Does PEMF cause membrane leaks?

This post examines the claim that PEMF opens up lipid bilayers making them more permeable. Phosphatidylcholine is a dominant phospholipid in the outer leaflet. The head groups have a positive and negative charged atoms. Egg phosphatidyl choline is part of the featured image as is phosphatidyl ethanolamine. My conclusion is that PEMF might be more of a means of giving a cell’s membrane a “massage” rather than opening up holes.

a visual of the claim [1]

The source of one image is a computer (Molecular Dynamics) simulation of sodium and potassium interacting with a dominate phospholipid, phosphatidylcholine. [1] Conventional wisdom is that bilayers separate the inside of the cell from the outside of the cell. True.

Left, an image of the Molecular Dynamics computer simulation with two bilayers of phosphatidyl choline. Right Ion gradients in real cells: There is more Na⁺and Cl^– outside the cell and more K⁺inside the cell. The balance of negative charges come from amino acid side chains on proteins.

ABSTRACT Differences of ionic concentrations across lipid bilayers are some of the primary energetic driving forces for cellular electrophysiology. While macroscopic models of asymmetric ionic solutions are well-developed, their connection to ion, water, and lipid interactions at the atomic scale are much more poorly understood. In this study, we used molecular dynamics to examine a system of two chambers of equal ionic strength, but differing amounts of NaCl and KCl, separated by a lipid bilayer. Our expectation was that the net electrostatic potential difference between the two chambers should be small or zero. Contrary to our expectation, a large potential difference (70 mV) slowly evolved across the two water chambers over the course of our 172-ns simulation. This potential primarily originated from strong Na⁺ binding to the carbonyls of the phosphatidylcholine lipids. This ion adsorption also led to significant structural and mechanical changes in the lipid bilayer. We discuss this surprising result in the context of indirect experimental evidence for Na⁺ interaction with bilayers as well as potential caveats in current biomembrane simulation methodology, including force-field parameters and finite size effects.”

Image from PubChem. The carbonyls and esters carrying partial negative charges are indicated.The location of Na+ is from text rather than MD coordinates

One of the unexpected findings of this study was that Na⁺ cations had a higher affinity for carbonyl -C=O groups of the glycerol backbone. The electron hungry oxygen atom of this group could have a partial charge of -0.07 leaving the carbon with a partial charge of +0.7. The ester oxygen that links the glycerol backbone to the two fatty acid groups. Figure 2 of this publication states that most PC is not complexed to K⁺ and 60% is not somplexed to Na⁺. About 20% of the PC might exist in complexes of four PC per one Na⁺. Naturally these are not covalent bonds, but rather electrostatic interactions. We must remember Faraday’s Law of Induction that states that a moving magnetic field will get charges moving. As a reminder, the Lee 2008 model predicted movement of water past the hydrophobic acyl groups of the PC fatty acids. PEMF making membranes leaky remains far fetched, increased osmotic flow of water maybe more plausible.

the message part

The message part comes from that layer of Na+ struggled next to the carbonyl groups being moved back and forth in the oscillating magnetic field as per Faraday’s Law of Induction.

Phospholipids align in mag field as per NMR

Scientists have know for a long time that the phospholipids in artificial bilayer vesicles will align in strong magnetic fields. Wikipedia has a good page on Nuclear Magnetic Resonance

NMR consists of three steps.

The alignment (polarization) of the magnetic nuclear spins in an applied, constant magnetic field B₀.
The perturbation of this alignment of the nuclear spins by a weak oscillating magnetic field, usually referred to as a radio frequency (RF) pulse. The oscillation frequency required for significant perturbation is dependent upon the static magnetic field (B₀) and the nuclei of observation. In order to interact with the magnetic field in the spectrometer, the nucleus must have an intrinsic angular momentum and nuclear magnetic dipole moment. This occurs when an isotope has a nonzero nuclear spin, meaning an odd number of protons and/or neutrons (see Isotope). Nuclides with even numbers of both have a total spin of zero and are therefore not NMR-active. Regular ³²P is inactive, ³¹P is good for imaging.
The detection of the NMR signal during or after the RF pulse, due to the voltage induced in a detection coil by precession of the nuclear spins around B₀. After an RF pulse, precession usually occurs with the nuclei’s…etc.

In short, scientists interested in studying how a ³¹P phosphorous nuclei in a phospholipid bilayer with or without paper have to be aware of how bilayers of said lipids behave in the strong magnetic field of step 1. Other physical properties of the molecules might give these lipids

Anisotrophy is a term we hear a lot in earlier papers. Anisotropy means that a physical object is not the same in all directions. The hydrophilic head groups of phospholipids haved a pretty obvious distribution of negative and positive charges. The van der Waals force, along with the hydrophobic effect hold the a acyl groups of phospholipids together. Hydrogen bonds, discussed in this YouTube video. In hydrogen bonding charge separation may be partial. In the hydrophobic fatty acid acyl groups the electrons might be to one side or another of the nuclei they “orbit” giving them a very temporary +/- dipole. Cohesion comes when those temporary +/- align in time, oscillating in unison. For those who think PEMF is pocking holes in membranes it probably isn’t happening, BUT, it might be shaking those phospholipids up. We know this by work done with strong, static magnetic fields in the early days of nuclear magnetic resonance that lead to magnetic resonance imaging. .

