BIOCHEMISTRY: A TEXTBOOK FOR MEDICAL UNIVERSITIES - E.S. Severin - 2004

CHAPTER 8. LIPID METABOLISM

VI. Eicosanoids

Eicosanoids are BIOLOGICALLY ACTIVE SUBSTANCES synthesized by most Cells from 20-carbon polyenic Fatty acids (the word "eicosa" comes from the Greek for 20).

Eicosanoids, which include Prostaglandins, thromboxanes, Leukotrienes, and several other substances, are highly active regulators of cellular Functions. They have a very short T_1/2, and therefore exert effects as "local Hormones," influencing the METABOLISM of The Cell that produces them via an autocrine mechanism, and affecting surrounding cells via a paracrine mechanism. Eicosanoids participate in numerous processes: they regulate the tone of vascular smooth Muscle and thereby affect Blood pressure, bronchial function, the intestines, and the Uterus. Eicosanoids regulate Water and sodium excretion by the Kidneys and influence blood clotting. Various types of eicosanoids are involved in The Development of the inflammatory response following tissue injury or infection. Such signs of inflammation as pain, edema, and fever are largely due to the action of eicosanoids. Excessive secretion of eicosanoids leads to A number of disorders, such as Bronchial Asthma and allergic reactions.

A. Substrates for eicosanoid synthesis

The primary substrate for eicosanoid synthesis in humans is arachidonic acid (20:4, ω-6), as its content in The Human Body is significantly higher than that of other polyenic eicosanoid precursors (see Table 8-1 above).

Eicosapentaenoic (20:5, ω-3) and dihomo-γ-linolenic (20:3, ω-6) Fatty acids are used in smaller amounts for eicosanoid synthesis.

Polyenic 20-carbon fatty acids enter the human body with food or are formed from essential 18-carbon fatty acids, which are also obtained through the diet (Fig. 8-44).

Class="center">Fig. 8-44. Synthesis of 20-carbon polyenic fatty acids in the human body.

Polyenic fatty acids that serve as substrates for eicosanoid synthesis are components of membrane Glycerophospholipids. Under the action of membrane-associated phospholipase A₂, the fatty acid is cleaved from the glycerophospholipid and utilized for eicosanoid synthesis.

B. Structure, nomenclature, and Biosynthesis of Prostaglandins and thromboxanes

Although the substrates for eicosanoid synthesis have a fairly simple structure (polyenic fatty acids), they give rise to a large and diverse group of substances. The most widespread in the human body are prostaglandins, which were first isolated from the Prostate Gland, hence their name. Later, it was shown that other body Tissues also synthesize prostaglandins and other eicosanoids.

1. STRUCTURE AND NOMENCLATURE of prostaglandins and thromboxanes

Prostaglandins (Fig. 8-45) are designated by symbols, such as PG A, where PG stands for "prostaglandin," and the letter A denotes the substituent in the five-membered ring of the eicosanoid molecule.

Fig. 8-45. Prostaglandin families.

Each of the specified prostaglandin groups consists of 3 types of molecules, differing in the number of double bonds in their side chains. The number of double bonds is indicated by a subscript digit, for example, PG E₂.

The number of double bonds in the side chains of prostaglandins depends on The structure of the precursor—the polyenic acid from which the prostaglandins are formed. Two double bonds of the polyenic acid are used to form the ring in the prostaglandin molecule, and the number of remaining double bonds in the radicals attached to the ring determines the prostaglandin series: 1 if there is one double bond, 2 if There are two double bonds, and 3 if there are three double bonds in the radicals.

PG I — prostacyclins. They contain 2 rings in their structure: one five-membered, like other prostaglandins, and another containing an oxygen atom. They are also subdivided depending on the number of double bonds in the radicals (PG I₂, PG I₃).

Thromboxanes. Unlike prostaglandins, thromboxanes are synthesized exclusively in platelets, hence their name, and they stimulate platelet aggregation during blood clot formation.

