AA OXIDOPATHY – PART 2 – Morphology

Majid Ali, M.D.



Below we describe our high-resolution, phase-contrast morphologic observations that comprise AA oxidopathy and oxidative coagulopathy. The degree and extent of oxidative changes, of course, varies over a broad range depending on the number and the nature of oxidative stressors. During the early months of our work with AA oxidopathy, we were concerned with the issue of whether the changes we observed involving the erythrocytes, granulocytes, platelets and plasma occurred in the circulating blood or were they artifacts caused by the process of preparing peripheral blood smears. We carefully examined fresh smears of several hundreds of apparently healthy individuals who sought our preventive medicine services—as well as those of many healthy volunteers—to assess the range of such morphologic changes in health. Thus, we were able to confidently differentiate semiquantitatively rather limited morphologic changes sometimes seen in healthy subjects from the frequently observed and pronounced abnormalities involving erythrocytes, platelets and plasma encountered in AA oxidopathy in a host of cardiovascular and noncardiovascular clinicopathologic entities.
In the context of IHD, important oxidant stressors include hyperadrenergic state, smoking, hyperglycemia, excess oxidized plasma lipids, obesity and cardiac arrhythmias. We have microscopically microscopically coagulopathy within the circulating blood to generally progress in the following seven morphologic stages:

1. Erythrocyte and leukocyte membrane deformities
2. Diaphanous congealing of plasma
3. Platelet aggregation and lysis
4. Filamentous coagulum (fibrin needles)
5. Lumpy coagulum
6. Microclots
7. Microplaques

    The patterns of oxidative coagulative injury described in this article were observed in extensive studies of blood morphology in a host of acute and chronic cardiovascular as well as non-cardiovascular disorders, including advanced IHD, unstable angina, congestive heart failure, cardiac arrhythmias, hypertensive crises, acute and chronic viral and bacterial infections, fungemia, acute and chronic atopic disorders, chemical sensitivity reactions, acute and chronic degenerative disorders and malignant diseases.

Erythrocyte Membrane Damage and Lysis in AA Oxidopathy
Erythrocytes, when observed with an ordinary bright-light microscope in stained smears of peripheral blood, appear as rigid, biconcave, disc-shaped corpuscles. When examined with a high-resolution, phase-contrast microscope in freshly prepared unstained smears, these cells are seen as pliable, round cells that readily change their shape to ovoid, triangular, dumbbell, or irregular outlines to squeeze past other erythrocytes in densely populated fields. Such cells resume their regular rounded contour as soon as they find open space.
Erythrocytes may be expected to show evidence of oxidative damage earlier than other blood corpuscles since these cells transport oxygen, the most important oxidizer in the body. Furthermore, unlike the leukocyte cell membrane which is sturdy and uniquely equipped with enzymatic antioxidant defenses against oxidative stresses of microbial invaders, the erythrocyte membrane is more permeable (to facilitate oxygen uptake and delivery) and, hence, may be deemed more vulnerable. Our microscopic findings provide some evidence for such theoretical considerations. The earliest and most common abnormalities we observed in AA oxidopathy are erythrocyte membrane irregularities and cell deformities. As oxidopathy progresses, an increasing number of red cells show morphologic abnormalities and some cells appear as ghost outlines. Many erythrocytes show surface wrinkling, teardrop deformity, sharp angulations and spike formations. Other changes include rouleaux formations and zones of plasma congealing around damaged erythrocytes.. Some zones of plasma congealing sometimes appear to form spontaneously (without a discernable cause) in close vicinity of damaged erythrocytes and leukocytes.
We established the oxidative nature of plasma and cellular abnormalities described above by demonstrating their reversibility with antioxidants such as vitamin E, taurine, vitamin A, and vitamin C, reported previously12 but not shown here. Parenthetically, we add that we have observed similar evidence of erythrocyte membrane injury in diverse clinical entities associated with accelerated molecular injury such as disabling chronic fatigue, fibromyalgia and a host of severe nutritional, ecologic and autoimmune disorders.

