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Fundamentals of Membrane Lipid Replacement: A Natural Medicine Approach to Repairing Cellular Membranes and Reducing Fatigue, Pain, and Other Symptoms While Restoring Function in Chronic Illnesses and Aging

  • The Institute for Molecular Medicine
  • Clinical Education
  • Sierra Productions Research, LLC


Membrane Lipid Replacement (MLR) uses natural membrane lipid supplements to safely replace damaged, oxidized lipids in membranes in order to restore membrane function, decrease symptoms and improve health. Oral MLR supplements contain mixtures of cell membrane glycerolphospholipids, fatty acids, and other lipids, and can be used to replace and remove damaged cellular and intracellular membrane lipids. Membrane injury, caused mainly by oxidative damage, occurs in essentially all chronic and acute medical conditions, including cancer and degenerative diseases, and in normal processes, such as aging and development. After ingestion, the protected MLR glycerolphospholipids and other lipids are dispersed, absorbed, and internalized in the small intestines, where they can be partitioned into circulating lipoproteins, globules, liposomes, micelles, membranes, and other carriers and transported in the lymphatics and blood circulation to tissues and cellular sites where they are taken in by cells and partitioned into various cellular membranes. Once inside cells, the glycerolphospholipids and other lipids are transferred to various intracellular membranes by lipid carriers, globules, liposomes, chylomicrons, or by direct membrane–membrane interactions. The entire process appears to be driven by ‘bulk flow’ or mass action principles, where surplus concentrations of replacement lipids can stimulate the natural exchange and removal of damaged membrane lipids while the replacement lipids undergo further enzymatic alterations. Clinical studies have demonstrated the advantages of MLR in restoring membrane and organelle function and reducing fatigue, pain, and other symptoms in chronic illness and aging patients.
Fundamentals of Membrane Lipid Replacement: A Natural
Medicine Approach to Repairing Cellular Membranes and
Reducing Fatigue, Pain, and Other Symptoms While Restoring
Function in Chronic Illnesses and Aging
Garth L. Nicolson 1, *, Gonzalo Ferreira de Mattos 2, Michael Ash 3, Robert Settineri 4and Pablo V. Escribá5
Citation: Nicolson, G.L.; Ferreira de
Mattos, G.; Ash, M.; Settineri, R.;
Escribá, P.V. Fundamentals of
Membrane Lipid Replacement: A
Natural Medicine Approach to
Repairing Cellular Membranes and
Reducing Fatigue, Pain, and Other
Symptoms While Restoring Function
in Chronic Illnesses and Aging.
Membranes 2021,11, 944. https://
Academic Editor: Francisco Monroy
Received: 24 October 2021
Accepted: 23 November 2021
Published: 29 November 2021
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Attribution (CC BY) license (https://
Department of Molecular Pathology, The Institute for Molecular Medicine, Huntington Beach, CA 92647, USA
2Laboratory of Ion Channels, Biological Membranes and Cell Signaling, Department of Biophysics,
Facultad de Medicina, Universidad de la República, Montevideo 11600, Uruguay;
3Clinical Education, Newton Abbot, Devon TQ12 4SG, UK;
4Sierra Productions Research, Mission Viejo, CA 92612, USA;
Laboratory of Molecular Cell Biomedicine, University of the Balearic Islands, 07122 Palma de Mallorca, Spain;
Membrane Lipid Replacement (MLR) uses natural membrane lipid supplements to safely
replace damaged, oxidized lipids in membranes in order to restore membrane function, decrease
symptoms and improve health. Oral MLR supplements contain mixtures of cell membrane glyc-
erolphospholipids, fatty acids, and other lipids, and can be used to replace and remove damaged
cellular and intracellular membrane lipids. Membrane injury, caused mainly by oxidative damage,
occurs in essentially all chronic and acute medical conditions, including cancer and degenerative
diseases, and in normal processes, such as aging and development. After ingestion, the protected
MLR glycerolphospholipids and other lipids are dispersed, absorbed, and internalized in the small
intestines, where they can be partitioned into circulating lipoproteins, globules, liposomes, micelles,
membranes, and other carriers and transported in the lymphatics and blood circulation to tissues
and cellular sites where they are taken in by cells and partitioned into various cellular membranes.
Once inside cells, the glycerolphospholipids and other lipids are transferred to various intracellular
membranes by lipid carriers, globules, liposomes, chylomicrons, or by direct membrane–membrane
interactions. The entire process appears to be driven by ‘bulk flow’ or mass action principles, where
surplus concentrations of replacement lipids can stimulate the natural exchange and removal of
damaged membrane lipids while the replacement lipids undergo further enzymatic alterations.
Clinical studies have demonstrated the advantages of MLR in restoring membrane and organelle
function and reducing fatigue, pain, and other symptoms in chronic illness and aging patients.
membrane phospholipids; membrane structure; lipid transport; lipid oxidation; lipid
exchange; mitochondrial function; fatigue; pain; chronic diseases
1. Introduction: Why Membrane Lipid Replacement (MLR)
Membrane lipids are essential to life and vital to cellular health [
]. They provide
cells with: (i) a matrix for all plasma and intracellular membranes, (ii) separation of chemi-
cal and enzymatic reactions into distinct cellular compartments; (iii) bioactive molecules
that are part of signal transduction and molecular recognition pathways; (iv) energy stor-
age reservoirs; and (v) molecules that interact with other membrane constituents, such as
proteins and glycoproteins [
]. The interactions of membrane lipids with membrane
proteins and glycoproteins is an absolute requirement for the formation, structure and
function of cellular membranes and thus for life itself [
]. In addition, peripheral signal-
ing proteins can translocate between the plasma membrane and cytoplasmatic aqueous or
Membranes 2021,11, 944.
Membranes 2021,11, 944 2 of 39
particulate (membrane) fractions. At the level of the lipid bilayer or membrane matrix, they
propagate intracellular signals only if they exert productive interactions with transmem-
brane receptors in specific membrane nanodomains or submicrodomains. Such signals
modulated by lipids can be replaced by pharma/nutraceutical interventions [
]. For
these reasons, lipid membranes and other lipid structures constitute an essential element
in a cell’s physiology, and lipid alterations within cells are likely to be involved in human
disease. Thus, lipid normalization could be an approach to counteract certain aspects of
pathological states.
During development, aging, and especially in chronic medical conditions and es-
sentially every acute illness, cellular membranes are damaged, in part due to the excess
production of free radical oxidants [
]. Free radical reactive oxygen species (ROS),
such as superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide, and reactive
nitrogen species (RNS), such as peroxynitrite anion, cause most of the chronic damage
to cellular membranes [
]. Cellular anti-oxidants usually neutralize excess free
radical oxidants, but in pathologic conditions the concentrations of free radical oxidants
are in such excess that normal anti-oxidants levels are ineffective and free radical oxidant
damage occurs [
]. Membrane glycerolphospholipids (GPL) and their unsaturated
fatty acids (FA) are especially sensitive to oxidative damage by ROS and RNS [
and they must be enzymatically repaired or replaced in order to maintain membrane
structure and function [14,7,9,12].
Under pathological conditions and normal aging, the use of Membrane Lipid Replace-
ment (MLR), the natural replacement of membrane glycerolphospholipids (GPL) and other
lipids mainly by oral dietary supplementation, can supply enough GPL and other lipids
to repair damaged lipids and return cellular membranes to normal function [
]. Thus,
membrane lipids can be maintained at optimal levels by complementing regular dietary
membrane lipid sources with MLR, and this should provide enough replacement molecules
for damaged cellular membrane lipids [
]. Membrane lipids and their derivatives can
also be used therapeutically to treat various conditions and diseases [15,12].
Oral GPLs and their unsaturated fatty acids (FA) are quickly and efficiently absorbed,
mainly in the upper small intestine, within hours of ingestion [
]. There are multiple
mechanisms for absorption of orally ingested GPL. The ingested phospholipids can be
degraded into their constituent parts and their component parts and separately absorbed;
they can be taken in as intact molecules without degradation, or they can be absorbed
in bulk in the form of small lipid micelles, liposomes, and globules [
]. When
present in great excess in the gastrointestinal system during oral MLR supplementation,
most membrane phospholipids are absorbed in undegraded bulk forms [
]. The intake
process appears to be driven by a mass action or ‘bulk flow’ process [
]. Thus, when in
large excess, intact MLR phospholipids have an advantage in being able to reach their
final destinations at high concentrations by bulk flow without significant degradation
or enzymatic alterations [
]. Once inserted at their ultimate cellular membrane and
intracellular sites, the GPL can be further enzymatically modified, such as by substitution
or modification of their FA side chains or head groups, in order to reflect the specific
compositional and functional needs at their membrane destinations. At the cellular level
the process of intracellular transport to organelles has been followed using spermatozoa.
For example, human sperm cells have been incubated with nanometer sized GPL micelles
loaded with some fluorescent-GPL molecules to follow their progress [
]. Fluorescence
was first found at the sperm surface, and this was accompanied by an increase in plasma
membrane surface area. Next, fluorescence could be found in the sperm intracellular
membranes as well as in mitochondria. The presence of MLR molecules in the sperm
mitochondria correlated with increases in sperm motility and resistance to oxidative
stress [33].
Membranes 2021,11, 944 3 of 39
2. Biological Membranes and GPL
GPL and other lipids are essential for biomembrane structure, properties, and func-
tion, and they form the matrix for all cellular membranes [
]. Along with GPL
head-group, the chain length and saturation of attached FA help determine GPL membrane
packing and membrane fluidity [
]. Unsaturated FA, such as oleic acid and linoleic
acid, confer a high degree of conformational flexibility to GPL. The unsaturated FA hy-
drocarbon chains cause GPL to occupy a slightly wedge-shaped space in the hydrophobic
interior of membranes, resulting in looser packing and a more fluid membrane [
For example, oleic acid provides special biophysical properties to membranes that are
involved in the interactions of amphitropic peripheral signaling proteins that control im-
portant physiological functions, such as blood pressure [
]. In contrast, saturated FA,
such as stearic acid and palmitic acid, confer membrane rigidity, and this results in a
less fluid or more organized membrane structure [
]. Different plasma and intra-
cellular membranes display GPL compositional differences that are characteristic of their
origins [8,9,34,35].
The actions of various agents and drugs can affect the compositions of plasma mem-
branes and in the process change their functional properties. For example, certain anti-
platelet drugs, such as ticagrelor, a membrane P2Y12 antagonist, and its active metabolite
significantly increased platelet cholesterol and PC with short FA acyl chains, suggesting
platelet membrane rigidification and modification of lipid–receptor interactions involved
in ADP-induced platelet activation [37].
There are also differences between the compositions of lipids on each side of the
lipid bilayer of a given biomembrane. Sphingolipids, such as gangliosides, are distributed
asymmetrically at much higher concentrations in the outer leaflets of plasma membranes.
Neutral GPL, such as phosphatidylcholine (PC), are also found preferentially on the outer
leaflets of plasma membranes, whereas anionic phospholipids, such as phosphatidylserine
(PS) and phosphatidylinositol (PI), are more concentrated on the plasma membrane inner
leaflet [
]. The asymmetric and cooperative distributions of lipids in inner
and outer membrane leaflets, as well as in the plane of the membrane, are important in
determining membrane physical properties, including membrane deformation, curvature,
compression and expansion, as well as their functional interactions with membrane pro-
teins [
]. These concepts have been incorporated in descriptions of membrane
structure, as in the Fluid–Mosaic Model of cell membrane structure [42,43].
As described above, an important characteristic of plasma membranes is the asso-
ciation of lipids (and under certain circumstances, proteins) into functional membrane
domains, such as lipid rafts [
]. This is due, in part, to lateral molecular interactions
and lipid domain formation, especially between PC and phosphatidylethanolamine (PE),
along with sphingomyelins and cholesterol [
]. Under physiological condi-
tions, membrane lipids are present in various fluid, semi-solid, and solid phases that are
organized into membrane domains that are characterized by different lipid compositions,
spatial arrangements, and rates of lipid rotational and lateral movements [
]. The
different lipid phases and domains in various membranes have profound functional signif-
icance in determining membrane properties, such as cell signaling and other important
membrane characteristics [40,4345].
Although cell membranes form functional ionic barriers, membrane ion channels and
transporters function as conductive pathways for ions or water across such hydrophobic
barriers, and biomembranes can thus behave as resistance-capacitor circuits with particular
time and spatial characteristics that regulate trans-membrane voltage kinetics. These prop-
erties are critically important in membrane signaling and energy production [
]. The FA
in membrane GPL that form most of the membrane hydrophobic barrier are present with
a variety of FA chain lengths and saturation states. Thus, FA are important components
of dietary MLR supplements, and among the most important FA for humans are unsat-
urated FA, such as oleic acid (18:1
9 or 18:1[n-9]), linoleic acid
(18:29,12 or 18:2[n-6])
alpha-linolenic acid (18:3
9,12,15 or 18:3[n-3]), and arachidonic acid (20:4
5,8,11,14 or
Membranes 2021,11, 944 4 of 39
20:4[n-6]) [
]. The FA with cis-double bonds are important in determining the melting
points of membrane GPL lipid mixtures, where increases in individual rotational properties
and solid-to-liquid phase transitions determine the separation of membrane regions into
fluid- and solid-phase domains [
]. Lipid lateral phase separations and lipid domain
development are important in the formation of membrane rafts and thus, as described
above, the establishment of membrane signaling domains [
]. Mammalian cells are
unable to synthesize or modify certain essential FAs with double bonds at specific positions,
and thus some specific unsaturated FA are considered essential dietary constituents [7].
