Download Identification of the peptide conjugate with toxic acetaminophen

CHARLES UNIVERSITY IN PRAGUE
FACULTY OF PHARMACY IN HRADEC KRÁLOVÉ
Department of Pharmacology and Toxicology
&
Identification of the peptide conjugate
with toxic acetaminophen metabolite Nacetyl-p-benzoquinone imine
DIPLOMA THESIS
Supervisors:
Prof. Markku Pasanen
Přemysl Mladěnka, Ph.D.
2009-2010
Student: Kateřina Landíková
I proclaim that this thesis is my original work. All literature and
other sources which I used during writing thesis are present in the
list of references and in work are properly quoted.
I would like to thank to The Department of Pharmacology and Toxicology,
University of Eastern Finland especially to: Prof. Markku Pasanen, Jaana
Laine and Jukka Leppanen for their professional leadership, support and
cooperation Last but not least I would like to thank to Přemysl
Mladěnka, Ph.D. for his professional leadership during writing of my diploma
thesis.
Content
ABSTRACT .......................................................................................................................... 6 ABSTRAKT ......................................................................................................................... 7 1. INTRODUCTION ....................................................................................................... 8 2. THEORETICAL PART .............................................................................................. 9 2.1 BASIC THEORY .......................................................................................................................................... 9 2.1.1 Biotransformation ...................................................................................................................... 9 2.1.1.1 Phase I ............................................................................................................................................ 10 2.1.1.2 Cytochrome P‐450 ......................................................................................................................... 12 2.1.1.3 Phase II ........................................................................................................................................... 14 2.1.2 Acetaminophen ........................................................................................................................ 15 2.1.2.1. Mechanism of toxicity .................................................................................................................... 17 2.2 PRINCIPLES METHODS USED ...................................................................................................................... 21 2.2.1 High performance liquid chromatography ............................................................................... 21 2.2.2 Mass spectrometry ................................................................................................................... 22 3. THE AIM OF THE STUDY ..................................................................................... 23 4. PRACTICAL PART .................................................................................................. 24 4.1 MATERIALS ........................................................................................................................................... 24 4.1.1 Chemicals ................................................................................................................................. 24 4.1.2 Equipment ................................................................................................................................ 25 4.2 ANIMALS AND ANIMAL LIVER MICROSOMES .................................................................................................. 26 4.3 METHODS ............................................................................................................................................. 27 4.3.1 Isolation of microsomes ........................................................................................................... 27 4.3.2 Incubation of NAPQI‐peptide conjugate .................................................................................. 27 4.3.3 The HPLC separation ................................................................................................................ 28 4.3.4 Specific details of LC/MS analysis for detection NAPQI‐peptide conjugate ............................. 29 4.4 RESULTS ............................................................................................................................................... 30 4.4.1 Separation of NAPQI‐peptide conjugate with the HPLC ........................................................... 31 4.4.2 LC/MS analysis of the NAPQI‐peptide conjugate ..................................................................... 33 4.4.2.1 The peptide spectra ....................................................................................................................... 34 4.4.2.2 The exact molecular weight of NAPQI‐peptide conjugate ............................................................. 35 4.4.2.3 Mass spectra of NAPQI‐peptide conjugate .................................................................................... 36 4
5. DISCUSSION ............................................................................................................. 39 6. CONCLUSION .......................................................................................................... 41 7. LIST OF ABBREVIATIONS ................................................................................... 42 8. REFERENCES........................................................................................................... 43 5
Abstract
Acetaminophen (paracetamol) is one of the most used analgesic drug. It is
considered as a safe drug, although its administration in large doses can lead to due to
known metabolism to impairment of hepatocytes with subsequent necrosis.
Acetaminophen is metabolized primarily in the liver where it is converted to inactive
compound by conjugation with sulphate or glucoronide. However a small proportion is
metabolized by hepatic cytochrome P450 enzymes to a minor but toxic intermediate
metabolite N-acetyl-p-benzoquinone imine (NAPQI). This metabolite binds with the
macromolecules of the hepatic cells causing dysfunction of the enzymatic systems,
structural and metabolic disarray and eventually necrotic cell death.