Erythrocyte Homogenate, Free Iron and AA Oxidopathy
In deliberations of atherogenesis, the issues of oxidative injury to erythrocytes and the presence in the plasma of free hemoglobin leached from damaged red cells—and the presence of excess iron in the plasma as a result of those factors—are seldom, if ever, addressed. Our morphologic findings lead us to propose that oxidative erythrocyte injury plays an important role in the genesis of AA oxidopathy and, hence, atherogenesis. We observed erythrocyte membrane damage and lysis with high frequency in many acute ischemic coronary syndromes and, less often, in patients with advanced IHD but without severe, acute coronary ischemia.
Iron, like oxygen, is a molecular Dr. Jekyll and Mr. Hyde. It is needed for molecular transport (in hemoglobin for oxygen), for storage (in myoglobin), for energy functions (in cytochrome oxidase and other cytochromes), for respiration (in non-heme-iron proteins), and for antioxidant defenses (in catalase). In its Mr. Hyde role, iron (in free form) is a potent oxidant and catalyzes the generation of many dangerous oxygen-derived radicals.175-182 In health, the Mr. Hyde roles of iron are minimized by transferrin, an iron-binding protein that rigidly limits the availability of free iron. In normal plasma, only 20 to 30 percent of transferrin occurs in a saturated state.
Free hemoglobin has been considered a dangerous protein—a biological Fenton catalyst.179 It rapidly quenches free radicals in a highly oxidizing environment and becomes oxidized, thus turning into a potent oxidant. It is readily degraded by H2O2 to release free iron, which initiates and propagates several free radical reactions.182-183Hemoglobin reacts with H2O2 to produce a protein-bound oxidizing species capable of causing lipid peroxidation.184Free hemoglobin also avidly binds with nitric oxide radicals and induces vasospasm, triggering yet other oxidizing events, which, in turn, feed the “oxidative fires” of AA oxidopathy.
Beyond ample evidence of the destructive oxidizing capacity of erythrocyte-derived factors discussed above, there is also direct evidence that red blood cells play a role in atherogenesis. Sambrano et al.185 and colleagues have shown that certain receptors on macrophages for oxidized LDL also bind to oxidatively-injured red cells prior to their internalization and lysis. Oxidatively-modified lipid, proteins, and carbohydrate moieties of erythrocyte membranes can be expected to play a host of roles in oxidative coagulopathy and AA oxidopathy, just as they do in attachment, endocytosis, membrane fusion, and viral hemagglutination in viral infections.186-189 We may point out in this context, as shown by Oda et al.189 that oxyradicals play the key pathogenetic roles in virus-induced illness. As we discuss in Part II of this article, a growing body of evidence points to the roles of strong inflammatory, infectious and autoimmune mechanisms in atherogenesis. It seems obvious to us that additional evidence for inflammatory and immunogenic roles of erythrocyte-derived factors in oxidative coagulopathy, AA oxidopathy, atherogenesis and IHD will be forthcoming as those areas are explored further in the future. Of considerable interest in this context is the matter of electrostatic interactions among oxidatively damaged erythrocyte membranes and other oxidized elements in the circulating blood ecosystem. Phospholipids and lipid components of LDL inhibit infectivity and hemagglutination of rhabdoviruses, probably because of structural similarity between such compounds and the receptors for viruses in cell membranes.190,191 In the case of vesicular stomatitis virus, phosphatidylinositol, phosphatidylserine and GM3 ganglioside show inhibitory activity.192 What are the mechanisms of action of such lipid moieties? Some light on this question is shed by studies of Mastromarino and colleagues193 in which removal of negatively charged molecules from membrane lipids by enzyme treatment significantly reduces their inhibitory activity, suggesting that electrostatic interactions play important roles in viral cell membrane dynamics. It seems highly likely that similar electrostatic roles involving platelets, monocytes and other elements in circulating blood ecology will also be discovered in the future.