Thromboxanes possess a six-membered ring containing an oxygen atom (Fig. 8-46). Just like other eicosanoids, thromboxanes can contain varying numbers of double bonds in their side chains, forming TX A₂ or TX A₃, which differ in activity. TX B₂ is a catabolic product of TX A₂ and lacks biological activity.

Fig. 8-46. Structure of thromboxanes. TX A₂ is synthesized from arachidonic acid; TX A₃ is synthesized from eicosapentaenoic acid.

2. Cyclooxygenase Pathway: Synthesis of Prostaglandins and Thromboxanes

Phospholipase activation. The synthesis of prostaglandins begins only after polyenoic acids are released from membrane Phospholipids through the action of Enzymes (Fig. 8-47). The activation of membrane-associated phospholipases is triggered by various factors, including hormones, histamine, cytokines, and mechanical stress.

Fig. 8-47. Release of arachidonic acid from glycerophospholipids. MAG — monoacylglycerol; IP₃ — Inositol trisphosphate.

The binding of a stimulating agent to its receptor can activate either phospholipase A₂ or phospholipase C, depending on the cell type and receptor class.

Once arachidonic acid is cleaved from the phospholipid, it enters the Cytosol and is converted into various eicosanoids depending on the cell type. There are two main pathways for arachidonic acid metabolism in cells: the cyclooxygenase pathway, which leads to the synthesis of prostaglandins, prostacyclins, and thromboxanes, and the lipoxygenase pathway, which results in The formation of leukotrienes and other eicosanoids (Fig. 8-48).

Fig. 8-48. Synthesis of eicosanoids from arachidonic acid. Glucocorticoids inhibit the synthesis of all types of eicosanoids by suppressing phospholipase A₂, thereby reducing The amount of substrate available for their production. Aspirin and other nonsteroidal anti-inflammatory drugs inhibit solely the cyclooxygenase pathway.

Synthesis of prostaglandins. The enzyme catalyzing the first step of prostaglandin synthesis is designated as PG H₂ synthase and possesses two catalytic centers. One is referred to as cyclooxygenase, and the other as peroxidase. This enzyme is a glycoprotein dimer composed of identical polypeptide chains. It features a hydrophobic domain embedded in The Lipid Bilayer of The Endoplasmic reticulum (ER) membrane and a catalytic domain facing the ER lumen. The active center of cyclooxygenase contains Tyrosine (385), while the active center of peroxidase contains a heme prosthetic group. The body contains Two Types of cyclooxygenases (PG H₂ synthases). Cyclooxygenase-1 is a constitutive enzyme synthesized at a constant rate, whereas the synthesis of cyclooxygenase-2 increases during inflammation and is induced by corresponding mediators such as cytokines.

Both types of cyclooxygenases catalyze the incorporation of 4 oxygen atoms into arachidonic acid and the formation of a five-membered ring. This yields an unstable hydroperoxide derivative known as PG G₂. The hydroperoxide group at carbon atom 15 is rapidly reduced to a hydroxyl group by peroxidase, forming PGH₂. Up to the formation of PG H₂, the synthesis pathway is identical for all types of prostaglandins. Subsequent transformations of PG H₂ are cell-type specific.

For instance, in smooth muscle cells (SMC), PG H₂ can be reduced by PG E synthase to yield PG E₂ or by PG D synthase to yield PG D₂. Platelets contain thromboxane synthase, an enzyme that converts the same precursor PG H₂ into TX A2, which exhibits potent vasoconstrictive activity. In endothelial cells, prostacyclin synthase converts PG H₂ into PG I₂ (prostacyclin), which acts as a vasodilator.

C. Structure and Synthesis of Leukotrienes, HETEs, and Lipoxins

Leukotrienes are also derived from eicosanoic acids; however, unlike prostaglandins, they lack ring structures and contain 3 conjugated double bonds, although the total number of double bonds in the molecule is greater (Fig. 8-49). Leukotrienes C₄, D₄, and E₄ feature substituents consisting of the tripeptide Glutathione, the dipeptide glycyl-Cysteine, or cysteine, respectively.

Fig. 8-49. Lipoxygenase pathway of eicosanoid synthesis.

