bio transformation bibek singh mahat rn07
TRANSCRIPT
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BIOTRANSFORMATION
Page1of20. Preparedandsubmittedby:BibekSinghMahat,RollNo.07,M.Pharm.(Industrial),May2010
BIOTRANSFORMATION
SUBMITTED FOR INTERNAL EVALUATION FOR
THE DEGREE IN MASTER IN PHARMACY
BY
BIBEK SINGH MAHAT, M. PHARM STUDENT,
2nd SEMESTER, BATCH OF 2009
SUBMITTED TO:
Dr. Dharma Prasad Khanal
DEPARTMENT OF PHARMACY
SCHOOL OF SCIENCE
KATHMANDU UNIVERSITY
DHULIKHEL, NEPAL
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BIOTRANSFORMATION
Page2of20. Preparedandsubmittedby:BibekSinghMahat,RollNo.07,M.Pharm.(Industrial),May2010
TABLEOFCONTENTS:
1. Introduction
a) Xenobiotics
b) Chemical Reactions
c) Categorization of the Biotransformation reactions
2. Phase I Reactionsa) Oxidation
b) Reduction
c) Hydrolysis
d) Cytochrome P450 system
3. Phase II Reactions
a) Glucuronide conjugation
b) Sulfate conjugation
4. Biotransformation Sites
5. Modifiers of Biotransformation
a) Ageb) Genetic variability in biotransforming capability
c) Poor nutrition
d) Enzyme inhibition and enzyme induction
e) Dose level
6. References
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BIOTRANSFORMATION
Page3of20. Preparedandsubmittedby:BibekSinghMahat,RollNo.07,M.Pharm.(Industrial),May2010
Introduction
Biotransformation is the process whereby a substance is changed from one chemical to another
(transformed) by a chemical reaction within the body. Metabolism or metabolic transformations
are terms frequently used for the biotransformation process. However, metabolism is sometimesnot specific for the transformation process but may include other phases of toxico-kinetics.
Biotransformation is vital to survival in that it transforms absorbed nutrients (food, oxygen, etc.)
into substances required for normal body functions. For some pharmaceuticals, it is a metabolite
that is therapeutic and not the absorbed drug. For example, phenoxy-benzamine, a drug given to
relieve hypertension, is biotransformed into a metabolite, which is the active agent.
Biotransformation also serves as an important defense mechanism in those toxic xenobiotics and
body wastes are converted into less harmful substances and substances that can be excreted from
the body.
Toxicants that are lipophilic, non-polar, and of low molecular weight are readily absorbed
through the cell membranes of the skin, gastrointestinal (GI) tract, and lungs. These same
chemical and physical properties control the distribution of a chemical throughout the body and
its penetration into tissue cells. Lipophilic toxicants are hard for the body to eliminate and can
accumulate to hazardous levels. However, most lipophilic toxicants can be transformed into
hydrophilic metabolites that are less likely to pass through membranes of critical cells.
Hydrophilic chemicals are easier for the body to eliminate than lipophilic substances.
Biotransformation is thus a key body defense mechanism.
Fortunately, the human body has a well-developed capacity to biotransform most xenobiotics aswell as body wastes. An example of a body waste that must be eliminated is hemoglobin, the
oxygen-carrying iron-protein complex in red blood cells. Hemoglobin is released during the
normal destruction of red blood cells. Under normal conditions hemoglobin is initially
biotransformed to bilirubin, one of a number of hemoglobin metabolites. Bilirubin is toxic to the
brain of newborns and, if present in high concentrations, may cause irreversible brain injury.
Biotransformation of the lipophilic bilirubin molecule in the liver results in the production of
water-soluble (hydrophilic) metabolites excreted into bile and eliminated via the feces. The
biotransformation process is not perfect. When biotransformation results in metabolites of lower
toxicity, the process is known as detoxification. In many cases, however, the metabolites are
more toxic than the parent substance. This is known as bio-activation.
Occasionally, biotransformation can produce an unusually reactive metabolite that may interact
with cellular macromolecules (e.g., DNA). This can lead to very serious health effects, for
example, cancer or birth defects. An example is the biotransformation of vinyl chloride to vinyl
chloride epoxide, which covalently binds to DNA and RNA, a step leading to cancer of the liver.
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BIOTRANSFORMATION
Page4of20. Preparedandsubmittedby:BibekSinghMahat,RollNo.07,M.Pharm.(Industrial),May2010
Xenobiotic
A xenobiotic is a chemical which is found in an organism but which is not normally produced or
expected to be present in it. It can also cover substances which are present in much higher
concentrations than are usual. Specifically, drugs such as antibiotics are xenobiotics in humans
because the human body does not produce them itself, nor are they part of a normal diet.