Granulocyte Clumping, Membrane Damage, and Lysis in AA Oxidopathy
The granulocyte is usually dismissed as inconsequential in discussions of atherogenesis. This surprises us for two reason: 1) we observe morphologic evidence of oxidative damage to granulocytes in AA oxidopathy with high frequency in patients with IHD; 2) it is known that granulocytes produce toxic oxidative species that degrade other intracellular and extracellular molecular species, inflict peroxidative injury to cytoplasmic and organelle membranes, enhance polymorphonuclear leukocyte-endothelial adhesion, and increase microvascular permeability.194-200Evidently, all of those factors can initiate, perpetuate and intensify oxidative phenomena that cause oxidative coagulopathy and AA oxidopathy and may result in IHD. Some oxidizing molecular species elaborated by granulocytes increase capillary permeability and enhance granulocyte-endothelial adhesiveness.202 It seems odd to us that the cell known to play initial and critical roles in oxidative tissue injury is ignored in conditions characterized by oxidative injury to the circulating blood that results in atherogenesis. We recognized that granulocytes would be found to play a central role in atherogenesis when the molecular dynamics of this cell in atherogenesis are eventually investigated. This, indeed, is beginning to happen.
The granulocyte, like the erythrocyte, is a victim of the current infatuation of cholesterol enthusiasts with cholesterol. Our microscopic findings show that granulocytes play pivotal roles in initiating and perpetuating oxidative cascades in the circulating blood. In freshly prepared, unstained peripheral blood smears of healthy subjects, we observe granulocytes as hunter cells that move like crabs on the ocean floor, their locomotion provided by streaming of their granules into little protrusions of their cytoplasm. These cells continuously change their shapes as they explore their microenvironment. Not uncommonly, we visualize active phagocytosis of bacteria and cellular debris by such cells. In AA oxidopathy, the earliest change involving granulocytes is loss of locomotion—the cells lie limp in pools of plasma, with diminished or absent granular streaming. In later stages, granulocytes exhibit clumping. As in the case of erythrocytes, some granulocytes in more advanced cases of AA oxidopathy show blurring of membranes while others appear as ghost outlines of cells. Eventually, badly damaged granulocyte show disintegration of segments of their walls, degranulation and lysis.
The cytoplasmic granules of human granulocytes are rich in many enzymes including proteases, such as elastase, which are capable of degrading proteins in intracellular as well extracellular fluids.202 Oxidative cell membrane injury may be expected to result in escape of proteases from granulocytes into the circulating blood. The destructive capacity of granulocytes represents an exaggerated physiologic response in which bursts of potent oxidative molecular species are produced during inflammatory and repair responses. Specifically, hydroxyl radical (OH.) derived from superoxide radical (O2-) produced by granulocytes are a major cause of cellular injury. Granulocytic myeloperoxidase generates hypochlorite radicals when exposed to H2O2 following phagocytic activation.203Hypochlorite, in turn, oxidizes protease inhibitors, thus leading to increased proteolytic tissue damage.
Granulocytes play a central role in the generation and function of oxidative species that control cellular signaling, regulate mediators of inflammatory and repair responses, and influence migration and replication of inflammatory cells.204-207 A spate of recent gene-activation studies show evidence of the involvement of granulocytes in atherogenesis. Transcription of many atheroscleroses-related genes is augmented by oxidant-sensitive regulatory pathways involving nuclear factor kB (NF-kB).207 Specifically, exposure to superoxide radicals produced in granulocytes—and to lesser degrees in other cells—activates the NF-kB regulatory complex,206,207 which, in turn, triggers transcription of genes that encode for a variety of proteins including leukocyte adhesion molecules, chemotactic cytokines and enzymes that regulate cellular and matrix metabolism.207 Indirect evidence of the relevance of granulocytic factors in coronary artery disease has been shown by Tanaka et al.204 who documented activation of vascular cells and leukocytes in the rabbit aorta after balloon injury. Direct evidence for activation of NF-kB in experimental injury has recently been shown by Lindner et al.209 Recent findings of Tardif et al.90 that probucol reduces the incidence of restenosis after coronary angioplasty is consistent with such considerations, since the drug is a potent antioxidant and would be expected to protect coronary arteries traumatized by the angioplasty procedure from granulocytic oxidative bursts.

