Life at the Cell and Below-Cell Level. The Hidden History of a Fundamental Revolution in Biology
Gilbert N. Ling, Ph.D.
Pacific Press
ISBN 0-9707322-0-1

"Dr. Ling is one of the most inventive biochemist I have ever met."
Prof. Albert Szent-Györgyi,
Nobel Laureate

Chapter 7.

Legacy of the
Nearly-Forgotten Pioneers
(p. 35-39)

The mistake of regarding all living cells as membrane-enclosed dilute solutions was fully recognized by micro-anatomists in the 1860's. Max Schultze announced his protoplasmic doctrine in 1861.11 Thomas Huxley lectured on protoplasm as the physical basis of life in 1868.72 Yet not until the turn of the century did a new generation of cell physiologists begin rising to the challenge. The research efforts of these early protoplasm-oriented cell physiologists centered around two subjects: (i) cell swelling, and (ii) the selective accumulation of potassium ion (K+) over sodium ion (Na+ in most living cellsa subject already briefly introduced in [4.3].

To explain the asymmetrical distribution of K+ and Na+ in red blood cells, the supporters of the membrane theory contended at first that the red blood cell membrane is impermeable to both ions.76

Benjamin Moore of the University of Liverpool objected briefly in 1906 to this impermeability interpretation.77 Two years later, he and Herbert Roaf further elaborated on their stand.77 First they reasoned that the content of K+ in the cell is more or less constant in the cell's life cycle. It would be very difficult to see, they argued, how the cell could maintain a constant K+ concentration inside the cell as it grows and multiplies, if at all times its membrane is impermeable to this ion.

As an alternative, Moore and Roaf suggested that the cell protoplasm possesses a special affinity, or adsorbing power for K+, but it has no adsorbing power for Na+. In support, they mentioned the preferential uptake of oxygen by living red blood cells (erythrocytes), and, even more to the point, selective uptake of + over Na+ by (inanimate) soils. However, they did not offer a molecular mechanism for the preferential adsorption in either the living cells as they hypothesized, or in soils as a known fact {see [10.1(3)]}.

. E. Overton, better known for his lipoidal membrane theory (to be discussed in [13.1(1)]), discovered in 1902 reasons to question that living cells are truly membrane-enclosed dilute solutions. Thus when he transferred a frog sartorius muscle from an isotonic 0.7% NaCl solution to a hypotonic NaCl solution at half of its strength (0.35%), the muscle did not swell to twice its initial weight as expected on the basis of the membrane theory. Instead an increase of only a third of its initial weight was observed. Overton concluded that at least a part of the cell water must be Quellungs-wasser (swelling water, imbibition water).25 p 273

In 1907 and 1909 Martin Fischer, then a professor of medicine in the Oakland School of Medicine in Oakland, California, argued that the swelling of living cells is not an osmotic membrane phenomenon as widely believed then [4.1], but a result of the strong affinity of protoplasmic colloids for waterseen also in fibrin (the protein of blood clots) and gelatin.546; 78 Along this line of reasoning, he proposed a theory of oedema and published a lengthy thesis on the subject.78 In this 1909 document he also offered briefly some new insights on the asymmetrical distribution of ions and other solutes in cells, which the distribution of K+ and Na+ exemplify.

Fischer pointed out that the dissolved substances may be either at higher or lower concentration in the colloidal mass (protoplasm) than in the surrounding medium. Adsorption could then account for its being at a higher concentration; the law of partition (also known as the Berthelot-Nernst distribution law,420 of which Henry's law for the distribution of gases in a liquid solvent is a special caseGL's addition) may account for its presence at a lower concentration.78 pp 545-546 But Fischer did not further exploit these important ideas.

His major scientific contribution to cell physiology aside, Martin Fischer should also be remembered for his kindliness and generosity, giving support out of his own pocket to even his scientific opponents in Germany at the end of World War I.79 Quoting Schopenhauer, colloid chemist Wolfgang Ostwald wrote on Fisher's 60th birthday: "As torches and fireworks pale and become invisible in the sunlight, so the mind and even genius and beauty are outshone and overshadowed by (the) goodness of heart."79 p 441

W. W. Lepeschkin also rejected the concept that living cells are membrane-enclosed dilute solutions. As mentioned earlier, when he crushed young cells of the plant, Bryopsis, in sea water, many water-immiscible little balls of protoplasm emerged62 pp 289-290not unlike that shown in Figure 3. On diluting the surrounding sea water with distilled water, these little balls swelled prodigiously and vacuoles formed within them. When these little balls were returned to sea water, they regained their original size and the vacuoles disappeared.

Lepeschkin estimated that the surface of these little balls created by shaking could be increased a thousand fold from the surface area of the intact cell. Because the total amount of (postulated) lipoids in the original membrane of the intact cell is limited, it could not be expanded to 1000 times its original dimension.62 p 275 Accordingly, he concluded that these findings contradict the ideaemerging from a synthesis of the lipoidal membrane theory of Overton21 and the theory of membrane regeneration [4.1(4)]that each of these little balls is covered with a continuous lipoid membrane as postulated by Overton for all living cells. By the same token, the surface of these little balls must be covered with materials that are abundant in the protoplasmic droplet as a whole, i.e., protoplasm62 p 276in harmony with Franz Leydig's, Max Schultze's belief that the cell surface is made of similar materials that make up the bulk of the cell's protoplasm. Surprising as it might seem, Wilhelm Pfeffer shared a similar view.18 p 156

In further support of Lepeschkin's idea, one may quote the observation of Pauli and Rona mentioned in [6.2]. They showed that a salt solution of (pure) gelatin warmed up to 30C forms immiscible coacervate. Since there is no other structure-forming material than gelatin here, the membrane of the coacervateif one calls the surface layer of coacervate a membranecan only be made of the same material, which makes up the bulk of the coacervate, i.e., gelatin.