The body removes xenobiotics by xenobiotic metabolism. This consists of the deactivation and
the secretion of xenobiotics, and happens mostly in the liver. Secretion routes are urine, faeces,
breath, and sweat. Hepatic enzymes are responsible for the metabolism of xenobiotics by first
activating them (oxidation, reduction, hydrolysis and/or hydration of the xenobiotic), and then
conjugating the active secondary metabolite with glucuronic or sulphuric acid, or glutathione,
followed by excretion in bile or urine. An example of a group of enzymes involved in xenobiotic
metabolism is hepatic microsomal cytochrome P450. These enzymes that metabolize xenobiotics
are very important for the pharmaceutical industry, because they are responsible for the
breakdown of medications.
Chemical Reactions
Chemical reactions are continually taking place in the body. They are a normal aspect of life,
participating in the building up of new tissue, tearing down of old tissue, conversion of food to
energy, disposal of waste materials, and elimination of toxic xenobiotics. Within the body is a
magnificent assembly of chemical reactions, which is well-orchestrated and called upon as
needed. Most of these chemical reactions occur at significant rates only because specific
proteins, known as enzymes, are present to catalyze them, that is, accelerate the reaction. A
catalyst is a substance that can accelerate a chemical reaction of another substance without itselfundergoing a permanent chemical change. Enzymes are the catalysts for nearly all biochemical
reactions in the body. Without these enzymes, essential biotransformation reactions would take
place slowly or not at all, causing major health problems.
An example is the inability of persons that have phenylketonuria (PKU) to use the artificial
sweetener, aspartame (in Equal). Aspartame is basically phenylalanine, a natural constituent of
most protein-containing foods. Some persons are born with a genetic condition in which the
enzyme that can biotransform phenylalanine to tyrosine (another amino acid), is defective. As
the result, phenylalanine can build up in the body and cause severe mental retardation. Babies areroutinely checked at birth for PKU. If they have PKU, they must be given a special diet to
restrict the intake of phenylalanine in infancy and childhood.
These enzymatic reactions are not always simple biochemical reactions. Some enzymes require
the presence of cofactors or co-enzymes in addition to the substrate before their catalytic activity
can be exerted.
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Page5of20.
These c
common
enzymes
co-enzy
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Page6of20.
The arra
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Example
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8/7/2019 Bio Transformation Bibek Singh Mahat RN07
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Page7of20.
acetamin
taken, th
excessiv
acetamin
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At high
to under
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frequentl
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amount
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ophen nor
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o reaction
xic to the li
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zation of t
ormation r
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ly classified
reactions ar
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ther substa
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ic can be
An exampl
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rmal level
y an additi
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situations
illustrates t
e Biotrans
actions are
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ANSFORMATIO
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BIOTRANSFORMATION
Page8of20. Preparedandsubmittedby:BibekSinghMahat,RollNo.07,M.Pharm.(Industrial),May2010
PhaseIReactions
Phase I biotransformation reactions are simple reactions as compared to Phase II reactions. In
Phase I reactions, a small polar group (containing both positive and negative charges) is either
exposed on the toxicant or added to the toxicant. The three main Phase I reactions are oxidation,reduction, and hydrolysis.
1. Oxidation
Oxidation is a chemical reaction in which a substrate loses electrons. There are a number of
reactions that can achieve the removal of electrons from the substrate. Addition of oxygen was
the first of these reactions discovered and thus the reaction was named oxidation. However,
many of the oxidizing reactions do not involve oxygen. The simplest type of oxidation reaction is
dehydrogenation that is the removal of hydrogen from the molecule. Another example of
oxidation is electron transfer that consists simply of the transfer of an electron from the substrate.Examples of these types of oxidizing reactions are illustrated below:
The specific oxidizing reactions and oxidizing enzymes are numerous. Most of the reactions are
self-evident from the name of the reaction or enzyme involved. Few of them are listed below:-
Oxidizing reactions.
Alcohol dehydrogenation
Aldehyde dehydrogenation
Alkyl/acyclic hydroxylation
Aromatic hydroxylation
Deamination / Desulfuration
N-hydroxylation
N-oxidation
Sulphoxidation
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Page9of20.