Platelet Aggregation and Lysis in AA Oxidopathy
The role of platelets in atherogenesis and coronary thrombosis has drawn much—and persistent—attention.210-222Yet, the atherogenic role of oxidatively damaged platelets in the circulating blood is ignored, just as the atherogenic consequences of oxidatively damaged circulating erythrocytes and granulocytes are neglected in deliberations of atherogenesis. In pathogenesis of atherosclerosis, the role of platelets is usually limited to the circumstances under which platelets adhere to endothelium or subendothelial stroma. This shifts the focus—to a great detriment to clear understanding of the pathogenesis of IHD—from initial molecular oxidative events taking place in the blood ecosystem to subsequent cellular oxidative events occurring in the vessel wall ecosystem.
In freshly prepared, unstained peripheral blood smears examined with a high-resolution, phase-contrast microscope, platelets appear as dark, round-to- ovoid, structureless bodies with poorly visualized granules, and without well-delineated plasma membranes. There is little or no tendency toward clumping and the plasma in their vicinity shows no evidence of congealing. Indeed even when smears are allowed to stand for 15 to 30 minutes, platelets remain discrete and do not cause congealing of fields of plasma that surround them in the central portions of the smears. (The peripheral portions of such smears often show early platelet clumping due to oxidative stress caused by exposure to the ambient oxygen.) Familiarity with the range of platelet morphology observed in health is essential before an observer can meaningfully interpret platelet changing seen in AA oxidopathy and oxidative coagulopathy.
In subjects with known atherogenic risk factors—especially in smokers, uncontrolled diabetics and those with chronic inflammatory conditions—we observe evidence of variable damage to platelet membranes and degranulation. The platelets in AA oxidopathy aggregate, change shape, degranulate and release various thrombogenic and atherogenic factors. Not unexpectedly, most platelet aggregates and clumps are surrounded zones of plasma congealing of variable widths. In more advanced stages of AA oxidopathy, platelet membranes become indistinct and lysis occurs. Parenthetically, we might add that we also observe similar damage in patients with disabling chronic fatigue, fibromyalgia, chemical sensitivity and a host of acute autoimmune disorders. Such changes are only rarely seen in apparently healthy subjects.
One of us (MA) established the oxidative redox nature of platelet aggregation and clot formation by addition of ascorbic acid and ethylenediaminetetraacetic acid (EDTA) to platelet aggregates induced by oxidizing agents such as collagen, epinephrine, ADP and ristocetin. We observed that both ascorbic acid and EDTA can readily break up platelet aggregates formed by addition of various aggregating agents.13 Those observations support our view that platelet aggregation and clot formation are oxidative phenomena and that antioxidants ascorbic acid and EDTA caused dispersal of platelet aggregates by protecting the platelet membranes from the oxidant stress. Interestingly, both ascorbic acid and EDTA failed to break up platelet aggregates caused by collagen, indicating a stronger—and perhaps irreversible—effect of collagen on platelet aggregation. From a teleologic perspective, it may be argued that collagen exerts a stronger aggregating influence than epinephrine because circulating platelets are exposed to collagen under more threatening conditions (bleeding from trauma to vessel walls) rather than to epinephrine (a common hyperadrenergic state created by lifestyle stressors).
Endothelial cells and platelets repel each other by their nonthrombogenic character—by their surface charges as well as their ability to generate antithrombotic molecules such as heparin and prostacyclin.220 Thus, adhesion of platelets to endothelial cells is prevented under ordinary conditions. Such electromagnetic and molecular conditions, however, are threatened continuously by the normal oxidative stress in healthy circulating blood. In states associated with accelerated oxidative injury, the normal nonthrombogenic capacity of platelets and endothelial cells is exceeded and platelets begin to agglutinate and adhere to endothelial cells. Examples of conditions of accelerated oxidative stress include catecholamine surges that accompany lifestyle stresses, hypercholesterolemia, denuding endothelial injury caused by intra-arterial catheters, and anastomotic sites of bypass surgery. Under such conditions, injury to platelets triggers chain reactions of oxidative coagulopathy, first in the blood and subsequently in the vascular wall affecting all four lines of cells involved in atherogenesis—endothelial cells, monocytes/macrophages, myocytes, and yet more platelets.221-226 Platelet degranulation releases several growth factors, including platelet-derived growth factor (PDGF),215-217 epidermal growth factor,227 nitric oxide,228 and transforming growth factor-beta.228 Some of these growth factors are powerful mitogens. But generation of all such growth factors is initiated by direct oxidative stress on platelets.
What is the common denominator in all platelet factors that are associated with IHD? Evidently, it is accelerated oxidative injury to elements in the circulating blood that leads to oxidative coagulopathy and AA oxidopathy. Again, as for erythrocytes and granulocytes, the patterns of oxidative damage to the components of the vascular wall that lead to plaque formation—and which have claimed enormous sums of research funds without significant benefit to those who suffer from IHD—clearly are consequences of changes in the circulating blood.