The biosynthesis or modification of non-essential FA occurs for the most part in the
ER in the cytosol, but some FA can be modified and assembled in the inner mitochondrial
membrane [
]. This includes some basic GPL construction and modification, such as
exchange of phospholipid headgroups and FA sidechains in mitochondria. The complete
synthesis of membrane GPL usually follows the sequence: (i) synthesis of a backbone
glycerol-3-phosphate molecule, (ii) attachment of FA to this backbone, (iii) dephosphoryla-
tion, and (iv) addition of a hydrophilic head group to produce various GPL. Some GPL are
synthesized by altering existing GPL molecules, such as phospholipid head group exchange
or methylation of ethanolamine groups to form PC [
]. Others are modified in their FA
side chains, producing an overall membrane GPL composition and saturation necessary
for protein remodeling and the formation of lipid rafts and other membrane domains.
The dynamic sorting of membrane lipid and protein components into domains of
specific compositions and mobilities was proposed to be primarily based on hydrophobic
and some hydrophilic interactions between membrane proteins and lipids [
]. Such
sorting can avoid hydrophobic mismatches between lipids and proteins, and this can
prevent membrane distortions that are unsustainable or eliminate areas of membrane weak-
ness [
]. Thus, the amounts and distributions of GPL and other lipids in biomembranes
are important for the structure and physical properties of membranes, but also functional
properties related to ion channels, transporters, and receptors. The original Fluid–Mosaic
model accounted for basic cell membrane structure, dynamics and asymmetry [
but over time newer models for plasma and intracellular membranes evolved into much
more complex and compact structures, but they still retained most of the basic nanometer
structure of the Fluid–Mosaic Membrane model [5356].
3. Mitochondria and Their Membranes
As an essential cellular organelle, mitochondria produce 80–90% of cellular energy
and play an indispensable role in redox balance, calcium signaling, energy storage and
immune functions, such as innate immunity. They possess a dual membrane structure sim-
ilar to bacteria, but the critical part of their structure is the mitochondrial inner membrane
(MIM) where electron transport and oxidative phosphorylation occur [
]. Between
the membranes of mitochondria is an intermembrane space, and inside the MIM is the
mitochondrial matrix. The matrix contains a mixture of enzymes, mitochondrial ribosomes,
tRNAs, mRNAs, and maternally dominant mitochondrial DNA (mtDNA) [
]. The
membranes of mitochondria have discrete lipid compositions that display bilayer asymme-
try and lateral heterogeneity. The MIM is the location of the respiratory chain complexes
or the electron transport chain (ETC), accessary proteins, and cofactors such as coenzyme
Q10 (CoQ10) [
]. The ETC essentially pumps protons into the intermembrane space,
creating an electrochemical gradient, and the energy stored in the resulting gradient is
used to drive oxidative phosphorylation and the synthesis of ATP [22,57,58,60].
Mitochondria have usual membrane concentrations of PE and PC, which are found
in all cellular and intracellular membranes. Mitochondria also contain some unique
membrane lipids, such as the tetra-acyl phospholipid known as cardiolipin (CL). CL
represents approximately 15–20% of the total mitochondrial phospholipid and is found
exclusively in the MIM where it is essential in maintaining MIM fluidity and osmotic
stability. Along with being functionally indispensable for MIM structure and ETC function,
CL is also important in the maintenance of MIM transmembrane potential [
]. CL
Membranes 2021,11, 944 5 of 39
molecules are highly sensitive to free-radical oxidation of their FA double bonds, resulting
in loss of MIM trans-membrane potential [
]. The appearance of oxidized CL or its
degradation products in blood is also associated with several pathological conditions,
including diabetes, heart failure, hyperthyroidism, neurodegeneration, and aging [
These conditions are characterized by excess oxidative stress, CL damage, and deficiency,
and increases in oxidative products such as docosahexaenoic acid (DHA) [65].
In order to function properly, mitochondria must respond to changes in MIM trans-
membrane potential. If mitochondria fail to adapt to variations in MIM trans-membrane
potential, the electrochemical gradient necessary for ATP synthesis could be lost along with
changes in the permeability of several mitochondrial transporters and channels. The loss
of the MIM electrochemical gradient creates mitochondrial dysfunction and can eventually
result in mitochondrial collapse (mitophagy) [
]. The release of cytochrome c through
the BCL-2 proapoptotic channels activates intracellular caspases, leading ultimately to cell
death by autophagy [
]. Balancing mitophagy and mitochondrial biogenesis are essential
for maintaining cellular homeostasis [
]. A fully-functioning ETC generates an obligatory
trans-membrane potential of
150 to
200 mV across the MIM, yielding an equivalent
field strength of about 30 million volt/m [
]. Failure to maintain MIM trans-membrane
potential results in loss of cellular energy production, increased free-radical ROS leakage,
decreased plasma membrane active transport, and suppression of energy-requiring cellular
processes. As the MIM is the primary location for production of excess ROS, eventually
this can result in cellular stress and affect cellular responses [70].
An increase in intracellular ROS and mitochondrial release of ‘danger signals’, such as
damage-associated molecular patterns (DAMPs), pathogen-associated molecular patterns
(PAMPs), mitochondrial nucleic acids, and other molecules, are typical of cellular stress
responses [
]. Some cellular stresses are also caused by K
efflux, uric-acid crystals
and the presence of extracellular ATP, among other stress molecules [
]. The presence
of stress agents can provoke the assembly of intracellular multi-protein inflammatory
complexes called inflammasomes, which are signaling platforms important in responses
against microorganisms and environmental toxins, and this can also result in triggering the
secretion of pro-inflammatory cytokines and other cellular responses [7074].
The products of oxidized-unsaturated FA are very important in inducing apoptosis
via reaction with mitochondrial permeability transition pores (mPTP). mPTP are voltage-
dependent channels that initiate calcium-dependent apoptosis and activate intracellular
calcium channels that promote Ca
release from endoplasmic reticulum and mitochondrial
loading of Ca
]. Increased mitochondrial ROS production results in oxidation of
unsaturated FA and also activates intracellular calcium channels [
]. mPTPs are opened
during oxidative stress, resulting in Ca
release and mitochondrial loading that leads to
loss of MIM trans-membrane potential and thus reduction in ATP production. The calcium
loading of mitochondria further increases ROS production, until mitochondria eventually
swell and bleb, degenerate, and undergo mitophagy [
]. Cell death eventually ensues [
However, dietary supplementation with unsaturated FA can repair and replace oxidized
GPL, modifying mitochondrial unsaturated FA composition and altering mitochondrial
calcium homeostasis, thus delaying mPTP damage and Ca2+-induced cell death [80].
4. Liposomes, Chylomicrons, Lipid Globules, Lipid Micelles, and GPL Transport
MLR GPL taken orally are usually absorbed in the upper small intestines as individual
GPL molecules or, if they are degraded, as constituent parts [
]. Although
some acid degradation of GPL occurs in the stomach, most enzymatic modification takes
place in the small intestine. There, FA and other fragments of degraded GPL are trans-
ported across the epithelium [
]. However, when present at high concentrations
where GPL are for the most part undegraded, GPL are absorbed as small lipid micelles,
globules, and liposomes in an endocytotic process and eventually delivered to the blood
circulation [28,29] (see Figure 1
of [
]). This process is also important in the co-transport of
other critical molecules that partition into lipid micelles, globules, and liposomes [
]. This
Membranes 2021,11, 944 6 of 39
evolutionary adaptation is likely present to capture as many GPL molecules as possible, as
historically high concentrations of essential GPL were only available at certain times.
Figure 1.
Some of the phospholipid transport systems involved in delivery of MLR phospholipids
and other lipids to intracellular membranes and the reverse of this process. A liver cell is shown with
internal lipid transport and storage systems, such as micelles, vesicles, globules, chylomicrons, and
lipid droplets. These various lipid transport structures can bind to different intracellular membranes
and transfer GPL and other lipids, and pick up damaged lipids for delivery outside of cells. Not
shown in the figure are lipid transport/transfer by direct adjacent membrane-to-membrane contact
and lipid droplet-, globule-, chylomicron- and vesicle-to-membrane contact or temporary fusion with
adjacent intracellular membranes. Both the forward and reverse processes appear to be driven by
mass action or bulk flow mechanisms. (Modified from Nicolson and Ash [1]).
GPL absorption in the small intestines is an efficient process. After a large meal,
over 90% of glycerolphospholipids are absorbed and transported into the blood within
six hours [
]. In the blood circulation, there is a limit to the amounts of GPL that can
be present in carrier molecules, such as lipoproteins, or in the membranes of erythrocytes.
However, when present in excess of the amounts that can be carrier-transported, GPL can
also be found in blood as lipid globules, liposomes, and other forms (discussed in [
Eventually the circulating GPL are delivered to major organs, such as liver (Figure 1), and
to cells where they are transferred by direct contact of lipoproteins and erythrocytes with
cell membranes or by endocytosis of GPL in micelles, globules, liposomes, chylomicrons,
and other forms. They can then transfer to other cells, and the process can be repeated.
Usually, dietary polyunsaturated GPL undergo oxidation and degradation during
their storage, ingestion, digestion, and adsorption in the small intestine. Thus, to increase
the amounts of intact oral MLR phospholipids that reach their final membrane destinations,
they must be protected from oxidation during storage and from acid degradation in the gut,
as well as disruption by bile salts and hydrolysis by phospholipases and other enzymes
released from the pancreas and gut microflora in the small intestines [
]. This has been
accomplished by complexing MLR phospholipids with specific fructooligosaccharides,
plant inulins which insert between the head groups of glycerolphospholipids and protect
them from excess temperatures, acidity, phospholipases, and bile salts [
]. The inulins
also protect GPL from oxidation and enzymatic degradation [
]. When fructooligosac-
charides (inulins) are used to protect GPL, MLR supplements can be protected during
storage and ingestion, while also being absorbed relatively intact by gastrointestinal brush
border cells [
]. Although some hydrolysis of GPL will occur during this multi-step
process, when MLR lipids are in excess, most of the micellar and globule phospholipids
are absorbed by brush border cells as unoxidized, undegraded molecules [1,85].
Membranes 2021,11, 944 7 of 39
The uptake of small GPL micelles, globules, or larger droplets by the small intestinal
epithelium has been examined using electron microscopy. Morphological studies have
shown that undigested dietary lipids and phospholipids present in small intestinal brush
border cells are mainly present as small lipid micelles, globules, chylomicrons or larger
lipid droplets of 50–1000 Å in diameter [
]. When in excess and protected by inulin,
the phospholipids are transported by endocytosis or pinocytosis into intestinal cells as
largely intact molecules. Even though ultrastructural methods cannot determine the lipid
compositions of the ingested lipid globules and droplets, or chylomicrons, these lipid forms
are not present in intestinal cells in fasting controls, indicating that they are derived from
ingested intestinal GPL [
]. Intestinal epithelial cells can also transport individual GPL
molecules and their degradation products, such as FAs, using specific membrane transport
systems, but not in the amounts necessary to absorb GPL and other lipids in quantities
found during ingestion of MLR supplements. Thus, after routine intestinal cell transport,
individual GPL molecules can form micelles, small liposomes, or phospholipid globules,
but not in the amounts found after MLR supplementation. It is thought that this routine
form of individual molecule transport is less important when GPL are present in excess
in the intestinal lumen, where they can associate into small micelles, globules, or larger
forms [89] (Figure 1).
Intestinal bush border plasma membranes can directly partition phospholipids into
their outer plasma membrane leaflets by membrane contact with GPL. Thus, it was ob-
served that intestinal microvillous plasma membranes became thicker on their outer sur-
faces during GPL absorption, and this was thought to be due to the direct insertion of GPL
molecules into the outer leaflets of brush border microvilli membranes [
]. Once GPL such
as PC are enriched in the outer leaflets of the plasma membranes of intestinal cells, they ap-
pear to protect this structure from pathogenic processes such as ulcerative colitis and other
chronic inflammatory conditions [
]. Individual GPL that are incorporated into the outer
plasma membrane leaflet of colonic brush border cells can be transported into these cells by
binding to trans-membrane translocases (flipases, flopases, and scramblases) that can trans-
fer the GPL to the opposite membrane surface [
]. Once the translocated GPL arrive
at the inner plasma membrane surface, they can then be partitioned into protein carriers
that transfer the GPL molecules to intracellular membranes or cellular organelles [
]. The
build-up of GPL at the inner membrane leaflet may also promote membrane blebs that are
then released as new intracellular lipid vesicles and globules [
]. Alternatively, GPL can
be transferred by partitioning into existing intracellular vesicles, globules, chylomicrons,
lipid transfer proteins, intracellular membranes, or other structures and subsequentially
transferred to various intracellular sites [1,94,95].
Once GPL and other lipids are moved to various intracellular membrane sites or
stored in lipid structures, they can be moved again [
]. They can also be transferred by
membrane–membrane contact. This involves transient membrane fusion or partitioning of
lipids from one closely apposed membrane to another membrane or lipid structure, and
this type of transfer appears to involve specific membrane transfer domains. For example,
ER and mitochondria can transfer membrane lipids by direct membrane contact-transfer
through specific ER membrane domains called the mitochondria-associated membrane
(MAM) domains [
]. Organelles such as mitochondria also have their own specific
internal lipid transport systems to move lipids from outer membrane to MIM. Once transfer
has occurred, enzymatic alterations can take place, eventually resulting in specific mem-
brane lipid compositions. The reverse of this mitochondrial transport process can exchange
damaged membrane GPL with undamaged GPL and removal of the former [
]. There
are also specific lipid transfer proteins in the intermembrane space between inner and outer
mitochondrial membranes, and these may also play a role in the maintenance of GPL and
FA composition between different mitochondrial compartments [101].
