A ROLE FOR WATER IN THE EXCLUSION OF CELLULAR SODIUM-IS A SODIUM PUMP NEEiDED?

Carlton F. Hazlewood, Ph. D.

INTRODUCTION

Water constitutes 60-90% of the weight of most biological tissues, andits importance in the living system is undeniable. Water has always been asubject of concern and curiosity to the biologist, yet very little basic knowl-edge is available on cellular water. An adequate model of the living cell,describing the unequal distribution of ions between the cellular inside andoutside, the dynamic aspects of solute and water movement into and outof cells, and the bioelectric phenomena of cells, requires that fundamentalassumptions be made relative to the physical state of cellular water. Theultimate understanding of cellular function will require concrete knowl-edge of the physical state of water.

Conventionally, biologists suggest that a living cell is equivalent to adilute aqueous solution surrounded by a semipermeable membrane.1-4Bioelectric phenomena of cells are also described classically by equationsevolving essentially from dilute solution theory. The history of osmoticpressure as the underlying mechanism for the movement of water inbiological cells dates from the 18th century.1 The greatest impetus to theapplication of osmotic theory to biological cells came, however, near theturn of the 20th century when chemists like Jacobus Hendricus van't Hoff(1852-1911) and Svante August Arrhenius (1859-1927) laid much of thefoundation for dilute solution theory. Biologists Hugo de Vries and Wil-helm Pfeffer capitalized on the substance of their work. For example, ac-cording to Smith ". . . it seems that the stars of science were in some sortof favorable conjunction over Amsterdam when de Vries, the isotonically-minded botanist, who was well informed on Pfeffer"s plasma membraneand copper ferrocyanide manometer, met van't Hoff . . . '" Bernstein'stheory5 of bioelectric phenomena evolves also from the use of ideal solu-tion theory. For example, Katz" states that ". . . in 1902 he (Bernstein)proposed an important theory which applied the physiochemical conceptsof Nernst and Ostwald to bioelectric phenomena."* Hodgkin and Huxley,7in 1939, challenged the Bernstein theory which argued for the impermea-bility of the cell to sodium. Later, Hodgkin and others modified and ex-tended the Bernstein theory to what is essentially the present concept ofthe excitable cell, yet the theory of dilute solutions remains fundamentalto the membrane concept.8

It is also known that since the middle of the 19th century, theories haveexisted which maintain that water in cytoplasm is not free as in dilute

*The generalizations of Ostwald stemmed largely from the work of van't Hoff and Arrhenius.1

From the Departments of Pediatrics (Section of Nutrition and Gastroenterology) and Physiology,Baylor College of Medicine, and Texas Children's Hospital; and the Department of Physics ofRice University, Houston, Texas 77025

Cardiovascular Diseases, Bulletin of the Texas Heart Institute. Vol. 2, No. 1. 1975 83

solutions but bound to macromolecular constituents.9 The concept of boundwater in living tissues was pursued actively during the 1920's and 1930's.Since the 1950's, a small but persistent group of biological scientists haveinsisted that a large part or all of the cellular water exists in a physicalstate significantly different from ordinary water.10" 3 Thus, two schools ofthought emerged concerning the physical state of cellular water, andhence two schools of thought concerning transport and accumulation ofsolutes in living cells. A review of this fundamental controversy was pre-sented by Oscar Hechter in 196514 whereupon he concluded the following:

"We have discussed the two opposing classical concepts of trans-port. Upon analysis, both ideas are shown to be right in part; andboth partially wrong. The proponents of the plasma membranethesis of transport were wrong in that they neglected the role ofthe cell interior, the holists were wrong in their de-emphasis of theplasma membrane and of intracellular membranes generally. Ifone considers that membrane systems throughout the cell are in-volved in transport, and that cells exhibit diversity as well as uni-formity, a pluralistic resolution is achieved."

To date, considerable evidence has been gathered by proponents of bothviews, but little has been done to bring about the pluralistic resolution soproposed by Hechter.

A fundamental understanding of the physical state of cellular waterwill lead to a resolution of these divergent views. It is, therefore, essentialthat we address ourselves to this task rather than consider the subjecteither unimportant or already solved.

STATEMENT OF A PROBLEM

Various laboratories throughout the world have reported dramaticchanges in the electrical properties of skeletal muscle and in the muscleelectrolyte concentrations in developing animals.'524 A summary of theresults published by our laboratory on the gastrocnemius muscle of therat will first be reviewed, followed by attempts to explain the remarkablereduction of cellular sodium associated with the normal developmentalprocess.