2. RedReductio
likely to
nitrogen
resultingcarbon t
tissues.
detoxific
illustrate
There ar
reactions
S
3. HydHydroly
fragment
and the
hydrazin
biotransf
illustrate
ction
n is a che
occur with
nitrogen d
amino cotrachloride
hus, reduc
ation. An
d below:
e fewer sp
is also self
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ehalogenati
isulfide red
itro reducti
-oxide redu
ulfoxide red
olysis
is is a che
s or smalle
ydrogen at
es, and car
ormation of
d below:
Prepared
ical reactio
xenobiotics
uble bonds
pounds arecan be red
tion reactio
xample of
ecific redu
evident fro
on
uction
n
ction
uction
mical reacti
r molecules
om is incor
amates are
procaine (l
BIOTR
ndsubmittedby:
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ANSFORMATIO
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the substra
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ly result in
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. Few of th
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at,RollNo.07,M.
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Page10of2
Toxicant
sufficien
biotransf
biotransf
more eff
Few Exa
1. Hyd
2. Hyd
COO
O
RCNH
.
s that have
tly ionized,
ormation o
ormation. T
ctive and i
mples of re
olysis of E
olysis of
HOCCH3
Oacetylsa
+
N
O
penicillins
Prepared
undergone
or hydrop
r converted
he interme
many case
ctions are li
ters :-
mides:-
licylic acid
H2O
SCH3
CH3
COOH
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andsubmittedb
Phase I biot
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to an int
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more toxic
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O
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tabolites th
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Page11of2
Cytoc
The prin
oxido-re
the cytoc
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romeipal reactio
uctases; c
hrome P45
Di
Prepared
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lled mixed-
system con
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BIOTRANSFORMATION
Page12of20. Preparedandsubmittedby:BibekSinghMahat,RollNo.07,M.Pharm.(Industrial),May2010
PhaseIIReactions
A xenobiotic that has undergone a Phase I reaction is now a new intermediate metabolite that
contains a reactive chemical group, e.g., hydroxyl (-OH), amino (-NH2), and carboxyl (-COOH).
Many of these intermediate metabolites do not possess sufficient hydrophilicity to permitelimination from the body. These metabolites must undergo additional biotransformation as a
Phase II reaction.
Phase II reactions are conjugation reactions, that is, a molecule normally present in the body is
added to the reactive site of the Phase I metabolite. The result is a conjugated metabolite that is
more water-soluble than the original xenobiotic or Phase I metabolite. Usually the Phase II
metabolite is quite hydrophilic and can be readily eliminated from the body.
The primary Phase II reactions are: Glucuronide conjugation - most important reaction
Sulfate conjugation - important reaction
Acetylation
Amino acid conjugation
Glutathione conjugation
Methylation
1. Glucuronide conjugation
Glucuronide conjugation is one of the most important and common Phase II reactions. One of themost popular molecules added directly to the toxicant or its phase I metabolite is glucuronic acid,
a molecule derived from glucose, a common carbohydrate (sugar) that is the primary source of
energy for cells.
The sites of glucuronidation reactions are substrates having an oxygen, nitrogen, or sulfur bond.
This includes a wide array of xenobiotics as well as endogenous substances, such as bilirubin,
steroid hormones and thyroid hormones. Glucuronidation is a high-capacity pathway for
xenobiotic conjugation.
Glucuronide conjugation usually decreases toxicity, although there are some notable exceptions,
for example, the production of carcinogenic substances. The glucuronide conjugates are
generally quite hydrophilic and are excreted by the kidney or bile, depending on the size of the
conjugate. The glucuronide conjugation of aniline is illustrated below:-
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Page13of2
Glucuro
.
nide forma
O
OH
O
OH
Olucose
Prepared
tion
H
H
H
gluconic a
glucuro
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C
OH
OH
OH
OH
O O
COOH
O
OH
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ANSFORMATIO
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OH
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H
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BIOTRANSFORMATION
Page14of20. Preparedandsubmittedby:BibekSinghMahat,RollNo.07,M.Pharm.(Industrial),May2010
2. Sulfate conjugation
Sulfate conjugation is another important Phase II reaction that occurs with many xenobiotics. In
general, sulfation decreases the toxicity of xenobiotics. Unlike glucuronic acid conjugates that
are often eliminated in the bile, the highly polar sulfate conjugates are readily secreted in the
urine. In general, sulfation is a low-capacity pathway for xenobiotic conjugation. Often
glucuronidation or sulfation can conjugate the same xenobiotics.
Sulfate ester formation
OH
COH
O
salicylic acid
+
a glucuronidederivative
O
H
HO
H
HO
H
H
OHHO
CO2H UDP
UDP-glucuronide
UDP
O
H
HO
H
HO
H
H
OHHO
CO2H
C
OOH
HO N
N N
N
NH2
O
SO
OHO
POOS
OO
OO
O
P OO
O
O
O
O
The enzymes catalyzing
this type of reaction are
called sulfotransferases.