The GPL in small vesicles, lipid globules, and chylomicrons can be transferred by
direct contact with intracellular membranes or other lipid structures, and eventually this
results in partitioning of their contents into various cellular and organelle membrane
Membranes 2021,11, 944 8 of 39
compartments [
]. During this process, GPL can undergo enzymatic mod-
ifications, such as head group substitution or modification of FA, to reflect the specific
membrane compositions at their final destinations. Not much is known about the roles
of small GPL micelles, vesicles, and globules inside cells, but additional information has
been published on larger lipid structures such as chylomicrons and lipid droplets [
Intracellular lipid droplets have been defined as structures composed of neutral lipid cores
that are surrounded by a layer of PC, PE, PI, and smaller amounts of other GPL and a pro-
tein outer surface. The outer surface protein coats of lipid droplets may be used to regulate
their size, structure, number, and fate of these lipid storage systems [
]. Lipid droplets
are used as the principle lipid storage system in certain cells: adipocytes, hepatocytes, and
other cells (Figure 1). They also play important roles in cellular lipogenesis and homeosta-
sis [
]. As lipid storage systems, lipid droplets are important in metabolic syndrome
(MetSyn), fatty liver diseases, steatohepatitis, atherosclerosis, and other diseases [99,108].
As important cellular lipid storage systems, chylomicrons and lipid droplets are
increased to the point of excess during excessive fat absorption and storage, for example, in
obesity [
]. In contrast to lipid droplets, chylomicrons are mostly GPL (70–75%), with
some cholesterol (5–10%), triglycerides (13–25%), FA, and other lipids [
]. Chylomicrons
can store some phospholipids in cells, but they are apparently used to transfer lipids to
various organelles and different intracellular compartments as well as to adjacent or distant
cells [
]. This type of transfer may initially involve small vesicles and globules released
from intracellular membranes, such as Golgi membranes, and these can be subsequently
formed into chylomicrons. In brush border cells, chylomicrons have been found to be
transported to the basolateral surfaces where they are released by a reverse pinocytosis
process. Eventually the cell-released chylomicrons find their way to the cells lining the
lymph or circulatory systems. At this juncture, endocytosis, transport, and eventually
exocytosis can be repeated until these lipid structures are eventually transferred from the
lymph and blood to the gastrointestinal system [
]. In addition to lipid transport
in micelles, vesicles, globules, chylomicrons, membranes, and other forms, individual
GPL can also be transported by protein lipid carriers or transfer proteins. Preference
for particular GPL by protein carriers include differences in GPL head group, saturation,
and length of FA acyl chains [
]. All of the lipid transport systems appear to
function on a mass action or ‘bulk flow’ basis.
As the overall lipid transport process appears to be driven by ‘bulk flow’ or mass
action mechanisms to deliver GPL and other lipids to particular membrane sites, the same
bulk flow/mass action process can also be used to remove oxidized or damaged lipids
from membranes and eventually send them to degradation sites where lipids are degraded
or to export sites where they can be exported from cells by exocytosis into the lymph or
blood for eventual delivery to the gastrointestinal system for excretion (reviews: [1,2]).
While in the lymph or blood circulation GPL, steroids, FAs, and other lipids can be
transferred to carrier molecules, such as high- and low-density lipoproteins (HDL and
LDL), or intercalated into the plasma membranes of blood cells, such as erythrocytes or
leukocytes. Circulatory lipoproteins are utilized as an important transport system for lipids
in the blood circulation. Once GPL and other lipids are bound to lipoproteins, they are
more protected from oxidation and enzymatic digestion during transport. In humans, the
amounts of membrane GPL exchanged and preferentially transported by HDL and LDL
are more than 20-times the amounts transported by erythrocytes [
]. An added bonus of
MLR is that the excess GPL in globules and vesicles can help remove cholesterol from the
circulation, in part, by partitioning the GPL into erythrocyte membranes and circulating
lipoproteins, and in the process enabling the partitioning and removal of excess cholesterol
and oxidized cholesterol [
]. Once excess cholesterol is removed from cells in tissues,
it can be partitioned into circulating lipoproteins and phospholipid globules and other lipid
forms, as well as circulating cells and delivered back to intestinal epithelium for eventual
export by the gastrointestinal system [1,3,99,111,114].
Membranes 2021,11, 944 9 of 39
As described above, the entire sequence of lipid transport appears to follow a ‘bulk
flow’ or ‘mass action’ process along a concentration gradient from the gut to tissues
and back again for damaged/oxidized phospholipids [
]. This natural removal process
can help slowly reduce oxidized cholesterol and excess cholesterol in membranes, cells,
and tissues.
5. MLR Formulations
Dietary or oral MLR supplements or intravenous introduction of MLR phospholipids
(also called “essential” phospholipids or EPL) have proven to be safe for replacement of
damaged membrane lipids, and there is apparently a limit to the amounts of lipids that can
be replaced by diet alone. In humans, the maximum total dietary membrane lipids that
can be consumed in foods are in the range of 2–6 g per day (reviewed in [
]). For example,
legumes are thought to be a good GPL source for dietary MLR
supplements [1,2,6]
. How-
ever, the amounts of, for example, soy beans required to deliver a dose of approximately
1.8 g of membrane GPL would be approximately 15 kg of legumes [
]. Therefore, the
consumption of sufficient amounts of plant products to obtain a clinically effective dose
of MLR and removal of damaged GPL, sterols, and other lipids would be impractical for
most diets [13].
If GPL and other lipids in foods or supplements are unprotected from oxidation,
disruption, and degradation, it would be difficult to maintain a clinically appropriate dose
level of GPL, which can be several g per day, without contamination of unoxidized GPL
with oxidized GPL (Table 1). Protected MLR supplements, on the other hand, can deliver
therapeutic doses of GPL, and certain MLR supplements, such as NTFactor Lipids
, are
fully protected from oxidation, bile disruption, and enzymatic digestion using protective
fructooligosaccharides and antioxidants [
]. MLR supplements containing GPL with
n-3 and n-6 polyunsaturated FA and other lipid components have been derived from
different sources: plants, fish, and krill, among other sources [
]. These
supplements have been tested in laboratory animals and humans for their effectiveness
and safety. For example, animals supplemented with n-3 unsaturated FA supplements
showed positive changes in mitochondrial membrane phospholipid FA composition, im-
proved mitochondrial function, delayed the opening of Ca
-induced mPTP, and increased
mitochondrial reserve [
]. Laboratory rats fed for 10 weeks with an unsaturated FA
supplement showed beneficial modifications of cardiac mitochondrial CL and improve-
ments in Ca2+-induced mitochondrial mPTP function [120].
Safe and cost effective MLR oral supplements for humans have utilized various
lecithins as a source of GPL [
]. Soy, eggs, or marine krill lecithin preparations have
been marketed, but most lack protection from oxidation and disruption by bile or acids.
Some oral MLR supplements, such as NTFactor
and NTFactor Lipids
, have long shelf-
life, are protected by fructooligosaccharides and antioxidants, are safe and contain a full
range of GPL, and have been tested for efficacy [
]. MLR supplements such as NTFactor
which also contains probiotic bacteria, growth media, and other ingredients, and NTFactor
, without the probiotic additives but with fructooligosaccharides and antioxidants,
come in several oral forms, and almost all contain 2 g or more of phospholipids per daily
dose (reviews: [13]).
Other forms of MLR supplements have utilized phospholipid-specific preparations.
For example, PS supplements have been used for specific purposes, such as to treat memory
loss in aged subjects or in Alzheimer’s disease (AD) patients. Supplementation with 300 mg
per day of bovine PS for 6 months resulted in cognitive improvements in AD patients
compared to controls [
]. However, another study in elderly subjects with age-associated
memory impairment that received 300–600 mg PS daily for 12 weeks did not show memory
improvements [
]. Although supplementing with a single class of glycerolphospholipid
alone, such as PS, has been shown to have health benefits, the use of more complex mixtures
of membrane GPL may be more beneficial, possibly as some of the PS does not have to be
enzymatically converted to other GPL [13,123,124].
Membranes 2021,11, 944 10 of 39
In addition to oral supplementation, MLR phospholipids have been introduced in-
travenously in hospital or out-patient settings [
]. Acute cases of liver toxicity, kidney
failure, hepatitis, dialysis, and other conditions have been treated with intravenous EPL [
Administration of EPL phospholipids IV have been used to deliver high concentrations
of GPL without the need for fructooligosaccharides to inhibit intestinal disruption. Any
intestinal adverse events can be minimized by IV administration, but it is expensive com-
pared to oral supplementation, and administration must be professionally supervised. GPL
IV preparations, such as Essentiale
, contain approximately 1 g of mainly PC (>75%) with
some minor amounts of other GPL and other ingredients in a single IV treatment [6].
6. Safety of MLR
MLR oral supplements are extraordinarily safe [
]. Preclinical and clinical safety
studies have not shown any acute or chronic MLR supplement toxicity, even at very high
daily doses levels. There has been no evidence of MLR perinatal and postnatal toxicity;
nor were there any mutagenic or carcinogenic effects of administering GPL. None of the
preclinical studies conducted in laboratory animals (mice, rats, and rabbits) demonstrated
any acute or chronic toxic effects of oral, subcutaneous, or intravenous GPL administration
(review: [
In vivo
studies in laboratory animals using potentially toxic or lethal doses
could not establish any toxic or lethal dose levels for MLR supplements. Doses up to 45 g
of MLR GPL have been given orally without evidence of any adverse effects [125].
Using mutagenicity and carcinogenicity animal models the effects of escalating doses
of MLR phospholipids were completely negative. For example, animals fed daily oral doses
up to 3.75 g/kg body weight produced no measurable toxic, mutagenic, or carcinogenic
events (review: [
]). In addition, GPL dose toxicity could not be established in young, adult,
pregnant, or fetal laboratory animals. Additionally, evidence of any GPL toxicity was not
found in pregnant rats or rabbits or in their newborns at doses up to and above 1 g/kg
(review: [
]). In addition, the co-administration of MLR GPL and carcinogens in laboratory
animals revealed no evidence that MLR can increase carcinogenicity. In fact, MLR with
GPL actually inhibited the formation of tumors in animals [126].
The effects of MLR on animal health has also been examined in life-long studies
(review: [
]). GPL have been given in chow or water to laboratory animals at daily doses
from 0.01–5 g/kg body weight, and there were no differences between test and control
animals in central or peripheral nervous systems, renal function, heart, and vascular
function, or other indicators of toxicity (reviewed in [
]). Lifetime administration of GPL
to laboratory rodents has demonstrated only beneficial effects. For example, feeding rats
in their chow over their lives reduced age-related hearing loss and deceased
mtDNA deletions associated with aging [
]. In addition, aged rats were fed NTFactor
or placebo for 6 months and their auditory brainstem responses, MIM potentials, and
mitochondrial DNA deletions were examined every 60 days. Auditory brainstem responses
were estimated by measuring hearing thresholds, and MIM membrane potentials were
estimated with redox dyes using blood leukocytes. DNA deletions in the aged rodents were
determined by extracting mtDNA from various brain regions and amplification of mtDNA
sequences. Deletions of known mtDNA sequences lost during aging were verified [
There were significant differences between the groups of animals receiving MLR GPL
and placebo groups in terms of auditory brainstem responses, MIM potential and the
presence of mtDNA deletions. With administration of NTFactor
in the chow of the rats,
there was significant preservations of hearing threshold at all frequencies tested in the
experimental animal group. NTFactor
also prevented the usual age-related decline in
MIM trans-membrane potential and reduced the incidence of common mtDNA deletions
in the aging rats [127].
High MLR doses of GPL have been taken orally by humans with no evidence of
any toxicity or adverse events (review: [
]). In cases of hepatic encephalopathy due to
decompensated liver cirrhosis, patients receiving EPL showed significant improvements
in their liver disease status and had prolonged survival compared to a control group that
Membranes 2021,11, 944 11 of 39
did not receive EPL [
]. The patients in this study also showed no evidence of any
toxic effects from the GPL. The use of high doses of MLR GPL in long-term studies in
humans has shown that subjects actually showed improvements in cardiovascular blood
markers. In one study, 35 elderly subjects received at least 4 g per day oral NTFactor
over 6 months. During this period participants showed no evidence of any adverse events,
and their blood cardiovascular risk markers, such as homocysteine, improved. In some
patients, homocysteine levels decreased from high-risk levels to normal ranges [
]. In
a different study, 58 middle-aged patients with fatiguing illnesses received doses of 4 g
per day oral NTFactor
for 2 months without any adverse incidents [
]. Some of these
patients continued using the MLR supplement for years without reporting any adverse
events. The long-term use of MLR GPL in clinical studies on cardiovascular diseases has
been reviewed, and there is no evidence of MLR GPL toxicity [
]. Safety studies, along
with the lack of observed toxicity in clinical trials likely supported the decision of the
U.S. Federal Drug Administration (FDA) classify oral MLR GPL as GRAS or ‘Generally
Recognized as Safe’ [
]. To date, there have been well over a million doses of MLR
supplements taken with no reported adverse effects.