Electrolyte composition in milliequivalents per kilogram fat-free wetweight as a function of animal age is given in Figure 1. Potassium con-centration increases, becoming constant between 32 and 64 days of age.Sodium and chloride concentrations exceed the potassium concentration atbirth, but rapidly decrease during the first 10 days of postnatal life. Again,the concentrations of these muscle electrolytes become constant between32 and 64 days of age. This general phenomenon was defined as chemicalmaturation by Moulton in 1923.25

During this same time period, changes in hydration are also observed.At birth the percentage of water is 90% decreasing to 77% in chemicallymature muscle. On a dry weight basis, this means a change in hydrationfrom over 7 liters/kg at 2 days of age to approximately 3.5 liters/kg at 65days of age.

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Because of the change in hydration, the electrolyte composition presentedin Figure 1 can be misleading. Therefore, these data were also reported interms of the skeletal muscle dry mass and are given in Figure 2. The po-tassium concentration, when related to the fat-free dry muscle mass, isrelatively invariant with age. Sodium and chloride show the same re-markable decrease.These data on water and electrolyte composition of whole tissue were

easily described by simple mathematical expressions, thereby makingtheoretical computations of intracellular concentrations possible.17 Esti-mates of upper and lower limits of the cellular concentrations were con-clusive for sodium.

Regardless of how the cellular concentrations were computed, the in-tracellular sodium decreases by a factor of almost ten (Fig. 3). In the caseof potassium, no conclusive statement could be made; however, one com-puter solution indicated that the potassium concentration might be con-stant over the same time period. These data were published in 1969. Sincethat time, we have been exploring better procedures for cellular analysis.During the past year, we have managed to standardize a procedure usingelectronprobe microanalysis.26'27 This was possible through a collabora-tive research effort with the Cellular Analytical Laboratory at the JohnsonSpace Center near Houston. In this particular study, small samples of the

200.

O *No+ zKx ci

150.

¢~~~~~~~~~~~~\L \ fK(t)=zIII-56e-O.075t> 100.

0

wE

50. D

Nao t)a 21 + 106,-0.132t

C I (t) - 13.5 + 54 a - 0.079 t

AGE IN DAYS

Fig. 1. Electrolyte composition of rat gastrocnemius muscle in milliequivalents/kilogram fat-freewet weight as a function of animal age in days. Tissue samples were taken at 2, 4, 8, 16,32 and 64 days of age. Each data point is the average of 5 to 11 samples.

85

it

a

300.1

0- Na+= K

X = Cl

K (t) = -0.67 t + 533

Na(t)- 99+ 1075 e-0 168 t

CI (t) - 72 + 567*-0.126t

AGE IN DAYS

Fig. 2. Electrolyte composition of rat gastrocnemius muscle in imiillie(juivalents per kilogram fat-free dry solids as a function of animal age in days. Ages of animals and sample numbersame as in FIGURE 1.

300n

_ 200-

3:04

ci' 00-

0-

(H0)0 =K [culi =0 Em(t)

I [Noli

10 20 30 40 5bAGE IN DAYS

60 7' 80 9*0 100

Fig. 3. Ordinate: Intracellular sodium concentration in millie(quivalents per kilogranm of water.Abscissa: Animal age in days. The three solid lines represent the computed values of[Na]1 by three different procedures (see Reference 3 for details of the computations).

86

af -

gastrocnemius muscle were taken and immediately frozen in liquid pro-pane and sectioned at 8 , in a cryostat. The samples were then lyophylized.Using a microprobe, the sample was visualized by an electron beam of ap-proximately 1.5 ,u which could scan single fibers. A 10 kev electron beamwas used to generate characteristic Kx X rays for Na and K. The X-raycounts were compared to calibrated gelatin standards to determine con-centration profiles. A summary of the results of one study is presented inTable I.

Intracellular concentrations of sodium and potassium are given in TableI in mEq/kg cells. Samples from gastrocnemii of 10- and 33-day-old ratsand those from adults confirmed our theoretical study that cellular K+ wasconstant and that cellular sodium decreased significantly.The question now is-how does one explain these data?

MECHANISMS: TRADITIONAL VIEW

At the outset it would appear that the classical membrane theory of thecell would be adequate to explain these data.8'28 Figure 4 will help to re-view the current "generally accepted" view of the living cell.

Some of the major contentions of the membrane or ionic theory are asfollows:

1. Polarized membrane (inside negative) that separates two SOLU-TIONS.

2. The inside SOLUTION is comprised of impermeable proteins, highconcentration of K+ and low concentration of sodium-this internalSOLUTION is equivalent to a DILUTE SOLUTION.

