COMPLEMENTARITY AND CONTRARIETY in Lipidomics

Majid Ali, M.D.


OUTLINE

I. Introduction

II. Lipidomics

III. Structure and Classification of Lipids

IV. Omega-3 and Omega-6 Fatty Acids

V. Metabolism of Lipids

VI. Phospholipids

VII. Lipid Ecosystems

VIII. The LDL-Oxidative Modification Hypothesis

IX. The Oxidative Model of HDL Dysregulation

X. Cholesterol as a Synapse Promoter

XI. Lipids in Biomembranes

XII. Eicosanoid Biology

XIII. Biology of Prostaglandins

XIV. Biology of Leukotrienes

XV. Drugs That Alter Eicosanoid Pathways

XVI. Endocytosis and Lipids

XVII. Attractive and Repulsive Interactions

XVIII. N-3 Fatty Acids and Sudden Cardiac Death

XIX. Lipids, Oxygen, and Obesity

XX. Lipid Nutrient Therapies

XXI. Summary

I. INTRODUCTION

The question that holds the most interest for me in the saga of Vioxx and Bextra—two highly profitable COX-2 inhibitors withdrawn from the market due to association with higher incidence of cardiovascular events—is this: Why should the use of putatively potent anti-inflammatory COX-2 inhibitors be associated with higher incidence of coronary heart disease and stroke, the disorders that are unequivocally caused by accelerated oxidative injury? As for me, this is but one reflection of Nature’s preoccupation with creating complementarity and contrariety in biologic systems. Clearly, COX-2 enzyme(s) must exert proinflammatory influences under certain conditions and serve antiinflammatory roles under others to account for the documented antiinflammatory consequences of their inhibition in joints and the proinflammatory effects of their inhibition in the cardiovascular system. Of course, COX-2 pathways are not the only instance in which biologic roles of lipids can be so contrarian. For instance, alpha-linolenic acid deficiency is associated with a broad range of symptoms that are relieved with appropriate supplementation. However, in experimental models, an excess of fatty acids is also associated with similar clinical manifestations. Indeed, such is Nature’s preoccupation with complementarity and contrariety that paradoxical and opposing biologic roles of lipids and lipoproteins are the norm rather than the exception in human biology.

Fats have been demonized in the Western countries. Poor understanding of the body energetics and the metabolic dynamics of sugars, proteins, and fats has led to the common—and mistaken—belief that natural (unoxidized and unrancid) fats are fattening. In clinical medicine, the interests of most physicians in those countries are confined to the potential roles of blood cholesterol and triglycerides in the pathogenesis of atherosclerosis. That compounds the problems caused by poor comprehension of the real issues involved in lipid homeostasis in the body. The regulatory influences of lipids on cellular development, differentiation, and demise are profound indeed.

Much time is devoted to the teaching of structure and function of the “lipid bilayer” in medical schools. By contrast, in clinical medicine little, if any, attention is paid to the crucial issue of oxidative damage to lipids of biomembranes and other cellular elements, nor to the dire clinical consequences of such oxidation denaturation. Nor is the important matter of employing lipids as nutrient therapies given due consideration. For decades the full focus has been on lowering the numerical value of cholesterol with pharmacologic agents for prevention and treatment of heart disease. The central issue of how to reduce oxidative stress on cholesterol—and other lipids—has been largely ignored. Atherosclerosis is initially triggered by free radical cascades that begin in the circulating blood and oxidatively modify lipids which, in turn, evoke endothelial responses that lead to damage to subepithelial tissues and plaque formation. At present, there is a great emphasis on lowering blood cholesterol with pharmacologic agents with the claim that the lower the cholesterol level, the greater the possibility of long, healthful life. Approximately twelve million Americans are taking those drugs. According to the current recommendations, 36 million Americans should be taking those drugs. The cholesterol enthusiasts seem to have little, if any, regard for myriad essential

physiologic roles of cholesterol, such as biosynthesis of several hormones, providing key assists to many membrane-associated and related proteins and cell-to-cell communication.

As in the case of chapters on glycomics and proteomics, a full discussion of the structures and functions of lipids is clearly outside the scope of this book. Chemistry texts, such as Medical Biochemistry1 by Baynes and Dominiczak, may be used for that purpose. For discussion of oxidative phenomena concerning lipids, I suggest Oxidology12 by Bradford and Allen. In this chapter, I focus on those aspects of lipids that illuminate the themes of molecular complementarity and contrariety in human biology.

II. LIPIDOMICS

Nature has been as preoccupied with its designs of complementarity and contrariety among lipids as it has been with those involving sugars and proteins. Lipids play as diverse and crucial roles in the various homeostatic mechanisms of human biology as do the members of the other two families. The central roles of phospholipids in preserving the structural and functional integrity of biomembranes have been recognized for decades. Those molecules also serve as “communicating molecules” for cells. But knowledge about the biologic roles of lipids is far from complete, and some additional aspects are being described literally every week. To cite an example especially appropriate for this subject, glial cells are necessary for neurons to develop functioning synapses. To that purpose glial cells produce heparin-binding apolipoprotein E (ApoE). What signaling molecule does Apo E employ to initiate and sustain synapse formation? This question remained unanswered until recently (November 9, 2001), when a report in Nature2 identified that factor to be cholesterol. I cite many other such examples in this and other chapters. Here, I introduce the term “lipidomics”—if it has not been already introduced by someone else—to give fats parity with sugars (glycomics) and proteins (proteomics).

III. STRUCTURE AND CLASSIFICATION OF LIPIDS

Lipids are a large group of organic molecules with the common characteristics of being water-insoluble and of being extractable from cells by fat solvents, such as ether, benzene, and other nonpolar solvent compounds. The lipids in the body are often categorized as follows: (1) neutral lipids or triglycerides; (2) phospholipids; (3) a group of miscellaneous lipids (including waxes); and (4) steroids. The basic moiety of the lipids in the first three categories is composed of straight-chain hydrocarbons containing carboxyl groups designated fatty acids. Fats are esters of the alcohol glycerol, with fatty acids accounting for most of the lipids in the body. Phospholipids are structurally similar to fats—they also contain condensates of glycerol with fatty acids—with the difference that they contain only two fatty acids and a phosphate group. Steroids are structurally distinct from the other three types of lipids, with the following important differences: (1) they contain three fused cyclohexane and one cyclopentane ring structure; and (2) many of them contain one, two, or three double bonds. Waxes are esters of long-chain fatty acids and monohydroxy alcohols. These lipid compounds are solid at ambient temperatures, but melt readily. They form protective waterproof coatings on surfaces of human and animal cells, as well as the skin of leaves and fruits. Ear wax and beeswax are two better-known examples.

Cholesterol does not contain fatty acids, but its sterol nucleus is derived from degradation products of fatty acids and hence shares many common characteristics of lipids. In the postabsorptive state, nearly 95 percent of lipids exist bound to proteins.

Figure 1. Condensation Reaction of Glycerol and Three Molecules of Oleic Acid

to Form Triglyceride

478 Tietz

 

There are three main types of lipoproteins: (1) very low-density lipoproteins that contain large amounts of triglycerides and smaller quantities of phospholipids and cholesterol; (2) low-density lipoproteins that contain a much higher percentage of cholesterol and scant triglycerides; and (3) high-density lipoproteins that contain nearly fifty percent protein. Strictly speaking, chylomicrons must also be considered as a fourth type of lipoprotein. Triglyceride, the most common fat in the body, is formed by condensation of glycerol with three molecules of oleic acid, as shown in Figure 1.

IV. OMEGA-3 AND OMEGA-6 FATTY ACIDS

Omega-3 and Omega-6 are structurally similar long chain, polyunsaturated fatty acids of great significance to chemists interested in human nutrition as well as to nutritionist physicians. The terms of Omega-3 (n3) and Omega-6 (n6) refer to the location of the first carbon-carbon double bond in the molecule, counting from the end of the carbon chain opposite the acid group. The biologic activities of Omega-3 and Omega-6 change dramatically with seemingly minor changes in their structure. For example, the biologic activities of eicosapentaenoic acid (EPA, an Omega-3 oil) are very different from those of arachidonic acid (AA, an Omega-6 oil), while the structural difference between the two involve just two hydrogen atoms (Figure 2). A double bond in fatty acids may be visualized as creating a kink in the carbon chain that permits many possible altered configurations which, in turn, profoundly alter their chemical characteristics as well as their biochemical activities. For example, double bonds seem to alter the molecular mobility and melting points. Thus, monounsaturated and polyunsaturated oils are liquid at ambient temperature—fluid and alive, but vulnerable to degradative influences, so to speak—and solidify readily at cooler temperatures. Examples include fish and olive oil, which are liquid at room temperature. The saturated fatty acids, by contrast, are solid—stable, with long shelf-lives—at ambient temperatures. Indeed, this explains why the oil industry hydrogenates oils—in margarines and some vegetable oils—to enhance profitability with longer shelf-life at the cost of adverse nutritional aspects. Tropical oils—such as coconut and palm oil—are rich in saturated fatty acids, and hence are not desirable from a nutritional standpoint.

