Focus: The Cell Membrane

The membrane of the cell and the organelles are composed of two fatty acid tails facing each other. The bilipid layer is minute in comparison to its vital role as cell protector with a thickness of 3 to 4.5 nm. It would take 10,000 membranes layered on top of each other to make up the thickness of a piece of paper. The dynamics that occur inside this tiny organic sliver is a microcosm of supportive cytoskeleton microtubules complete with internal roadways and has fostered many hypotheses, one of the most interesting from Stuart Hameroff (Hameroff et ai, 2002). Joining with Roger Penrose, Hameroff orchestrated an objective reduction model suggesting a cognitive role conveyed on the inside, the hole in the center of microtubule structures, which is -10 nm and which acts as a quantum wave carrier of cellular information. Hameroff, an anesthesiologist and professor at the University of Arizona at Tucson, describes dynamic activities within every cell which are regulated by the cell cytoskeleton, particularly microtubules, which are cylindrical lattice polymers of the protein tubulin. Recent evidence indicates signaling, communication and conductivity in microtubules. There is a marriage between the soft flexible membrane and the structural rigidity of the microtubules. Not so within the body of the cell but significantly as the cell tries to extend its reach out into the outside world seeking nourishment in the gut using cilia, seeking oxygen in the trachea and lungs with micro-villi, and signaling in the brain with a vast array of dendrites. All are similarly constructed principally of membrane and microtubules.

Assessing the cell membrane through the examination of red cell lipids can lead the clinician into a deeper level of understanding of metabolic strategies to influence treatment outcome in a wide range of degenerative disorders. Essential (EFAs) and non-essential lipids are incorporated into the bilipid layer of the membrane of every cell in the body and brain. There is virtually no system of the body that does not require attenuation of specific fatty acid substrates and coenzymes to maintain health and repair of bodily tissues. The human cell membrane must be continually fed with the correct lipid substrates to enable the organism to function ideally, yet fatty acid metabolism has been poorly delineated in treatment protocols. Exploration of lipid metabolism brings a striking new intervention that unlocks the systemic nature of disorders as well as an exquisite capacity to impact the brain architecture.

The membrane of every cell and organelle is a lipid envelope that encases and protects the internal working cellular components. The bilipid layer is far more than isolation and protection, for linked and interlocked within the membrane are literally thousands of proteins (peptides) that are the windows and doors of the cell. They form the gates for ingress and egress; the multitudinous array of receptors and ion channels that perform the vast metabolic functions of life. In addition, select lipids, the Eicosanoids, after set free from the membrane, metabolize up to an intercellular communication and information system through their prostaglandin regulatory activity. Prostaglandins, thromboxanes, and leukotrienes may have evolved to be the basic information broadcasting and control mechanism that permitted metazoa, the grouping of cells, which we are, to advance to our present level. The mere thought of multi-cellular activity, and especially the evolution of humankind is, at our present level of knowledge, not possible without essential fatty acids (EFAs), which are the precursors to the regulatory prostaglandins, which provide cell to cell communication and basic molecular initiation. Before one can advance beyond protozoa, or a single cell organism, into multi-cell metazoa, there must be both communication and a means of regulation. This is the world of the prostaglandins (PGs), the “local hormones” that control the interactions without which there is no complex life form.

This powerful role that lipids play is of prime importance for the clinician to understand metabolism. One of the greatest biochemical advances came about with the understanding of the energy cycle for plants and animals. Looking at oxidative energy through a lipid lens can help to adjust our view of their importance. Peter Mitchell’s earlier research on membrane transport led him towards a construct that explained oxidative phosphorylation (Mitchell 1961). Mitchell realized that the bilipid membrane would have to be a key factor in any hypothesis to explain that substances (hydrogen ions) were moved from one side of a membrane to the other to accumulate potential energy.

In his chemiosmosis theory, Mitchell proposed that the movement of electrons down the chain of oxidation chemistry results in the translocation (shifting) of protons (H-ions) from one side of the membrane to the other, (Mitchell 1963). In essence, the hydrogen atom is separated with the electron moving down the chain on the inside of the membrane while the H-ion is shuttled to the outside using the membrane as an insulator for a momentary separation. In his hypothesis, Mitchell stressed the importance of the spatial arrangement of the various carriers within the energy-transducing membrane. Spatial arrangement refers to the positioning of the molecules involved that sit on and span the membrane and carry out the chemistry. He suggested that those carriers that bind both electrons and protons must be facing on the inside of the membrane, while the carriers facing the outside would only accept electrons, leaving the protons to accumulate resulting in an electrochemical gradient. The accumulation of ions (hydrogen protons) is then the motive force required to link substrate oxidation to phosphorylation and drive the ATP energy cycle.