7. MLR in Aging
The aging process is thought to be linked to the progressive accumulation of physio-
logical changes, such as declining cognitive and immune function, reductions in cellular
activities due to accumulations of cellular damage, and enhanced likelihood of contracting
chronic and lethal diseases. The foundations of normal aging appear to be complex, with
no single mechanism explaining every aspect of this complicated phenomenon. There are
multiple effects of aging that can be seen at the cellular level. Some age-related changes
have been related to genomic instability, telomere shortening, mitochondrial dysfunction,
cellular senescence, epigenetic alterations, dysregulated nutrient sensing, stem cell exhaus-
tion, altered intercellular communication, changes in calcium stores, and inflammation,
among other changes [
]. One characteristic of human aging is the change in a
type of inflammation—chronic, progressive inflammation—that is related to mitochondrial
responses [
]. These types of inflammation responses are often linked to age-related
diseases and disorders [133,136].
Mitochondria are especially important in aging, and their decline in function and
numbers with age involves a number of possible mechanisms, including: (i) increased
mitochondrial uncoupling and production of ROS; (ii) insufficient antioxidative enzymes
to counter the increased production of ROS; (iii) increased disorganization of mitochon-
drial structure, especially mitochondrial membranes; (iv) changes in intracellular Ca
stores; (v) decline in mitochondrial oxidative phosphorylation and production of ATP;
(vi) accumulation of mtDNA deletions and mutations; and (vii) increased damage to mi-
tochondrial proteins and lipids [
]. These events are not mutually exclusive and
likely contribute to the overall decline in mitochondrial number and function with age.
An aspect of aging relating to cellular organelles, such as mitochondria, is the ac-
cumulation of endogenous debris, such as age-related accumulation of membrane and
mitochondrial debris. This accumulation may reduce the ability of mitochondria to remove
the debris by mitochondrial autophagy and mitophagy [
]. The accumulation of
endogenous debris can lead to protein aggregation and accrual of damaged proteins that
eventually results in reductions in mitochondrial function. Some of the observations on
aging and mitochondrial function, such as age-related reductions in oxidative phosphoryla-
tion, reduced MIM trans-membrane potential and increased permeabilization of the outer
mitochondrial membrane, among other changes, are consistent this hypothesis [
In addition, the increases in ROS/RNS production and resulting oxidative stress found
with aging cause further oxidative damage, causing reductions in membrane fluidity and
damage to mitochondrial CL due to lipid peroxidation [
]. The age-related func-
tional decline seen during aging, along with changes in many other markers of aging could
also be related, to a large degree, to losses in mitochondrial function and number [
Membranes 2021,11, 944 12 of 39
An obvious example is fatigue. Aged individuals universally complain about this prob-
lem [
]. The role of age-related membrane changes to fatigue will be considered in more
detail in Section 8.
MLR can be used to modify the usual decline in mitochondrial function seen during ag-
ing. In one clinical study on aging, daily use of MLR with NTFactor
resulted in improved
MIM trans-membrane potential, which has been linked to mitochondrial function, reduced
fatigue, and increased cognition measured using a validated survey instrument [
]. The
use of oral MLR and the improvements in MIM trans-membrane potential suggest that
mitochondrial membranes were functionally improved in aged individuals by oral MLR
supplementation. Interestingly, the MIM trans-membrane potential in the aged group was
improved over time and actually reached the levels found in healthy 30-year-olds [
MLR supplementation has also been used to improve cognition in aged individuals. Daily
oral administration of 300 mg of PS in aged patients was found to deliver significant
improvements in cognition compared to controls [121].
Multiple clinical studies have linked the process of aging to mitochondrial decline.
For example, studies have shown that aging is inversely related to the cellular content
and number of mitochondria and maintenance of their function [
]. The loss in the
number of mitochondria in each cell correlated with age along with loss of mitochondrial
CL content and changes in membrane fluidity due to lipid peroxidation. This latter process
affects MIM trans-membrane potential, which requires an appropriate redox balance to
be fully functional, and this is a significant challenge during aging [
]. These changes
result in total cellular reductions in ETC activity and ATP production [
Nonetheless, such changes can be ameliorated by MLR interventions. Indeed, patient
improvements in subsets of mitochondrial FA oxidation disorders have been seen during
MLR interventions [
]. The regulation of the biosynthesis and degradation of GPL in
mitochondria, and their transport into and out of the outer and inner mitochondrial mem-
branes play important roles in maintaining cellular health and delaying age-related declines
in mitochondrial function. As the mitochondrial production of ATP is directly linked to
the regulation of the synthesis, degradation, and transport of GPL and of maintenance of
MIM trans-membrane potential [
], the provision of fully-functional GPL substrates by
the use of oral MLR supplements or nutritional means represents a safe option for dealing
with age-related mitochondrial decline [13].
The use of MLR to inhibit CL peroxidation and its cellular relocation, as well as
MLR to provide precursor molecules for CL synthesis, can also reduce the risk for age-
related damage to mitochondria. In addition, CL is also involved in the import and
assembly of mitochondrial proteins [
], and the majority of such mitochondrial proteins
are nuclear-encoded and require protein translocases to be transported to their mitochon-
drial membrane targets. This transport system relies on ATP, and mitochondria provide
80–90% of the cellular ATP necessary for cellular function, including membrane function
and reorganization [134,150,151].
Another age-related process affecting cells and originating within mitochondria is
programmed cell death. This involves the specific, organized destruction of mitochondria
known as mitophagy, which in turn is highly sensitive to redox potential and the concen-
trations of GPL in mitochondrial membranes [
]. Some age-related changes are,
in turn, related to the accumulation of dysfunctional mitochondria within cells, resulting
from a combination of impaired clearance by autophagy and inadequate mitochondrial
replenishment [
]. The reduced regenerative capacity or turnover of healthy cells
and mitochondria in this state can be the result of mtDNA damage and mitochondrial
dysfunction, further adding to the vicious cycle of inflammation and age-related disease
onset [
]. For example, increased production of ROS/RNS associated with aging can
stimulate NLRP3 inflammasome formation by reacting with mtDNA, and this can promote
chronic inflammation [
]. Although ROS/RNS are also important in regulating and
maintaining normal homeostatic processes in living organisms, their excess during aging
can lead to sustained inflammasome production of IL-1
and IL-18 [
]. MLR with
Membranes 2021,11, 944 13 of 39
dietary FA can affect the over-expression of inflammasomes during aging. For example,
n-3 unsaturated FA, such as DHA and EPA, exhibit anti-inflammatory properties by inhibit-
ing the production of inflammasomes [
]. Multiple mechanisms may be involved,
including the manipulation of cell membrane lipids as well as inhibiting primary and
secondary factors, such as modifying Ca
stores, the expression of NF
B, and modifying
cholesterol metabolism [
]. Using MLR dietary lipids to inhibit chronic inflammation
may be useful in reducing age-related chronic inflammation and maintaining a functional
balance between inflammatory and anti-inflammatory responses.
Mitochondria can be functionally improved by the several cellular mechanisms, in-
cluding mitochondrial fission and fusion, and their numbers can also be increased by
mitochondrial biogenesis [
]. Mitochondrial fission is normally associated with DNA
replication and is essential for mitochondrial duplication and biogenesis. Mitochondrial
fission is also essential for mitophagy, the recycling of mitochondria that have become
dysfunctional or damaged [168]. Mitochondrial fusion can repair damaged mitochondria
and maintain their function [
]. It can occur rather quickly, and it is mediated by a
three-stage process, involving: (i) end-to-end alignment of mitochondria; (ii) fusion of the
outer membranes of mitochondria; and (iii) fusion of the inner membranes, forming a larger
intact mitochondrion [
]. Mitochondrial fusion can rescue impaired mitochondria
by reorganizing their contents and unifying the mitochondrial compartment [
MLR can be used to help support mitochondrial fission and fusion and to increase ATP pro-
duction, decrease membrane permeability, increase MIM trans-membrane potential, reduce
inflammasome production and eventually improve organ function [124,126,145,164168].
How cellular function can be enhanced by MLR supplementation to improve age-
related changes has been visited in a randomized, double-blind, placebo-controlled study
in middle-aged, pre-, and post-menopausal women who complained of some fatigue but
were relatively healthy [
]. The participants (32 patients in each arm) were given MLR
with NTFactor (0.8 or 1.2 g per day or placebo) for 4 or 8 weeks, and fatigue, vigor, mood,
and menopause symptoms were monitored. The women receiving 1.2 g per day GPL
showed greater reductions in fatigue (compared to placebo) using the Chalder fatigue scale
but this did not reach significance. There were significant improvements in patient vigor
in the GPL group. Sleep, confusion, anger, and menopausal symptoms were evaluated
using Quality of Life instruments, and were found to improve, but again this did not
reach significance. Cardiovascular parameters improved, and reductions in diastolic blood
pressure and improvements in cardio-ankle vascular index were significant [174].
MLR use, either as a sole supplement or as part of a nutrient and lifestyle interven-
tion, including fasting, exercise, and increased intake of plant-based foods, can improve
membrane functionality and mitochondrial function, and make meaningful improvements
in age-related disorders [
]. Some elements of functional decline associated
with aging appear to be more susceptible to MLR supplementation than others. For ex-
ample, there are particular morbidities such as elements of coronary heart age-associated
diseases, stroke, neurodegeneration and some metabolic disorders, that are at least par-
tially to MLR used either as a stand-alone supplement to improve outcome and reduce
age-related morbidity or as part of comprehensive program of dietary and lifestyle changes.
8. MLR in Fatiguing Illnesses
Fatigue is a widely found indicator or symptom in various diseases, conditions, and
during normal aging, but it nonetheless remains poorly understood. It is a complex,
multi-dimensional sensation that is perceived as loss of overall energy and feeling of
exhaustion and inability to perform tasks without excessive exertion [
]. Mild
fatigue can be attributed to several mental and physiological disorders, such as depression,
but moderate to severe fatigue is generally related to loss of mitochondrial function and
reduced production of cellular ATP [124,175,182,183].
Mitochondrial stress, and especially damage to mitochondrial membranes by ROS/RNS,
occurs naturally during aging and in essentially all chronic and acute medical condi-
Membranes 2021,11, 944 14 of 39
tions [
]. Patients with severe fatigue, such as in chronic fatigue syndrome
or myalgic encephalomyelitis, evidence of free-radical oxidative damage to the DNA and
lipids, especially membrane and mitochondrial lipids, is commonly found [
]. Pa-
tients with severe fatigue also test positive for oxidized blood markers
such as oxidized
membrane lipids, that are indicators of excess oxidative stress [
]. Patients with
chronic fatigue syndrome also have sustained elevated levels of peroxynitrite due to excess
release of nitric oxide. An overabundance of peroxynitrite also results in excessive lipid
peroxidation and loss of mitochondrial function. Peroxynitrite is a potent stimulator of
increases in cytokine levels that can have a positive feedback effect and increase nitric oxide
production even further [188].
As the most commonly found symptom in chronic medical conditions, fatigue is
also one of the most familiar symptoms seen in cancer patients [
]. In cancer
patients, fatigue can occur at the onset of disease, and it is certainly present in the most
progressive forms of metastatic disease [
]. The most universal symptoms found
in cancer patients are fatigue, pain, and nausea, and these are often found as disabling
symptoms [
]. Similar to fatigue found in other diagnoses, cancer-associated fatigue
is also not well understood. It is thought to be a combination of the effects of the cancer plus
the effects of cancer treatments. Most cancer therapies are known to cause fatigue [
Until recently cancer-associated fatigue was largely considered to be an untreatable
condition and was thought to be unavoidable [
]. As cancer has often been found to
be associated with symptoms of depression and anxiety [
], cancer-associated fatigue
was believed to be a psychological problem that was only secondarily associated with can-
cer [
]. Indeed, malaise, fatigue, and even loss of vitality were considered associated
characteristics with depression, and fatigue and its manifestations are now considered
primary elements in the diagnosis of depression [
]. As stated above, fatigue and
depression are often diagnosed together in cancer patients, especially in patients with
severe disease; thus, they can be considered cancer co-morbidities or as part of a symptom
cluster in cancer patients [194].
Therapy-associated fatigue is often seen during the treatment of cancer, and such treat-
ments often utilize techniques that result in the generation of free radical
oxidants [189,191,195]
Fatigue can be so severe during cancer therapy that it is often the most common reason
why patients discontinue their therapy [
]. Up to 96% of patients receiving chemother-
apy and up to 93% receiving radiotherapy recount moderate to severe therapy-associated
fatigue, and such fatigue can continue for months or even years after the completion of
therapy [196].
MLR can lessen cancer-associated fatigue and reduce the fatigue caused by can-
cer therapy [
]. MLR supplements, such as NTFactor
, reduced cancer-
associated fatigue approximately 30–40% [
]. In addition, MLR supplements
have also been used to reduce the adverse effects of cancer therapy, such as fatigue, nau-
sea, vomiting, malaise, diarrhea, headaches, insomnia, constipation, and other adverse
events [
]. Using a combination MLR supplement mixture containing NTFactor
Colodny et al. [
] were able to reduce several common adverse events during and after
cancer chemotherapy of colon, rectal, and pancreatic cancers. MLR supplements, such
as Propax
with NTFactor
, have also been used to reduce the adverse effects of treat-
ment using multiple chemotherapy agents. MLR supplementation resulted in significantly
fewer episodes of fatigue, nausea, vomiting, diarrhea, constipation, insomnia, and other
effects. Cancer patients on combination chemotherapy that used the MLR supplement
also experienced an overall improvement in quality of life indicators. In a
double-blind, placebo-controlled, cross-over study on patients with advanced cancers
undergoing combination chemotherapy a MLR supplement containing NTFactor
used. During anti-cancer therapy patients had fewer and less severe adverse effects due to
the combination chemotherapy. For example, there were improvements in the incidence
of fatigue, nausea, diarrhea, impaired taste, constipation, insomnia, and other symptoms
compared to controls [199].