3. The outside solution is high in sodium and low in potassium andprotein.

4. Originally, it was thought that sodium did not permeate the cell.Once it was found that it did-a dilemma existed. The cellular so-

TABLE I

INTRACELLULAR CONCENTRATIONS OF SODIUM AND POTASSIUMIN mEq/kg OF CELLS

(M + S.D.)

{K}i [Naji % HoO10 days (5) 135 ±29 54 6 84.8+ .433 days (3) 121 14 29 4 80.1 ±+.8Adult (3) 126 ± 16 26 ± 3 77.1 ± .5

NOTE: Intracellular electrolyte concentrations are mean values in milli-equivalents per liter ± standard error of the mean. The percentageof water is in grams of H,0 per 100 grams of fat-free wet weight.

87

dium ions then must be moved from a dilute solution of low concen-tration within the cell through a polarized membrane to a dilutesolution of high sodium concentration outside the cell. This met thedefinition of Active Transport. Therefore, Dean,29 in 1941, stated that". . . there must be some sort of a pump, possibly located in the fibermembrane, which can pump out the sodium, or what is equivalent,a pump in the potassium." With this, the pump was invented.

A brief survey of the history leading to the pump concept is appropriatehere. Such a review is given in Figure 5 and the bibliographical materialgiving rise to this figure may be found in references 1-8, and 28-36.

In a review of the literature, the evidence for active cellular transportis, at best, equivocal. To be more explicit, a report of a definitive experi-ment demonstrating the movement of sodium or any other ion from a freesolution of low concentration within a cell to a free solution of high con-centration outside the cell was not found (Fig. 5).: Even the experiments

INSIDE SOLUTION - + OUTSIDE SOLUTION

No+ K

CELL MEMBRANE

HERE LIES THEPUTATIVE SODIUM PUMP

INVENWTED IN 1941

Fig. 4. Classical view of the cell. Inside the cell is a dilute solution of ions and proteins separatedfrom the external solution by a semi-permeable polarized (inside negative) membrane.The external solution is high in sodium and low in potassium, whereas the opposite holdsfor the internal solution. In general, the membrane separates two dilute soultions and thedissimilar electrolyte concentrations are maintained by the putative pump and the mem-brane potential.

88

with extruded squid axons32-34 and red blood cell ghosts38 have failed todemonstrate active transport.**

It is true that in the context of membrane theory the presence of anenzyme system such as the Na+ - K+ ATPase is mandatory and that thedemonstration of the same would, at first glance, appear to be proof of

Menbrane Theory of Pfeffer, de Vries, Bernstein, etc.evolved fran Dilute Solution Theory of van't Hoff and Arrhenius

The existence of a cell membrane postulated.Bernstein maintained the impermeability of the membrane to sodium.

View held until 1939-1941 when sodium was indirectlyfound to penetrate the cell

Concept of active transport oi sodium in cells proposedand "Pump" created by Dean, 1941.

Confirmation of theexistence of cellrrmembranes

Na-K ATPASE activity in membrane fragments 1957 by Skou

1~Active Transport of cellular sodium now identified by demonstrating

potassium and ouabain sensitive sodium efflux.

Fig. 5: A flow diagram depicting the major events in the evolution of the membrane or ionictheory. One purpose of this diagram is to point out that the actual demonstration of activecellular transport never occurred but has been taken to be synonymous with the demon-stration of Na+-K+ ATPase activity or potassium and ouabain sensitive sodium efflux.

**The earlier studies of the extruded squid axons32,33 were concerned primarily with restingand active electrical activity and its relationship to ion activity. No data concerning active iontransport or maintenance of ionic gradients were reported. Unfortunately, these papers have beenquoted as demonstrating the maintenance of ionic gradients.4' The failure, however, to dem-onstrate ion selectivity in squid axons has been reported. 42 In a later publication by Baker.Foster, Gilbert and Shaw, they demonstrated the downhill diffusion of radioactive sodium andits modulation by ouabain and external potassium.34 Again, active ion transport was not dem-onstrated. For example, in their paper entitled "Sodium Transport by Perfused Giant Axonsof Loligo," appearing in the Journal of Physiology (London) in 1971, they reported that "Thefirmest criterion for the demonstration of a sodium transport system in the cell is a demon-stration of a net movement of sodium ions against their electrochemical gradient. However, agreat deal is already known about the sodium transport system in squid fibres, enough . . . toplace considerable confidence in identifying it with potassium, and ouabain-sensitive sodiumefflux, as has been done in the present paper." (The italics are mine.) The explanation thatthe efflux of sodium may be modified by external potassium and ouabain is in no way uniqueto the membrane theory.10The non-existence of a definitive experiment with red cell ghosts was reviewed by Freedman,38and his manuscript was reviewed by several prominent membranologists before publication-theaccuracy of the review was recognized.