Ali M, Ali O. AA Oxidopathy:
the core pathogenetic mechanism of ischemic heart disease.
J Integrative Medicine 1997;1:1-112.

Erythrocyte Morphology in Health
and Early Stages of AA Oxidopathy

Figure 1 (top): All erythrocytes and two lymphocytes shown in the photomicrograph keep their distance from each other (due to negative electrostatic surface charges) and show regular outlines. Note that none of the well-preserved erythrocytes are discoid in shape. Figure 2 (bottom) shows early changes of AA oxidopathy with many damaged erythrocytes.

Severe Erythrocyte Damage and Leukocyte
Clumping in AA Oxidopathy

Figure 3 (top) shows a more advanced stage of erythrocyte damage in a 66-year old hypertensive female than in figure 2. Only a few cells show well-preserved, completely regular outlines. Some cells appear as ghost outlines of leached cells. Figure 4 (bottom) shows clumped leukocytes surrounded by some faded cells.

Zones of Congealed Plasma Surrounding
Platelets and a Damaged Leukocyte

Figure 5 (top): A spreading zone of congealed plasma representing initial changes of oxidative coagulopathy in a smoker is seen near the center of the field. Note rouleaux formation of erythrocytes. Figure 6 (bottom) shows a similar zone of congealed plasma surrounding a damaged leukocyte. We confirmed the spreading nature of these zones of congealing by observing these zones over time.

Erythrocyte-Induced and “Spontaneous”
Plasma Congealing

Figure 7 (top) illustrates severely damaged erythrocytes in a 52-year-old man with persistent atrial fibrillation. Close examination shows some zones of congealing surrounding many damaged red blood cells. Figure 8 (bottom) illustrates a zone of plasma congealing unaccompanied by any cellular elements of the blood (seemingly a “spontaneous” phenomenon) in a diabetic with IHD. In our view, such congealing represents accelerated oxidative stress on plasma.

Needle-like and Amorphous Microclots
And “Dirty” Field of AA Oxidopathy

Figure 9 (top) shows some needle-like and amorphous granular microclots in a patient with unstable angina. Figure 10 (bottom) shows a “dirty” blood smear of a man with severe peripheral vascular disease and extensive bilateral leg ulcerations, showing zones of plasma congealing and lumpiness, platelet clumping, and some other zones of plasma congealing unaccompanied by any blood corpuscular elements, representing diffuse changes of AA oxidopathy.

 A Large Platelet Clot And a Meshwork of Clots

Figure 11 (top) shows a microclot formed by a large aggregate of platelets and congealed plasma in a patient five days after angioplasty. Figure 12 (bottom) shows another field from the same smear and illustrates how microclots in oxidative coagulopathy grow in size when oxidative stress persists.

Microplaque Formation In AA Oxidopathy

Figure 13 (top) and figure 14 (bottom) show two microplaques in a patient who had received three unsuccessful angioplasties for advanced IHD. Photomicrographs were taken the day after a major nosebleed. Note the compaction of necrotic debris and blood elements in microplaques as contrasted with loose structure of microclots in figure 11.