This thesis is a part of larger work intended for preparation polyclonal antibodies
which can be used for further research in vivo / in vitro. The objectives of this study are a)
the confirmation that NAPQI is the toxic intermediate that can react with free thiol groups
and b) the in vitro production of the formed NAPQI-peptide conjugate.
Using dexamethasone-induced rat liver microsomal fraction led to the expected
production of NAPQI, that was linked on a synthetic peptide containing free thiol group.
This adduct was collected and purified by HPLC with gradient elution. The outcome of
this work is the final identification of the NAPQI-peptide conjugate by liquid
chromatography/mass spectrometry (LC/MS) system.
6
Abstrakt
Acetaminofen (paracetamol) je jedním z nejpoužívanějších léčiv. Je považován za
lék bezpečný, i když ve větších dávkách může, v důsledku známého metabolismu, vést
k poškození až nekróze hepatocytů.
Přednostně je metabolizován v játrech, kde je konjugačními reakcemi se sulfátem
nebo kyselinou glukuronidovou přeměňován na neúčinné sloučeniny. Nicméně malá část
je metabolizována pomocí cytochromu P450 na toxický metabolit N-acetyl-p-benzochinon
imin (NAPQI). Tento metabolit se váže na makromolekuly jaterních buněk a způsobuje
strukturní a metabolické poruchy a nakonec až nekrózu buněk.
Tato diplomová práce je součástí rozsáhlejšího výzkumu směřujícího k přípravě
polyklonálních protilátek, které budou použity pro další výzkum in vivo/in vitro.
Předmětem této práce bylo a) potvrzení, že NAPQI je toxický intermediát
acetaminofenového metabolismu, který váže volné thiolové skupiny a b) vytvořit in vitro
NAPQI-peptidový konjugát.
Použitím mikrosomální frakce z krysích jater, indukované dexametasonem, bylo
skutečně dosaženo produkce NAPQI intermediátu, který byl navázán na syntetický
polypeptid s volnou thiolovou skupinou. Tato sloučenina byla získána a vyčištěna pomocí
HPLC s gradientovou elucí. Následně byl tento adukt identifikován kapalinovou
chromatografií ve spojení s hmotnostní spektrometrií.
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1. Introduction
Acetaminophen (or commonly known in the Czech republic as paracetamol) is a
very common non-prescription analgesic widely used for pain relief and as an antipyretic.
It is recommended for adults, adolescents and children to calm the pain of various origins
such as headaches, teeth, musculoskeletal system, painful menstruation, and to reduce
elevated body temperature.
It is a safe drug but in large doses can lead to liver damage. Acetaminophen is one
of the most widely drugs cause hepatotoxicity in the United States, Great Britain and most
of Europe. Acetaminophen toxicity accounts for approximately 50 percents of all cases
of liver failure in US and carries 30 percents of mortality.
More than 90% of acetaminophen in the body is metabolized in the liver by the way
of conjugation, two thirds through glucuronidation and one third through sulphating.
Approximately 5 - 9% undergoes oxidative conversion by CYP 450 to the toxic metabolite
N-acetyl-p-benzoquinone imine (NAPQI).
This reactive electrophile NAPQI is detoxified mainly by conjugation with
glutathione, which can lead in large doses of acetaminophen to depletion of glutathione. In
that case, NAPQI binds to cellular macromolecules causing damage and necrosis to the
hepatocytes.
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2. Theoretical part
2.1 Basic theory
2.1.1 Biotransformation
Biotransformation is a part of process by which organism eliminate xenobiotics
which are both pharmaceuticals and toxic substances. In general lipophilic xenobiotics are
transformed to more polar and therefore more readily excretable products. Some
xenobiotics are innocuous, but many of them can provoke biologic response. Without
biotransformation potentially toxic lipophilic xenobiotics would be excreted very slowly
and cause marked impairment in the organism (Klaassen, 2001).
The outcomes of biotransformation may include, except for mentioned facilitation
of excretion, conversion of toxic parent compounds to nontoxic metabolites (known as
detoxification), conversion of nontoxic parent compounds to toxic metabolites (known as
bioactivation), and conversion of nonreactive compounds to reactive metabolites
(pharmacological bioactivation) (Barile, 2004).