As mentioned above, Lepeschkin was among the earliest in maintaining that protoplasm is a coacervate.324 He also offered a theory of the fundamental substance of living matter, a loose chemical complex of proteins and lipids, which he called vitaproteids or vitaids.325 This specific hypothesis was severely criticized332; 92 p 62 and not further defended.

Then there was Ross Gortner (1885-1942), for more than a quarter of a century the Chief of the Division of Agricultural Biochemistry at the University of Minnesota and a strong advocate for the existence of bound water in living cells. In an address given at the Faraday Society meeting to a group of scientists with diverse first-hand knowledge on various aspects of waterthe printed version of which bears the title: "The State of Water in Colloidal and Living Systems" and was published in the Transactions of the Faraday Society80Gortner presented his view that at least a part of the water in living cells is not normal liquid water and is referred to as bound. As one of the criteria that sets bound water apart from normal water, Gortner suggested that bound water does not dissolve substances soluble in normal liquid waterhence an alternative name for bound water in his and his coworkers' vocabulary is "non-solvent water."

In anticipation of what follows in Chapter 11, I mention that Gortner referred to experimental evidence in the literature for the existence of multilayers of adsorbed water in inanimate systems. However, Gortner then backed away in these words: "Unfortunately the properties of water in oriented adsorption films have not been sufficiently characterized to enable us to state whether or not this may be the type of water which the biologist is coming to call bound water."80 pp 684-685

Gortner's paper presented at the Faraday Society meeting was well received. A majority of the participants expressed interest, some very enthusiastically. However, there was one notable exception, i.e., the same Professor A. V. Hill, whose evidence for free cell water has already been briefly presented in [5.1].

Not mentioned earlier is the fact that A. V. Hill almost single-handedly extinguished the enthusiasm generated for the "bound water" concept, and put to rout the entire colloidal approach to cell physiology. He achieved this feat by presenting just a single set of experimental data demonstrating that water in frog muscle accommodates the same concentration of urea as in the surrounding fluid.81 Accordingly, there is no "nonsolvent" water. (In years following, others showed that ethylene glycol also distributes equally across the cell membrane of rectus muscle and of red blood cells.82) Since cell water is thus proven to be normal liquid water, the components in the cells high enough in concentration to match osmotically the free Na+ and Cl- in a Ringer's solution, can only be free K+ and free cell anion(s). Ergo, no bound water, nor bound K+ in living cells81in affirmation of the clinching twin tenets of the membrane theory.

Hungarian biophysicist, Professor E. Ernst, a witness to these events, recalled how the opinion makers of the day, including Rudolf Höber (despite his own electric conductance study pointing to the contrary [5.2]), W. O. Fenn, and F. Buchthal all renounced the concept of bound water and bound K+, and subscribed in toto to the idea that living cells are membrane-enclosed dilute solutions following van't Hoffs osmotic law.83 p 112 Each acknowledged that his new position was based on Hill's crucial experimental finding of equal urea distribution and his compelling logic.

In 1940 the major English-language Journal of Colloidal Chemistry was combined with the Journal of Physical Chemistry. For a few years, the combined journal bore the new title Journal of Physical and Colloidal Chemistry. Then the words "and Colloidal" were quietly dropped. The demise of the main English-language colloid magazine did not signal the end of colloid chemistry per se. Other journals like Zeitschrifts für Kolloid Chemie, Kolloid Beihefts and even Protoplasma continued publication. It was only the fledgling colloid- or protoplasm-oriented cell physiology that suffered an (undeserved) near-fatal blow.

"Life at the Cell and Below-Cell Level.
The Hidden History of a Fundamental Revolution in Biology":

Contents (PDF 218 Kb)
Preface (
PDF 155 Kb)
Answers to Reader's Queries (Read First!) (
PDF 120 Kb)

1. How It Began on the Wrong Foot---Perhaps Inescapably
2. The Same Mistake Repeated in Cell Physiology
3. How the Membrane Theory Began
4. Evidence for a Cell Membrane Covering All Living Cells
5. Evidence for the Cell Content as a Dilute Solution
6. Colloid, the Brain Child of a Chemist
7. Legacy of the Nearly Forgotten Pioneers
8. Aftermath of the Rout
9. Troshin's Sorption Theory for Solute Distribution
10. Ling's Fixed Charge Hypothesis (LFCH)
11. The Polarized Multilayer Theory of Cell Water
12. The Membrane-Pump Theory and Grave Contradictions
13. The Physico-chemical Makeup of the Cell Membrane
14. The Living State: Electronic Mechanisms for its Maintenance and Control
15. Physiological Activities: Electronic Mechanisms and Their Control by ATP, Drugs, Hormones and Other Cardinal Adsorbents
16. Summary Plus
17. Epilogue 

A Super-Glossary
List of Abbreviations
List of Figures, Tables and Equations
References (
PDF 193 Kb)
Subject Index

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