The n3 family of fatty acids includes the following :

* EPA: eicosapentaenoic acid (20:5n3).

* DHA: docosahexaenoic acid (22:6n3 /carbon-long, six double bonds)

* DPA: docosapentaenoic acid (22:5n3)

EPA (produced from alpha-linoleic acid or ALA) is the parent lipid for the generation of the 3-series prostanoids and leukotrienes that moderate the proinflammatory influences of the 2-series derived from AA. The generation of EPA from ALA requires delta-6 desaturase (which requires magnesium, zinc, vitamins B3, B6, and C). Conversion of ALA is inhibited by trans fatty acids, as well as highly saturated (even monounsaturated) fatty acids. Good sources of essential alpha-linolenic acid (ALA, 18:3n3) include flax, hemp, canola, soybean, walnut, and dark green vegetables.

The n6 family of fatty acids includes the following:

* Linolenic acid, LA (18:2n6)

* Gamma linolenic acid, GLA: (18:3n6)

* Dihomogammalinolenic acid, DGLA

(18:3n6)

* Arachidonic acid, AA (18:4n6)

* Docosatetraenoic acid, DTA (22:4n6)

* Docosadienoic acid (22:2n6)

Linolenic acid, an essential acid, is the most abundant polyunsaturated fatty acid in human biology. The human fatty acid synthetic pathways cannot generate LA because the double bond at the n6 position is beyond the reach of the human desaturase. Deficiency of LA can have far-reaching effects, since it is the starting point of 1-series eicosanoids. True to its theme of complementarity and contrariety, Nature has also built in a mechanism for tissue injury if LA concentrations rise beyond the physiologic limits. Thus excessive amounts of LA are also associated with overproduction of proinflammatory 2-series local moieties.

GLA is produced from LA by the action of desaturase. In turn, it is the precursor of DGLA (the parent of 1-series prostanoids), as well as the precursor of AA (the parent of 2-series prostanoids). Good sources of this oil in clinical nutrition include black currant, evening primrose, borage, and hemp.

Saturated Fatty Acids

The following is the list of saturated fatty acids:

* Capric acid (10:0)

* Lauric acid (12:0)

* Myristic acid (14:0)

* Palmitic acid (16:0)

* Stearic acid (18:0)

* Arachidonic acid (20:0)

* Behenic acid (22:0)

* Lignoceric acid (24:0)

* Hexacosanoic acid (26:0)

Monounsaturated Acids

The following is the list of monounsaturated fatty acids:

* Myristoleic acid (14:1n5)

* Palmitoleic acid (16:1n7)

* Vaccenic acid (18:1n11)

* Oleic acid (18:1n9)

* Erucic acid (22:1n9)

* Nervonic acid (24:1n9)

Trans Fatty Acids

Elaidic acid is the most common trans fatty acid in the standard American diet. A partial list of trans fatty acids is given below:

* Elaidic acid (18:1n9t)

* Palmitoleic acid (16:1n9t

* Petroselaidic acid (18:1n12t)

* Trans-vaccenic acid (18:1n6t)

Trans fatty acids are created in the process of dehydrogenation that are even more stable. However, trans structural configuration is quite distinct from the natural cis fatty acid chain and seemingly “clogs” the physiological lipid pathways (Figure 2). The biologic cost of adding such synthetic components to natural oils is a greater risk to the structural and functional integrity of biomembranes and other cellular organelles, as demonstrated by higher risk of ischemic coronary artery disease. It may be added here that fat in wild game contains nearly three times as many polyunsaturated fatty acids as fat in the species raised for commercial use.

Fish, marine mammals, and other aquatic species, as well as some uncommon plants, are rich sources of Omega-3 oils. An example of the latter source is purslane, a spinach-like plant, that was a favorite of physicians of the Hippocratic school for neurologic, cardiovascular, and gastrointestinal disorders. Omega-6 oils are found in corn, canola, flaxseed, and other oils. The importance of Omega-3 oil was demonstrated in experiments in which monkeys—but not rats and mice—fed a diet that exclusively contains corn oil (rich in Omega-6 essential fatty acid linoleic acid) developed dermatitis, diarrhea, dementia, and other disorders. The health of the ill monkeys was restored within weeks of flaxseed oil supplementation as a source of Omega-3 oils.

The available evidence suggests that there was a preponderance of Omega-3 fatty acids over Omega-6 fatty acids in the diet during the early evolutionary period of Homo sapiens. The ratio of Omega-3 to Omega-6 fatty acids in the indigenous diets of the Alaskan Eskimos and Australian Aborigines is much higher than in the standard American diet. There are little, if any, diabetes and cardiovascular disorders in Eskimos and Aborigines in their native environments.

It has also been estimated that the supply of Omega-3 has dwindled significantly during several decades due to changes in agriculture, as well as methods of food processing, with significant adverse effects. Such changes in the fatty acid supply in the diet were accompanied by steep rises in the incidence of diabetes, cardiovascular disorders, and many degenerative disorders. It is notable that there is a direct competition between Omega-3 and Omega-6 fatty acids for enzymes involved in the desaturation and delongation steps. Thus, chronic excess of one type of acid can be expected to stress and interfere with the metabolic pathways of the other type. For example, higher levels of Omega-6 fatty acids lead to greater production of vasoconstrictive thromboxane A2, while excess of Omega-3 fatty acids results in the production of larger quantities of vasodilatory thromboxane A3.

There is a near-complete agreement among the clinical nutritionists that the average American diet carries a suboptimal n-3 to n-6 ratio. However, there is divergence of opinion about how that ratio might be optimized—whether by increasing n-3, reducing n-6, or both. Increasing the n-3 content by increasing fish and other marine species is most desirable, though it creates issues of freshness and cost for many persons. Most nutritionists prescribe liberal flaxseed supplements to achieve that objective. N-6 fatty acids are known to reduce the risk of ischemic coronary artery disease, and deliberate attempts to reduce their dietary content seem open to question. Of course, an important step that everyone should agree upon is to replace hydrogenated oils with natural liquid oils having a rich content of mono- and polyunsaturated fatty acids. Major sources of linoleic acid include soy, corn, sunflower and safflower oils, whereas flaxseed oil and walnut oil contain a substantial amount of alpha-linoleic acid.

The biology of prostaglandins and leukotrienes derived from Omega-3 and Omega-6 oils is presented later in this chapter.

Figure 2. Representations of saturated and unsaturated (trans and cis forms) fatty acids are illustrated. Note the shortest and most

“bent”shape of cis form.

435 Teitz

 

The Odd-Numbered Fatty Acids

Propionic acid (three-carbon) is a normal metabolic intermediate that can take the place of the two-carbon acetyl group in the first step of fatty acid synthesis. Biotin is required for that step. The odd-numbered fatty acids are produced in smaller amounts in the body largely because the supply of two-carbon precursors is much larger. Acetyl-CoA, of course, is generated from carbohydrates, proteins, and fatty acids.

In biotin deficiency, conversion of acetyl-CoA into malonyl-CoA is diminished, allowing propionic acid to enter fat synthetic pathways in larger amounts and to occur in human plasma phospholipids in that deficiency state (Figure 3).

The Triene/Tetraene (T/T) Ratio

The ratio of Mead acid (20:3n9 to AA (20:4n6) is used as an indication of essential fatty acid deficiency. The red cell stearic/oleic index reflects the efficiency of desaturase enzyme. In cancer, stearic acid is converted into oleic acid at a faster rate, thus decreasing the ratio (as well as membrane fluidity). The ratio rises with tumor regression.