Membranes 2021,11, 944 15 of 39
Lipids are important in cell proliferation, and thus their use in cancer therapy and
support of cancer patients has been considered [
]. There are over 100 clinical
cancer trials that have used the polyunsaturated fatty acids EPA and DHA, and over 60 tri-
als have used other fatty acids and phospholipids ( website, accessed on
1 November 2021). For example, the efficacy and safety of 2-hydroxyoleic (LAM561) and
2-hydrocylinoleic (ABTL0812) acids have been demonstrated in clinical trials (Clinicaltri- identifiers #NCT01792310 and #NCT03366480, respectively). They exert their MLR
effects after their incorporation in cell membranes, both in the original forms or via their
metabolites, and they induce relevant changes in the interactions of membrane proteins
that regulate cell signaling [
] in ways that reduce cancer cell proliferation and induce
autophagy [201,202].
The most common clinical use of MLR with GPL is to treat fatigue not associated
with depression or cancer. For example, MLR has been used in several studies in pa-
tients with fatiguing illnesses to reduce moderate to severe chronic fatigue. Patients
complaining of fatigue lasting more than 6 months are considered patients with chronic
fatigue [123,124,145,175,189]
. The effects of NTFactor
on chronic fatigue in moderately
fatigued subjects were documented to determine if mitochondrial function improved si-
multaneous with reductions in fatigue in patients taking oral NTFactor
]. In this
cross-over clinical study, there was good correspondence between the reductions in fatigue
and gains in mitochondrial function as determined by increases in MIM trans-membrane
potential in leukocytes using a redox dye and fluorescent-activated cell sorting. As dis-
cussed above, mitochondrial function is directly related to MIM trans-membrane potential.
After 8 weeks of MLR with NTFactor
, mitochondrial function had improved significantly,
and after 12 weeks of NTFactor
supplementation, mitochondrial function was found
to be similar to the levels found in young healthy adults [
]. Specifically, there was a
significant increase (+26.8%) in mitochondrial function, measured by increases in MIM
trans-membrane potential. After 12 weeks of supplement use, subjects were switched from
MLR to placebo without their knowledge for an additional 12 weeks, and their fatigue
and mitochondrial function were again measured. After the 12-week placebo period, fa-
tigue and mitochondrial function were intermediate between the initial starting values
and those found after 8 or 12 weeks on the MLR supplement, suggesting that continued
MLR supplementation is probably required for further improvements in mitochondrial
function and maintenance of lower fatigue scores [
]. Similar results on the effects of
on fatigue were found in patients with chronic fatigue syndrome (CFS/ME)
and/or fibromyalgia syndrome, Gulf War Illness, chronic Lyme disease, mycoplasmal
infections, or various other conditions [123,145,175,203205].
MLR combination supplements have proved advantageous and safe for treatment of
fatiguing illnesses [
]. MLR supplements containing NTFactor
have also
been used in combination with other ingredients to treat long-term chronic illness patients
with moderate to severe fatigue [
]. In one of these studies, patients had been
ill with intractable fatigue for an average of over 17 years and had been seen by an average
of more than 15 physicians. In addition, they had taken an average of over 35 supplements
and drugs with no improvements in their fatigue scores [
]. Within a few weeks on
the combination MLR supplement ATP Fuel
, they responded and showed significant
reductions (30.7%) in fatigue. Regression analysis of the data indicated that the reductions
in fatigue were consistent, occurred with a high degree of confidence (R
= 0.960 for overall
fatigue) and were gradual. Thus, the ATP Fuel
proved to be a safe and effective in
significantly reducing fatigue in patients with intractable chronic fatigue [
]. A recent
follow-on, open label study using fewer subjects and a second-generation ATP Fuel
supplement (ATP360
) found similar results [
]. Chronic fatigue was reduced within one
week and the results were statistically significant throughout the 8-week study. They also
monitored blood pressure and found that the ATP360 supplement reduced significantly
diastolic blood pressure and pain (discussed below). Wellness scores were collected during
the study, and participants on ATP360
were found to have significant improvements in
Membranes 2021,11, 944 16 of 39
sleep and vitality and mental function scores by the end of 8 weeks. Cytokine TNF
serum was reduced significantly by week 4. They also examined mitochondrial resistance
to inflammatory stress ex vivo in blood leukocytes from chronic fatigue patients and
controls treated with the endotoxin lipopolysaccharide. After 4 weeks consumption of the
combination supplement, leukocyte subpopulations responded to the ex vivo inflammation
stress by producing a similar increase in mitochondrial mass per cell as leukocytes from
healthy controls, and monocyte trans-membrane MIM potential increased by 51% in cells
from chronic fatigue patients, but this was not statistically significant [206].
Another type of oral MLR combination supplement containing NTFactor Lipids
low-dose, controlled-release caffeine was used to reduce fatigue, pain (to be discussed
below), gastrointestinal symptoms and improve Quality of Life (QOL) indicators in fi-
bromyalgia patients [
]. Previously, it was noted that low-dose caffeine, equivalent to
1–2.5 cups of coffee, had a modest effect in reducing some chronic pain and some fatigue,
but among non-opioid users these effects were not significant [
]. By combining NT-
Factor Lipids
plus low-dose, control-release caffeine (equivalent to one cup of coffee
maintained over 8 h) in a chewable wafer a preliminary open label study was initiated
to study its effects in group of fibromyalgia patients during an 8-day open-label trial. By
the end of the study patients showed significant reductions in fatigue, pain, and gastroin-
testinal symptoms and improved abilities to complete tasks and participate in activities
(Quality of Life indicators) [207].
Mitochondria-supporting combination supplements have been used to treat mi-
tochondrial cytopathies and to support mitochondrial function [
]. The
use of combination supplements containing antioxidants, alpha-lipoic acid, vitamins,
CoQ10, lycopene, creatine, riboflavin, and other ingredients have been proposed [
Gonzales et al. [210]
have discussed in detail the use of various ingredients, including MLR,
in supporting mitochondrial function and improving health outcomes.
9. MLR in Pain Control
Chronic pain is a complex symptom involving nerve membrane channelopathies
(damage to membrane channels in nerve cells that initiate the transmission of pain signals),
autoimmune responses, microvascular damage, and other factors [
]. There are sev-
eral types of pain, but the chronic pain under consideration here is nociceptive pain, which
is pain originated by depolarization of nociceptors in sensory nerve fibers in response to
noxious stimuli [
]. In terminal nociceptors noxious stimuli can elicit membrane currents
that, if they surpass a certain threshold, send signals from the nerve fibers to the spinal
cord. The conducting fibers from the periphery to the Central Nervous System involved in
pain perception are usually of the A or C type [
]. MLR supplements such as NTFactor
Lipids®have been used to reduce widespread and chronic pain in patients [214,215]. The
MLR lipids are not thought to be acting by repairing mitochondrial membranes; they are
thought to act by modulating and restoring the functions of cell membrane ion channels
(for example, TRPV, P2X, or ASIC channels), maintaining intracellular Ca
or other effects [
]. Indeed, nerve cell membrane ion channels related to pain path-
ways can be dramatically modulated in their function by particular MLR GPL [
In addition, other properties, such as stimulation of free radical oxidative damage, loss of
mitochondrial function or the presence of proapoptotic enzymes such as caspases, could
also be modulated in nerve cells by MLR GPL [218].
MLR supplements, such as NTFactor Lipids
, have been used to reduce pain, such as
widespread pain, gastrointestinal pain, and other symptoms in chronic illness patients. For
example, in fibromyalgia patients 4.8 g per day of oral NTFactor Lipids
for 8 days signifi-
cantly reduced pain (p< 0.001), fatigue (p< 0.001), gastrointestinal symptoms (p< 0.001)
and improved Quality of Life assessments (p< 0.001) in an open label clinical trial [
]. The
MLR supplement NTFactor Lipids
has been shown in case studies to reduce widespread
pain in fibromyalgia, gunshot wounds, and other chronic conditions [
]. This new
area of MLR use will continue to grow in the future, but there is one important limitation.
Membranes 2021,11, 944 17 of 39
The pain responses to MLR appear to be limited to higher daily doses of MLR supplements.
Patients responded better and their responses were more durable at higher daily doses
6 g per day) of the MLR supplement. For example, oral NTFactor Lipids
was found
to be most effective at reducing widespread pain, specific musculoskeletal pain, abdom-
inal pain, or other pain if used at or above 6 g per day. In these case studies, the MLR
supplement also showed benefits in sleep, digestion, hypertension, headaches, and other
signs and symptoms [
]. This dose requirement may reflect the concentrations of
GPL necessary to modulate membrane channels that mediate pain transmission or other
mechanisms. As found in previous MLR studies, when the higher daily oral doses of
NTFactor Lipids
were lowered or stopped, widespread and other pain as well as other
signs and symptoms returned to levels found before MLR supplementation, consistent
with the bulk or mass action delivery of MLR to peripheral and central nerve cells and
other cells involved in pain transmission.
10. MLR in Degenerative Diseases
As mentioned above, the proper maintenance of mitochondrial health is essential
in order to prevent the formation of degenerative diseases [
]. Non-infectious,
non-communicable degenerative diseases (NCD) are age-related diseases whose incidence
increase with age or certain behaviors. The most common NCD are: cardiovascular diseases
(CVD) (hypertension, cardiopathies, coronary heart disease, myocardial infarction, and
stroke), neoplastic diseases (various cancers), neurodegenerative diseases (Alzheimer’s
disease, Parkinson’s disease, and multiple sclerosis, among others), autoimmune disease,
rheumatic diseases, and chronic respiratory diseases. Mitochondrial changes, such as
mitochondrial fission and fusion to improve function, play critical roles in ensuring and
maintaining mitochondrial health. However, if high concentrations of fats or other nu-
trients are consumed in excess, molecular damage may occur that can spread through
mitochondria and affect their collective performance [
]. Mitochondria can optimally
produce ATP when they are undamaged or only marginally damaged, or alternatively if
the damage is repaired, for example with MLR. When mitochondria are damaged by free
radical oxidants, their dynamics can be unfavorable, and fission and fusion often fail to
recover function. This suggests that nutritional support and lifestyle changes that favor
optimal mitochondrial dynamics, for example, with appropriate MLR, antioxidants, and
other nutrients along with caloric restriction, can result in enhancement of mitochondrial
function, increased cellular energy, and improved health [
]. Defects in mitochon-
drial membranes, systemic inflammation, and oxidative stress are thought to be the most
likely causes of most NCD. Therefore, nutritional interventions, repair of mitochondrial
damage, and improvements in lifestyle have the potential to improve health [226228].
Nutritional, pharmacological, and lifestyle approaches can potentially slow down age-
related functional decline and damage that predisposes an individual to
NCD [220,225228]
Such approaches include the use of insulin sensitizers [
], exercise to promote mitochon-
drial fusion [
], and targeted antioxidant treatments to reduce ROS/RNS damage to
membranes [
]. However, the effectiveness of many nutritional approaches, such as
the general use of antioxidants, has been questioned [
]. Most antioxidants are not
selective for mitochondria, and they fail to complex with lipophilic transporters and do
not reach the MIM [
]. Other possible therapeutic approaches, such as caloric restriction
or reduction in caloric intake without malnutrition or intermittent fasting continue to be
examined [233235]
. Use of combination supplements with protected GPL and antioxi-
dants has been shown to improve mitochondrial bioenergetics [
]. This has
the added benefit of potentially reducing inflammation [
]. Unfortunately, high caloric
western diets, with excessive consumption of sugar and saturated fats, along with environ-
mental insults, can result in mitochondrial dysfunction and higher susceptibilities to NCD,
premature aging, and carcinogenesis [
]. Therefore, lifestyle and behavioral changes
combined with nutrition and MLR can provide essential mitochondrial support and may
prevent or forestall the development of NCD [238,239].
Membranes 2021,11, 944 18 of 39
One of the pathways involved in regulating mitochondrial function involves the
enzyme ‘mammalian target of rapamycin’ (mTOR), a serine/threonine kinase that regu-
lates cell growth responses to nutrients [
]. There are two important mTOR complexes,
and dysregulation of their activities is associated with aging and various NCD, includ-
ing diabetes, cancers, and neurodegenerative diseases [
]. Signaling by one of these
complexes (TORC1) regulates mitochondrial biogenesis, oxidative stress, and turnover in
mammals and lower organisms. Defects in the clearance of damaged mitochondria by
TORC1-regulated autophagy contributes to accumulation of cellular ROS/RNS [
]. The
TORC1 signaling pathway is involved in the sensing the abundance of various nutrients
and regulating the activity of critical processes, such as autophagy. Caloric restriction
without malnutrition and periodic short-term nutrient excess with use of specific inhibitors
of mTOR can affect mitochondrial function [
]. This suggests that mitochondria
are highly responsive nutrient sensors and effectors, and this could potentially be used to
suppress the generation of NCD.