The lipoxygenase synthesis pathway, which gives rise to A wide variety of eicosanoids, initiates with The addition of an oxygen molecule to one of the carbon atoms adjacent to a double bond, yielding hydroperoxides known as hydroperoxyeicosatetraenoic acids (HPETEs). These hydroperoxides are subsequently converted into the corresponding hydroxyeicosatetraenoic acids (HETEs).

Structure and Synthesis of Leukotrienes and HETEs

Leukotrienes are synthesized via a pathway distinct from that of prostaglandins, starting with the formation of hydroperoxides—specifically, hydroperoxyeicosatetraenoic acids (HPETEs). These substances are either reduced to form hydroxyeicosatetraenoic acids (HETEs) or converted into leukotrienes or lipoxins. HETEs differ in THE POSITION OF the hydroxyl group at carbon 5, 12, or 15 (e.g., 5-HETE, 12-HETE).

Lipoxygenases act at the 5th, 12th, or 15th position of arachidonic acid depending on the tissue type. For example, polymorphonuclear leukocytes (PMNL) predominantly contain 5-lipoxygenase, platelets contain 12-lipoxygenase, and eosinophils contain 15-lipoxygenase.

In leukocytes and mast cells, 5-HPETE is converted into the epoxide leukotriene A₄ (LTA₄), where the subscript 4 denotes the total number of double bonds. The presence of 3 conjugated double bonds gives rise to the name "leukotriene."

Other types of leukotrienes are derived from LT A₄. LT B₄ is produced through the action of epoxide hydrolase in leukocytes and vascular endothelial cells. Another branch leads to the formation of a group of leukotrienes comprising LT C₄, LT D₄, and LT E₄. Their synthesis begins with the attachment of the tripeptide glutathione to carbon atom 6 to form LT C₄ in a reaction catalyzed by glutathione S-transferase. In the next step, glutamate is cleaved, leaving LT D₄ with the dipeptide glycylcysteine. In The final stage, Glycine is removed, leaving LT E₄ containing only cysteine.

Lipoxins (such as the primary lipoxin A₄) contain 4 conjugated double bonds and 3 hydroxyl groups.

The synthesis of lipoxins begins with the action of 15-lipoxygenase on arachidonic acid, followed by a series of reactions that lead to the formation of lipoxin A₄ (Fig. 8-50).

Fig. 8-50. Structure and synthesis of lipoxin A₄.

G. Mechanisms of eicosanoid action and major biological effects

Eicosanoids function as local hormones based on several criteria:

✵ they are synthesized in various tissues and Organs rather than solely in Endocrine glands;

✵ they act via autocrine or paracrine mechanisms;

✵ the concentration of eicosanoids in the blood is lower than that required to elicit a response in target cells.

Eicosanoids can exert a systemic effect only under certain pathological conditions, when their blood concentration increases to levels sufficient to affect the smooth muscle cells (SMCs) of an entire organ, such as the intestine, Lungs, or Blood Vessels.

Mechanisms of eicosanoid action

The same type of eicosanoid can act via both paracrine and autocrine mechanisms. For instance, TX A₂, produced by activated platelets, acts on the platelets themselves to enhance their aggregation capacity while simultaneously acting on surrounding vascular SMCs to promote their contraction. This creates conditions conducive to thrombus formation and prevents Hemorrhage at the site of vascular injury.

Eicosanoids act on cells through specific receptors. Some eicosanoid receptors are coupled to the adenylate cyclase system and protein kinase A—these are the receptors for PGE, PG D, and PC I. PG F₂α, TX A₂, endoperoxides (HPETE), and leukotrienes act through mechanisms that increase cytosolic calcium levels in target cells. In many cells, eicosanoids modulate the degree of adenylate cyclase activation in response to other factors, such as hormones. In such cases, eicosanoids affect the conformation of G Proteins in The Plasma Membrane. When an eicosanoid binds to stimulatory G_s proteins, The Effect of the primary stimulating agent is enhanced; when it binds to inhibitory G_i proteins, the effect is diminished. Eicosanoids act on cells across nearly all tissues in the body. Excessive eicosanoid production is observed in numerous diseases.