After 15 years of controversy and experimentation, mostly designed to prove him wrong, Mitchell’s hypothesis coupling oxidation to phosphorylation earned him a Nobel Prize. The awarding of this Nobel Prize was probably the most significant in Nobel history since the hypothesis, now universally accepted, explains the creation of energy for life in plants and animals.

The details of oxidation chemistry is not a prerequisite for clinicians. Every cell biology text covers the subject of oxaloacetate to citric acid to C02 and H20 with the ultimate end product of phosphorylation of ADP to ATP, (Karp 1999). The Krebs or TCA (citric acid) cycle is an example of cellular beauty in the creation of energy for the biochemistry of life. The elegance however, lies in the lipid membrane that embraces the enzymes, manipulating them to their most optimal position to allow it all to occur. The preciseness of the various peptides and lipids and their relation to one another is critical for the chemistry to play out.

The significant role of the membrane and the lipids is hidden in the details of the chemistry. The high energy phosphate heads and their lipid tails provide the structure for the trans-membrane peptides (carriers), which are positioned in that lipid membrane sea in correct juxtaposition to carry out the energy production, most of which is little understood. The membrane is one of the most elegant structures in the universe. The lipids themselves are one of the smallest molecules in biochemistry, which may contribute to their mystery, and possibly the reason for their late prominence in biochemistry. In comparison, proteins and nucleic acids are much larger and more photographable, which often provides important clues as to their function. The most famous example is when Watson snuck a look at Rosalind Franklin’s photographs which led to the discovery of DNA. Not the most honorable event but certainly an important one. Lipids however are too small with frequencies too high to capture. Their performance lies in their enormous numbers and their resonance for communication and metabolic influence which moves us into the world of Quantum Mechanics.

Without the view of the lipid membrane as the insulator that permitted charge separation and the transfer of ions to the opposite side of the membrane from the site of the actual oxidation, Mitchell, or anyone else, could not have developed the TCA cycle hypothesis. All of the TCA cycle chemistry, as well as much of metabolism in both plants and animals, occurs on one or both sides of the membrane, be it in the cell, the mitochondria, the ER, the golgi, the nuclear membrane, or the vast neuronal network of the body and the brain. All thought, all sensory transmission, and all motion, involves the lipid membrane which carries the signals and information.

Mitochondria are tiny energy organelles often described as miniature power plants for the TCA energy producing cycle. There are -200-500 per cell, with 10,000 or more in a heart myocyte. The citric acid chemistry occurs on the inside of the second bilipid membrane (there are 2); with the space between the first and second layer (intermembrane space) the collection area of the hydrogen ions. Oxidation produces the separation of ions, which are accumulated and guided to ATP synthase, the enzyme responsible for phosphorylation of ATP. The hydrogen ions are passed back into the matrix by the ATP pump (ATP synthase) sitting on the inside membrane, after which they combine with oxygen (with H20 as a byproduct).

Both of the bilipid membranes of mitochondria are the typical “unit membrane” (railroad track) type in structure. Between the two membranes is the space where the H ions accumulate. Inside the 2nd membrane is the matrix, which appears moderately dense and one may find strands of DNA, ribosomes, or small granules. The outer and inner membranes have very different properties. The outer membrane is composed of approximately 50 percent lipid by weight. In contrast the inner membrane contains more than 100 different polypeptides and has a very high proteinllipid ratio, more than 3: 1 by weight, which corresponds to about one protein molecule for every 15 phospholipids, (Karp 1999). The outer membrane has porins, which permit / accommodate molecules up to - 5000 Daltons for the passage of ATP, NAD, and coenzyme A. The inner membrane however is highly impermeable; virtually all molecules and ions require special transporters situated in the inner membrane space to facilitate entrance through the inner membrane to the matrix, i.e. L-carnitine for the transport of long chain fatty acids.

In addition there is a phospholipid peculiar to the inner leaflet of mitochondria that is found nowhere else in the body called cardiolipin (CL). As the drawing shows CL is a joining of the head groups of two phospholipids. It looks something like Siamese Twins --- actually it is one. The two head groups are chemically linked together at the sides of the head groups. It has the compulsory two lipid tails on each PL and can be positioned comfortably in the membrane but now with the odd connection contains four lipid tails instead of two. CL is found only in the inner matrix of the mitochondria, nowhere else, and appears to have one specific function, that of hindering, slowing down the high activity level characteristic of all normal active membrane PLs.