Dissociation of Platelet Aggregates by Vitamin C

Figure 15 (top) shows patterns of aggregation of platelets induced by oxidative stress of epinephrine, collagen, ADP and ristocetin. Such aggregation is the in vitro counterpart of in-vivo platelet clumping seen in AA oxidopathy and shown in figure 11. Figure 16 (bottom) shows patterns of dissociation of platelet aggregates obtained with the four aggregating agents on addition (arrow) of a 0.5 percent solution of ascorbic acid. Note that ascorbic acid completely dissociates epinephrine-induced aggregates (top), while its effect on collagen-induced aggregation (bottom line) is minimal.13

Reversal of Early Changes of AA Oxidopathy

Figures 17 (top) and 18 (bottom) show abnormal erythrocyte morphology in a highly stressed 53-year-old man before and after addition of 1:50 dilution of mycelized vitamin E solution. Note how vitamin E normalizes red cell morphology and establishes the oxidative nature of erythrocyte injury.

Reversal of Early Changes of AA Oxidopathy with Taurine

Figures 19 (top) and 20 (bottom) illustrate AA oxidopathy changes involving erythrocytes before (top) and after (bottom) addition of taurine (1 mg/50 ml) in a 63-year-old man in congestive heart failure. Taurine is a powerful cell membrane stabilizer that is known for its cardio- and neuroprotective roles. This simple experiment establishes the oxidative nature of red cell abnormalities seen in congestive heart failure.

Diaphanous Congealing of Plasma
We observed diaphanous zones of plasma congealing surrounding platelets, fragments of leukocytes, and fungal organisms. In many cases we observed areas of plasma congealing without any involvement of platelets, leukocytes and fungal organisms. That some free radical activity exists in plasma in health must be accepted on teleologic grounds alone. Such oxidative stress is generated by normal metabolic activity of red and white blood corpuscles as well as of platelets, oxidation of catecholamines, enzymatic glucose breakdown, nonenzymatic autoxidation of blood glucose in hyperglycemic states, and mechanical shearing stress on endothelial cells. Furthermore, zones of plasma congealing and microclots produced by physiologic redox dynamics may be expected to be dissolved as soon as they form by normal plasma fibrinolytic activity. Notwithstanding such physiologic fibrinolytic activity, some free radical damage to the endothelium and subendothelial matrix would be expected to ensue. Indeed, the presence of fatty-streak lesions in children attests to the existence of such insidious and clinically silent oxidative coagulopathy.
It is to be expected that normal oxidative stresses on blood plasma are markedly increased during a host of pathologic states of the cardiovascular system, as well as of other body organ ecosystems, accompanied by accelerated oxidative injury. This includes advanced IHD, unstable angina, congestive heart failure, cardiac arrhythmias, hypertensive crises, hyperglycemia, and during smoking.

Lumpy Coagulum and Fibrin Needles
Intravascular coagulation has long been assumed to be an uncommon and potentially life-threatening state. Our high-resolution, phase-contrast microscopy observations of peripheral blood in a host of cardiovascular and non-cardiovascular entities challenge this assumption. In health, plasma in peripheral blood smears appears as clear liquid that bathes cells. In states of accelerated oxidative molecular injury, damaged plasma proteins begin to congeal, and such zones of clotted plasma spread as thin diaphanous films. As the oxidative process advances, cross-linked fibrin appears as filamentous and lumpy coagulum. Some platelets can usually be recognized trapped within filamentous and lumpy fibrin deposits, undoubtedly contributing oxidized phospholipids and glycolipids to the protein coagulum. Such needles and masses of oxidized, coagulated proteins and peroxidized lipids grow by triggering the chain reactions of plasma lipid peroxidation and protein coagulation. We have consistently documented the presence of fibrin needles and lumpy coagulum of protein in freshly prepared and unstained blood smears in states of accelerated oxidative damage. By comparing peripheral blood morphology before and after intravenous infusions of EDTA and ascorbic acid, both administered with magnesium, we have repeatedly observed dissolution of fibrin needles and lumpy protein after the infusions in cases in which such evidence of oxidative coagulopathy was clearly discerned.

The presence in circulating blood of microclots formed by oxidative stress of normal blood ecology, and an excess of such clots in states of accelerated oxidative stress, may be reasonably deduced from the foregoing discussion of redox dynamics of plasma components and blood corpuscles in health and disease. Congealing of plasma, erythrocyte and leukocyte membrane damage and platelet clumping may be expected to add to the oxidizing capacity of blood by triggering fibrinogenic and lipid peroxidation chain reactions. Furthermore, such changes may be expected to initiate oxidative chain reactions, thus increasing oxidative stress and enlarging zones of plasma congealing into microclots. We document such progressive changes of AA oxidopathy.