Figure 3. Generation of Odd-Numbered Fatty Acids in Vitamin Deficiency

Acetyl-CoA Propionate

Biotin

Malonyl-CoA Methylmalonyl CoA

B12

Succinyl CoA

Even-Numbered Odd-Numbered

Fatty Acids Fatty Acids

V. METABOLISM OF LIPIDS

Except in the brain and erythrocytes, lipids are the major source of energy in the body. Triglycerides are the principal form of transport and storage of fats, whereas fatty acids are the immediate source of energy. Fatty acids are released from adipose tissue and are transported in loose association with albumin to other tissues where they achieve equilibrium for cellular metabolism. Catabolism of fatty acids is entirely oxidative. Once within the cytoplasm of cells, oxidative breakdown of fatty acids proceeds both in mitochondria and peroxisomes, mainly by a cycle of reaction designated as â-oxidation. In this process, carbon atoms are released in pairs from the carboxyl end of the fatty acids to produce acetyl coenzyme (CoA) and reduced forms of nucleotides, flavin adenine dinucleotide (FADH2) and nicotinamide adenine dinucleotide (NADH). In the liver, acetyl CoA is metabolized via the tricarboxylic acid (TCA) cycle and oxidative phosphorylation to produce ATP. In the muscle, acetyl CoA is largely shunted to produce ketones—water-soluble lipid derivatives that are exported to other tissues for further breakdown. Unsaturated status of fatty acids means they are partially oxidized. Thus, their oxidation yields less FADH2 and correspondingly less ATP. There is also another important difference that affects the fatty acid energetics: The intermediates of â-oxidation have a cis geometry, while the double bonds of polyunsaturated fatty acids have a trans geometry and occur at three-carbon intervals. Thus, the metabolism of unsaturated fatty acids requires several additional enzymatic steps—at some energy costs—to change the geometry of double bonds and to shift their position.

Following are the major stages of lipoproteins:

1. Assembly: Chylomicrons are assembled in the gut and VLDL in the liver.

2. Triglyceride off-loading: Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) are the two major enzymes involved at this stage. The resulting particles are called “remnants.”

3. Processing of remnants: Cellular receptors are involved in binding, internalization, and breakdown of remnants.

4. LDL generation: Some remnants are transformed into LDL, which is then bound to its receptors and internalized.

5. Component exchange: Lecithin cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) are the enzymes involved in exchange of components between various remnant particles.

During fasting and starvation, the liver metabolizes fatty acids to produce energy and for gluconeogenesis. Under those two conditions, the concentrations of lipid-derived ATP and NADH rises sharply in hepatic mitochondria. That inhibits isocitrate dehydrogenase reaction and affects the malate shuttle, shifting the oxaloacetate-malate equilibrium. (See chapter entitled “Oxidative-Dysoxygenative Model of Insulin Dysfunction” for further details.) Concurrently, amino acids are released from muscle to cope with the energy deficit associated with those two conditions. Certain intermediates of the Krebs cycle are produced from such amino acids and are also converted into malate, increasing the malate supply. Finally, malate leaves mitochondria to participate in gluconeogenesis. An adverse effect of those changes is that a relative deficiency of oxaloacetate so produced in mitochondria inhibits the Krebs cycle, resulting in failure to process acetyl CoA in the cycle.

Lipid metabolism is primarily regulated by the rate of triglyceride hydrolysis—also called lipolysis—under a variety of hormonal influences. This subject is discussed further in the chapter entitled “Beyond Insulin Resistance: Oxidative-Dysoxygenative Model of Insulin Dysfunction.”

VI. PHOSPHOLIPIDS

An enormous variety of phospholipids is found in nature with wide structural and functional diversity, involving both polar and apolar moieties of lipids.3-7 To underscore that richness, I include here an incomplete list of the major biologically significant phospholipids: phosphatidic acid, phosphatidylcholine, phosphatidyl- ethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylsulfocholine, lysophosphatidylcholine, lysophosphatidyl-ethanolamine, lysophosphatidylserine, lysophosphatidylinositol, lysophosphatidylglycerol, phosphoryl-N,N-dimethylethanolamine, and sphingomyelin. In biosynthesis of phospholipids, fatty acids are modified in the following four ways:

1. desaturation;

2. chain elongation;

3. chain shortening; and

4. reduction to fatty alcohol.

In mammalian cells, fatty acid desaturation is oxygen-dependent. Double bonds are introduced at one or more of the positions nine, six, five, and four from the carboxyl end of a fatty acyl-CoA thioester. Membrane-bound enzymes catalyzing those reactions are named as follows: 9 , 6, 5, and 4 desaturases respectively. It may be pointed out here that in some bacterial species, such as E. coli, formation of polyunsaturated fatty acids is oxygen-independent. Chain elongation is a process by which the molecules of fatty acids are lengthened in two-carbon increments from the carboxylic acid end. Fatty acid shortening occurs in peroxisomes by â-oxidation function that is similar to â-oxidation function in mitochondria.

Nearly all phospholipids of biologic significance are constructed from two combinations of apolar and ‘backbone’ moieties: a glycerol—or other polyol— moiety substituted with one or two acyl or alkyl chains, or an N-acylated sphingoid base (ceramide). Following are some of the salient features of phospholipids of biologic significance: (1) they are ubiquitous; (2) within specific organisms, they have multiple loci; and (3) their composition varies in response to environmental stressors, including age, light, temperature, pH, pressure, salt concentration, and infection. To subserve those functions, polar moieties of phospholipids cover a broad range.*

Phospholipids serve myriad cellular messenger functions. For example, phosphatidylcholine comprises nearly 40% of human cellular phospholipids. It is hydrolyzed by specific phospholipases to generate a variety of lipid second messengers. Three notable examples of such messengers are: arachidonic acid generated by phospholipase A2 (PLA2); different species of diaclyglycerol (DAG) generated by phospholipase C (PLC); and phospholipid generated by phospholipase D (PLD). Some phosphatidylethanolamine-PLD activities are also stimulated by certain hormones.

Phospholipids decrease the surface tension of the aqueous surfaces of cells—they confer upon cells their surfactant characteristic. In the lung, phospholipids facilitate opening of alveoli during inspiration and their lack causes the lungs to collapse during expiration.Specifically, dipalmitoylphosphatidylcholine (DPPC), an important phospholipid, accounts for nearly 80% of the surfactant on the type II epithelial cells lining pulmonary surfaces. The deficiency of this surfactant has been clearly related to the respiratory distress syndrome in neonates.

VII. LIPID ECOSYSTEMS

Lipids in plasma membranes are essential for membrane fluidity, surface potentials, surface ligand activity, and transport functions.8-12 To serve their diverse functions, lipids exist in blood, plasma membranes, and within cells not as discrete molecular species—as it might seem from the conventional description of lipid chemistry—but as dynamic “lipid redox ecosystems” in which external pro-oxidant influences are vigorously counterbalanced by antioxidant defenses that exist within the lipid particles. For example, low-density lipoprotein (LDL) particles are found as spherical particles with diameters ranging from 19-25 nm, molecular weight varying over a broad range from 1.8 to 2.8 million daltons, and the density ranging from 1.019 to 1.063 g/ml. LDL is a large lipoprotein complex that includes the following: cholesterol moieties (estimated 1600 and 600 molecules of cholesterol esters and free cholesterol respectively), triglycerides (estimated 170 molecules), phospholipids (estimated 700 molecules), apolipoprotein B, neutral and polar lipids including polyunsaturated fatty acids, and lipophilic antioxidant species such as beta carotene and vitamin E. Predictably, the antioxidant content of LDL varies over a broad range and appears to be diet-related. Lipoprotein (a) [Lp (a)] is structurally similar to LDL, but is distinguished from it by the presence in it of a highly glycosylated protein designated apolipoprotein (a).13 It binds to apolipoprotein B-containing lipoproteins and proteoglycans.14 It has a complex relationship between fibrin, platelets, and atherogenesis. By its high affinity for and binding with fibrin, it activates plasminogen,15-17 while its binding to platelet receptors leads to plasminogen binding and activation. Lp(a) is considered atherogenic because it is taken up by foam cells; however, elevated levels are associated with ischemic heart disease (IHD) in most, but not all, reports.

VIII. THE LDL-OXIDATIVE MODIFICATION HYPOTHESIS

The LDL-oxidative modification hypothesis of the pathogenesis of ischemic heart disease (IHD) fails to address several important issues.18-20 First, this hypothesis assumes that oxidative modification of LDL occurs within sequestrated regions of the vascular wall. This assumption, as we stressed earlier in this article, is not warranted in view of our morphologic observations. Second, this hypothesis completely ignores the consequences of accelerated oxidative stress on erythrocytes in the bloodstream. The erythrocyte is the cell most vulnerable to high oxygen tension because it is the primary oxygen transport cell in the body. Third, this hypothesis fails to account for the contribution to atherogenesis of oxidative stress on platelets. Fourth, it ignores the atherogenic role of oxidative bursts of healthy and oxidatively damaged granulocytes, both insidiously during slowly progressive atherogenesis and acutely following intimal injury inflicted during angioplasty and coronary bypass surgery. Fifth, it ignores susceptibility of plasma proteins (including those of coagulation pathways) to redox dysregulation within the circulating blood. Sixth, the vulnerability of circulating plasma and cellular enzymes (and other functional proteins) is ignored by the LDL hypothesis.