Because of its bulky shape its activity level would be severely restricted. Active PLs are in constant motion including rotation. Placing a bulky CL strategically could restrict normal PL movement. Placed in the right spot with its inability to spin as most all PLs do --- could, quite possibly --- be the restricting agent that the inner mitochondrial membrane needs to fix the citric acid participants in juxtaposition and keep them steady as they perform the critical electron handoff. (PIC)

Normally, the accurate positioning of the necessary proteins within that crowded lipid sea involve a combination of phospholipids with a high concentration of double bonds (think high energy). The high frequency character of the double bonds guarantees the energy required to perform the accurate peptide positioning. There is also a wide assortment of many other proteins as well as the ones involved in the chemiosmotic production of energy. The precise arrangement of those proteins is not known, however, the requirement for a tight control and still maintaining fluidity is paramount and the main element lost with age and disease.

The membrane surrounds and protects every cell of every organ including the tissues of the heart and the neurons of the brain. It is a remarkably thin insulator, the protective outer skin, with a carbon copy duplicated over and over surrounding the tiny organelles inside each cell. Bruce Lipton puts the lipid cell membrane in perspective, in his book ‘The Biology of Belief’ when he compares the function of the membrane to the DNA, currently the darling of medical science. Dr. Lipton calls the DNA the gonads of the cell and compares it to the hard drive of a computer, while comparing the lipid membrane to the keyboard. He describes the DNA as a storehouse of information, a personal library housing a pattern (copy) of every protein molecule specific to each of us. The DNA of each cell contains a duplicate set of genetic instructions for the development and function of every organ of our body.

However, the concept we are led to believe is that we are controlled by our genes, by our hard drive, but this is inaccurate. The DNA, like a central processor, is a library of information. A cell can actually exist for a few weeks or even months without its DNA. Lipton performed this in his lab with cells in a petri dish. By surgically extracting the contents of the nucleolus, the DNA, and continuing to provide nourishment, the cell could last for several months. The same does not occur with a fractured membrane, the cell dies instantaneously without it’s outer coat. It’s a bit sacrilegious to call our libraries dumb, but they are certainly not smart. Some form of energy must exist to extract the information from the library and direct it to perform a necessary function. Even though the DNA holds the program for the production of all the intricate proteins we need, it acts exactly like a library. It holds all the intelligence, but initiates no activity.

The membrane is continuously collecting pertinent information from the outside world sending instructions inward to the cytosol and the DNA. In part, it is both in position to collect knowledge and equipped with the energy component within the essential fats to initiate and direct cellular activity, the details of which is yet unknown. Dr. Lipton says, as the keyboard, we should refer to it as our mem-Brain --- and, since it is -70% fatty acids, our lipid membranes now take on a whole new level of importance.

Renowned lipid researcher Michael Crawford defines the dry weight of the human brain as 60% lipid, with the dendrites and synapses up to 80% composed predominantly of the Highly Unsaturated w6 and w3 Fatty Acids (HUFAs). Phospholipids, cholesterol, cerebrosides, gangliosides and sulfatides are the lipids residing within the bilayers in the brain (Bazan et aI., 1992). The phospholipids and their fatty acid tails provide second messengers and signal mediators (Schachter et aI., 1983). Those phospholipids play a vital role in the cell signaling systems in the neuron (Rapoport, 1999). The functional behavior of neuronal membranes depend largely on the ways in which individual phospholipids are aligned and interspersed with both cholesterol and the necessary functional proteins. Neurotransmitters are wrapped up in phospholipid vesicles waiting for the right moment of release. The release and uptake of the neurotransmitters is dependent upon the realignment of the phospholipid molecules containing a high concentration of n-3 DHA. The energetic nature of the phospholipids is a vital factor in determining how efficiently the neurotransmitters are delivered since the violent expUlsion at the synaptic cleft can only be accomplished efficiently with a ready supply of the highest energy lipid, DHA. Re-modeling of the phospholipids may be accomplished by supplying both oral and intravenous phosphatidylcholine with a balance of the w 6 and w 3 oils at the preferred ratio of 80% w 6 to 20% w 3 (Yehuda 1993).