89

the pump concept. It must be remembered, however, that the first andsubsequent pumps were invented out of necessity stemming from the as-sumption that the cellular ions and water were in free solution.The sodium pump is presented anthropomorphically in Figure 6. The

creation of a membrane-situated energy-requiring pump endowed withthe capacity to transfer ions across membranes against concentration grad-ients and, sometimes, electrical gradients, requires first the demonstrationof an adequate energy supply. Twelve years ago, Gilbert Ling publishedhis book entitled "A Physical Theory of the Living State: The Association-Induction Hypothesis." I call to your attention Figure 7, which is in Chap-ter 8, Table 8.9, page 211 of Ling's book.10

This table is an energy balance sheet for the Na pump in frog sartoriusmuscle (00). The minimum rate of energy delivery required to operate aNa pump according to the membrane-pump theory was calculated fromintegrated values of the measured rates of Na+ --ion exchange and theenergy needed to pump each mole of Na+ ion out against the measuredelectrical and concentration gradients. The maximum energy deliveryrate was calculated from the measured hydrolysis of CrP, ATP, the onlyeffective energy sources available to the muscles which were poisonedwith IAA and N2. Total inhibition of respiration and of glycolysis was

Fig. 6. Anthropomorphic representation of the sodium "pump". The "pump" is membrane situate(land energy requiring. The invention of a "pump" follows the use of dilute solution theory.Demonstration of an adequate energy supply is mandatory.

90

assured by the simultaneous presence of 10-3 M NaCN and verified by theactual measurement of residual lactate production. We should also be re-minded that the ratios between the required and available energy given inthis table are underestimations.

According to this ledger sheet, a rather large discrepancy exists be-tween energy required and energy available-indeed an alert to an im-pending energy crisis for the cell. Although Ling's book is often quoted,the data pointing to the energy crisis is largely ignored. One major rebuttalby R. D. Keynes published in a symposium43 stated:

"One of the principal arguments used by Ling against the conceptof the cell membrane is concerned with the energy requirement forextrusion of sodium from frog muscle fibres. He measured therate at which labelled sodium emerged from frog muscles at 0°Cafter they had been poisoned to block both glycolysis and oxidativephosphorylation, and calculated the amount of work that wouldhave been necessary to transport sodium actively at this rate. Hethen showed that this rate of consumption of energy was muchgreater than the actual rate at which the reserves of phosphate-bond energy in the muscle decreased, and concluded that thepostulate of a sodium-pumping mechanism in the membrane wasuntenable. This is not, however, a legitimate argument, because noproponent of membranes would claim that the exchange of la-belled ions observed in a muscle at 0°C results wholly from the op-eration of an energy-consuming process; it seems much more likelythat under these conditions, labelled sodium mainly crosses themembrane through the type of mechanism that has been termed"exchange diffusion." I have to admit that there is rather little di-rect experimental evidence on the validity of the exchange diffu-

MinimumRate of rate ofNa energy Maximum Minimum required

exchange, # + EN.1/ required rate of energyintegrated integrated for Na energy

Duration, average, average, pump, delivery, Maximum availableDate hr M/kg/hr mn cal/kg/hr cal/kg/hr energy

9-12*-56 10 0.138 111 353 11.57 3060%(highest value,22.19)

9-20-56 4 0.121 123 3413 22.25 1542%(h)ighest value,33.71)

9-26-56 4-.5 0.131 122 368 20.47 1800%(htighest valuc,

26.10)

Fig. 7. Copy of Table 8.9 page 211 from Ling's book"+. Data from three different frog skeletalmuscles are presented. All parameters needed to make the calculations (i.e., flux, voltageand energy available from creatine phosphate, adenosine triphosphate and adenosinediphosphate) were measured on each muscle.

91

sion idea, but I am nevertheless sure that Ling's argument does notrest on a satisfactory basis."*

These criticisms are inadequate for two primary reasons: 1) no unequiv-ocal evidence exists for exchange diffusion in skeletal muscle and 2)Keynes' final criticism that Ling's argument is not satisfactory is no morethan opinion.