1978 egg lecithin vesicles align with a magnetic field [2]

Egg lecithin vesicles were prepared by spontaneous swelling. Some of these vesicles were cylinder shaped. They were placed in air tight containers that were placed on a microscope stage. A homogeneous field of 15 kG, 1.5 T, was applied parallel to the slides. The vesicle movements, translation, and rotation, could be observed by eye or photographed

Magneto orientation of dipalmitoyl phosphatidyl-choline [3]

This group used dipalmitoyl-L-alpha-lecithin. They prepared bilayers from a xylene mixture. Anisotropy was measured by visible light optical means.

FIG. 2. Typical results of variation of transmitted-light intensity in the magnetic field when the field has been turned on or off.

A. Dipalmitoyl phosphatidyl choline when the polarizer was oriented parallel to the magnetic field. In the minute that the 0.16-0.9 T magnetic field was turned on the light intensity decreased. When the field was turned off, the light intensity increased with a given time constant.
B Dipalmitoyl phosphatidyl choline when the polarizer was oriented to make an angle of 45° with the field. When the 0.41 to 0.9 T magnetic field was turned on, the polarized light intensity increased with a given rise time. Likewise, when the field was turned off, light decreased.
C Polyethylene crystals suspended in xylene; the polarizer was oriented parallel to the field direction. vesicles. Turning the 0.32-0.9 T magnetic field on and off decreased and increased the light transmitted.

Polarizer parallel to the magnetic. Experiment at 22^oC. Two of many examples, See text for original copyright image

The stronger the magnetic field, the quicker the reorientation upon turning on and off. Note that this is pretty far from physiological 37^oC. The next step would be to recreate this experiment under more physiological salts, temperature, and lipid composition. Under conditions under which even the more powerful PEMF is performed we can speculate that PEMF only “shakes” things up without reorientation the lipids in a huge way.

A look at different forms of phosphatidylcholine [4]

Lipid composition helps determine the type of shape phospholipid vesicles take on: cigars or balls. The balls are under two sorts of strain in a magnetic field:

involves lipid molecules at the equator which resist the magnetically induced reorientation force, and strain-2
involves the lipid molecules at the apex that experience the highest magnetically induced reorientation force which torques lipid molecules at the liposome-north (or south) pole towards the liposome equator. the strain is relieved when the liposome is distorted into a cigar shape; the cigar shape minimizes only strain-2 o

Lipid bilayers prepared from natural phospholipids orient in magnetic fields with the long axis of the lipid molecules perpendicular to the magnetic field.
The field strengths were 4.68-9.36 T, much stronger than most commercial PEMF machines.
Phosphatidycholine analogs containing saturated diacylated chains (12 to 16 carbons/chain) exhibited extensive orientation of the lipid when bilayer formation occurred by gentle hydration conditions.
The method of bilayer formation significantly influenced the amount of lipid that orients in magnetic fields.
The concept of melting temperature, Tm, was introduced. This refers to membrane fluidity. The supramolecular structures (and % orientation) above Tm in an 11.7 T field of dimyristoylphosphatidylcholine (DMPC) bilayers are SUV (0%), LUV (~ 15%), SPLV (~ 40%),vortexed-MLV (~ 60%) and non-vortexed MLV (~ 90%).
Single layered vesicles prepared by the reverse
phase evaporation vesicle REV method exhibited orientation at 11.7 T similar to LUV prepared by freeze thaw cycles.
Aqueous dispersion of egg PC prepared by gentle hydration exhibit ~ 40% orientation at 11.7 T which decreased to ~ 30% orientation if 30% cholesterol is added to the membrane.

Magnetic orientation of bilayers thus appears to be a general phenomenon for both saturated and unsaturated natural phospholipids either with or without cholesterol in the membrane.

N-palmitoylsphingomyelin and lyso phosphatidyl-choline [5-7]

In a magnetic field molecules may orient due to the presence of an anisotropy in their diamagnetic susceptibility.[5]

β=NΔx H²/k_BT

β magnetic susceptibility
N number of molecules in the cluster
Δx diamagnetic anisotropy
H² the square of the magnetic field
k_B the Boltzman constant
T absolute temperature in degree Kelvin

The degree of orientation is usually quite small even for molecules with large values of Δx. However, in a lipid bilayer, with liquid and crystal characteristics, anisotropy is additive. Negligible orientations were observed by previous authors using nuclear magnetic resonance and electron spin resonance.