One of the most important biochemical changes regarding aging is a change in membrane phospholipid composition. Phosphatidylcholine (PC), is the predominate head group PL in the outer leaflet of the membrane, which as discussed above, is composed of two phospholipid groups opposing each other. PC also tends to incorporate a predominance of HUFAs, especially arachidonic acid (AA) on the Sn2 position, thus the outer leaflet is composed of a grouping of higher energy lipids than the inner leaflet. This varies with the curvature demands of the membrane since the tighter the curve would necessitate the preference for a smaller head group i.e. PC over PE, since PC has a larger dimension. In mammalian plasma membranes, the main variation occurs in the relative composition of phosphatidylcholine (PC), and both sphingomyelin (SM) and cholesterol. PC decreases with age while SM and cholesterol increase with age (Schacter et aI., 1983). The impact of this shift in the outer membrane is difficult to envision. It involves every cell of the body. Every sensory neuron, touch, smell, taste, sight, hearing; skin, blood cells, brain neurons, endothelium, alveoli, immune cells, bone cells, etc. It involves the organelles within the cell such as the mitochondria the peroxisomes and the nuclear membrane. The concept of aging and PC decline is a dramatic shift in the body’s homeostatic ability.

The changes in the relative amounts of PC and SM are especially great in tissues, which have a low phospholipid turnover. For example, plasma membranes associated with the aorta and arterial wall show a 6-fold decrease in PC/SM ratio with aging. SM also increases in several diseases, including atherosclerosis. The SM content can be as high as 70-80% of the total phospholipids in advanced aortic lesion (Yechiel et al.,1985), (Yechiel and Barenholz, 1985), (Yechiel et al.,1986), (Yechiel and Barenholz, 1986; Barenholz 2004), (Cohen and Barenholz, 1984). Both sphingomyelin (SM) and cholesterol are structurally similar to saturated fats. They are rigid, with the concommitment decline in fluidity and lower metabolic performance. The loss of those dynamic double bonds of the high energy lipids could be the major cause of the aging disease.

In 1985 Yechiel and Barenholtz, from Hebrew University, authored a significant paper highlighting phosphatidylcholine and its relationship to aging and disease (Yechiel 1985a.b, 1986; Muscona-Amir 1986). Using rat myocytes (heart cells) in a 20 day in vitro study, they demonstrated the ability of PC to completely rejuvenate cells that were all but expired. Heart cells can be separated in a dish in vitro, but with proper feeding within a few days, they self agglomerate (gather together) and beat in unison at a rate of - 160 beats per minute.

To demonstrate the importance of PC, they fed one group of cultures (the A Groups) egg yolk PC and continued to do so for the life of the experiment (20 days), while two other groups (the Bs and Cs) were deprived of PC and later had it added back into their feed.

The Group A cultures are represented with a straight line (green) at the top of the chart. They had been given egg PC after day 6 and for the entire 20 days they maintained a constant beating rate of - 160 beats/ min. Group B (Red) cultures were not as fortunate and were denied PC until day 16. The chart shows the result for after 6 days the B groups started to weaken and by day 8 began a precipitous decline in beating rate until day 12 wherein some of the B groups were only beating at - 20 beats/min. and others not beating at all. The Group Cs (Blue) were given PC as the A groups, but only to day 11, after which PC was removed from their feed. As you can see on the chart, almost immediately the Cs started a decline in their beating rate and mirrored the decline of group B with a 5 day drop to - 20 beats/min. In addition both Band C groups suffered a variety of cellular distortions in size and production of protein.

On day 16 all cultures, including groups Band C were given PC, and within 24 hours, the Bs and Cs recovered their beating rate to - 160 beats/min and continued so until the study was concluded at day 20. In addition, they also recovered most of their distorted chemistry. This was a remarkable demonstration of the power of phosphatidylcholine, or to be more precise, of the absolute necessity of it.

A medline search on ‘Phosphatidylcholine’ will reward you, or inundate you, with 37,471 citations. To review them would easily take a year or two, but it speaks volumes of the importance of PC. In all of our studies, we have yet to uncover a report as powerful as that of Yechiel and Barenholtz. However, there are two that are noteworthy. Tile review by Cui and Howeling, PC and Cell Death 2002, that focuses on the ability of PC to reverse a number of biochemical distortions and prevent cellular necrosis and / or apoptosis. Apoptosis is a controlled, regulated death, while necrosis is a rupture of membranes with the release of vital components into the surrounding blood stream. Cui et al presented their prior biochemical studies and many others demonstrating that perturbation of PC leads to cell death, and the subsequent replacement of PC reestablishes homeostasis.

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