Additional studies coming later from Ling's laboratory"'40'44 (conductedat room temperature) have also demonstrated an inadequate supply ofenergy for the sodium pump.** Some other laboratories, however, havereported the requirements of the sodium pump to be from 10-45%5 of theTOTAL available energy.35'36'45'46 Historically, the invention of the pumpconcept was initiated by the need to explain the asymmetrical distributionof sodium. It might be reasonable to expect a cell to spend as much as 10%0of its energy, or even 45%, to transport sodium; however, since 1941 therehas been something of a population explosion in the pump world (Fig. 8).

There exists quite a large number of postulated pumps now, and evenif we assume only modest needs for each, an adequate energy supply issuspect. Therefore, one may conclude that if pumps exist, they will die ofmalnutrition (Fig. 9). This is a conclusion similar to that of Minkoff andDamadian who reported an insufficient energy supply for pumps in E. Coliin a paper entitled "Caloric Catastrophe" appearing in The BiophysicalJournal in February, 1974.50

MECHANISMS: ALTERNATE VIEWS

I have digressed somewhat but I did so to indicate that the classicalmembrane pump view of the living cell has been considered as a possibleexplanation of our data. In attempting to explain our data, we looked foralternatives and found that some do exist: for example, the SorptionTheory of Troshin,'2 The Theory of Ernst,'3 The Biostructure Theory ofMacovski,5' The Solid State Theory of Cope,52 The Ion Exchanger ResinTheory of Damadian,53 and the Association-Induction Hypothesis ofLing.'0,"",54

All these theories, though differing in specifics, agree in general that thecell cytoplasm should not be considered a dilute solution nor should themembrane be considered the universal rate limiting step to the entry ofsolutes. Furthermore, these theories consider that the cytoplasmic waterexists in a state different from pure water-e.g., the solvent properties aredifferent.

*Italics mine.* *It should be pointed out that authors, when reviewing Ling's work, often fail to review hiswork in depth. For example, he may have papers published which are addressed to a givencriticism yet these are not referred to. (See Caldwell,28 Stein,47 DeVoe,48 and Black,49-noneof these authors quote any of Ling's work beyond 1962.) Furthermore, the evidence accumu-lated by numerous laboratories (other than Ling's) strongly suggest that alternate views areneeded (for references, see body of this text). It would seem only reasonable that future reviewson the "popular view (i.e., membrane or ionic theory) of the cell must include a seriousreview of the evidence not in support of the fundamental bases from which the theory wasderivedL

92

Na PUMPMg PUMP

Ca PUMP

CI PUMP

SUGAR PERMEASES

<AMINO ACID PUMPINSULIN PUMP

DNA PUMP PROT -EIN PUMPFig. 8. A list of some of the pumps reported. Note that sugar, amiiino aci(ls. and(l proteins are

categories within which are several species. Each of these requires a finite amount ofenergy to operate. Is the energy supply adequate?

N~~~~~~~~~~~~

Fig. 9. The sodiunm pump was invented out of a need to explain the "non-equilibrium" distributiomiof sodium. Since 1941, the large number of pumps postulated thus far. exceed the totalenergy supply of the cell. These demands exist because of the fundamental assuniption tllatthe cell is a dilute solution. If one assumes that the cell is not a solution, then the"membrane pumps" are unnecessary. If one assumes they do exist, then an energy crisisfollows.

93

Among these views, the association-induction hypothesis of Ling hasbeen worked out in most detail. (Incidentally, Ling was the first to sug-gest that cellular water may be a primary determinant of cellular per-meability.9" 1,54.55) In Figures 10 and 11, I have taken considerable libertyin interpreting Ling's view.

First, consider Figure 10: According to this hypothesis, the cell is con-sidered a highly complex system, wherein many of the biological processesare controlled by interactions between water, ions, and macromolecules.

(I 11111 IX 11111)

0000000000oQooQoooooooooooooo,000 m00

Fig. 10. Alternate view of the living cell. Potassium is preferentially adsorbed to a major fractionof the cellular miiacromolecules. A small amount of the total sodium may also b)e adsorbedto cellular macromolecules. The cytoplasmic water exists in a different physical statesuch that sodium is not readily dissolved and therefore, entropically excluded.

94

The intracellular concentration of any ion will be determined by its asso-ciation energy (attraction) to macromolecular fixed charge sites and thesolvent properties of the cellular water. For example, potassium is prefer-entially associated* with the fixed charges on proteins within the cell,and the sodium concentration is low, primarily because the cellular wateris more structured than ordinary water and, therefore, less sodium isdissolved in it.In Figure 11, one might visualize how water structure and electrolytecomposition may be modulated.