The use of MLR along with lifestyle changes may present an opportunity to enhance
mitochondrial fusion, while reducing inflammation and oxidative stress, and this could
ultimately assist in the management, prevention, and treatment of NCD. As an example
of this approach, a preliminary clinical trial in which MLR supplementation was used to
modify metabolism through weight reduction and appetite restraint, was initiated [
Thirty middle-aged adults (mean age, 56.8; 24 females and 6 males) used an oral MLR
supplement (HealthyCurb
, containing NTFactor
and alpha-amylase inhibitor) 30 min
before each meal. Participants were told to eat and exercise normally. In the study, weight,
waist, and hip measurements were taken weekly, and blood samples were taken prior to
and at the end of the study for lipid and chemical analyses. Appetite and sweet cravings
were assessed weekly by standard methods, and fatigue was determined using a fatigue
instrument. A majority (63%) of the participants lost an average of 2.77
0.12 kg, along
with significant average reductions of 6.4
0.13 cm and 3.8
0.10 cm inches from waist
and hip circumferences, respectively. The entire group lost an average of 1.65
0.11 kg
(p< 0.001) with significant average reductions of 4.04
0.06 cm and 2.87
0.05 cm in
waist and hip circumferences, respectively. Weight loss and body measurement decreases
were gradual, consistent, and significant, along with reductions in body mass index and
basal metabolic rate. Hunger was significantly reduced (44.5%), and there was decreased
cravings for sweets and fats. There was a 23.9% reduction in fatigue during the study.
There was also a 26.8% improvement in cognition as well as improvements in the ability
to think clearly, concentrate, and remember details. At the end of the study blood lipid
profiles showed improvements, with better cardiovascular lipid profiles (lipoprotein ratios).
During and after the study no adverse events were reported [
]. The effects of MLR on
weight loss in the clinical studies could be due to the replacement of damaged GPL with
undamaged GPL and support of mitochondrial function. In animal weight-loss studies,
rats were fed a diet high in stearic, elaidic, oleic, linoleic, and 2-hydroxyoleic acids for
7 days. On this diet, rats showed less food intake and there was weight loss, mainly due to
reductions in adipose fat [
]. Subsequently, they found that only the chow containing
excess oleic acid or 2-hydroxyoleic acid induced body weight loss [
]. The clinical trial
and animal study indicated that MLR supplements can reduce the urge to eat, and this
may be related to their positive, systemic effects on mitochondrial function.
MLR with GPL have been used to modify NCD-related functional decline in patients,
such as supplementation with oral PS to improve memory loss and cognition. For example,
in one 12-week study 30 male and female subjects (age 50–90 years) with memory impair-
ments not related to neurological disease, stroke, intracranial hemorrhage, brain lesions,
diabetes, infections, or inflammatory processes were used to ascertain if
300 mg
PS per
day modified the outcomes of 6 tests of memory and cognition [
]. At the end of the
study participants exhibited significant improvements in memory recognition, recall, total
learning, executive functions, mental flexibility, and visual spatial learning. Participants
also showed reductions in mean systolic and diastolic blood pressure compared to baseline
Membranes 2021,11, 944 19 of 39
pressures, and there were no adverse events reported [
]. Similarly, a double-blind, ran-
domized clinical trial on 78 subjects (50–69 years) was initiated to determine if
100–300 mg
oral PS per day versus placebo affected memory. In this study, PS supplementation sig-
nificantly improved behavioral memory functions, especially short-term memory and
cognitive function (measured by delayed word recall). The study was completed without
any adverse events or changes in vital signs or laboratory tests [
]. The clinical results
using MLR supplements universally did not result in any adverse events.
The studies discussed above support the relevance of membrane lipids in the pathophys-
iology of neurodegeneration. In this context, recent studies demonstrate that Alzheimer’s
disease is instigated, in part, by alterations in membrane lipids that alter the functions of
important membrane proteins [
]. Thus, membrane GPL and other membrane lipids
regulate the nanodomain localization and functional activity of many if not most mem-
brane proteins, and changes in lipid species present in the plasma membrane appear to be
associated with mild or severe deteriorations associated with pathophysiological cognitive
decline [
]. In this context, treatment with 2-hydroxydocosahexaenoic acid (DHAH)
induced marked increases in the levels of polyunsaturated fatty acids and PE in brain
membranes in a model of Alzheimer’s disease; this showed that MLR can modulate the
biophysical properties of neuronal membranes [
]. Interestingly,
- and
-secretases that
process the amyloid precursor protein (APP) to form
-amyloid are embedded in the cell
membrane, and their activities depend on the membrane lipid composition and structure.
These facts suggest that the amyloidogenic processing of APP could be facilitated by alter-
ations in the membrane lipid bilayer, and MLR could constitute an innovative approach
to interdict this process [
]. Several studies carried out in cellula and in animal models
of human Alzheimer’s disease (5XFAD mice) using the hydroxyalated polyunsaturated
fatty acid DHAH and other fatty acids support this hypothesis and shed light on the
molecular and cellular mechanisms involved in their therapeutic action. The changes in
neuronal membrane lipids induced by DHAH cause dramatic inhibitions of amyloidogenic
secretases activities, followed by reductions in amyloid loads, amyloid binding to mem-
branes, and Tau protein phosphorylation. Moreover, 5XFAD mice treated with DHAH
show normal cognitive scores and increased neurospore (neuronal stem cells) proliferation
in the brain [
]. These studies indicate that MLR and related membrane lipid therapy
(melitherapy) approaches can be used in the prevention and treatment of neurodegen-
erative processes associated with aging. Moreover, in homozygotic APOE-
4 people (at
highest risk for Alzheimer’s disease), diet supplementation with DHA is related to better
neural and cerebrovascular neuroimaging (magnetic resonance imaging), consistent with a
benefit against neurodegeneration [254].
Nutritional approaches to preventing neurodegeneration have also been applied to
Parkinson’s disease. In a rat model of Parkinson’s disease, the omega-3 fatty acids DHA
and DHAH induced important improvements in behavioral parameters associated with
reductions in astrogliosis, microgliosis, and GFAP staining in brain striatum and substantia
nigra [
]. These changes were markedly and significantly more pronounced in animals
treated with DHAH, as this compound has a greater ability to incorporate into membrane
GPL through its metabolite heneicosapentaenoic acid (21:5 n-3) [
]. Thus, lipid alterations
and normalization can counter the development of pathophysiology of neurodegenerative
diseases, such as Alzheimer’s and Parkinson’s diseases. Lipid composition influences
membrane lipid bilayer structure, and the interactions of membrane proteins involved in
normal and pathological protein functions and cell signaling, which justifies the use of
MLR to prevent neurodegeneration [11,249].
11. MLR in Metabolic and Cardiovascular Diseases
As a common pre-disease condition found predominantly in the aging population,
metabolic syndrome (MetSyn) is thought to be a precursor condition for several important
age-related diseases, most such as type 2 diabetes (T2D). MetSyn is characterized by several
interrelated blood disturbances of glucose and lipid homeostasis that are generally but not
Membranes 2021,11, 944 20 of 39
always found in obese, middle-aged individuals. These changes include: insulin resistance,
changes in blood lipid profiles, abnormal glucose tolerance, hypertension, and vascular
inflammation, as well as a background of multiple genetic abnormalities [
]. There
are a number of major risk factors and blood markers routinely found in most MetSyn
patients: (i) obesity or excess abdominal fat; (ii) elevated fasting plasma glucose levels;
(iii) dyslipidemia, such as increased triacylglyerols, increased LDL, and reduced HDL in
blood; (iv) elevated blood pressure; and (v) the presence of circulatory prothrombotic and
proinflammatory molecules [
]. MetSyn (also called Syndrome X [
] or insulin-
resistance syndrome [
]) is estimated to be present in over 40% of the over-60 age group
in North America [
]. The same list of important risk factors in MetSyn patients are also
found in NCD, such as hypertension, CVD, and other conditions [257,261,262].
Although there several blood markers and characteristics of MetSyn (listed above),
the most important risk factors in the initiation of MetSyn are considered to be genetic
abnormalities, obesity, and metabolic susceptibility (insulin resistance and other charac-
teristics) [
]. Summarizing the factors that are involved in insulin resistance
and MetSyn: (i) multiple genes; (ii) epigenetics, such as nutrition, low birth weight, and
others; (iii) visceral obesity; (iv) body-mass index; (v) caloric and carbohydrate intake;
(vi) sedentary lifestyle; (vii) age; (viii) ethnicity; (ix) gender; (x) menopausal status; (xi) al-
cohol consumption; (xii) inflammation; and (xiii) dysbiosis [
]. Thus, multiple risk
factors, signs, symptoms, and abnormal laboratory test results can be used to generate a
MetSyn diagnosis, but the relative merits of using the MetSyn diagnosis in clinical practice
to direct patient management remains in development [263,264,267,268].
One of the lesser studied aspects of metabolic disorders are inflammation-associated
changes involved in the generation of insulin resistance and MetSyn. These can result in
the activation of NLRP3 inflammasomes by various mechanisms, including generation of
mitochondrial DAMPs [
], lysosomal membrane disruption [
], and high fat diets [
The activation of inflammation complexes such as the NLRP3 inflammasome complex
contributes to the development of visceral adiposity, endothelial dysfunction and insulin
resistance, precursors to type 2 diabetes (T2D) [
]. The NLRP3 inflammasome complex
may represent a signaling pathway that facilitates multiple organ metabolic damage [
The NLRP3 inflammasome also regulates the gastrointestinal microbiome and is activated
by pathobionts and associated dysbiosis, which can affect host susceptibility to metabolic
disease onset and progression [
]. Obesity, non-alcoholic fatty liver disease, and T2D, as
well as changes in the integrity of the gastrointestinal barrier all affect lipid balance and
homeostasis [275].
Lipids are critically involved in MetSyn and associated diseases, and defects in the
capacity to utilize and metabolize lipids and glucose are especially important in insulin
resistance and MetSyn [
]. Accumulations of DAG, triacylglycerol, and free FAs in non-
adipose tissues correlate strongly with insulin resistance [
], and increases in released,
free fatty acids may block insulin signal transduction and contribute to MetSyn [
Lipids, such as DAG, have been implicated in insulin resistance by activating isoforms of
protein kinase C, which in turn can directly modulate insulin signaling by phosphorylating
and inhibiting the insulin receptor tyrosine kinase and activating genes responsible for
FA-induced inhibition of insulin activity [
]. Differences in gene expression in adipose
tissue may be responsible for increasing the secretion of MetSyn-related factors, such as
the pro-inflammatory cytokine TNF
and the tissue-specific protein adiponectin [
In muscle tissue, decreased oxidative capacity and fat accumulation may also induce
skeletal muscle insulin resistance and contribute to the development of MetSyn, and even-
tually T2D [
]. Although MetSyn, T2D, and their associated diseases are known to be
caused by multiple events, various studies point to mitochondrial dysfunction as a major
component [265,268,269]
. This has been shown in experiments where microarrays have
been used to monitor gene expression in T2D. Such experiments revealed that several mito-
chondrial genes were down-regulated in T2D, supporting the notion that mitochondrial
dysfunction is important in T2D [
]. Additionally, in muscle cells decreased ETC activity
Membranes 2021,11, 944 21 of 39
as well as decreased whole body anaerobic capacity are present in T2D patients, indicating
involvement of mitochondrial dysfunction [271].
In T2D patients’ genetic polymorphisms have been found that are involved in FA
oxidation and in factors that control transcriptional activities important in mitochondrial
function [
]. Reduced fat oxidative capacities and increases in release of FA are found in
MetSyn and T2D patients [
]. In obese, pre-diabetic, and diabetic patients, free FA levels
are increased together with decreases in fat oxidative capacity, and this causes accumulation
of FA and acylglyerols in pancreatic beta cells and other tissues. These changes correlate
strongly with insulin resistance, MetSyn, and pre-diabetes [
]. In MetSyn, T2D, CVD,
and renal diseases free FA can accumulate inside cells and in mitochondria, where they
are prone to peroxidation, resulting in the formation of lipid peroxides. Lipid peroxides
can be cytotoxic and result in free-radical damage to lipids, proteins, and DNA, especially
in mitochondria. When the MIM is oxidatively damaged, MIM proton leakage and ETC
function and mtDNA damage occurs. This type of damage can also result in activation of
the NLRP3 inflammasome [281].
T2D is thought to develop from MetSyn due to persistent hyperglycemia, which in
turn causes: (i) formation of advanced glycation end-products (AGEs), their accumulation
and oxidative interactions with cell receptors; (ii) activation of various isoforms of protein
kinase C; (iii) induction of the polyol pathway; and (iv) increased hexosamine pathway
flux [
]. Most of these pathways are associated with elevated oxidative stress
and over-production of ROS/RNS during hyperglycemia. This can result in MetSyn, and
eventually the development of T2D [
]. Obesity cannot be underestimated in this process,
as it results in hyperlipidemia, an increase in FA oxidation products that stimulate insulin
secretion, and hyperinsulinemia. Hyperinsulinemia can, in turn, down-regulate insulin
receptors and increase blood glucose levels [
]. Excess oxidative stress contributes to the
progression of MetSyn to T2D by disrupting the ability of pancreatic beta cells to respond
to elevated blood glucose levels [
]. The excess ROS/RNS eventually results in death
of pancreatic beta cells by apoptosis, and this further reduces production of insulin [286].