____________________________________________

*Polar moieties of phospholipids include the following: glycerol (snGro); phosphatidyl (Ptd); sphingosine (Sph); sphingoid base (Spd); ceramide (Cer); choline (Cho); ethanolamine (Etn); serine (Ser); myoinositol (Ins); phosphate in mono- or diester-linkage (P); glucose (Glc); N-acetylglucosamine (GlcNAc); galactose (Gal); N-acetylgalactosamine (GalNAc); and 2-aminoethyphosphono (PetNH2).

Seventh, this hypothesis assumes—again without justification—that oxidative injury to the vascular intima (and, hence, to subendothelial stroma and myocytes) is inflicted only by oxidatively modified LDL. Eighth, vitamin E significantly increases the resistance of LDL to oxidation without inhibiting atherogenesis in the same animals.21-23 Ninth, at least one antioxidant (beta carotene) decreases atherogenesis in cholesterol-fed rabbits without reducing susceptibility of LDL to oxidation.21 Tenth, in cholesterol-fed rabbits impaired nitric-oxide-mediated vasodilatation is due to increased endothelial generation of superoxide, which inactivates nitric oxide.24,25

There are yet other considerations of coronary vascular dynamics and clinical expressions of atherosclerosis that may not be explained by the LDL-modification hypothesis. The clinical course of IHD is determined not only by atherogenesis, but also by diverse elements such as vasoconstriction, accretion of circulating microclots and microplaques on the intimal surface, thrombosis, plaque rupture, and release of proteolytic enzymes from ruptured and necrotic plaques, which further contribute to the degree of oxidative coagulopathy.19,20,26,27 The release of such proteolytic enzymes has been thought to contribute to lysis of fibrous caps of plaques, with resulting plaque rupture and thrombotic occlusions.28,29 Indeed, a large body of experimental evidence in atherogenesis points to etiologic roles of a multitude of oxidant phenomena involving synthesis of connective tissue macromolecules,30 secretion of substances with platelet-derived growth factor (PDGF)-like activity by intimal smooth muscle cells,31,32 ozone induction of cytokine-induced neutrophil chemoattractants and nuclear factor kB,33 endothelial cell replication,34 cytokine-inducible nitric oxide synthesis,35 elaboration of circulating and tissue immunoreactivity, 36 endothelial cell activity and its relationship with oxidation of LDL, 37 and the role of oxidized LDL in recruitment of monocytes and macrophages.38 Evidently, all of the above biochemical and cellular responses can be accentuated by oxidized LDL. However, the essential point here is that none of them depends on oxidized LDL for its initiation and propagation.

The key unanswered questions in the context of the cholesterol hypothesis are: (1) Why does the blood cholesterol level go up in the first place? (2) What are the molecular events that lead to a decrease in the number of LDL receptors? (3) How do elevated levels of cholesterol cause vessel wall injury and initiate atheroma formation? Our morphologic studies of peripheral blood presented in this article, though not addressing the first two questions directly, strongly suggest that hypercholesterolemia develops as an antioxidant defense adaptation to accelerated, chronic, and persistent oxidative stress on the circulating blood—the events that create and perpetuate oxidative coagulopathy. Indirect evidence to support our view derives from the fact that raised blood cholesterol levels in many persons living highly stressed lives return to a normal range when lifestyle stressors are brought under control (unpublished personal data). Furthermore, it seems to us that a decrease in the number of LDL receptors is an adaptive response to hypercholesterolemia. I discuss this subject at length in a subsequent volume of this textbook.

As regards the third question, several mechanisms by which hypercholesterolemia leads to atherosclerosis have been proposed. One such mechanism focuses on possible subtle endothelial injury caused by excess blood cholesterol that might increase endothelial cell membrane viscosity by altering its cholesterol-phospholipid ratio. Some other proposed mechanisms include the following: (1) the effect of hyperviscous, and hence less malleable, endothelial membrane on monocyte adhesion and chemotaxis; (2) the induction by excess cholesterol of growth factors in endothelial cells; and (3) the direct effects of cholesterol on platelets, monocyte/macrophage transformations, and accumulation of lipids in myocytes.31, 34-38 Of greater interest are the observations that LDL exposed to all major cell types involved in atherogenesis (monocytes, macrophages, platelets, endothelial cells, and smooth muscle cells) is oxidized and triggers generation of a vast array of molecules that perpetuate oxidative chain reactions and inflict cellular injury in the vascular wall.37 This is fully consistent with the core tenets of oxidative coagulopathy.

How may the association between elevated Lp(a) and IHD be explained in the context of oxidative coagulopathy? Lp(a) is structurally similar to plasminogen and is known to bind to fibrin.39-41 Thus, when present in the blood in elevated levels, it may be expected to exert a procoagulant effect and compound the procoagulant effects of oxidants in circulating blood, thus tipping the balance in favor of the clotting side of clotting-unclotting equilibrium in health. In addition, Lp(a) can be expected to increase the thrombogenic character of blood in oxidative coagulopathy by its known antifibrinolytic actions.

In summary, what is the common denominator of all initial lipid-related factors that are involved with atherogenesis and clinical ischemic coronary heart disease? Evidently it is accelerated oxidative injury to all lipids, including lipoproteins and glycolipids. Hypercholesterolemia plays a role in atherogenesis to the degree that higher concentrations of cholesterol lead to generation of greater amounts of oxidized LDL, and hence greater oxidative stress on the circulating blood. It is reasonable to conclude that all of the known molecular dynamics of dyslipidemia are consistent with the essential concept of oxidative coagulopathy.

IX. THE OXIDATIVE MODEL OF HDL DYSREGULATION

In 1997 my colleague, Omar Ali, and I introduced an oxidative model of HDL dysregulation.19 In this model, alterations of blood HDL cholesterol levels are related to the cumulative oxidative stress on the lipid metabolism. Specifically, incremental stress lowers blood HDL values, while reduction in such stress restores the levels to pre-stress levels.

A low level of HDL cholesterol is a recognized risk of IHD.42-44 Accelerated atherosclerosis is seen in most genetic HDL-deficiency syndromes. However, the mechanisms by which low HDL becomes a risk factor for IHD remain unelucidated. Factors that lower HDL are also recognized risk factors for IHD and include obesity, lack of physical exercise, tobacco smoking, abstinence from alcohol, and male gender.45 Dietary sugars and starches lower plasma levels of HDL,46 and the levels stay low for as long as a high-carbohydrate—and low-fat—diet is consumed.47 Physical exercise increases HDL levels.48

None of the commonly prescribed drugs for managing dyslipidemias raise plasma HDL levels. Indeed, some drugs (such as probucol and EDTA) with potent antioxidant effects are antiatherogenic. For example, probucol, a powerful antioxidant, significantly reduces restenosis rate after coronary angioplasty.49-52 Yet, it reduced HDL levels by approximately 40% and thus may not be useful long term. By contrast, we have found EDTA chelation therapy to raise HDL cholesterol, while it lowers the total cholesterol levels in many patients (personal unpublished observations).

What is the molecular basis of HDL dysregulation? We propose that reduction in plasma HDL levels observed in various clinicopathologic states is caused by oxidative dysregulation of lipid metabolism that occurs in oxidative coagulopathy.19,20,53,54 Such lipid dysregulation may involve one or more of the following: (1) accumulation in blood of oxidized and denatured lipids, which leads to raised levels of LDL and VLDL (very low-density lipoprotein particles); (2) accumulation of oxidized and denatured lipids in tissues; (3) down-regulation of lipoprotein lipase; (4) increased oxidizability of blood and tissue fats; and, as a result of all those factors, (5) expanding surface area of LDL and VLDL particles, which “sucks” in yet more cholesterol. We hold that this view is consistent with all known aspects of HDL metabolism referred to above. Below, I reproduce a paragraph from a previous article19 to elaborate the oxidative HDL hypothesis.

High-density lipoprotein, in contrast to low-density lipoprotein, as the names imply, has a higher density as determined by ultracentrifugation. We hold that HDL has a higher density than LDL because it has a higher protein content. We propose any or all factors that cause AA oxidopathy and lead to lipid dysregulation result in accumulation of peroxidized lipids and, of necessity, reduced amounts of protein moieties in the lipid molecular species. This hypothesis—that the level of HDL is a function of the degree of oxidative lipid dysregulation associated with oxidative coagulopathy—is consistent with all known aspects of HDL dysregulation. Plasma HDL levels are reduced, as we indicate earlier, in obesity, tobacco smoking, high intake of sugar and starches, abstinence from alcohol in males, and during periods of physical inactivity. The underlying mechanism in all of those states is chronic lipid dysregulation that is caused, as we demonstrate in this article, by oxidative coagulopathy.

