Bio Chemistry: Bio Chemical Mechanism by ClassOf1

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									              Sub: Chemistry                                                                          Topic: Bio Chemistry

              Question:
              Scenario: An individual eats meats with much sodium nitrite, also drinks ethanol (alcohol),
              taking vitamins E and C, while also taking four tablets of 500 mg Tylenol.
              • Develop the following components of the question with brief organic chemistry explanations
              of what is happening and integrate the interactions of these ingested items.

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              1. Acetaminophen
              Phase I- Detoxification [show this to be occurring at the smooth ER] (cytochrome P 450)
              Phase II- Detoxification [show this occurring in the cytoplasm] (Glutathione –SH)
              [Be sure to include GSH and explanation of why the conjugate basically stays in the blood]
              Pain reduction by blocking conversion of arachidonic acid.
              2. Alcohol Detoxification
              Alcohol Dehyrdogenase
              NADH
              GSH
              3. Sodium nitrite
              Formation of carbocations
              Vitamin C
              4. Potential Lipid Radicals formed from superoxide [may form from converting 02 into water] or
              hydroxyl radicals that could be affected by the sodium nitrite’s utilization of vitamin C.
              Show how a fatty acid is made and then show the assembly of a triglyceride
              Synthesis of fatty acid
              Triglyceride
              Vitamin E
              Vitamin C
              Integration: How can liver damage result due to the intake of ethanol and Tylenol, but also how
              could liver damage occur even if ethanol was not ingested?

              Solution:


              General Drug Metabolism in Liver:

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              Sub: Chemistry                                                                          Topic: Bio Chemistry
                      A xenobiotic is a chemical which is found in an organism but which is not normally
              produced or expected to be present in it. Specifically, drugs such as antibiotics are xenobiotics
              in humans because the human body does not produce them itself nor would they be expected
              to be present as part of a normal diet.
                      The human body identifies almost all drugs as foreign substances (i.e. Xenobiotics) and
              subjects them to various chemical processes (i.e. metabolism) to make them suitable for
              elimination. This involves chemical transformations to
                               (a) reduce fat solubility and
                              (b) to change biological activity
                      Although almost all tissue in the body have some ability to metabolize chemicals,
              smooth endoplasmic reticulum in liver is the principal "metabolic clearing house" for both
              endogenous chemicals (e.g., cholesterol, steroid hormones, fatty acids, and proteins), and
              exogenous substances (e.g. drugs). The central role played by liver in the clearance and
              transformation of chemicals also makes it susceptible to drug induced injury.
                      Drug metabolism is usually divided into two phases: phase 1 and phase 2. Phase 1
              reaction is thought to prepare a drug for phase 2. However many compounds can be
              metabolised by phase 2 directly.
                      Phase I and Phase II reactions are biotransformations of chemicals that occur during
              drug metabolism.
                      Phase I reactions usually precedes Phase II, though not necessarily. During these
              reactions, polar bodies are either introduced or unmasked, which results in (more) polar
              metabolites of the original chemicals. In the case of pharmaceutical drugs, Phase I reactions can
              lead either to activation or inactivation of the drug.
                      Phase I reactions (also termed nonsynthetic reactions) may occur by oxidation,
              reduction, hydrolysis, cyclization, and decyclization reactions. Oxidation involves the enzymatic
              addition of oxygen or removal of hydrogen, carried out by mixed function oxidases, often in the
              liver. These oxidative reactions typically involve a cytochrome P450 haemoprotein, NADPH and
              oxygen. The classes of pharmaceutical drugs that utilize this method for their metabolism
              include phenothiazines, paracetamol and steroids. If the metabolites of phase I reactions are
              sufficiently polar, they may be readily excreted at this point. However, many phase I products
              are not eliminated rapidly and undergo a subsequent reaction in which an endogenous
              substrate combines with the newly incorporated functional group to form a highly polar
              conjugate (Hodjegan, 2007).