The Role of eicosanoids in the development of inflammation

Inflammation is the body's response to injury or infection, aimed at eliminating the infectious agent and repairing damaged tissues. The production of inflammatory mediators—eicosanoids, histamine, and kinins (local Peptide Hormones)—is triggered by reaction cascades initiated by invading infectious agents or tissue injury. The rate-limiting factor in eicosanoid synthesis is the release of fatty acids mediated by phospholipase. Phospholipase A₂ is associated with cell membranes and is activated by various factors, including histamine, kinins, mechanical stress on the cell, and the interaction of antigen-antibody complexes with the cell surface. The activation of phospholipase A₂ leads to increased eicosanoid synthesis.

Many eicosanoids function as inflammatory mediators and operate at all stages of inflammation. This results in increased capillary permeability, allowing transudate and leukocytes to migrate across the vascular wall. Leukotriene B₄ and lipoxin A₄ are potent chemotactic factors; by interacting with receptors, they stimulate leukocyte migration to the site of inflammation, as well as the secretion of lysosomal enzymes and the phagocytosis of foreign particles.

The symptoms of inflammation are redness, heat, Swelling, and pain. Redness and heat are caused by factors that increase blood flow to the injury site. Swelling results from increased fluid extravasation from capillaries and the migration of white Blood Cells into the inflammatory focus. Pain is caused by chemical components (tissue breakdown products, protons) and the compression of nerve endings. Various types of eicosanoids are involved in the development of these inflammatory signs (Table 8-8).

Table 8-8. CHARACTERISTICS OF THE BIOLOGICAL EFFECTS OF major eicosanoid types

The role of eicosanoids in thrombus formation

Blood Coagulation can be viewed as a process maintained in equilibrium by opposing systems: procoagulant and anticoagulant. Under pathological conditions or upon pharmacological intervention, this balance may shift in either direction. Normally, vascular endothelial cells produce prostacyclin I₂, which prevents platelet aggregation and vasoconstriction (Fig. 8-51). Upon endothelial cell damage (e.g., due to atherosclerotic plaque formation), PG I₂ synthesis declines. Platelets come into contact with the damaged vessel wall, resulting in the activation of phospholipase A₂. This leads to increased secretion of TX A₂, which stimulates platelet aggregation and thrombus formation at the site of vascular injury (Fig. 8-52), frequently precipitating myocardial infarction.

Fig. 8-51. The role of prostacyclins in regulating the tone of vascular smooth muscle cells and platelet aggregation. Under normal conditions, endothelial cells produce PG I₂, which induces SMC relaxation and inhibits platelet aggregation. Resting platelets do not produce thromboxanes. NO (nitric oxide) is a vasodilator.

Fig. 8-52. Impairment of eicosanoid synthesis in the area of endothelial injury. The action of TX A₂, which stimulates platelet aggregation and vasoconstriction, predominates at the site of vascular wall damage. Consequently, a thrombus forms in the damaged area, accompanied by a marked narrowing of the vascular lumen. In the myocardium, this can lead to myocardial infarction.

Studies on risk factors for myocardial infarction have demonstrated that individuals consuming large amounts of fish oil have a significantly lower incidence of this disease due to a reduced frequency of cardiac thrombus formation. It has been found that the family of eicosanoids synthesized in the body is influenced by the dietary fatty acid composition (see Table 8-3 above). If the diet provides an excess of eicosapentaenoic acid (20:5, ω-3), which is abundant in fish oil, this acid is preferentially incorporated into membrane phospholipids (replacing arachidonic acid) and subsequently

serves as the primary substrate for eicosanoid synthesis following the action of phospholipase A₂. This exerts a substantial effect on blood coagulation.

On a standard diet with a predominance of arachidonic acid (20:4, ω-6) over eicosapentaenoic acid, the action of TX A₂ is balanced by that of PG I₂ (Fig. 8-53) and other prostaglandins. In contrast, a diet rich in ω-3 fatty acids leads to the synthesis of more potent inhibitors of thrombosis (PG I₃, PG E₃, PG D₃) in endothelial cells, thereby reducing the risk of thrombus formation and myocardial infarction.