The effect of physical exercise on plasma HDL levels presents an apparent paradox as well as strong support for our proposed HDL hypothesis. Physical exercise requires expenditure of energy, which is derived from oxidative metabolism of sugars, fats and proteins and, of course, is associated with increased free radical activity. Thus, exercise may be expected to fan the fires of oxidative coagulopathy. But exercise has other important counterbalancing metabolic functions. Specifically, during exercise myocytes are “hungry for fat” and their hunger is satisfied by upregulation of the activity of lipoprotein lipase, which breaks up triglycerides contained within LDL and VLDL particles and makes them available to myocytes for utilization in energy generation. The LDL and VLDL particles depleted of their triglyceride contents shrink with loss of the particle surface. The reduced surface area of LDL and VLDL particles diminishes their capacity for carrying cholesterol molecules. Such lipid particles shed cholesterol, which is avidly picked up by HDL particles for delivery to the liver for further metabolism. This explains how exercise simultaneously raises blood HDL and lowers blood LDL and VLDL levels. Beyond these effects, exercise, by increasing oxidant stress temporarily, brings about a compensatory upregulation of antioxidant defenses that seems to outlast the oxidant stress created by it. This view is supported by observations of Kujala, who reported diminished oxidative modification of LDL in veteran endurance athletes.55

Table 1 shows the formula of several important fatty acids. Table 2 lists classes of lipoproteins. Table 3 provides information on phenotypic familial dyslipidemias. Table 4 gives genetic dyslipidemias.

X. CHOLESTEROL AS A SYNAPSE PROMOTER

For decades, the majority of physicians have dismissed the matrix (connective tissue) in the various body organs as a mere scaffold for propping up the parenchymal cells. No attempts have been made to investigate the role of matrix in the pathogenesis of disease processes. The so-called collagen disorders, in reality, are autoimmune disorders that are treated with different types of immunosuppressant therapies. By contrast, the practitioners of holistic medicine—and now integrative medicine—have taken keen interest in the structure and function of matrix.56 They have long considered connective tissue derangements of great significance in the pathogenesis of a host of clinicopathologic entities. They have also focused on specific therapies to normalize the structure and function of matrix.56,57

For decades, neurologists, by and large, also have dismissed the glial cells—the matrix cells of brain connective tissues—as silent bystanders in the formation and operations of neural circuitry. The roles which the glia might play in the pathogenesis of a host of lesions in the brain parenchyma have escaped their notice. Researchers, however, have been taking hard looks at this issue. It is recognized that the glial cell plays critical roles in the development, differentiation, and demise of the parenchymatous cells of the brain. Astrocytes, a type of glial cell, secrete factors that promote neuronal survival.58 Neurons communicate with each other through synapses, the points of contact between their processes. The formation of synapses and contacts among neurons is essential for the full development of neurons as well as for maintaining long-term synaptic plasticity. The glial cells are also essential for synaptogenesis. Specifically, neurons by themselves form few synapses without the involvement of glia.59,60-62 Synapses that do form in neuron cultures supported by astrocytes develop poorly or die when the glial cells are removed. In the brain, apolipoprotein E (apoE)—a soluble protein that binds to heparin—is primarily produced by astrocytes. Those cells release apoE in the medium to promote the formation of synapses between neuronal cells. ApoE is a constituent of many large lipoprotein particles that ferry lipids to and from various locations in the body. Though produced mainly by glial cells, ApoE is abundantly expressed by neurons.

An important question that remained unanswered until recently concerns the molecular basis of the glial support for synaptogenesis. Neurons in the brain generate enough cholesterol to flourish and function, but the formation of a normal complement of synapses requires additional amounts provided by glial cells.2 Thus, cholesterol emerged as the primary regulatory influence over the formation and sustenance of synapses. This observation may provide a molecular mechanism also for delayed onset of synaptogenesis in the central nervous system after glial differentiation, as well as neurobehavioral symptomatology encountered in cholesterol and lipoprotein derangements. For example, the incidence of postpartum depression is higher among women with lower levels of blood cholesterol.

XI. LIPIDS IN BIOMEMBRANES

Biomembranes are dynamic structures with diverse functions. In a large measure, such membranes self-regulate their fluidity, surface potentials, surface ligand activity, and transport functions.62-65 Phospholipids form the basic structure of biomembranes and largely create the dynamic lipid redox ecosystems, which are vigorously defended by a host of enzymatic and nonenzymatic antioxidant systems. Phospholipids are amphipathic molecules because they are composed of both hydrophobic fatty acids and hydrophilic or polar head groups. Characteristic groups of membrane phospholipids are choline, serine, and ethanolamine. When hydrated, phospholipids form micelles and lamellar structures, which under certain conditions spontaneously self-organize into single-lipid bilayer structures as well as vesicular structures, called liposomes. Indeed, liposomes are being intensely investigated as delivery system carriers of drugs. The classical Singer and Nicolson model projects cell membranes as mosaics of globular proteins embedded in a fluid phospholipid bilayer. In this model the polar head groups of phospholipids are exposed on the external surface of membranes. There is an enormous structural and functional range of phospholipids in nature.3 Specific functions of various phospholipids in various organs of the human body, as well as in animal species, have been described. Beyond that, specific alterations in their structure and functions have been described in health and disease.

Lipids are required for enzymatic activities at biomembranes.63-65 Sometimes lipids are assigned two functions in this context: (1) as a physical entity that represents a dispersing medium in which proteins are randomly dissolved yet can move freely to serve their various functions; and (2) as specific chemicals to facilitate specific catalytic functions of various proteins and enzymes. Such a distinction, as elsewhere, may have some value in teaching students lipid chemistry. However, it is clearly not valid in a clinical setting since separate lipids do not serve mechanical and chemical functions in the lipid bilayer—or triple layer, to be precise, in view of the presence of sugars in it. The dispersing effects of lipids are well-delineated in the interaction of biomembrane protein complexes inserted in multi-component electron transfer pathways.66 In redox homeostasis, membrane lipids transfer electrons between relatively immobile redox complexes. In such events, ubiquinone appears to mediate the electron transfer from dehydrogenases to redox complexes.67

As for hormone-receptor dynamics at biomembranes, lipids are not required for hormone binding. However, lipids are necessary for transferring the signal from the receptor to the active site of adenylate cyclase on the opposite side of the biomembrane.68 Thus, the receptor-adenylate cyclase system has three components: (1) a catalytic site in the cell interior; (2) a regulatory site provided by the receptor; and (3) a coupling site required to transfer the signal, which is lipid-dependent.69 This subject is further discussed in the chapter entitled “Oxidative Biomembrane Dysfunction.”

Table 1. Molecular Formulas of Important Fatty Acids

Fatty Acid

Molecular Formula

Food Source

Unsaturated

Oleic acid

Linoleic acid

Linolenic acid

Saturated

Butyric acid

Caproic acid

Lauric acid

Stearic acid

Arachidic acid

CH3—-(CH2)—CH=CH—(CH2)7—COOH

CH3—-(CH2)—CH=CH—CH2—CH=CH—(CH2)4—COOH

CH3—-(CH2)—CH=CH—CH2—CH=CH=CH—CH2—CH=CH—(CH2—COOH

CH3—-(CH2)8—COOH

CH3—-(CH2)4—COOH

CH3—-(CH2)10—COOH

CH3—-(CH2)16—COOH

CH3—-(CH2)18—COOH

Corn oil

Linseed oil

Linseed oil

Butter

Butter

Coconut oil

Animal & vegetable fats

Peanut oil

Table 2. Classes of Lipoproteins

Particle

Apoproteins

Main

Component

Density

(kg/L)

Diameter

Chylomicrons

B48

(A,C,E)

Triglycerides

<0.95

75-1200

VLDL

B100

(A,C,E)

Triglycerides

0.95-1.006

30-80

IDL

B100, E

Triglycerides & cholesterol

1.006-1.019

25-35

LDL

B100

Cholesterol

1.019-1.063

18-25

HDL

AI, AII,

(C,E)

Protein

1.063-1.210

5-12

Table 3. Phenotypic Classification of Dyslipidemias

Dyslipidemia type

Increased electrophoretic fraction (lipoproteins)

