User:PaulGWiki/sandbox/AIH Criticisms

Criticisms of the theory
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 lipid 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 degree Centigrade. 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 00 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 1971-1973  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 surrouding 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 surrouding 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) thoery.

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.