A natural consequence of oxidant microclots—oxidative coals, in our terminology—circulating in blood would be for them to grow in size as the plasma at their periphery continues to congeal and as an increasing number of platelets and other blood corpuscles are entrapped into or stick to them. With ongoing oxidative stress, such microclots coalesce to make yet larger and lumpier microclots. With time, such loosely bound microclots are compacted in form layered structures with dead and dying cells and other necrotic debris trapped between layers of fibrin that we call microplaques. Such microclots and microplaques float in the bloodstream as simmering oxidative coals, lighting up oxidative fires and inflicting further oxidative damage to blood corpuscles, endothelial cells and subendothelial collagen matrix wherever the lining cells of the vascular lumen have been denuded by the shearing mechanical stress of circulating blood. We have observed microclots grow into microplaques that measure as much as several hundred microns.
All oxidants in circulating blood trigger oxidative coagulative phenomena involving blood corpuscles and plasma contents. Our clinical and high-resolution microscopic observations lead us to consider the following groups of causes of accelerated oxidative stress on the circulating blood that lead to oxidative coagulopathy:

1. Adrenergic hypervigilence associated with lifestyle stressors
2. Rapid glucose-insulin and adrenergic shifts
3. Mycotoxicity and, to lesser degrees, toxins from other microbes
4. Increased oxidizability of blood associated with obesity
5. Diminished dietary intake of natural antioxidants
6. Increased body burden of prooxidants such as iron, copper and mercury
7. Inflammatory factors
8. Infectious agents
9. Excess of oxidized and denatured lipids
10. Autoimmune factors
11. Oxidative stress of cigarette smoking
12. Hyperhomocysteinemia
13. Mechanical shearing stress associated with hypertension.

Ali M, Ali O. AA Oxidopathy:
the core pathogenetic mechanism of ischemic heart disease.
J Integrative Medicine 1997;1:1-112.

Smoking and AA Oxidopathy

Figure 21 (top) before smoking and Figure 22 (bottom) after smoking show changes of AA oxidopathy observed in a volunteer who abstained from smoking overnight and then smoked three cigarettes in three minutes.

AA Oxidopathy and Fungemia

There are four important questions here:

1. How often are fungal organisms seen in the circulating blood of nonfebrile ambulatory persons?

2. What roles do such organisms play in the pathogenesis of oxidative coagulopathy and AA oxidopathy?

3. What are the possible mechanisms of action of mycotoxins and other fungal proteins?

4. What roles do fungal organisms play in the inflammatory and autoimmune processes that are known to be atherogenic and involved in other aspects of IHD?

As to the first question of how frequently fungal organisms may be observed in afebrile ambulatory patients, there is wide divergence of opinion among those who routinely use high-resolution (15,000 x) phase-contrast microscopy and those who never use such technology. We have documented the presence of fungal organisms in peripheral blood of severely immunocompromised individuals with high frequency (over 95%).229 As a part of our study of the phenomena of oxidative coagulopathy and AA oxidopathy, we also examined the peripheral blood smears of 50 consecutive patients with advanced IHD (including those recovering from angioplasty and coronary bypass operations) and detected the presence of fungal organisms in many microscopic fields in 19. Identification of specific fungal species cannot be done with such microscopy. However, employing anticandida antibodies labeled with horseradish peroxidase, we have documented the presence of Candida species in peripheral smears in some cases.230,231 We have previously published the specificity characteristics of the anti-candida antibodies we employed in such studies.232,233 Such observations may be challenged by those unfamiliar with high-resolution microscopy on the ground that if true fungemia did exist in such patients, they would be critically ill. This requires further comment.
The clinical distinction between benign bacteremia and potentially life-threatening septicemia is well recognized; the former occurs after tooth brushing and is clinically insignificant. It is noteworthy that no such clinical distinction is made in the prevailing medical thinking between insidious and clinically silent fungemia and potentially life-threatening fungal invasion of the bloodstream. Fungemia, the presence of fungi in circulating blood, is always considered a serious pathologic entity. This is clearly erroneous in view of the direct evidence to the contrary that we present here. Regrettably, many physicians who have not taken the time to learn the use of high-resolution microscopy—and hence are uninformed about the prevalence of fungal organisms in the peripheral blood of immunocompromised individuals—make irresponsible and derogatory statements about those who use such technology. Indeed, some licensing boards controlled by such uninformed physicians have taken serious disciplinary actions, including suspension of medical licenses, against holistic practitioners who diagnosed fungemia with high-resolution phase-contrast microscopy and treated clinical yeast syndromes.234