Increased triglyceride

Increased cholesterol

I

chylomicrons

yes

yes

IIa

beta (LDL)

no

yes

IIb

pre-beta & beta (VLDL & LDL)

yes

yes

III

“broad beta” band (IDL)

yes

yes

IV

pre-beta (VLDL)

yes

no

V

pre-beta (VLDL) plus chylomicrons

yes

yes

Table 4. Major Genetic Dyslipidemias

Dyslipidemia

Increased cardiovascular risk

Plasma lipid pattern

Defect

Frequency/

Inheritance

Familial

hypercholesterolemia

Yes

hypercholesterolemia or mixed hyperlipidemia

(IIa or IIb)

LDL receptor

1:500

autosomal

dominant

Familial combined

hyperlipidemia

Yes

hypercholesterolemia or mixed hyperlipidemia

(IIa or IIb)

Overproduction of apoB100

1:50

autosomal

dominant

Familial dysbetalipoproteinemia (type III hyperlipidemia)

Yes

mixed hyperlipidemia (III)

Presence of E2/E2 isoform defective remnant binding to LDL receptor

1:5000

autosomal

dominant

XII. EICOSANOID BIOLOGY

Eicosanoids are a family of lipid mediators that serve a broad range of biologic roles.4 Their name is derived from the Greek eicosa—meaning twentyand they are comprised of twenty carbon fatty acids and their derivatives. The eicosanoid story began in 1930 with two seminal discoveries. The first involved a set of observations by Burr, Kurzrock, and colleagues concerning the effects of fat-free diet on experimental animals.70 Such animals showed retarded growth, reproductive disturbances, scaly skin, excess water intake, and renal lesions. In the second discovery, “prostaglandin”—a lipid with vasopressive and muscle-stimulatory effects—was described by Bergstrom and colleagues.71 The follow-up research in the first case paved the way for definition of the nutritional roles of lipids in general and recognition of essential fatty acids in particular. The work in the prostaglandin field led to characterization of the classical prostaglandin pathways and the recognition of arachidonic acid (AA) as a fatty acid of pivotal importance in inflammation, allergy, and numerous homeostatic pathways. Subsequent work revealed the biologic impact of prostaglandins on all organ systems and delineated the central link between the two discoveries. The next important discovery was made by Vance, who delineated pathways for inhibition of prostaglandin synthesis by aspirin-like drugs. That was followed by recognition of the proaggregatory effects of thromboxane A2 and the counterregulatory influence of prostacyclin. The work on delineation of leukotriene pathways was completed soon after, and it became evident that that family of eicosanoids plays critical roles in allergy, asthma, and the inflammatory response.

Despite recent and impressive strides in research, eicosanoid pathways remain a conundrum and we are far from knowing when these lipids are friends and when they turn foes. A simplified—and, of necessity, simplistic—schema of basic relationships is shown in Figure 5.

The American diet is laced with hydrogenated fats, which contain large amounts of trans fatty acids which, in turn, impair the desaturation and elongation steps involved in the conversion of linoleic acid to gammalinoleic acid and dihydrogammalinoleic acid.

Figure 5. Simplified and Generic Schema of Eicosanoid Biosynthesis

 

Linoleic Acid Alpha-Linolenic Acid

LA ALA

GLA EPA

DGLA

Arachidonic Acid*

Leukotrienes

Proinflammatory Antiinflammatory

*Arachidonic acid metabolism favorably influenced by aspirin and only temporarily improved by steroids.

The latter two polyunsaturates support the arachidonic axis which, in turn, sustains the many pathways for biosynthesis of prostaglandins and leukotrienes. Those eicosanoids mediate many alpha and beta adrenergic and cholinergic receptor functions. The activity of delta 6 desaturase is impaired in many disorders caused by—or associated with—chronic oxidosis and leads to disturbances in the arachidonic axis. That leads to impaired desaturation and elongation steps in the metabolism of linoleic and alpha-linolenic acids in patients with nutritional, ecologic, and autoimmune disorders. Thus, this enzyme is of considerable interest in the clinical practice of nutritional medicine. Normalization of its activity by nutritional and other antioxidant therapies creates opportunities for designing nonpharmacologic therapies that restore arachidonic, prostaglandin, and leukotriene pathways. Details of such therapies are given in the volume devoted to nutritional therapies.

XIII. BIOLOGY OF PROSTAGLANDINS

Prostaglandins (PGs) are autocrine and paracrine lipid mediators—they signal at or in the immediate vicinity of the sites of production.72-80 The cells do not contain any preformed prostaglandins. Rather, they are produced de novo from biomembrane-released arachidonic acid (AA) in times of need when cells respond to specific stimulation, such as cytokines, growth factors, collagen, adenosine diphosphate (ADP) in platelets, bradykinin and thrombin from endothelial cells, and trauma. Over thirty different prostaglandins have been characterized. The difference in their structure accrue from the number and location of their double bonds. There is a remarkable breadth of the range of molecular complementarity and contrariety among the members of this family of mediators.

Prostaglandins are synthesized from arachidonic acid by the action of lipoxygenase isoforms in most cells of the body. AA is maintained in an ester form by an exquisite regulation by a host of phospholipase enzymes (PLA2). The relative importance of their roles has gone through several paradigm shifts, with ongoing discoveries of newer members of this family of enzymes. At this time, type IV cytosolic PLA2 (cPLA2) is generally regarded as the preeminent member of the family. Cell-specific and agonist-dependent events deliver cPLA2 to the endoplasmic reticulum (ER), nuclear envelope, and Golgi apparatus.

At the ER and nuclear biomembranes, AA released by cPLA2 is presented to prostaglandin H synthase—PGHS, colloquially referred to as COX for cyclooxygenase. COX occurs in two isoforms, COX-1 and COX-2. In a generic sense, COX-1 moderates basal constitutive PG synthesis, whereas COX-2 is responsible for “induced” synthesis in the allergic, inflammatory, and autoimmune states. The COX enzymes are monotropically inserted into the nuclear and ER membranes so that the substrate-binding domain (“side pocket”) is precisely oriented for expeditiously receiving released AA. The crystalline structure studies have revealed a remarkable similarity between the two COX enzymes, with a single amino acid difference that accounts for a broader side pocket in COX-2. Thromboxane and prostacyclin have very short half-lives—from a few seconds to a few minutes—and act at or near their sites of production. Prostaglandin transporter (PGT), a member of the organic anion transporter polypeptide family, facilitates the release of prostaglandins.

There are nine known prostaglandin receptor forms, as well as several additional splice variants with divergent carboxy termini. Four of the receptor subtypes bind PGE2, two bind PGD2, and a receptor binds each of PGF2a, PGI2, TxA2, and TP respectively. These receptors belong to a distinct subfamily of the G protein-coupled receptor (GPCR) superfamily of seven transmembrane-spanning proteins.

AA is converted into an intermediate prostaglandin PGH2, the metabolism of which is subsequently coupled with downstream enzymes in a cell-specific fashion. Specifically:

1. Thromboxane (Tx) is produced in platelets and macrophages.

2. Prostacyclin synthesis occurs in endothelial cells.

3. Two types of PGD are made in mast cells and in the brain.

4. PGE production is under the influence of microsomal PGE synthase (mPGES), a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family.

5. PGF is generated in the uterus.

6. PGH2, as indicated above, is an early metabolite of AA.

In a generic sense, the Dr. Jekyll role of COX-1 may be regarded as a health promoter, while the Mr. Hyde role of COX-2 plays a dominant role in times of cellular stress, the inflammatory response, immune responses, allergic phenomena, and some homeostatic mechanisms. COX-1-related metabolites participate in myriad homeostatic mechanisms, including:

1. reduction of platelet adhesiveness;

2. enhancement of the rheologic characteristics of the circulating blood, improving cardiovascular dynamics and renal perfusion;

3. facilitation of diuresis;

4. stabilization of biomembranes;

5. prevention of abnormal and excess release of arachidonic acid;

6. suppression of excess production of cholesterol;

7. participation in the regulation of calcium homeostasis;

8. regulation of T cell functions; and

9. participation in diverse pathways for cellular replication and differentiation.

The COX-2-related metabolites, in many ways, counter the effects of those of COX-1. This family of prostaglandins promotes platelet aggregation and so supports the initial events that lead to atherogenesis. It triggers the inflammatory response. In the kidney, it causes sodium and water retention, hence its role in the causation and perpetuation of hypertension. However, some members of the prostaglandins play contrarian roles. Specifically, PGI2 inhibits the release of arachidonic acid from the cell membrane and so counterbalances many of the activities of PGE2.