Bioregulants, such as ATP, insulin or ouabain, compete for receptor,allosteric or cardinal sites at the cell surface or within the cell. This asso-ciation induces both short and long range effects on the protein polymersdetermining the specificity of charged sites and modulating the extent ofpolarization of the water molecules.Ouabain, for example, could have its effect at the surface, competing

successfully with ATP for the receptor, and thereby, changing the polymerto a sodium preferring state. There might or might not be long rangechanges in the water structure.Now let us return to the problem. How does one explain the large drop

in muscle fiber sodium that accompanies early postnatal growth? We mightargue that the decrease in sodium is primary due to a change in the physicalstate of water. That is, the cytoplasmic water may become less solvent tosodium through enhanced interaction between water and the cellularproteins. If such wild speculation should be remotely possible, what is theevidence?

Please consider the following: Nuclear magnetic resonance (NMR)spectroscopy may be used to obtain insight into certain physical propertiesof water. We have used NMR spectroscopy to study the state of water inmuscle tissue.67 NMR is well suited to this task because the width of thesignal produced by water hydrogens is dependent on the motional freedom

*The notion that potassium is associated to fixed charges in the cell has been a subject of somecontroversy.28'43 The major piece of work cited as proof that potassium is free in the cell is thatof Hodgkin and Keynes.56 Ling discussed these findings and offered an alternative explanation in1969.54 Also, in 1969, Jones and Karreman57,58 reported that the cellular uptake of potassium incarotid artery smooth muscle could be described by a cooperative absorption isotherm derived byLing and Yang.59'60 In addition, Ling and Bohr61 and Gulati and Reisin62 have shown that potas-sium uptake in frog muscle and taenia coli can be described by the same adsorption isotherms. Gu-lati and Jones demonstrated that the potassium cooperative adsorption isotherm could be altered byvarying concentrations of ouabain.39 These studies were extended to include a number of metabolicpoisons and it was shown that cellular potassium is directly proportioned to the ATP content ofskeletal muscle.63 More importantly, Ling64 has reported a detailed study of the diffusion co-efficient of potassium (in a "membraneless" preparation) and found that the diffusion coefficientof potassium in the cytoplasm is significantly reduced in a viable cell. As the muscle preparationdeteriorates, the value for the potassium diffusion coefficient approaches those values reported byHodgkin and Keynes56 and Kushmerick and Podolsky.65 (it may be of interest to note thatHodgkin and Keynes used the electrical activity as an index of the viability of the axoplasm ofthe squid axon, yet in 1962, Baker, Hodgkin and Shaw32'33 demonstrated that electrical activ-ity of the membrane was independent of the axoplasm.) Recent NMR studies of the potassiumnucleus show that the molecular motion of potassium is reduced in several tissues.66The findings of most of the publications cited in this footnote seriously question yet anotherfundamental assumption of the membrane theory and need some response from those who areproponents of that theory.

95

of the water molecules. As the mobility of the water molecules increases,the line width decreases (Fig. 12).

The NMR signal for muscle water shown on the left side of Figure 12is at least ten times broader than that of pure water, and heat denaturationof the muscle leads to a narrowing of the NMR water signal. We in-terpreted these data, along with a large number of other NMR studies, tomean that water molecules significantly interact with cellular macro-molecules such that water in skeletal muscle could be considered to existin a more ordered or structured state than pure water.67 68 This interpreta-tion of high resolution NMR spectroscopy was supported also by observa-tions utilizing pulsed or spin echo NMR.OiO*

The question is then raised: Do the NMR water signals for skeletalmuscle change with normal development? Figure 13 shows the results ofsuch a study where the ordinate is the line width of the high resolution

4. +

Fig. 11. My interpretation of Ling's association-induction hypotheses. The surface (membrane)of the cell as well as the interior of the cell contain receptors (special or cardinal sites)which, when appropriate molecules (solutes) adsorb or "associate" with these cardinalsites, induce changes in the macromolecules that may extend over larger distances. Thatsolute which associates with a cardinal site is called a cardinal adsorbant or bioregulant.The water structure of the cell is determined primarily by the cellular proteins and thewater structure may be modulated by the proteins as influenced by the interaction withspecific bioregulants.

*Considerable non-NMR evidence supports this interpretation and an extensive review of inter-facial water has been given by Drost-Hansen.70

96

+ HEAT

Fig. 12. High resolution NMR spectra of water protons in a rat gastrocnemius muscle before (A)and after (B) protein denaturation. The line width decreased from 14.5 Hz to 3 Hz.

tNj

I-

L&J

z

-J

17

15

13

11

9

7

5

3

0 10 20 30 40 50 60 70 80 90 100

AGE IN DAYSFig. 13. Ordinate: NMR line width at half amplitude in hertz. Abscissa: Animal age in days.

