Chapter 9: The Physical State of Water in Living Cells
Bound intracellular K+ necessitates bound intracellular water.
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Today’s Substack will continue with Chapter 9, The Physical State of Water in Living Cells. I had requests to define terms more throughout, even if I have in prior chapter reviews - I tried to accommodate that with this review. Please let me know if it was sufficient or if more is needed, my goal is to make Ling’s work as accessible as possible to non-scientists and for those who do not have the time to look up definitions, especially the context in which they are used.
Please feel free to skip to the parts you wish to read.
Summary:
This chapter is research data heavy so I will try to stick to the main takeaways.
Chapter 8 argued that K+ is in an adsorbed state (bound to proteins or other macromolecules) in cells. This means something different than osmotic pressure (basic def: solvent pressure to equalize solute concentration across a “membrane”) is needed to equalize the cell (volume, etc.) to the environment. “Water in the state of polarized multilayers provides this mechanism.”
9.1. Introduction
Ling starts the chapter off with a powerful statement about what follows from the antithesis of the prevailing view that intracellular K+ is free, which means the intracellular water is free - “If K+ is not available to reduce the thermodynamic activity of cell water to equal that of the environment, then the cell volume can be maintained only if the water is associated with intracellular molecules.” Here free can be thought of putting “no salt” (KCl) in a glass of water and not free/ structured/ adsorbed/ etc. more like a gelatin state with K+ associating with the amino acids, etc. Intracellular K+ is approximately 30+x that of extracellular. Thermodynamics, the study of energy, entropy, heat work, etc., and their transformations in systems necessitates equal intracellular:extracellular K+ concentrations. The gradient difference and lack of diffusion to correct the disparity impacts things like osmotic balance (equalizing the concentration gradient). This would affect cell size due to pressure differences intracellular:extracellular. To overcome this and to maintain cell size, intracellular water would need to be structured - see chapter six’s review for more information on how Ling theorizes this structure with his “theory of the multilayer polarization.”
9.2. Solvent Properties
The polarized multilayer theory postulates that the water multilayers create a water lattice (sugar and diamonds are crystalline materials exhibiting lattice structures). Based on the specific structure of the lattice, things can fit in between the water molecules. These things would be expected to have “near-normal solubility” (solute dissolves in a solvent to form a homogenous solution). “Molecules that cannot easily fit into the water lattice are excluded to varying degrees in direct proportion to their size and complexity.”
9.3. Freezing Points
Cells have lower freezing points than expected. The freezing point is the temperature a liquid solidifies. Ling suggests that the freezing point depression in living systems is primarily influenced by the presence of solutes, particularly potassium ions (K+). He explains that when solutes are dissolved in water, they disrupt the formation of ice crystals and lower the freezing point. The theoretical expectations are that the concentration of solutes within cells, primarily K+, would substantially reduce the freezing point compared to pure water. This is analogous to salt increasing water’s boiling point. Ling proposes that the adsorption of K+ onto the surfaces of proteins and other macromolecules in cells plays a crucial role in the freezing point depression. This adsorption reduces the thermodynamic activity of water, preventing ice crystal formation and further lowering the freezing point. “Water in normal resting cells exists in a different physical state. Its structure resists freezing in a manner different from that of supercooled liquid water, because no matter how rapidly liquid water is called, it has not been possible to freeze it without crystal formation.” Experiments show gelatin gel and muscle cells do not form ice crystals when rapidly frozen, unlike free water.
9.4. Vapor Sorption Isotherms
Vapor sorption isotherms describe the relationship between the amount of water vapor absorbed by a material and the relative humidity of the surrounding environment. Experimental studies have shown that biological materials, including living cells, exhibit unique vapor sorption isotherms compared to non-biological materials. The sorption behavior of biological materials is often characterized by non-linear isotherms, indicating complex interactions between water molecules and the material’s surface. Ling suggests that the sorption behavior of biological materials can be attributed to the adsorption of water molecules onto “extended proteins with their polypeptide chains” within cells. Water adsorption alters the material’s physical properties, including its swelling behavior and water-holding capacity.
9.5. Infrared and Raman Spectra
IR and Raman spectroscopy provide valuable information about the molecular vibrations and interactions within biological materials. These techniques involve the interaction of materials with infrared or laser radiation, which leads to the scattering or absorption of photons. The IR and Raman spectra can be used to study the effects of solutes, such as potassium ions (K+), on the vibrational properties of biological materials. Ling proposes that solute interactions with macro- and water molecules influence the observed spectral features that differ from free water spectra.