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              Sub: Chemistry                                                                          Topic: Bio Chemistry
                     Phase II reactions — usually known as conjugation reactions (e.g., with glucuronic acid,
              sulfonates (commonly known as sulfation) , glutathione or amino acids) — are usually
              detoxication in nature, and involve the interactions of the polar functional groups of phase I
              metabolites (Katzung).
                     A group of enzymes located in the endoplasmic reticulum, known as cytochrome, is the
              most important family of metabolizing enzymes in the liver. Cytochrome P-450 is the terminal
              oxidase component of an electron transport chain. It is not a single enzyme, rather consists of a
              family of closely related 50 isoforms, six of them metabolize 90% of drugs (Bailey and Dresser,
              2004; Nelson, 2003). There is a tremendous diversity of individual P-450 gene products and this
              heterogeneity allows the liver to perform oxidation on a vast array of chemicals (including
              almost all drugs) in phase 1. Three important characteristics of the P450 system have roles in
              drug induced toxicity:




              Drug metabolism in liver: transferases are : glutathione, sulfate, acetate, glucoronic acid. P-
              450 is cytochrome P-450 enzymes. 3 different pathways are depicted for Drugs A, B and C.
              1. Genetic diversity:
                      Each of the P-450 proteins is unique and accounts to some extent for the variation in
              drug metabolism between individuals. Genetic variations (polymorphism) in CYP450
              metabolism should be considered when patients exhibit unusual sensitivity or resistance to
              drug effects at normal doses. Such polymorphism is also responsible for variable drug response
              among patients of differing ethnic backgrounds (Nelson, 2003).
              2. Change in enzyme activity:



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              Sub: Chemistry                                                                          Topic: Bio Chemistry
                      Many substances can influence P-450 enzyme mechanism. Drugs interact with the
              enzyme family in several ways. Drugs that modify Cytochrome P-450 enzyme are referred to as
              either inhibitors or inducers. Enzyme inhibitors block the metabolic activity of one or several P-
              450 enzymes. This effect usually occurs immediately. On the other hand inducers increase P-
              450 activity by increasing its synthesis. Depending on inducing drug's half life, there is usually a
              delay before enzyme activity increases (Narhi, 1986).
              3. Competitive inhibition:
                      Some drugs may share the same P-450 specificity and thus competitively block their bio
              transformation. This may lead to accumulation of drugs metabolized by the enzyme. This type
              of drug interaction may also reduce the rate of generation of toxic substrate.
              Mechanism of liver damage:
                      Drugs continue to be taken off the market due to late discovery of hepatotoxicity. Due
              to its unique metabolism and close relationship with the gastrointestinal tract, the liver is
              susceptible to injury from drugs and other substances. 75% of blood coming to the liver arrives
              directly from gastrointestinal organs and then spleen via portal veins which bring drugs and
              xenobiotics in concentrated form. Several mechanisms are responsible for either inducing
              hepatic injury or worsening the damage process. Many chemicals damage mitochondria, an
              intracellular organelle that produce energy. Its dysfunction releases excessive amount of
              oxidants which in turn injures hepatic cells. Activation of some enzymes in the cytochrome P-
              450 system such as CYP2E1 also lead to oxidative stress.[11] Injury to hepatocyte and bile duct
              cells lead to accumulation of bile acid inside liver. This promotes further liver damage (Patel et
              al., 1998). Non-parenchymal cells such as Kupffer cells, fat storing stellate cells and leukocytes
              (i.e. neutrophil and monocyte) also have role in the mechanism.
              Acetaminophen:
                      Acetaminophen (Tylenol; N-acetyl-p-aminophenol; 4-hydroxyanilide; paracetamol), a p-
              aminophenol derivative is an active metabolite of both acetanilide and phenacetin. Acetanilide,
              the parent compound was first introduced as an antipyretic and analgesic in 1886 but its use
              was limited by its toxicity. Consequently other p-aminophenol derivatives were tested which
              led to the introduction of phenacetin in 1887 followed by acetaminophen in 1893, which was
              first used in medicine by von Mering (Insel, 1990). In the USA, acetaminophen has been
              available since 1952 and in the UK it has steadily gained in popularity as an analgesic from 1956
              onwards. It is available without prescription and when used at the recommended dosage has