Dietary supplements can prevent some of the damage to cellular and mitochondrial
membranes seen during MetSyn development and potentially slow the process of progres-
sion to T2D. Such supplements are also important in preventing loss of ETC function seen
in MetSyn and T2D [
]. Dietary use of various types of antioxidants or other supplements
that increase free-radical scavenging systems have been employed to reduce or prevent the
process of development of MetSyn and T2D [
]. In MetSyn, T2D, CVD, and dis-
eases caused or promoted by continuing excess ROS/RNS, dietary supplementation with
low molecular weight antioxidants, plus some replacements of accessory molecules have
been employed. By adding enzyme cofactors such as zinc, manganese, copper, vanadium,
chromium, and selenium ions necessary for antioxidant and some enzyme function plus
certain vitamins with antioxidant properties and coenzymes (examples: C, E, A, CoQ
such combinations can be used to maintain antioxidant levels and free-radical scavenging
systems [
]. As discussed in previous sections of this review, supplemen-
tation with oral antioxidants, enzymes, vitamins, and other cofactors alone may not be
sufficient to maintain cellular components and eliminate ROS/RNS damage. Thus, supple-
mentation with antioxidants and needed cofactors alone cannot replace damaged cellular
components, especially the GPL in cell and mitochondrial membranes [
]. Thus,
as stated previously in this review, addition of MLR ingredients to the usual antioxidant
supplements may be necessary for optimal maintenance of membrane health.
MLR should also be useful in replacing membrane and blood components damaged by
MetSyn, such as restoring unoxidized GPL in blood lipoproteins [
]. The administration
of MLR phospholipids, along with changes in diet and lifestyle, can help to remove oxidized
phospholipids and cholesterol from HDL and LDL [
]. Treating T2D patients with
oral MLR supplements resulted in decreased serum triglyceride levels and reduced lipid
peroxidation products compared to placebo controls [
]. There was also a significant
reduction in the levels of acyl-hydroperoxides, Schiff’s bases, diene/triene conjugates, and
Membranes 2021,11, 944 22 of 39
MDA in patients taking oral MLR phospholipids [
]. Some studies found a significant
reduction in blood sugar levels in patients with T2D given 1.2 g of oral MLR for 60 days
compared to a control diet group [
]. Despite the evidence for a connection between
excess oxidative stress in MetSyn, T2D, and associated diseases, an association between
the intake of high concentrations of oral antioxidant nutrients and the prevention or delay
of MetSyn progression to T2D and other diseases has not been realized [
Controlled clinical trials, for the most part containing single oral antioxidants, failed to
show significant prevention benefits [
]. This should not be unexpected, as these complex
phenomena involve multiple, complex mechanisms and pathways, and it is unlikely that a
simple, singular approach could be effective.
An example of this complexity is the involvement of inflammation and vascular
function in the development of metabolic diseases. For example, an important condition
that usually precedes the development of MetSyn and T2D, hypertension, is directly
related to vascular dysfunction, and this can be present with insulin resistance for many
years before the development of T2D [
]. Hypertension is linked to insulin resistance,
excess oxidative stress, mediated mainly by ROS/RNS, and changes in endothelial and
smooth muscle cells. These are also known to be involved in vascular inflammation and
initiation of apoptosis [
]. Chronic inflammatory damage to blood vessels due to
lipid accumulation, inflammatory responses, endothelial cell death, and thrombosis, can
eventually result in atherosclerosis and CVD.
Atherosclerosis is characterized by a number of risk factors, including abnormalities
in lipoproteins, increases in vascular acute phase response proteins, changes in vascu-
lar endothelial cell adhesion molecules, and the presence of certain inflammatory cy-
tokines [
]. ROS/RNS plays an important physiological role in maintaining vascular
integrity, but when present in excess, ROS/RNS can be involved in pathological processes.
As mentioned above, excess production of ROS/RNS is associated with the development of
MetSyn, T2D, hypertension, atherosclerosis, and CVD [
]. Endothelial and adipose
dysfunction, along with insulin resistance, are thought to be among the most basic physio-
logic abnormalities that link MetSyn and CVD [
]. Vascular damage associated with
excess oxidation, inflammation, and thrombosis is a primary event in the development of
MetSyn, CVD, and other diseases [
]. Macrophages are also recruited to adipose tissue,
and changes occur in adipose cells in parallel with changes in endothelial cells, such as
induction of secreted adipokines and cytokines [301].
That MLR alone can modify or reverse the conditions described above and result
in reversal of T2D, atherosclerosis, CVD, and other diseases remains extremely unlikely,
if not completely impossible. However, the use of MLR phospholipids can change the
composition and oxidation state of circulating lipoproteins and lipids [
]. Thus, this
has the potential to modify pathologic processes and limit the progression of more lethal
forms of metabolic diseases. In addition, MLR can also have other benefits. For example, the
administration of MLR has resulted in the removal of cholesterol from serum lipoproteins
and membranes [
]. In a 10-year study using rhesus monkeys fed high cholesterol
diets, seven weeks of oral MLR with dietary lecithin lowered significantly total cholesterol,
LDL cholesterol, and triglyceride blood levels [
]. Other studies have also shown that
MLR supplements can reduce blood cholesterol, LDL-cholesterol, and triglycerides [
Another example is the MLR treatment of pigs that were previously fed a high cholesterol
and coconut oil diet for 24 weeks as an experimental model of atherosclerosis. The pigs
were then administered a MLR product (EPL) [
]. Without EPL, the serum levels of
triglycerides, cholesterol free FAs, and beta-lipoproteins gradually increased with time, but
with 8-weeks of EPL (up to 280 mg/kg body weight) there was a dose-related reduction in
total lipids, cholesterol esters, free cholesterol, and triglycerides. At the highest EPL dose
levels, there was a reduction in atherosclerotic plaques observed in the aortas and heart
valves of the animals [306].
Interestingly, glycemia and blood pressure have been improved in elderly people who
follow diets enriched in olive oil, and this occurs in connection with significant membrane
Membranes 2021,11, 944 23 of 39
lipid regulation and subsequent modulation of membrane PKC and G protein levels [
Olive oil is rich in unsaturated fatty acid, such as oleic acid (70–80%), which increases in
content in membranes upon chronic supplementation with olive oil [
]. This and other
studies showed that oleic acid regulates the interactions of PKC and G proteins with cell
membranes, and these changes are associated with the health benefits seen in populations
that have higher levels of olive oil consumption [307,308].
Excessive lipoprotein lipid peroxidation is one of the first changes associated with
the development of MetSyn, and it is thought to also be important in the development of
hypertension, T2D, atherosclerosis, and CVD. Thus, agents that reduce lipoprotein lipid
oxidation may inhibit or attenuate the development of these diseases [
]. Or this
could at least be a good marker for inhibition of progression to more lethal diseases. MLR
has been shown to reduce lipid peroxidation in patients with ischemic heart disease. For
example, patients with angina pectoris took an oral MLR supplement containing 1.8 g GPL
per day for 3 weeks. At the end of treatment, there was a significant reduction in oxidized
serum lipids, an increase in HDL cholesterol, and a reduction in erythrocyte hemolysis
due to peroxidation [
]. Although these patients were not followed long enough to
determine the long-term benefits of MLR, the prospect of long-term benefits of MLR needs
to be examined in controlled clinical studies.
The use of MLR in clinical settings has demonstrated that blood levels of choles-
terol, LDL-cholesterol, and triglycerides can be slowly reduced over time, and this should
eventually result in health benefits. In long-term dialysis patients who are at risk for
ischemic cardiovascular complications, patients tend to have high blood lipid values. In a
double-blind, randomized study, groups of ten patients who had hyperlipidemia (serum
cholesterol >260 mg/dL, LDL-cholesterol >180 mg/dL, and triglyerides >200 mg/dL)
were given 2.7 g per day oral PC or placebo for 6-weeks [
]. The 6-week treatment was
followed by a two-week wash-out period, and lipid parameters were determined at 2, 4,
and 6 weeks of treatment. Two weeks after PC administration, there was a significant
reduction in LDL-cholesterol of 32 mg/dL compared to the stable placebo controls. By
4 weeks, triglycerides were significantly decreased by 58.2 mg/dL, and by 6 weeks. there
was a reduction in triglycerides of 43.3 mg/dL compared to the placebo group (at 4 weeks,
5.7 mg/dL and at 6 weeks,
11.4 mg/dL) [
]. Thus, MLR supplementation can al-
ter blood lipid markers in hyperlipidemia to more favorable values. In a double-blind
study of type II hyperlipidemia patients, participants received either three doses of oral
polyenylphosphatidylcholine (0.9 g per day) or placebo, and their blood lipid levels deter-
mined over time. Total cholesterol and LDL-cholesterol were lowered significantly, and
there was a downward trend in apoprotein B, triglycerides, and VLDL-cholesterol, and an
upward trend in apoprotein A1 content compared to the placebo group [
]. As discussed
above, MLR supplementation or treatment can be useful in altering markers of pathologic
changes. Thus, adding MLR supplementation with alterations in diet and appropriate
caloric restriction and macro- and micro-nutrient adjustments can reduce and replace
oxidized phospholipids and cholesterol from HDL and LDL. The long-term consequences
of such interventions need to be determined.
Thus, MLR supplementation has the potential to reverse some of the lipid changes
that are important in MetSyn development and possibly prevent the formation of MetSyn-
associated diseases and their progression to more lethal outcomes. It is interesting that the
long-term use of MLR in the form of oral NTFactor
and vitamins can significantly reduce
blood markers for CVD risk. In one such study, CVD risk factors, such as homocyteine
and fasting insulin levels, were brought to normal ranges found in controls within 6-
months [
]. In a group of these patients with homocyteine levels above the threshold
levels for high-risk for CVD, such as heart attack, hospitalization, and death, MLR with
oral NTFactor
resulted in a reduction in blood test results to the normal ‘safe’ ranges
within 6 months [
]. This rather short-term study did not allow the necessary time to
determine if the use of MLR and vitamin supplementation resulted in long-term health
benefits, such as survival and avoidance of CVD-related hospitalization, but it was a
Membranes 2021,11, 944 24 of 39
promising start. Future studies should document whether MLR can impede or reverse the
course of development and progression of MetSyn and its associated diseases and their
long-term consequences.
12. MLR in Other Clinical Conditions
In addition to the many uses of MLR described in this review and elsewhere [
], and
shown in Table 1for NTFactor
and NTFactor Lipids
, MLR has been used in laboratory
animals and humans to treat other life-threatening conditions, such as extensive chronic
and acute liver and kidney disease. These include: toxic liver and kidney damage caused
by carbontetrachloride, alcohol, galactosamine, acetaminophen, tetracycline, solvents, de-
tergents, thioacetamide, indomethacin, anesthetics, ionizing radiation, immune-mediated
hepatitis, and others. MLR, mainly with EPL, was shown to reduce the toxic effects of these
agents and promote organ repair and regeneration (reviewed by Gundermann [
]). In addi-
tion, there are other exposures and infections in humans where MLR has been useful, such
as in the treatment of damage caused by fat embolism, non-steroidal anti-inflammatory
drugs, liver-damaging anti-microbial drugs, lethal hepatic toxins, fatty liver conditions due
to malnutrition, and infections such as hepatitis [
]. MLR could also be useful in alleviating
some of the symptoms of COVID-19, especially in cases where symptoms persist long after
there is no longer evidence for an active viral infection [312].
The treatment of viral hepatitis and liver cirrhosis using MLR GPL have been studied
mainly in uncontrolled and in some controlled clinical trials [
]. Hepatitis patients
treated with MLR using intravenous EPL reported improvements in dyspepsia, nausea, epi-
gastric pain, fullness in the epigastrium, and other symptoms, as well as improvements in
hepatomegaly and presence of ascites. Laboratory test results also showed improvements,
and histological analysis of liver biopsies indicated some regeneration of hepatocytes [
In a controlled clinical study, patients with chronic hepatitis were treated for one year with
intravenous EPL, resulting in significant reductions in hepatomegaly, liver enzymes, hep-
atic excretory capacity, and improvements in gamma-globulin and serum albumin levels
compared to controls [
]. Patients with advanced liver cirrhosis were also treated with
oral MLR phospholipids. After 3 months treatment, most blood tests improved and were
found to be within the normal ranges. Symptoms also improved along with reductions in
hepatomegaly and ascites [
]. In other studies, patients with moderately severe to severe
cirrhosis caused by hepatitis B virus were treated with IV EPL for 3 months and compared
to patients who only received a vitamin preparation. In the control group liver function
remained similar to pre-treatment values, whereas in the EPL treatment group there were
significant improvements in liver function in a majority of patients [316].
MLR has also been successfully used in chronic ambulatory peritoneal dialysis (CAPD).
MLR GPL-treated CAPD patients showed increases in ultrafiltration with more electrolytes,
creatinine, urea, and phospholipids released into the ascites fluid, which were removed by
dialysis. MLR phospholipids were able to restore normal physiological conditions in CAPD
patients with abnormal ultrafiltration rates. Symptoms improved and patients generally
benefited significantly from the treatment [317].
MLR is safe to employ during pregnancy, and thus MLR has been used to treat
pregnancy-associated health conditions. For example, during pregnancy, gestosis or tox-
emia can occur where patients present with hypertension, edema, and proteinuria. This is
thought to be caused by chronic intravascular clotting and fibrin deposition in the uteropla-
cental bloodstream, which can affect uteroplacental perfusion and fetal development [
In the more severe cases, high levels of lipid peroxidation products have been found in the
serum and erythrocyte membranes [
]. In these patients, IV or oral MLR phospholipids
were administered twice daily at a dose of 500 mg per day during the last trimester of preg-
nancy. In the treated patients, edema subsided, liver and kidney function tests normalized,
and other symptoms eventually subsided [320].