9.6. Dielectric Dispersion
“In a vacuum, electric charges on a parallel plate condenser give rise to a potential difference, V. The introduction of water into the space between the plates causes a reduction of V by a factor of 81; this is the static dielectric constant (epsilon_s) of water. Water molecules have an asymmetric charge distribution and are polarized by the electric field of the charged condenser in such a way as to almost completely annul the electric field produced by the charges originally present on the parallel plate condenser.” That was probably a lot for most, so let me explain. A parallel plate condenser is a capacitor that has two parallel plates. The parallel plate capacitor stores opposite charges, +q and -q, on opposite plates (so if plate 1 is +q, plate 2 is -q). This charge separation creates an electric field, and that electric field stores electrical energy. Voltage, also known as the electric potential difference, is high level understood as the electric field over some distance. In a static electric field, the voltage is the amount of work needed per unit of charge, q, to move a test charge, q, between two points in space. The asymmetric charge distribution of water has to do with the partial +q on the two H and the partial -q on the O. Water’s asymmetric charge distribution cancels out the charge separation of the plates, which is why there is a reduction in voltage.
The dielectric constant, also known as relative permittivity, is a property that characterizes the ability of a material to store electrical energy in an electric field. It is defined as the ratio of the electric displacement produced in a material to the electric field applied to it. The dielectric constant represents how easily a material can be polarized when subjected to an electric field. It quantifies how much the material can store electrical charge and electrically insulate or screen against the electric field.
There are slight differences and more math when the electric field is not static but fluctuates with time. These differences exhibit inertia which can be considered a recall of the past. As the electric field changes in time, “the polarization of water can follow until the frequency reaches a level so high that the polarization can no longer keep up. At this point, the dielectric constant begins to decrease with further increase of frequency to reach another stead low level designated as epsilon_infiniti. The decrease of dielectric constant with increase of frequency is called dielectric dispersion.”
A difficulty in measuring dielectric properties in living cells “is the deterioration of tissue during the measurement.” Also, getting to the cells themselves removes them from the organism that is needed to keeping them “living cells.” NMR studies of dielectric relaxation show that cell water dielectric constants are different from free. Nuclear magnetic imaging, NMR, is colloquially the MRI but for research purposes.
9.7. NMR Relaxation Times of Water Protons and Other Nuclei
“The great advantage of NMR lies in its nondestructiveness. The static magnetic field and weak radio frequency signals applied to the specimen do not harm it.” NMR and the theory underpinning its use, was still being developed in most of the research Ling reviews so he did not believe it was “capable of providing decisive information on the physical state of water in living cells,” at the time. Water in different phases will have different relaxation times.
NMR relaxation times refer to the characteristic timescales on which the nuclear spins of atoms return to equilibrium after being perturbed by an external magnetic field. Two critical parameters associated with NMR relaxation are the longitudinal relaxation time (T1) and the transverse relaxation time (T2). Ling explains that the longitudinal relaxation time (T1) is related to the rate at which nuclear spins return to equilibrium with the surrounding environment. It provides information about the spin-lattice interactions’ dynamics and the energy exchange between the nuclear spins and their surroundings. The transverse relaxation time (T2) is associated with the loss of phase coherence among the nuclear spins due to interactions with their local environments. T2 reflects the decay of the NMR signal and provides insights into the mobility and interactions of the nuclei in the sample.
Early NMR studies led researchers to conclude most of the cell water was “normal liquid water” with sole “solid” water. However, there was a splitting signal for which many have offered different explanations. Ling argues, “Thus far, NMR studies cannot provide proof for or against the concept that the bulk of cell water is different from normal water. It is my opinion that part of the indecisiveness in interpretation of NMR studies on the nature of the bulk of cell water lies in a lack of studies of a clearly demonstrable example or model of water in the state of polarized multilayers.”
9.8. Quasielastic Neutron Scattering
Quasielastic neutron scattering, QNS, is a spectroscopic technique that provides information about the dynamics of atoms and molecules in materials. It involves the scattering of neutrons by the sample, and the analysis of the scattered neutrons provides insights into the motions and interactions of the sample’s constituents. “When slow neutrons impinge on liquids like water, they are scattered, largely by the H atoms of water and on a time scale comparable to the time that the water molecules take to jump from one position to another. As a result, the observed scattering of neutrons contains information about the diffusive motions of water molecules involved.” Fission reactors can produce slow or fast neutrons. What differentiates slow from fast is their energy. If there is no energy change during the scattering process, this is known as elastic scattering. Inelastic scattering occurs with an energy change. Most liquids exhibit QNS. “QNS study of water in the cells of the cysts of brine shrimp… came to the conclusion that the bulk of cell water has strongly reduced translational and rotational diffusion coefficients that are not due to obstruction, compartments, or exchange with a minor phase. Their important findings have therefore confirmed the predictions of reduced rotational and translational mobility of the bulk of cell water based on the polarized multilayer theory of cell water.”