This is a period of great expectation about the clinical benefits of the available COX-2 inhibitors (coxcibs). Undoubtedly, continued progress in this class of drugs will expand the pharmacologic armamentarium of blocker drugs for acute illness. However, in the integrative model, we need to be equally enthusiastic about the restorative therapies that normalize COX-1 pathways and that have stood the test of time. I reiterate this point several times in this volume because it clearly is the primary reason for my writing it.

XIV. BIOLOGY OF LEUKOTRIENES

Leukotrienes are primarily produced in inflammatory cells, including polymorphonuclear leukocytes, macrophages, and mast cells.81-88 The major enzymes involved in the biosynthetic pathways are 5-lipooxygenase (5-LO) and cPLA2. The former, a nonheme dioxygenase, is found in the nuclei in some cells and in cytosol in others. It is the key enzyme translocated to the nuclear envelope by cellular response to bacterial peptides, immune complexes, and other stimuli. 5-LO contains a NH2 terminal domain that binds two calcium ions—similar to the C2 domains of PLA2 and protein kinases—and a large catalytic domain that binds iron. In concert with the 5-lipoxygenase-activating protein (FLAP), 5-LO converts released AA into the epoxide LTA4.

LTA4 undergoes one of three possible fates: (1) hydrolysis; (2) conjugation with glutathione, and (3) transformation by transcellular processes that produce other bioactive eicosanoids. Hydrolysis is mediated by leukotriene A4 hydrolase (LTA4H), a zinc-containing bifunctional enzyme with aminopeptidase and epoxide hydrolase activities. LTA4H occurs in yeast and evolutionarily seems to predate other enzymes of leukotriene biosynthetic pathways. LTA4H in the cytoplasm, and possibly in the nucleus, produces LTB4—a powerful leukocyte chemoattractant that also stimulates leukocyte adhesion to endothelial cells.

LTA4 also couples with glutathione to yield LTC4. This reaction at the nuclear envelope is catalyzed by leukotriene C4 hydrolase (LTC4H), another member of the MAPEG family. LTC4 is ferried out of the cell by transporters, such as multidrug resistance-associated protein (MRP1). This transport process seems to be involved in dendritic cell migration to lymph nodes. Extracellularly, the peptide moiety of LTC4 T is metabolized to LTD4 and LTE4. Together the three molecular species are designated the cysteinyl leukotrienes, a biochemical entity that was named the slow-reacting substance of anaphylaxis (SRA) for its ability to cause slow and sustained smooth muscle contractility nearly six decades ago.

Leukotrienes, like prostaglandins, act at four distinct G protein-coupled receptors (GPCR)s. B-LT1 receptor on leukocytes binds LTB4 in the subnanomolar range and evokes a strong chemotactic response. Signaling through Gq is also involved in this pathway. B-LT2 is a second low-affinity receptor for LTB4. Two subtypes of cysteinyl leukotriene receptors, CysLT1 and CysLT2 mediate the actions of LTC4 and LTD4. Leukotrienes, again like prostaglandins, can also bind and be activated by peroxisomal proliferator-activated receptors (PPARs).

XV. DRUGS THAT ALTER EICOSANOID PATHWAYS

The potential for pharmacologic modification of eicosanoid pathways is being vigorously explored. Aspirin, the best-known modifier of prostaglandin metabolism, blocks the action of COX with a unique mechanism that involves covalent acetylation of a serine residue. That blocks proper orientation and ready substrate access at the active site. The other established drugs with COX-blocking ability include ibuprofen and indomethicin. All three agents block PGHS-derived prostaglandin synthesis, and their potent anti-inflammatory and analgesic effects are commonly known. Coxibs are selective inhibitors of COX-2 and represent newer eicosanoid modifiers that have been used for treatment of arthritis. This group includes celecoxib (Celebrex) and rofecoxib (Vioxx). The second generation coxibs under trials are valdecoxib (Bextra) and etoricoxib (Arcoxia).

The essential message of crucial clinical significance of this first volume of The Principles and Practice of Integrative Medicine is this: Nature has had preoccupation with complementarity and contrariety for over two billion years. The result of that is an order of natural phenomena that has little patience with molecular tinkering with synthetic molecules. Thus, notwithstanding the spectacular advances in pharmacologic therapeutics for achieving short-term objectives in acute illness, the notion that we can block one or more biologic pathways for reversing chronic degenerative diseases is fundamentally flawed. COX-2 blockers — Vioxx, Celebrex, and Bextra — were introduced with great fanfare and claims of ‘limited side effects.’ Of course, what were dismissed as ‘acceptable’ side effects were, in reality, serious consequences of disrupting eicosanoid chemistry. So I was not surprised when those drugs were withdrawn some years later because of cardiovascular fatalities.89-93

5-lipoxygenase inhibitors modify the leukotriene arm of eicosanoid metabolism.94-98 This class of agents has been employed for treatment of asthma and include zileuton (Zyflo), montelukast (Singulair), and CysLT4 receptor antagonist zafirlukast (Accolate). Early reports show that their bronchodilatory benefits exceed those of â agonist effects. Reduction in the number of eosinophils in the sputum has also been reported. 5-LO blockers have shown some steroid-sparing benefits. However, their clinical efficacy for most cases of asthma has been challenged, though they appear to be valuable in exercise-induced and aspirin-intolerant cases of asthma. Not unexpectedly, the therapeutic response to 5-LO inhibitors can vary with 5-LO promoter polymorphism.

XVI. ENDOCYTOSIS AND LIPIDS

As should be amply evident from the preceding sections on glycomics and proteomics, sugars and proteins need lipids for their survival as much as lipids need those molecular species. Below, I cite the example of membrane lipid phosphatidylinositol-4,5-biphosphate (PIP2) to illustrate how dependent lipids are on the protein pathways that were, until recently, seen as discrete and functionally self-contained.99-100

Essential cellular processes such as acquisition of nutrients, discharge of waste, movement across membranes of inorganic ions, regulation of cell membrane receptors (vital for communication between extracellular and intracellular environments), passage of drugs and other compounds into the cells, and the transmission of nerve impulses all depend on endocytosis—inward budding of the plasma membrane followed by scission of the bud to form discrete structures called vesicles. The protein dynamics of endocytosis have been extensively investigated. Vesicle formation begins with the formation of a lattice, comprising protein clathrin, at the cytosolic side of the plasma membrane. This latticework seems necessary for recruitment of receptors as well as cargo molecules into clathrin-coated indentations (pits) in the membrane that eventually are nipped off to form vesicles. Several other proteins involved in those processes include CALM, its brain-specific homolog AP180, and epsin. All three contain a conserved amino-terminal ENTH (epsin N-terminal homology) domain. ENTH domains are structurally similar to yet other proteins, including â-catenin and karyopherin-â. I briefly outline those components of protein networks involved in vesiculation to furnish some sense of the complexities of that process. Returning to the subject of phospholipids, recent studies have remarkably revealed a vital role of PIP2 in vesicle formation and all cellular functions dependent on it mentioned earlier. It has been established that functionalities of clathrin, CALM, and other proteins, are dependent on phospholipid PIP2, which serves a vital role in recruitment of various proteins in the vesiculation process.

Dissection of molecular pathways involved in the health/dis-ease/disease continuum undoubtedly will continue with important advances in our knowledge of those molecular dynamics. However, it seems certain to me that after we exhaust all possible molecular relationships, clinicians will discover that proper health management is simply impossible without integrating the dissected parts into the whole. For a part can be understood well only in relationship to the whole.

XVII. ATTRACTIVE AND REPULSIVE INTERACTIONS

Many attractive and repulsive interactions have been recognized in the complex energetics that preserve the structural and functional integrity of the multilamellar arrays in living membranes (the so-called lipid bilayer). Four repulsive factors are the electrostatic, solvation, steric, and undulation influences. Attractive forces include short-range bonds between molecules in opposing layers (such as hydrogen bonds and bridges formed by divalent salts) and relatively long-range van der Waals pressures. The sum total of those forces is profoundly influenced by the state of the membrane hydration. The amount of water taken up in those multilamellar arrays, in turn, is determined by interactions between the lipid molecules—those that are perpendicular to the plane of the membrane (the so-called interbilayer forces) and those that are in the plane of the membrane (the so-called intrabilayer forces). Phospholipids appear to be the primary molecules generating and maintaining the attractant and repulsive forces. I include the preceding brief comments about the “phospholipid energetics” of membranes to emphasize the need for thinking of the dynamic energy of living membranes rather than thinking of them as static structures formed by molecular bricks aligned in layers.