Membranes 2021,11, 944 25 of 39
Table 1. Current and potential uses of oral MLR supplements and revised dose levels a.
Use Subjects/Patients
Age MLR Lipid NTFL Dose bNTFL Dose cExample
Group Supplement Range
(g/day) Range (g/day) Reference
(Original) (Revised)
General health Aged senior NTFactor/L d2 2–3 Nicolson et al. [3]
Fatigue Aged senior NTFactor/L 3 4 Agadjanyan et al. [145]
Fatigue CFS/ME adult/teen NTFactor/L 2–4 4 Nicolson & Ellithorpe [203]
Fatigue CFS/ME adult ATP Fuel 4 4 Nicolson et al. [123]
Inflammation Chronic fatigue adult ATP360 0.4 N/A eHamilton & Jensen [206]
Fatigue Fibromyalgia adult NTFactor/L 3–4 4 Nicolson et al. [215]
Fatigue Menopause adult NTFactor/L 1.2 3 Hirose et al. [174]
Weight loss Obesity, fatigue dadult NTFactor 2 3–4 Ellithrope et al. [245]
Brain health Neurodegen. dis. adult NTFactor/L 3–4 4 Nicolson et al. [175]
CD health CD risk/CD dis. adult NTFactor/L 2–4 4 Ellithorpe et al. [128]
Metabolic health MetSyn/diabetes adult NTFactor/L 2–4 4 Nicolson [289]
Metabolic health Diabetes adult ATP Fuel 4 4 Nicolson et al. [123]
Neurobehavior Autism Spectrum dis. child NTFactor/L 1–2 1–3 Nicolson et al. [175]
Infections Lyme/mycoplasma adult ATP Fuel 4 4 Nicolson et al. [321]
Fertility Fertility Diseases adult NTFactor/L 2–3 4 Ferreira et al. [33]
Fatigue Cancer adult NTFactor/L 2–3 4 Nicolson & Conklin [197]
Anemia Anemia adult NTFactor/L 1–2 4 Nicolson et al. [123]
Injury Spinal injury adult NTFactor/L 1–2 4 Ellithorpe et al. [123]
Autoimmune Rheumatoid arthritis adult ATP Fuel 3 4 Nicolson et al. [123]
General health Pregnancy adult NTFactor/L 1–2 2–3 Ellithorpe et al. [205]
Chemical detox GW Illnesses adult NTFactor/L >4 >6 Nicolson & Breeding [214]
Modified from Nicolson et al. [
Dose range in grams per day based on NTFactor Lipids
Revised dose range in grams per day
based on NTFactor Lipids®.dNTFactor®or NTFactor Lipids®.eNot Available.
Finally, oral MLR with NTFactor
or NTFactor Lipids
has been used to reduce symp-
tom severities associated with complex chronic infections such as Mycoplasma species and
Lyme disease-associated multiple co-infections [
]. The use of MLR in these chronic
bacterial infections was mainly administered as patient support dietary supplements,
not as treatments for the infections themselves. It was proposed that MLR supplementa-
tion should be considered in such chronic infectious diseases as part of a normal patient
support protocol.
13. MLR: Future Directions
MLR supplements containing GPL, antioxidants, and other ingredients are being used
to repair and replace oxidatively-damaged membrane GPL in order to restore activities
and functions of cellular membranes, organelles, cells, tissues, and organs, and generally
improve quality of life in aging and chronic illnesses. Many cellular activities require
energy supplied primarily by ATP produced in mitochondria, and damage to mitochondria,
specifically mitochondrial membranes and ETC complexes, reduces overall cellular energy
production [
]. MLR can be used to increase and restore MIM trans-membrane potential
and recover mitochondrial membrane function and production of ATP. It can also be used to
repair plasma and other membranes and also to remove damaged membrane components
and other hydrophobic toxic molecules. Clinical trials have shown the usefulness of MLR
supplements in reducing symptoms associated with loss of mitochondrial and other cellular
functions and improving the quality of life in patients with a variety of chronic illnesses
and normal age-related loss of function [
]. Recent efforts have focused on the effects
Membranes 2021,11, 944 26 of 39
of MLR supplements on reducing pain, gastrointestinal, and other symptoms, as well as
age-related functional loss. In these more recent studies, the recommended daily doses of
MLR supplements, such as NTFactor Lipids, have generally increased in amounts
(Table 1
In addition to the use of MLR in slowing or reversing functional loss in advanced age, MLR
supplements can also be useful at the earliest stage of life, for example, in enhancing sperm
motility, increasing fertility, and reducing the effects of oxidative stress on sperm and ova
membranes and protecting gametes during cryogenic storage [33,322].
An important new use of MLR GPL is the potential of MLR to remove damaged
lipids and toxins by an exchange process that is driven by bulk flow or mass action
principals. This process and its dependence on mass action have the potential to reverse the
presence of damaged GPL in organs, tissues, cells, and cellular membranes by exchange and
transport to the lymph and blood circulation and eventual deposition in the gastrointestinal
system and removal in stool [
]. The exchange and replacement of GPL in cellular
membranes releases the damaged GPL, and after partitioning in lipid globules, lipid
vesicles, chylomicrons, and lipoproteins, and exportation from cells into the circulation,
the partitioned GPL can be transported by lipoproteins, blood cells, and other carriers and
deposited in the gastrointestinal system. Eventually, the damaged GPL can be eliminated
by excretion in stool. This mass action process explains the usefulness of MLR in the slow,
bulk flow removal of molecules such as oxidized GPL, oxidized cholesterol, and oxidized
fragments of other hydrophobic molecules as well as toxic, fat-soluble chemicals from
hydrophobic cellular stores and their elimination.
The use of MLR phospholipids for repairing and replacing membrane GPL and
returning oxidized GPL and cholesterol to the gastrointestinal system appears to be dose-
dependent and requires some time to accomplish. Cross-over clinical trials have suggested
that the use of MLR supplements needs to be continued as a long-term strategy for support
of chronic diseases (or age-related membrane damage), as temporary changes due to MLR
supplementation can be slowly reversed [
]. Even in cases of hydrophobic toxic insult
or removal of hydrophobic toxins that have been partitioned into membranes and other
hydrophobic centers, MLR administration must be a slow, long-term process, as mobilizing
and removal of toxic molecules too quickly can result in symptomatic effects. Thus, the
removal of deeply embedded toxic molecules must occur at a slow, steady pace that could
require many months or even years of MLR supplementation to accomplish. However,
some uses of MLR may not take more than several minutes, such as the ex vivo treatment
of sperm or ova.
Some benefits of MLR may not be related to removal of GPL, but instead are likely to
be related instead to simple lipid substitution in cellular membranes, such as modulation
of ion channels in membranes by MLR supplements to reduce pain and other symptoms.
For example, exchanged GPL can modulate the functions of many, if not most cell surface
ion channels, transporters, and receptors [
]. Among numerous possible membrane
channels, voltage-gated calcium channels, as well as intracellular calcium channels are
particularly important in nerve cells in maintaining trans-membrane potentials and home-
ostasis [
]. Such effects of GPL may explain the effects of MLR on pain and other
symptoms [214,215].
MLR may also be useful as an adjunct to more traditional drug approaches, either
to enhance cell and tissue absorption or increase circulatory transport of drugs, modify
cellular energy requirements, or change drug and messenger membrane interactions [
The design of new MLR formulations, and especially the use of MLR phospholipids in
combination with other health supplements, will prove to be extremely useful for future
specific health uses.
Author Contributions:
G.L.N. drafted the manuscript and the other authors contributed writing and
editing. All authors have read and agreed to the published version of the manuscript.
Membranes 2021,11, 944 27 of 39
G.L.N. acknowledges support from the Institute for Molecular Medicine and Nutritional
Therapeutics, Inc. G.F.d.M. has support from Research and Development Funds CSIC 91 and
137 from Universidad de la Republica, Montevideo. P.V.E. acknowledges support from the European
Commission (H2020 Framework Programme Project CLINGLIO 755179-pve) and the Marathon
Foundation (Grant MF2018-pve).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank John Michael for artwork assistance.
Conflicts of Interest:
G.L.N. and R.S. are part-time consultants to Nutritional Therapeutics, Inc. No
other possible conflicts of interest are reported.
ABR auditory brainstem responses
AD Alzheimers disease
AGEs advanced glycation end products
CAPD chronic ambulatory peritoneal dialysis
CFS chronic fatigue syndrome
CL cardiolipin
CoQ10 coenzyme Q10
CVD cardiovascular disease
DAG diacylglycerol
DAMPs damage associated molecular patterns
DHA docosahexaenoic acid
EPA eicosapentaenoic acid
EPL essential phospholipids
ETC electron transport chain
FA fatty acid
FDA US Federal Drug Administration
GPL glycerolphospholipids
GRAS generally recognized as safe
HDL high density lipoproteins; HNE 4-hydroxynonenal
IL interleukin
MDA malondialdehyde
MetSyn metabolic syndrome
MIM mitochondrial inner membrane
MLR membrane lipid replacement
mPTP mitochondrial permeability transition pores
mtDNA mitochondrial DNA
NCD non-communicable diseases
NF-κB nuclear factor kappa B
PC phosphatidylcholine
PE phosphatidylethanolamine
PG phosphatidylglycerol
PI phosphatidylinositol
PS phosphatidylserine
RNS reactive nitrogen species
ROS reactive oxygen species
TNFαtumor necrosis factor alpha
T2D type 2 diabetes
Membranes 2021,11, 944 28 of 39
Nicolson, G.L.; Ash, M.E. Membrane Lipid Replacement for chronic illnesses, aging and cancer using oral glycerolphospholipid
formulations with fructooligosaccharides to restore phospholipid function in cellular membranes, organelles, cells and tissues.
Biochim. Biophys. Acta Biomembr. 2017,1859, 1704–1724. [CrossRef]
Nicolson, G.L. Membrane Lipid Replacement: Clinical studies using a natural medicine approach to restoring membrane function
and improving health. Intern. J. Clin. Med. 2016,7, 133–143. [CrossRef]
Nicolson, G.L.; Rosenblatt, S.; Ferreira de Mattos, G.; Settineri, R.; Breeding, P.C.; Ellithorpe, R.R.; Ash, M.E. Clinical uses of
Membrane Lipid Replacement supplements in restoring membrane function and reducing fatigue in chronic illnesses and cancer.
Discoveries 2016,4, e54. [CrossRef]
4. Escribá, P.V. Membrane-lipid therapy: A new approach in molecular medicine. Trends Mol. Med. 2006,12, 34–43. [CrossRef]
Ibarguren, M.; López, D.J.; Escribá, P.V. The effect of natural and synthetic fatty acids on membrane structure, cellular functions
and human health. Biochim. Biophys. Acta 2014,1838, 1518–1528. [CrossRef] [PubMed]
Gundermann, K.J. The “Essential” Phospholipids as a Membrane Therapeutic; European Society of Biochemical Pharmacology:
Szcecin, Poland, 1993.
Küllenberg, D.; Taylor, L.A.; Schneider, M.; Massing, U. Health effects of dietary phospholipids. Lipids Health Dis.
,11, 3.
Edidin, M. Lipids on the frontier: A quarter century of cell-membrane bilayers. Nat. Rev. Mol. Cell Biol.
,4, 414–418.
Tarnopolsky, M.; van Meer, G.; Voelker, D.R.; Feigenson, G.W. Membrane lipids: Where they are and how they behave. Nat. Rev.
Mol. Cell Biol. 2008,9, 112–124.
Ibarguaren, M.; López, D.J.; Escribá, P.V.; Tekpli, X.; Holme, J.A.; Sergent, O.; Lagadic-Gossmann, D. Role for membrane in cell
death: Implication for health and disease. Toxicology 2013,304, 141–157.
Alvarez, R.; López, D.J.; Casas, J.; Liadó, V.; Higuera, M.; Barceló, M.; Busquets, X.; Escribá, P.V. G protein-membrane interactions:
Gai1 myristoyl and palmitoyl modifications in protein-lipid interactions and its implications in membrane microdomain
localization. Biochim. Biophys. Acta 2015,1851, 1511–1520. [CrossRef] [PubMed]
Escribá, P.V.; González-Ros, J.M.; Goñi, F.M.; Kinnunen, J.; Vigh, L.; Sánchez-Magraner, L.; Fernández, A.M.; Busquets, X.;
Horváth, I.; Barceló-Coblijn, G. Membranes: A meeting point for lipids, proteins and therapies. J. Cell Mol. Med.
,12, 829–875.
13. Knight, J.A. Diseases related to oxygen-derived free radicals. Ann. Clin. Lab. Sci. 1995,25, 111–121. [PubMed]
Gutteridge, J.M.C. Free radicals in disease processes: A compilation of cause and consequence. Free Rad. Res. Commun.
141–158. [CrossRef]
15. Harman, D. Aging: A theory based on free radical and radiation chemistry. J. Gerontol. 1953,11, 298–300. [CrossRef] [PubMed]
Aruoma, O.I.; Kaur, H.; Halliwell, B. Oxygen free radicals and human diseases. J. Royal Soc. Health
,111, 172–177. [CrossRef]