The dashed line curve generated from the following equation: W 12.81-7.0 exp

(-0.55*t) where W represents line width in hertz and t represents animal age in daysThe equation was determined by a procedure reported in Reference 71. Reprinted withpermission.

97

* 9 - - - -- -- - - - - -

S 0

//

5 ///_,**

/ #00 /

0

/-.

I IIII

NMR signals in hertz for muscle water protons and the abscissa is animalage in days.71 The line width increases monotonically with normal de-velopment and we have interpreted these data to mean that there is anincrease in the order or structure of muscle water with normal develop-ment.

Next, we looked for a correlation between these changes in the NMRwater signals and the changes in tissue sodium concentration. Figure 14summarizes the results of this study. The ordinate is the muscle sodiumconcentration in mEq/Kg FFWW, and the abscissa is the NMR line widthfor muscle water protons in hertz. The narrow water signals are correlatedwith high tissue sodium concentration. Broad water signals are correlatedwith low tissue sodium concentration. We hypothesized from this thatmuscle water exists in a less ordered state in the younger animals andthat a large fraction of sodium can dissolve in the water, and with de-velopment, the water assumes more structure due to the increase in theconcentration of the cellular macromolecules. As a result, the increase inwater structure reduced the sodium concentration. This high resolution

8

_-

oSU-8

z

a

8a; I I . . I

0w 7.00 98.0 9.00 10.00 12.00 12.00 13.00 1'4.00NMR LINE WIDTH IN HERTZ

Fig. 14. Ordinate: Skeletal muscle sodium concentration in milliequivalents per kilogram of fat-free wet weight. Abscissa: The NMR line width for skeletal muscle water protons inhertz. The solid line represents the expected relationship between tissue sodium concen-tration and the NMR water signals as determined by the techniques reported in Reference71. Narrow water signals are associated with high tissue sodium concentrations.

98

study of muscle water protons was also corroborated by another NMRstudy. (A detailed account of the studies shown in Figures 13 and 14 arepresented in references 71 and 72.)

A schematic representation of what is a reasonable interpretation of themechanism of sodium exclusion associated with normal development isgiven in Figure 15.

Potassium. Potassium is associated with fixed charges within the musclefibers in both immature and mature skeletal muscle. A large subset of thetotal set of cellular macromolecules (probably proteins) exists in aphysical state that preferentially adsorbs potassium over sodium. Thissubset of cellular macromolecules increases with normal development andthe accumulation of potassium is proportional. It is likely that most ofthe adsorbed fraction of sodium reported for mature muscle is also asso-ciated with this major macromolecular subset.

Water. Skeletal muscles taken from newborn animals contain as muchas 90% water. The percentage of water decreases to approximately 77%0in the mature animal. Therefore, the cellular macromolecules increaseduring development at the expense of tissue water. The increase in thesurface to volume ratio between birth and 65 days of age may accountfor the increase in the cellular water structure that has been proposed.

0o CHEMICAL0000000000

00000o0 MATURATION) 0°00oo°00 ~~~~~~~~0000000000o 0

0

Fig. 15. During the first sixty-five days of postnatal life, the concentration of potassium in muscle-is relatively constant. The sodium concentration of muscle, however, decreases by almost afactor of 10. It is suggested that the decrease in cellular sodium is a result of a changein the solvent properties of the myoplasmic water. This figure depicts the major macro-molecule as adsorbing potassium in the same proportion throughout the maturationalprocess. The minor fraction which preferentially binds sodium is shown to undergo a con-formational change during the developmental period. Both the increase in the proportionalmass of the dry solids and, perhaps, a conformational change in one or more macromo-lecular fractions could account for the changes in the NMR water proton signals.

99

It is possible, however, that during early postnatal development somemodification in the structure of the contractile proteins occurs. For example,Trayer, et al, have shown the presence of 3-methylhistidine in adult rabbitskeletal muscle and its absence in fetal muscle.3 The possibility that aconformational change may occur in one or more of the contractile proteinsand that this change may effect a change in long-range order of thecellular water cannot be ruled out.*

Sodium. It is proposed that the decrease in cellular sodium concentrationresults primarily from a relative increase in the structure of cellular water.

The data gathered so far are consistent with this view. Obviously, it doesnot represent a truth. Yet, this general concept of the events occurringin developing muscle leads one to consider the following: In a general sense,we are observing events associated with controlled differentiation. If thedifferentiation process should falter as in tumorgenesis, would one expectto see differences in the NMR water signals?

