorption of K+ in cells.]
12. (1977) "Potassium adsorption sites in frog muscle visualized by cesium and thallium under the transmission electron microscope" by Edelmann, L. [on selective ion adsorption in cells]
13. (1973) "Caloric catastrophe" by Minkoff, L. and Damadian, R. [on cooperative K+ adsorption in cells]
14. (1972) "A nuclear magnetic resonance study of water in two Morris hepatomas" by Hollis, D., Saryan, L.A. and Morris, H. P. [on cell water]
15. (1971) "Preferential accumulation of cesium 137 in the mammalian organism, in comparison with potassium accumulation. I. Accumulation of potassium, rubidium and cesium in the perfused guinea pig heart" by Edelmann, L., Pfleger, K. and Matt, K.H. [on resting potential of living cells]
16. (1971) "Rapid exchange of cellular K+, Rb+, and Cs+ and its relation to the resting potential of guinea pig papillary muscle cells" by Edelmann, L. and Baldauf, J.H. [on resting potential of living cells]
17. (1971) "Tumor detection by nuclear magnetic resonance" by Raymond, D., [on cell water]
18. (1969) "Evidence for the Existence of a Minimum of Two Phases of Ordered Water in Skeletal Muscle" by Hazelwood, C.F., Nichols, B.L.and Chamberlain, N.F. [on cell water]
19. (1969) "Nuclear magnetic resonance evidence using D2O for structured water in muscle and brain" by Cope, F.W. [on cell water]

List of some books that discuss the Association Induction Hypothesis

1. (2013) The Fourth Phase of Water: Beyond Solid, Liquid, and Vapor by Gerald H Pollack
2. (2012) Living Rainbow H2O By Mae-Wan Ho
3. (2011) Science in Society 52 By Dr. Mae-Wan Ho, Prof. Peter Saunders, Dr. Eva Sirinathsinghji
4. (2010) Proceedings of the First Interdisciplinary CHESS Interactions Conference, Saskatoon, Saskatchewan, Canada, 17–20 August 2009
5. (2009) Introduction to Bioregulatory Medicine
6. (2007) Water and the Cell edited by Gerald H. Pollack, Ivan L. Cameron, Denys N. Wheatle
7. (2006) Bioelectromagnetics Current Concepts: the mechanisms of the biological effect of extremely high power pulses edited by S. N. Ayrapetyan, Marko S. Markov
8. (2005) Phase Transitions in Cell Biology edited by Gerald H. Pollack
9. (2005) Rhetoric and Incommensurability edited by Randy Allen Harris
10. (2001) Global Brain: The Evolution of Mass Mind from the Big Bang to the 21st Century by Howard Bloom
11. (2001) Cell, Gells and the Engines of Life by Gerald H Pollack.
12. (1991) Phylogenetic and Biochemical Perspectives edited by T.P. Mommsen, Peter W. Hochachka
13. (1985) New Scientist April 1985 Article by Stephen Mason.
14. (1981) Tissues and Organs: Hierarchical and Functional Integration By G. A. Chauvet
15. (1980) Cooperative Phenomena in Biology edited by George Karreman
16. (1971) Chemistry of the Cell Interface, Part 2 edited by Harry Brown

See also

• Membrane potential
• Sodium potassium pump
• Cell biology
• Cell physiology
• Molecular biology
• Cell membrane
• History of the cell membrane theory

External links

• Dr. Gilbert Ling's website

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