Fungemia, Mycelia Formation and Fungal Budding
In figures 27 through 30, we illustrate the replication, mycelia formation and fungal budding in peripheral blood smears observed over a period of five hours for two reasons: 1) to provide additional proof that the bodies we recognize as fungal organisms are indeed fungi (shown by their ability to form mycelia and the ability of the mycelia to show budding); and 2) to document the rapidity with which fungal organisms multiply as oxygen tension falls and acidity increases in their microenvironment—the two conditions under which fungi would be expected to grow luxuriantly.
As to the second question concerning the possible roles of fungal proteins and mycotoxins in the pathogenesis of oxidative coagulopathy and AA oxidopathy, we illustrate some of the observable phenomenon of zones of plasma congealing surrounding fungal organisms.. We observed this phenomenon to occur within ten to sixty minutes in almost all instances in which we studied the morphology of fungal organisms continuously in freshly prepared unstained peripheral blood smears. We also observed fungal spores to germinate within one to ten hours in most such cases. The zones of plasma congealing surrounding fungal organisms increase in area, trap platelets and cellular debris, and grow into microclots, and finally into micro-plaques. Such findings suggest that fungal organisms play a role in the pathogenesis of oxidative coagulopathy and AA oxidopathy. We return to this subject later in this article.
If fungemia occurs frequently in chronic immune disorders, why can’t the fungal organisms be cultured from blood in such cases? This is a valid question. We have addressed this issue at length elsewhere.235 It is noteworthy that negative blood cultures are frequently seen in patients with documented invasive tissue fungal infections. In one study of such patients, a Candida enzyme called enolase was detected in 42 percent of patients with proven tissue candidiasis.236
The third and fourth questions concern the possible molecular mechanisms by which fungi cause AA oxidopathy and might play etiologic roles in the pathogenesis of IHD. We return to this subject after discussing AA oxidopathy in relationship to the known molecular dynamics of IHD.

Fungemia and AA Oxidopathy Phase-Contrast and Darkfield Views

Figure 23 (top) shows clusters of round-to-ovoid white fungal bodies that contrast with dark erythrocytes in a high-resolution (15,000X) phase-contrast photomicrograph. Figure 24 (bottom) shows the same microscopic field in darkfield. Note that unlike erythrocyte lipid membranes that reflect light, fungal membranes contain disaccharides that absorb light and do not appear as bright as red cell membranes.

Immunostaining of Candida Organisms
in Peripheral Smears

Figure 25 (top) and figure 26 (bottom) show unstained and immunostained Candida organisms in phase-contrast and darkfield fields. In this procedure, human anti-candida IgG antibodies labeled with horseradish peroxidase were used to specifically stain Candida organisms. For procedural details and antibody specificity characteristics, see references #230 through 233.

In Vitro Fungal Growth

Figure 27 (top) is a photomicrograph of a freshly prepared peripheral blood smear of a diabetic with leg ulcers and severe fatigue and shows several fungal organisms. Figure 28 (bottom) represents the same smear photographed 37 minutes later showing a luxuriant growth of fungal organisms as the oxygen tension of the smear under a coverslip falls and acidosis develops due to continued glycolysis(the two conditions that are known to support rapid fungal replication.

Mycelia Formation and Building

Figures 29 (top) and 30 (bottom) are photomicrographs of the smear shown in figures 27 and 28 taken 3 1/2 and 4 hours later respectively. Note how yeast grow mycelia with profusion and how some mycelia grow buds.

Fungemia and Oxidative Coagulopathy Earliest Changes





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