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              Sub: Chemistry                                                                          Topic: Bio Chemistry
              few side effects and is considered safer than aspirin. In the UK and USA, there are more
              proprietary preparations of acetaminophen and multi-ingredient acetaminophen preparations.
                      The hepatotoxic effects of acetaminophen were first reported by
              Eder (1964) during long-term toxicity studies in cats and two years later, extensive centrilobular
              necrosis was observed in rats given high doses of acetaminophen (Boyd and Bereczky, 1966).
              The first cases of severe and fatal liver damage in man following acetaminophen overdose were
              reported in the UK in the same year (Davidson and Eastham, 1966; Thomson and Prescott,
              1966).
              Incidence
                      Acetaminophen overdose, with deliberate suicidal intent is increasing in most Western
              countries. In the USA during 1987, there were 60,000 calls to Poison Control Centers concerning
              acetaminophen overdoses (Smilkstein et al., 1988) and in 1993 over 90,000 were recorded,
              although in the same year only 92 deaths were directly related to overdose of acetaminophen
              (Litovitz et al., 1994). In the UK, the available figures indicate that acetaminophen overdose
              caused about 150 deaths in England and Wales in 1992. Acetaminophen-induced hepatotoxicity
              remains the commonest cause of acute liver failure accounting for between 50 and 60% of the
              cases seen (O’Grady et al., 1989). It has been recently reported that acetaminophen induced
              hepatic failure is the second leading cause of liver transplantation and accounts to considerable
              levels of morbidity and mortality (Lee, 2004).
              Clinical signs and symptoms
                      Acute hepatotoxicity often follows ingestion of more than 10 g of acetaminophen as is
              described in four clinical phases. The first phase begins shortly after ingestion and mainly
              involves gastrointestinal irritability. The latent phase (Phase 2) of 24 - 48 hours may extend
              upto 4 days and involves a transition from the patient feeling well to early clinical indications of
              hepatic dysfunction. Resolution of mild injury may occur at this point or severe hepatic necrosis
              may develop. Signs and symptoms may vary with liver failure and exhibit coma, encephalopathy
              and renal failure (Anker and Smilkstein, 1994).
              Acetaminophen-induced toxicity
                      Acetaminophen is a major cause of drug-related morbidity and mortality in humans,
              producing massive hepatic necrosis after a single toxic dose. A similar pathological picture is
              observed in rodents. Toxicity is essentially dose-dependent but there is interindividual
              variability in susceptibility with alcoholics and patients on enzyme-inducing drugs perhaps being
              more susceptible.



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              Sub: Chemistry                                                                          Topic: Bio Chemistry
                      Overdoses of the analgesic and antipyretic acetaminophen represent use of the most
              common pharmaceutical product poisonings. Although considered safe therapeutic doses, in
              overdose, acetaminophen produces a centrilobular hepatic necrosis that can be fatal (Prescott,
              1980). Whereas, the initial biochemical and metabolic events that occur in the early stages of
              toxicity have been well described, the precise mechanisms of hepatocyte death are poorly
              understood. Necrosis is recognized as the mode of cell death and apoptosis has been ruled out
              (Lawson et al., 1999; Gujral et al., 2002).
                      The role of covalent binding in toxicity as well as other factors recently identified
              contributes to the toxicity. These factors include oxidative stress, nitrotyrosine formation,
              inflammatory cytokines and the possible importance of mitochondrial permeability transition.
              Mechanism of biotransformation of xenobiotics:
              Phase I reactions:
                      The majority of xenobiotics have liophilic properties that facilitate absorption through
              the skin, lungs or mucosal surfaces. These require metabolic transformations to more water-
              soulble compounds before they can be excreted. These transformations are mediated by phase
              I and phase II biotransformation reactions. Phase I reactions results in the synthesis of a more
              chemically reactive metabolite of a lipophilic xenobiotic. In most , cases, these are followed by
              phase II reactions that conjugate the product in the phase I reaction with another molecule that
              detoxifies it, renders it significantly more stable and facilitates its elimination.