The prediction of Damadian was yes, and he experimentally verified hisprediction in dedifferentiated tissue in 1971.74 We extended his obser-vation to an animal model system in which three morphological statescould be defined. (These data are presented in Figure 16 and in reference

Tissue and Dnumber of (cm2/sec Xsamples T1 (sec) T2 (sec) lo-6)

Pure water 3.1 1.43 4 0.27 2.38 ± 0.016Tumor (5) 0.920 ± 0.047 0.091 ± 0.008 0.78 ± 0.05Nodule (5) 0.451 ± 0.021 0.053 ± 0.001 0.44 ± 0.03Normal preg-

nant mam-mary gland(5) 0.380 ± 0.041 0.039 ± 0.002 0.34 ± 0.04

Fig. 16. Relaxation times and diffusion coefficient of water protons in pure water, normalmammary gland, preneoplastic nodule outgrowth, and neoplastic 'tissues at 25°C. The purewater data were from four samples of water except for T,, where only two samples wereanalyzed. All values for tissue-water protons are given as the average + the standarderror. For a detailed description of this study, see Reference 75.

*In evaluating data on developing skeletal muscle which we have accumulated over the past 10years by way of various methods, I have developed the notion that some macromolecular struc-ture (protein, glycoprotein, mucopolysaccharide, etc.) may be synthesized during early postnatalor late prenatal development which can induce long-range ordered structure in the cellularwater. The early changes in the electrolyte concentration and in the water structure could beexplained as a cooperative process and once stabilized (i.e., by 32 days of age) differentiationwould be stopped. Later, external influences such as trace minerals, hormones, drugs, carcinogens,etc., could turn the system on, which would result in changes in hydration and in electrolyteconcentration. Although this speculation is stimulating, the evidence is, at present, circum-stantial. (A more formal description of the cooperative process in development and in tumor-genesis is given in Chapter 18 of reference 10.)

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75.) The preneoplastic nodule and the malignant tissue were distinguishablefrom normal mammary tissue. Also, all three morphological states weredistinguishable from pure water. Furthermore, significant differenceswere found in each of the NMR parameter studies. That is, T1, the longi-tudinal relaxation time, T2, the transverse relaxation time which is pro-portional to the line width, and the diffusion coefficient for water protonswere all distinguishable.72 (A simplified description of the relaxation timesand the diffusion coefficient are given in reference 72, and further reviewof the literature on NMR and malignant tissues is presented in other chap-ters in this referenced book.)The speculation about water has been extended to human breast cancer.

This is presented in Figure 17. The mean values of mammary adenocar-cinoma (AC) can clearly be distinguished from normal (N) and diseased

7876747270686664626058

0 56Li 54

_J 52- 50

48464442403836343230

I I I I

N FC FA ACI I I IN FC FA AC

The values are the means ± standard error. The abbreviations used areN - Normal. FC - Fibrocystic, FA - Fibroadenoma, AC Adenocarcinoma.

Fig. 17. Nuclear magnetic resonance relaxation times T1 and T2 of human breast tissues. Thevalues are the means + standard error. The abbreviations used are N Normal,FC Fibrocystic. FA Fibroadenoma, AC=Adenocarcinoma.

101

1.1001.0801.060-1,040 _1,0201,000 _980 _960UO _940

900880860840820800_780760740720700680660640620600

0

LJ

-J

I-

-4

or fibrocystic breast tissue (FC). The mean values of the benign fibroad-enoma (FA) can be distinguished from the malignant adenocarcinomaif one takes T1/T., ratio.

Once again the predictions of the new view of the living cell are borneout. Furthermore, it seems evident that NMR spectroscopy should beevaluated in detail relative to its utility as a tool for cancer detection andfor studying tumorgenesis. In addition, it should be pointed out that thisnew view predicts a physical role for water in fundamental cellular pro-cesses not predicted by the classical view. The cellular changes in waterand electrolyte composition associated with congestive heart failure andother cardiovascular diseases related to edema formation, for example,are explained by the new view. Experiments in this domain are justbeginning.

ACKNOWLEDGEMENTS

Supported by grants from the Robert A. Welch Foundation, the LibertyMuscular Dystrophy Research Foundation, and USPHS Research GrantsGM-20154 and RR-0188 from the General Clinical Research Centers Pro-gram of the Division of Research Resources, National Institutes of Health,Bethesda, Maryland. The electronmicroprobe used in this study was pro-vided through NASA Grant NGR-44003053. The assistance of Mrs. MarthaSimmons and Miss Debbie Severs is acknowledged.

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