The liver represents the major target organ for many xenobiotics (Pelkonen, 2008).
Although other organs, e.g. - the kidneys, the lungs, the gastrointestinal tract and the skin,
have also an important though smaller metabolic activity. Moreover, most substances can
reach systemic circulation after absorption from gastrointestinal tract only through the liver
(Jaeschke et al., 2002). The first pass effect, which is the term used for the first line
metabolism carried by the liver, significantly limits the bioavailability of many
pharmaceuticals.
Biotransformation reactions can be divided into two major categories called phase
I and phase II reactions.
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2.1.1.1 Phase I
Phase I reactions involve hydrolysis, reduction, and oxidation. These reactions
usually convert an initial drug to a more polar metabolite by addition of a hydrophilic
functional group (-OH, -NH2, -SH, -COOH). Often these metabolites are inactive, but this
is not a general rule. Polar metabolites may be excreted directly, but most of these
metabolites are not eliminated rapidly and undergo a subsequent reaction in which
an endogenous substrate (such as glucuronid acid, sulphuric acid) combines with the newly
established functional group to form a highly polar conjugate (phase II). In some instances,
the parent drug may already possess a functional group necessary for the formation
of conjugate (Katzung, 2001).
Drug-metabolising enzymes are not structurally specific and therefore may
advantageously cause biotransformation of many, even previously unknown, compounds.
The majority of oxidative biotransformation reactions are catalyzed by cytochrome P-450
(CYP). Other enzymes and their localization are shown in the Table I.
10
Table I General pathways of xenobiotic biotransformation and their major subcellular location
according to Klaassen, 2001.
REACTION
ENZYME
LOCALIZATION
Phase I
Hydrolysis
Reduction
Oxidation
Esterase
Microsomes, cytosol, lysosomes, blood
Peptidase
Blood, lysosomes
Epoxide hydrolase
Microsomes, cytosol
Azo- and nitro-reduction
Microflora, microsomes, cytosol
Carbonyl reduction
Cytosol, blood, microsomes
Disulfide reduction
Cytosol
Sulfoxide reduction
Cytosol
Quinone reduction
Cytosol. microsomes
Reductive dehalogenation
Microsomes
Alcohol dehydrogenase
Cytosol
Aldehyde dehydrogenase
Mitochondria, cytosol
Aldehyde oxidase
Cytosol
Xanthine oxidase
Cytosol
Monoamine oxidase
Mitochondria
Diamine oxidase
Cytosol
Prostaglandin H synthase
Microsomes
Flavin-monooxygenases
Microsomes
Cytochrome P450
Microsomes
Phase II
Glucuronide conjugation
Microsomes
Sulfate conjugation
Cytosol
Glutathione conjugation
Cytosol, microsomes
Amino acid conjugation
Mitochondria, cytosol
Acylation
Mitochondria, cytosol
Methylation
Cytosol, microsomes, blood
11
2.1.1.2 Cytochrome P-450
Many drug-metabolizing enzymes are located in the lipophilic membranes of
the endoplasmic reticulum of the liver and other tissues. When these lamelar membranes
are isolated by homogenization and fractionation of the cell, they re-form into vesicles
called microsomes. Cytochromes P-450 located in the microsomal fraction are an enzymes
family mediating most of oxidative processes in the liver. They require reduced
nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen (Gibaldi,
1991).
Cytochrome P-450 is a superfamily of hemoproteins that take part in oxidative
metabolism of many endogenous compounds as well. These enzymes with unique redox
properties are located on a smooth endoplasmic reticulum in practically all tissues. The
highest amount is present in the liver (Pelkonen et al., 2008). There are many CYP forms,
the classification of these forms is based on similar structural sequences. Individual family
members show distinct, but often overlapping substrate specificity (Gonzalez, 1988;
Klaassen, 2001). The main forms responsible for the metabolism of most drugs are: CYP
1A2, CYP 3A4, CYP 2B6, CYP 2C9, CYP 2D6 and CYP 2C19 (Pelkonen et al., 2008;
Dong et al. 2000).