XVIII. N-3 FATTY ACIDS AND SUDDEN CARDIAC DEATH

The blood levels of long-chain n-3 fatty acids derived from fish are strongly associated with a diminished risk of sudden death among men without recognized prior cardiovascular disease.101-103 Specifically, the content of eicosapentaenoic acid and docosahexaenoic acid in fish oils appears to confer the protective effect. Among the survivors of myocardial infarction, supplementation with n-3 fatty acids was reported to reduce the risk of sudden death by 45%, though no effect on the overall nonfatal infarction was observed.104 Some evidence derived from animal experimental studies suggests that n-3 fatty acids carry antiarrhythmic properties.105-108 The putative mechanisms underlying those effects involve:

1. modulation of sodium, potassium, and L-type calcium channels101-103,109,110

2. inhibition of thromboxane production111;

3. stabilization of the heart rate by beneficial effects on heart rate variability112,113; and

4. lowering of the concentration of nonesterified fatty acids—which have strong prorhythmic properties—in biomembranes.114

The shorter-chain n-3 fatty acid, á-linolenic acid, probably has a similar beneficial effect in reducing the risk of sudden death, since it is largely metabolized after elongation and conversion into eicosapentaenoic acid and docosahexaenoic acid. Also, when stored for later utilization, á-linolenic acid is also stored as its longer-chain counterparts.115

Lipid Peroxides Are Elevated in Eclampsia

The serum and placental concentrations of lipid peroxides are significantly elevated in chronic hypertension during pregnancy with or without superimposed preeclampsia (5.5 +- 0.8 nmol/L in controls versus 7.6+-2.3). In contrast and not surprisingly, the blood levels of vitamin E are higher in normal controls than in patients with preeclampsia and eclampsia (38.1 +-9.1 mg/mL in controls versus 29.+-5.1 in eclampsia).116 Both sets of observations are consistent with the oxidative coagulopathy model of incremental stress on the circulating blood (associated with similar stress on the matrix and cellular ecosystems of the body. This subject is addressed at length in the chapter on oxidative coagulopathy. These findings concerning peroxidized lipids also fit well with the dysox model of the pathogenesis of cardiovascular disease presented in Integrative Cardiology, the sixth volume of The Principles and Practice of Integrative Medicine.117

XIX. LIPIDS, OXYGEN, AND OBESITY

Lipids and oxygen share mutual disrespect for each other. Oxygen has ‘a thing’ for fats; it spoils to hurt fats as soon as it sees them. Fats also have ‘a thing’ for oxygen. Though fats do not have a ready weapon to block oxygen’s evil designs on them, they nonetheless employ clever molecular mechanisms not only to protect themselves from their nemesis, but also to blunt the weapons of oxygen against other molecular species. Simply stated, lipids cope with the vicissitudes of oxygen by assuming proinflammatory roles and building protective shields for themselves.

Specifically, the fat cells produce and release into the blood a number of potent proinflammatory substances, including tumor necrosis factor (TNF-á). The higher the number of fat cells and the larger the amount of their contained lipids, the greater the quantities of the proinflammatory moieties produced by them. In obesity, the production of those factors is markedly increased. The examples of JNKs and c-JUN amino-terminal kinases may be cited here. Both types of kinases interfere with insulin action and are activated by free fatty acids as well as inflammatory cytokines, including TNF-a. In both dietary and genetic (ob/ob) models of obesity, total JNK activity is markedly increased in the liver, muscle, and adipose tissue.118 Those are among the many observations that support my view regarding the fundamental molecular mechanisms in the twin epidemics of obesity and diabetes (diabesity seems like a good term for it). I present this subject at length in the chapter entitled “Obesity Is Cellular Oxygen Deficiency State” in Integrative Nutritional Medicine, the fifth volume of The Principles and Practice of Integrative Medicine.119

XX. LIPID NUTRIENT THERAPIES

In the mainstream pharmacologic model, natural lipids are seldom, if ever, used as specific therapies for specific disorders. When asked why that is so, doctors who limit their clinical work to prescriptions for drugs often state that there is no scientific evidence of their clinical efficacy. This is surprising, considering the enormous body of literature validating the clinical efficacy of essential fatty acid supplementation.103-114,119-130 In integrative medicine, by contrast, most clinicians prescribe “lipid therapies” as their pharmacologic agents. They offer many reports to support their view of the clinical benefits of cold-pressed oils, lecithin, and some other fats. Indeed, the literature concerning the lipid dynamics of biomembranes, matrix, and intracellular organelles—providing the scientific basis and rationale for such therapies—is voluminous.131-141 For me, the reasons for employing lipids as supplements to prevent disease and to reverse chronic degenerative and autoimmune disorders are threefold: (1) As discussed in earlier chapters, oxygen and redox homeostasis are the major mechanisms involved in health preservation; (2) There is incontrovertible evidence that oxidative injury is the initial molecular event in the pathogenesis of those disorders; and (3) There is extensive empirical evidence for the efficacy of lipid therapies that normalize oxygen metabolism and restore redox homeostasis with the use of appropriate lipids administered in optimal doses. I furnish details of lipid therapies that were found to be safe and effective at the Institute in the third volume of this textbook.

The terms lipidomics and proteomics are introduced in this volume largely for ease of expression—just as proteomics is employed by the champions of proteins and glycomics and glycobiology are used by the sugar scions. But such terminology must be seen for what it really is: good for organizing the teaching material for the teachers and useful for learning by the students. None of those terms have any validity for clinicians. It must be recognized that proteins cannot function without lipids, lipids cannot be lipids without sugars, and sugars can be neither utilized nor serve any of their cell recognition roles without vitamins. The clinical outcome of the treatment of a given individual is determined by how effectively the clinician integrates in his management plan all the operant oxidative-dysoxygenative stressors and how successful he is in restoring the various homeostatic pathways and ecologic systems of the body.

The subject of the application of the science of lipidomics to clinical integrative medicine is addressed in Integrative Nutritional MedicineLooking Through the Prism of Oxygen Homeostasis, the fifth volume of The Principles and Practice of Integrative Medicine.118 In this section, I include brief comments about essential oil supplementation in various disorders. The biochemistry and functionalities of lipids are exceedingly complex. Fortunately, there are simple approaches to essential oil supplementation that are quite effective clinically in the long term.

The first order of business for essential oil therapeutics is to reduce the need for such supplementation by an optimal dietary approach. The reason for that is self-evident. In a highly chemicalized society as ours, lipid chemistry is under unrelenting oxidizing stress. Oxidized, de-natured fats literally clog biomembranes and severely interfere with their functionalities. Even copious essential oil supplementation under those conditions does not allow the needed oils to dislodge the unnecessary oils in those membranes, nor in matrix and cellular organelles.

I strongly recommend the following breakfast for my patients on five days a week:

Two tablespoons of freshly ground flaxseed;

Two tablespoons of lecithin;

Twelve ounces of organic vegetable juice;

One and a half tablespoons of suitable protein drink concentrate (containing 80% to 90% calories in amino acids).

For the remaining two days, an egg breakfast may be substituted for the protein drink. Again, I discussed the theoretical and practical aspects of such a breakfast in Integrative Nutritional Medicine.

For additional essential oil supplementation, I recommend one to two tablespoons of one of the following oils to be taken with steamed vegetables or salads: extra-virgin olive oil, cold-pressed avocado, pumpkin, or sesame oils.

XXI. SUMMARY

An overview of the classification, structure, and function of lipids is given to provide a framework for presenting the main theme of complementarity and contrariety in lipidomics. A special focus is on the redox homeostatic aspects of lipids. Blood lipids are recognized as lipid ecosystems. Specifically, the pathophysiologic aspects of various types of cholesterol are discussed, and the oxidative models of LDL and HDL dysregulation are presented. The case for oxidative coagulopathy as the primary event in the pathogenesis of arteriosclerosis is made. Examples are furnished to support the view that the functions of sugars, proteins, and lipids may not be seen as discrete molecular events. Rather, lipids ‘regulate’ the functions of sugars and proteins, just as those molecules ‘regulate’ the functions of lipids. The rationale for employing lipid therapies (described at length in the third volume of this textbook) is offered.

This is a time of pregnant enthusiasm about advances in “lipid blockers”—drugs that block one or more lipid pathways—that bring about short-term clinical benefits, such as statins and COX-2 inhibitors. There is no doubt that future work will yield highly effective drugs, expanding the pharmacologic options in acute illness. However, in the integrative model, there is a clear need for enthusiastic support of natural restorative therapies that normalize the vast and intricate network of lipid pathways.

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