Sulfates are carried as
phosphoadenosine-
phosphosulfate derivatives
(PAPS) - a high energy form.
+
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BIOTRANSFORMATION
Page15of20. Preparedandsubmittedby:BibekSinghMahat,RollNo.07,M.Pharm.(Industrial),May2010
Few Examples of other Phase II reactions are listed below:-
1.Methylation
2.Amino acid conjugation
OH
COH
H2N CH2COH
O
O OH
CNHCH2COH
O
O
salicylic acid
+
glycine
HO
HO
CHCH2NH2
OH
HO
HO
CHCH2NHCH3 CH3O
HO
CHCH2NH2
OH
dimethylmercury
(CH3)2HgCH3Hg+Hg2+
metanephrineepinephrinenorepinephrine
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BIOTRANSFORMATION
Page16of20. Preparedandsubmittedby:BibekSinghMahat,RollNo.07,M.Pharm.(Industrial),May2010
Few Examples of biotransformation reactions are listed below:-
ACTIVE(more potent)ACTIVE narcotic analgesicnarcotic analgesicMorphineCodeine
OH3CO OH
H
H N CH3
OHO OH
H
H N CH3
ACTIVE analgesicanalgesic
Salicylic acidAcetylsalicylic Acid
ACTIVE
OCCH3
CO2H
O
OH
CO2H
ACTIVE TOXICTOXICCNS depressantFormic AcidFormaldehydeMethanol
() ()
CH3OH HCH
O
HCOH
O
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BIOTRANSFORMATION
Page17of20. Preparedandsubmittedby:BibekSinghMahat,RollNo.07,M.Pharm.(Industrial),May2010
BiotransformationSites
Biotransforming enzymes are widely distributed throughout the body. However, the liver is the
primary biotransforming organ due to its large size and high concentration of biotransforming
enzymes. The kidneys and lungs are next with 10-30% of the liver's capacity. A low capacityexists in the skin, intestines, testes, and placenta. Since the liver is the primary site for
biotransformation, it is also potentially quite vulnerable to the toxic action of a xenobiotic that is
activated to a more toxic compound.
Within the liver cell, the primary subcellular components that contain the transforming enzymes
are the microsomes (small vesicles) of the endoplasmic reticulum and the soluble fraction of the
cytoplasm (cytosol). The mitochondria, nuclei, and lysosomes contain a small level of
transforming activity.
Microsomal enzymes are associated with most Phase I reactions. Glucuronidation enzymes,
however, are contained in microsomes. Cytosolic enzymes are non-membrane-bound and occur
free within the cytoplasm. They are generally associated with Phase II reactions, although some
oxidation and reduction enzymes are contained in the cytosol. The most important enzyme
system involved in Phase I reactions it the cytochrome P-450 enzyme system. This system is
frequently referred to as the "mixed function oxidase (MFO) system. It is found in microsomes
and is responsible for oxidation reactions of a wide array of chemicals.
The fact that the liver biotransforms most xenobiotics and that it receive blood directly from the
gastrointestinal tract renders it particularly susceptible to damage by ingested toxicants. Blood
leaving the gastrointestinal tract does not directly flow into the general circulatory system.
Instead, it flows into the liver first via the portal vein. This is known as the "first pass"
phenomena. Blood leaving the liver is eventually distributed to all other areas of the body;
however, much of the absorbed xenobiotic has undergone detoxication or bioactivation. Thus,
the liver may have removed most of the potentially toxic chemical. On the other hand, some
toxic metabolites are in high concentration in the liver.
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ModifiersofBiotransformation
The relative effectiveness of biotransformation depends on several factors, including species,
age, gender, genetic variability, nutrition, disease, exposure to other chemicals that can inhibit or
induce enzymes, and dose levels. Differences in species capability to biotransform specificchemicals are well known. Such differences are normally the basis for selective toxicity, used to
develop chemicals effective as pesticides but relatively safe in humans. For example, malathion
in mammals is biotransformed by hydrolysis to relatively safe metabolites, but in insects, it is
oxidized to malaoxon, which is lethal to insects.
Safety testing of pharmaceuticals, environmental and occupational substances is conducted with
laboratory animals. Often, differences between animal and human biotransformation are not
known at the time of initial laboratory testing since information is lacking in humans. Humans
have a higher capacity for glutamine conjugation than laboratory rodents. Otherwise, the types ofenzymes and biotransforming reactions are basically comparable. For this reason, determination
of biotransformation of drugs and other chemicals using laboratory animals is an accepted
procedure in safety testing.
