Drugs and Poisons Chapter 2 - 1 hapter 2: Metabolism In an example cited previously you saw that pentobarbital had an extraordinarily long half-life if we considered only its binding to plasma proteins. In reality it is metabolized in 4 to 6 hours. The process of metabolism is also called biotransformation. All living organisms have developed enzymatic schemes to alter natural materials and xenobiotics for the purpose of using them for energy or building blocks and/or eliminating them or their by-products from the body. Since the major route of elimination for animal species is through the kidneys, most molecules have to be hydrophilic or capable of being converted to hydrophilic species in order to dissolve in the water leaving the body. This metabolic, biotransformative, process is meant to convert lipophilic substances to hydrophilic ones for subsequent excretion through the kidneys. An example of the endogenous molecules that are converted by this means are the steroid hormones, bile acids, cholesterol, neurotransmitters, and vitamins. Those substances not converted will most likely be excreted unchanged in the feces. A. Sites of biotransformation Metabolic reactions can occur almost anywhere in the body. However, there are certain organs and tissues that are the primary sites for the processes involved. 1. liver - The liver is the largest organ in the body weighing about 3 pounds. It is the major site of metabolism, and the formation of bile acids and other important biomolecules. When foods and other xenobiotics are ingested they pass through the walls of the intestine into the blood and travel via the large portal vein to the liver. Materials can also reach the liver after skin and lung absorption. If the dose of a drug or poison is large, it may take several passes through the liver in order to be completely absorbed by the liver and metabolized. C GlaxoWelcomeWeb Site Figure 2.1Drugs and Poisons Chapter 2 - 2 The hepatic (liver) intracellular sites of biotransformation are the cytosol and the surface of the organelle known as the smooth endoplasmic reticulum (SER). When attempts are made to isolate this organelle by disruption and differential centrifugation, the extensive membranous structure breaks up into tiny vesicles (like liposomes). These vesicles are called the microsomes or the microsomal fraction of a liver homogenate. When nerve cells are disrupted in a similar manner, they form vesicles known as synaptosomes. As we discuss further the process of metabolism we will refer to microsomal sites and nonmicrosomal sites, that is, those associated with the smooth endoplasmic reticulum and those not so associated, respectively. 2. other sites - There are many tissues and body fluids that contain enzymes of biotransformation. For example, the intestines contain a major oxidative enzyme system as do fractions of a liver homogenate, the brain (review the section on passing the blood-brain barrier), blood plasma, lungs, kidneys, and the skin; all contain nonmicrosomal enzymes or membrane-bound systems which can begin the transformation of lipophilic materials into lipophobic ones. B. Overview of Reactions The enzyme-catalyzed reactions of biotransformation can be categorized as two general types: Phase I - nonsynthetic and Phase 2 - synthetic (or conjugative). The nonsynthetic reactions are further delineated as oxidation, reduction, and hydrolysis. The nonsynthetic reactions can assist in preparing a highly lipophilic molecule for the synthetic pathways by introducing lipophobic and reactive functional groups such as -OH, -COOH, -SH, or NH2.. For example, an aromatic ring (lipophilic) can be converted to a phenol so that it can undergo esterification. The nonsynthetic reactions are usually nonmicrosomal and occur in many fluids and tissues as a xenbiotic first enters the body or bloodstream. The synthetic reactions involve the combination of the xenobiotic with another molecule prior to elimination via the urine or feces. Since these are synthesis reactions they require energy to be expended in the form of ATP or some other high energy intermediate. Among the conjugation reactions, glucuronide formation is exclusively microsomal. Other synthetic reactions may be associated with the membranes of the endoplasmic reticulum, mitochondria, or Golgi vesicles. The syntheses are divided into groups according to the species that is being used to derivatize the metabolite. C. Nonsynthetic Reactions 1. oxidation - Recall that the term oxidation refers to the loss of electrons which may also be identified by the loss of hydrogen atoms (formation of a multiple bond) or the addition of oxygen atoms. Oxidation cannot proceed without a substance to receive the lost electrons. The species receiving electrons is undergoing reduction. Converse Figure 2.2 The Endoplasmic ReticulumDrugs and Poisons Chapter 2 - 3 to the case of oxidation, reduction may involve the gain of electrons or hydrogen atoms (decrease in the number of multiple bonds) or the loss of oxygen atoms. The most prevalent nonsynthetic biotransformation reaction is OXIDATION. And the principle oxidative enzyme system is CYTOCHROME P450 (CYP450). If the cytochrome name is familiar, you may remember that cytochromes are part of the electron transport system. In fact they are part of a protein superfamily of heme proteins and are in a specific class of enzymes, the oxido-reductases. The figure below shows the specific subdivisions of this E.C.C. enzyme class of which the cytochrome P450 system is a part (from BBA, Hannemann, F. et al, "Cytochrome P450 systems-biological variations of electron transport chains") a. The Cytochrome P450 System CYP450 is a group of iron-heme proteins and occurs as isoforms throughout the animal, plant, and insect kingdoms. The 450 refers to the absorption maximum at λ=450 when iron in the reduced state (II) and bound to CO. In contrast to other heme proteins, the fifth ligand to the heme iron is a thiolate, so the group is more distinctly referred to as heme-thiolate proteins. There is sequence homology for the primary protein structures. Isoforms are identified using CYP for the enzyme, then a number for the family (>40% amino acid sequence homology), a letter for a subfamily (>55% sequence homology), and finally another number for the individual gene location. There are 14