Fig. 8-53. Synthesis of thromboxanes and prostaglandins from arachidonic and eicosapentaenoic acids.

Inactivation of Eicosanoids

All types of eicosanoids undergo rapid inactivation. The half-life (T_1/2) of eicosanoids ranges from several seconds to a few minutes. Prostaglandins are inactivated by oxidation of the hydroxyl group at the C-15 position, which is essential for their activity, converting it into a keto group. The double bond at position 13 is then reduced. This is followed by β-oxidation of the side chain and subsequently ω-oxidation. The end products (dicarboxylic acids) are excreted in the urine. Active TX A₂

is rapidly converted into the biologically inactive TX B₂ through the Cleavage of the oxygen bridge between the 9th and 11th carbon atoms, resulting in the formation of hydroxyl groups.

D. Drugs as Inhibitors of Eicosanoid Synthesis

MECHANISM OF ACTION of Aspirin and Other Non-Steroidal Anti-Inflammatory Drugs

Aspirin is a drug that suppresses the primary signs of inflammation. The Mechanism of aspirin's anti-inflammatory action became understood when it was discovered that it inhibits cyclooxygenase. Consequently, it reduces the synthesis of inflammatory mediators and thereby dampens the inflammatory response. Cyclooxygenase is irreversibly inhibited via the Acetylation of Serine at position 530 within the Active Site (Fig. 8-54). However, the effect of aspirin is not very prolonged, as the Gene Expression of this enzyme remains unimpaired and new enzyme molecules continue to be produced. Other non-steroidal anti-inflammatory drugs (such as ibuprofen and acetaminophen) act via a competitive mechanism by binding to the enzyme's active site, thereby also decreasing prostaglandin synthesis.

Fig. 8-54. Mechanism of cyclooxygenase inactivation by aspirin. The acetyl residue is transferred from the aspirin molecule to the enzyme's OH group, resulting in its irreversible inhibition.

Mechanism of Action of Steroidal Anti-Inflammatory Drugs on Eicosanoid Synthesis

Steroidal drugs exhibit a significantly more potent anti-inflammatory effect than non-steroidal agents. Their mechanism of action involves the Induction of the synthesis of proteins known as lipocortins (or macrocortins). These proteins inhibit The activity of phospholipase A₂ and reduce the synthesis of all types of eicosanoids by preventing the release of arachidonic acid (or its analogue), which serves as the substrate for eicosanoid synthesis.

The Use of steroidal anti-inflammatory drugs is particularly crucial for patients suffering from bronchial asthma. The development of symptoms in this condition (bronchospasm and mucus exudation into the bronchial lumen) is driven, in part, by the overproduction of leukotrienes by mast cells, leukocytes, and bronchial epithelial cells. Administration of aspirin to patients possessing a high-activity lipoxygenase isoform can trigger an asthma attack. The reason for "aspirin-induced" asthma is that aspirin and other non-steroidal anti-inflammatory drugs inhibit solely the cyclooxygenase pathway of arachidonic acid metabolism, thereby increasing substrate availability for the lipoxygenase pathway and, consequently, leukotriene synthesis. Steroidal drugs inhibit the utilization of arachidonic acid through both the lipoxygenase and cyclooxygenase pathways, and thus they cannot induce bronchospasm.

Therapeutic Applications of Eicosanoid Derivatives

Although the Physiological effects of all eicosanoids are not yet fully elucidated, there are successful Examples of using eicosanoid analogues as medications for various diseases. For instance, analogues of PG E₁ and PG E₂ suppress gastric Hydrochloric acid secretion by blocking type II histamine receptors in gastric mucosal cells. These drugs, commonly known as H₂-blockers, accelerate the healing of gastric and duodenal ulcers. Furthermore, the ability of PG E₂ and PG F₂α to stimulate uterine smooth Muscle contraction is utilized to induce labor.