1. Age:
Age may affect the efficiency of biotransformation. In general, human fetuses and neonates
(newborns) have limited abilities for xenobiotic biotransformations. This is due to inherent
deficiencies in many, but not all, of the enzymes responsible for catalyzing Phase I and Phase II
biotransformations. While the capacity for biotransformation fluctuates with age in adolescents,
by early adulthood the enzyme activities have essentially stabilized. Biotransformation capability
is also decreased in the aged. Gender may influence the efficiency of biotransformation for
specific xenobiotics. This is usually limited to hormone-related differences in the oxidizing
cytochrome P-450 enzymes.
2. Genetic variability in biotransforming capability :
Genetic variability in biotransforming capabilityaccounts for most of the large variation among
humans. The Phase II acetylation reaction in particular is influenced by genetic differences in
humans. Some persons are rapid and some are slow acetylators. The most serious drug-related
toxicity occurs in the slow acetylators, often referred to as "slow metabolizers". With slowacetylators, acetylation is so slow that blood or tissue levels of certain drugs (or Phase I
metabolites) exceeds their toxic threshold.
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Examples of drugs that build up to toxic levels in slow metabolizers that have specific genetic-
related defects in biotransforming enzymes are listed below:
3. Poor nutrition:
Poor nutrition can have a detrimental effect on biotransforming ability. This is related to
inadequate levels of protein, vitamins, and essential metals. These deficiencies can decrease the
ability to synthesize biotransforming enzymes. Many diseases can impair an individual's capacity
to biotransform xenobiotics. A good example, is hepatitis (a liver disease), which is well known
to reduce hepatic biotransformation to less than half normal capacity.
4. Enzyme inhibition and enzyme induction:
Enzyme inhibition and enzyme induction can be caused by prior or simultaneous exposure to
xenobiotics. In some situations exposure to a substance will inhibit the biotransformation
capacity for another chemical due to inhibition of specific enzymes. A major mechanism for the
inhibition is competition between the two substances for the available oxidizing or conjugating
enzymes is the presence of one substance uses up the enzyme that is needed to metabolize the
second substance.
Enzyme induction is a situation where prior exposure to certain environmental chemicals and
drugs results in an enhanced capability for biotransforming a xenobiotic. The prior exposures
stimulate the body to increase the production of some enzymes. This increased level of enzyme
activity results in increased biotransformation of a chemical subsequently absorbed. Examples of
enzyme inducers are alcohol, isoniazid, polycyclic halogenated aromatic hydrocarbons (e.g.,
dioxin), phenobarbital, and cigarette smoke. The most commonly induced enzyme reactions
involve the cytochrome P-450 enzymes.
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5. Dose level:
Dose level can affect the nature of the biotransformation. In certain situations, the
biotransformation may be quite different at high doses versus that seen at low dose levels. This
contributes to the existence of a dose threshold for toxicity. The mechanism that causes this
dose-related difference in biotransformation usually can be explained by the existence ofdifferent biotransformation pathways. At low doses, a xenobiotic may follow a biotransformation
pathway that detoxifies the substance. However, if the amount of xenobiotic exceeds the specific
enzyme capacity, the biotransformation pathway is "saturated". In that case, it is possible that the
level of parent toxin builds up. In other cases, the xenobiotic may enter a different
biotransformation pathway that may result in the production of a toxic metabolite.
An example of a dose-related difference in biotransformation occurs with acetaminophen. At
normal doses, approximately 96% of acetaminophen is biotransformed to non-toxic metabolites
by sulfate and glucuronide conjugation. At the normal dose, about 4% of the acetaminophen is
oxidized to a toxic metabolite; however, that toxic metabolite is conjugated with glutathione and
excreted. With 7-10 times the recommended therapeutic level, the sulphate and glucuronide
conjugation pathways become saturated and more of the toxic metabolite is formed. In addition,
the glutathione in the liver may also be depleted so that the toxic metabolite is not detoxified and
eliminated. It can react with liver proteins and cause fatal liver damage.
References:
1. National Library of Medicine; Emily Monosson ; 2008 "Biotransformation.
2. Encyclopedia of Earth; Eds. Cutler J. Cleveland, Washington, D.C.: Environmental
Information Coalition,
3. National Council for Science and the
Environment
4. Diaz E (editor). (2008). Microbial Biodegradation: Genomics and Molecular Biology (1st ed.
ed.). Caister Academic Press. ISBN 978-1-904455-17-2. http://www.horizonpress.com/biod.
5. www. wikipedia, the free encyclopedia.