                      Phase I reactions are primarily oxidation-reduction reactions. Oxidation involves the
              extraction of electrons from a substrate and their transfer to molecular oxygen or to another
              electron-seeking (electrophilic) substance such as NAD+. Reduction involves the transfer of
              electrons to a substrate. Phase I reactions result in the addition of more polar, reactive groups
              such as hydroxyl (-OH), sulfhydryl (-SH), amino (-NH2), aldehyde (-COH) or carboxyl (-COOH).
              The most common biochemical oxidation-reduction reactions are mediated by three types of
              enzyme systems: (a) membrane bound iron-containing P450 cytochrome that are oxidized
              (Fe3+) or reduced (Fe2+) during their transfer of electrons from one substarte to another; (b)
              membrane-bound flavin-associated NADPH-dependent monoxygenases and (c) cytosolic NADH
              or NADPH-linked dehydrogenases, which oxidizes or reduce substrates by transfer of electron
              between the oxidized (NAD+, NADP+) and reduced (NADH, NADPH) forms of these nucleotides.
              Prostaglandin H synthase, found in abundance in the renal tubules, participates in the
              biotransformation of xenobiotics. It metabolizes acetaminophen to a highly reactive
              semiquinoneimine.


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              Sub: Chemistry                                                                          Topic: Bio Chemistry
                      The cytochrome P450 enzymes locate primarily in the mitochondrial endoplasmic
              reticulum are the most numerous and important of the enzymes involved in phase I oxidation
              reactions. They are heme proteins whose name derives from spectrophotometric
              characteristics of the heme molecule. When reduced cytochrome P450 (Fe 2+) binds to carbon
              monoxide, its maximal absorption spectrum occurs at 450 nm. The cytochrome P450 system is
              actually a coupled system containing an NADPH-dependent reductase that facilitates the
              transfer of electrons from NADPH to the enzyme-substrate complex and a heme-containing
              cytochrome that allows oxidation of complex by molecular oxygen and transfer of electrons to
              oxygen to form a water molecule. These enzymes mediate many different types of oxidation
              reactions. A general example is illustarted by the oxidation of a foreign compound R-H to R-OH:

                                 RH + NADH + O2                        NAD+ + H2O + ROH

                        This reaction occasionally proceeds through a reactive radical intermediate:

                                 (FeO)3+ + RH [(FeOH)3+R]                                  Fe3+ + ROH

              In other cases, electron transfer mar result in the production of an activated metabolites.

                               Fe3+ + ROOH RO- + (FeOH)3+
                               Fe3+ + ROOH RO + (FeOH)2+
                       Reduction biotransformation is facilitated by the P450 system when the electron
              donated by NADPH are transferred to the substarte, rather than to moleuclar oxygen. The
              reduction of a nitrogroup R-NO2 to an amine R-NH2 is an example of reductive
              biotransformation.
                       Other important phase I reactions are mediated by the alcohol, aldehyde and ketone
              oxidation system. These are predominantly cytosolic enzymes that depend on NAD + for
              oxidation reactions. A clinically familiar example is the metabolism of ethanol to acetaldehyde
              by alcohol dehydrogenase (ADH) followed by the rapid metabolism of ethanol to acetic acid by
              aldehyde dehydrogenase. (Fig) Alcohol dehydrogenase (ADH) is a cytosolic enzyme found in the
              liver, lungs, kidney and gastric mucosa that oxidizes many different alcohols.
                       Pharmacokinetic properties of these enzymes determine the extent of their involvement
              in the metabolism of a substrate. The ability of an enzyme to metabolize a substrate in a test
              tube does not predict its role in the cell. If two cellular enzymes are able to metabolize a
              substrate in order to function and needs only a low concentration, the enzyme that works at


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              Sub: Chemistry                                                                          Topic: Bio Chemistry
              the lower concentration will be responsible for most of the metabolism of that substrate. The
              Km which is defined as the concentration of the enzyme that results in 50% of maximal enzyme
              activity, describes this property of enzymes. For example, liver AD
								
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