These authors used hydrated phospholipid mixture that exhibits a high degree of orientation in a magnetic field. The mixture is composed of 60 mol% N-palmitoylsphingomyelin (NPSM) and 40 mol% 1,2-dimyristoylsn-glycero-3-phosphocholine(DMPC) and hydrated with 50 wt% water. The percentage of DMPC seemed to matter about orientation perhaps because of the size of the vesicles and whether they could form rods to add another component of anisotropy. It should be pointed out that there are more lipids than just DMPC and NPSM in the lipid membranes. When one of the fatty acids is removed from the middle carbon of the glycerol backbone by the action of phospholipase A₂ the result is lysophosphatidylcholine. A publication using nuclear magnetic resonance found that lysophosphatidylcholine changed membrane dynamics. [6] While NMR uses a strong static magnetic field and radiofrequencies for the resonance, the implication is different membrane compositions may respond in tiny ways to very low frequency PEMF.

A follow up study looked at excised spinal cord samples, rich in white matter containing these phospholipids, in a 7 T MRI. [7] Inside this MRI was a torque balance. All samples demonstrated orientating effects. Myelin sheaths and white fiber bundles were argued to be highly ordered. This ordered structure leads to an orientation preference of specific molecular bonds, which in turn leads to an orientation dependence of the magnetic susceptibility [7]

the mesage part

While even very low frequency PEMF is probably too short lived to realign whole cells. Plus, cells in our tissues are surrounded by other cells. It is tempting to imagine slight and gentle swaying phospholipids in our cell membranes. Surely something like this happens when we get a real message!

In cancer cells [8]

Some of this fact checking on the claim that PEMF causes increases in membrane permeability has been a bit of a journey. I school we were taught that phosphatidyl choline ends to be in the outer leaflet and phosphatidyl ethanolamine in the inner leaflet towards the inside of the cell. Bigger head groups tilt the radius of curvature to convex out. Lipid holes can theoretically form in membranes. These holes are referred to hexagonal I and II.

Image of lamellar to hexagonal phase transitions

Tumor cells express a unique cell surface glycocalyx with up regulation of sulfated glycosaminoglycans and charged glycoproteins. The human lung carcinoma cell line A549, like other carcinoma cells, is known to over express the sulfated GAG heparan sulfate (HS)
These authors applied a pulsed 20 mT magnetic field with rate of rise (dB/dt) in the msec range. The target was cultured tumor cells.
Figure 2 A 10-min exposure of A549 human lung cancer cells to sequential 50- and 385-Hz oscillating magnetic fields was sufficient to induce intracellular protease release, suggesting altered membrane integrity after the field exposure.
Figure 4 PEMF induced plasma membrane leak in MDA-MB-231 human breast carcinoma cells that is partially sialic acid dependent Figure 1, see panel C multiple points is shown (i.e., at glucosamine-uronic-acid linkage, in which uronic acid is relatively unsulfated/uncharged; arrows). This fragments HS chains with essentially glycan-denuded (and charge-denuded) proteoglycan core proteins (shown to right of H’ase III arrow), which are unable to confer forces (and thus torque) by pulsed-magnet-induced (dB/dt) EMFs at the point of core protein attachment to the membrane.
Heparinase, which digests anionic sulfated glycan polymers, before exposure rendered cells insensitive to this effect. Fig 6 is a summary cartoon that shows sulfated proteoglycans acting as an antenna for the PEMF. This is a thought provoking cartoon: Are proteases released by a twisting motion or some other mechanism?
Figure 3 a non-neoplastic human primary cell line (lung lymphatic endothelial cells) did not lose membrane integrity upon exposure to magnetic fields.
PEMFexposure induced protesase release from human breast cancer cells, that express a sialic-acid rich glycocalyx. Sialidase pretreatment mitigated this PEMF relase of protease. , which removes cell surface anionic sialic acid.
Figure 5 Scanning electron microscopy showed that field exposure may induce unique membrane “rippling” along with nanoscale pores on A549 cells. These holes were abut 50 nm in diameter and were thought to be consistent with endocytic events. .

CSPG are glycosyled membrane proteins. A Wikimedia Commons user, Blar3, released the image on the right of CSPG in perineural nets are made of chondroitin sulfate proteoglycans neuroscan, veriscan, brevican, and aggrecan Tenascin, in turn, binds to CS glycosaminoglycans (red lines) as well as cell surface CSPGs. Phosphacan can also bind to cell surface receptors such as NCAM. B. Application of chondroitinase ABC (ChABC) degrades all the CS glycosaminoglycans (red lines) as well as hyaluronan (pink line), causing major disruptions in the structure of the perineuronal net. These disruptions may allow axons to penetrate the vacated space and allow restoration of neuronal plasticity.

Some Wikimedia commons images of proteins that bind to chondroitin sulfate and anchor it to membranes. VS is highly charged and likely to have counter cations that could move around in PEMF. Note that is a neuronal model

The possibility of PEMF doing something selective to tumor cells expressing chondroitin sulfate is an interesting one. PEMF is well documented as a means of healing joints.