Additional enzyme NADPH-cytochrome P450 reductase, a reducing agent
(NADPH) and molecular oxygen are necessary for the accomplishment of oxidationreduction processes. One oxygen atom is inserted in the product and the other into
a molecule of water (Katzung, 2001). A scheme of the whole oxidative cycle is presented
in Figure 1.
12
Figure 1 Oxidation of drugs by cytochrome P450
(R-H parent drug; R-OH oxidized metabolite; e- electron) Taken from Katzung, 2001.
step 
Oxidized (Fe3+) cytochrome P450 combines with a drug to form a binary complex.
step 
NADPH donates an electron to the flavoprotein reductase, which in turn reduces
the oxidized cytochrome P450-drug complex.
step 
A second electron is introduced from NADPH via the same flavoprotein reductase, which
serves to reduce molecular oxygen and to form an „activated oxygen“ cytochrome P450substrate complex.
step 
This complex in turn transfers
“activated“ oxygen
to the drug substrate to form
the oxidized product.
This enzyme complex has very low substrate specificity, the only common
character of the wide variety structurally unrelated drugs and chemicals as substrates are
high solubility in lipids (Katzung, 2001).
13
2.1.1.3 Phase II
A metabolite from phase I or rarely an original drug undergoes conjugation reaction
with an endogenous substrate. Mechanism of conjugation reactions demand high-energy
sources and is catalyzed by enzymes named tranferases. Trasferases catalyze the coupling
of an activated endogenous substance with a drug or its metabolite. Types of transferases
and their location are shown in Table II with types of substrate and specific examples.
Glucuronide formation is the most common conjugation process. It involves the reaction
between uridine diphosphate glucuronic acid and drugs containing hydroxyl, carboxyl or
amine groups. The second most common conjugation of hydroxyl containing compounds
represents conjugation with sulphate (Gibaldi, 1991).
Conjugates are polar molecules that are readily excreted. They are often inactive,
but in some cases may lead to the formation of reactive compounds responsible for
the toxicity of the drug e.g. acyl glucuronidation of nonsteroidal anti-inflammatory drugs
or N-acetylation of isoniazid (Katzung, 2001).
Table II Phase II reactions taken from Katzung, 2001.
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2.1.2 Acetaminophen
Acetaminophen (paracetamol) is an effective antipyretic and analgesic with
minimal anti-inflammatory properties. The absence of anti-inflammatory properties
distinguishes it from nonsteroidal anti-inflammatory drugs (NSAIDs) (Schilling et al.
2010). Acetaminophen is commonly used for the relief of moderate pains and for
reduction of a body temperature in fever. It is a major ingredient of numerous cold and flu
remedies. Acetaminophen is the major metabolite of two potent analgetic parent
compounds, acetanilid and phenacetin (Figure 2). The antipyretic activity of the molecule
resides in the aminobenzene structure (Barile, 2004).
The mechanism of action has not been satisfactorily explained so far. Antipyretic
effect of acetaminophen is mediated by the hypothalamus, the analgesic effect is mediated
according to current knowledge via central nervous system and but has a peripheral
component too (Lüllmann et al., 2004). In the periphery, acetaminophen is a weak inhibitor
of cyclooxygenases (COX), both COX-1 and COX-2, in comparison to NSAIDs. Therefore
its action is not associated with adverse gastrointestinal effects in contrast to NSAIDs. In
2002, a new COX isoenzyme was discovered, which can be the target of acetaminophen. It
was provisionally named COX-3. However, the idea that the mechanism of action of
acetaminophen is selective inhibition of COX-3 is not entirely clear. Some experimental
data say that the so-called COX-3 is just a variant of the COX-1 (Botting, 2000; Kis at al.,
2005).
Acetaminophen is recommended as an analgesic and antipyretic in the presence of
aspirin allergy, is also recommended for children (it is not associated with a risk of Reye's
syndrome in children) and for patients with blood coagulation disorders. It is also preferred
over NSAIDs to some patients because it carries a lower risk of gastrointestinal toxicity
(e.g., ulceration, bleeding) and therefore has better toleration. However it cannot be used
in patients allergic to acetaminophen or in patients with impaired of hepatic or kidney
function (Schilling et al., 2010).
The usual dosage is 325 to 650 mg orally or rectally every 4 to 6 hours in adults and
in children over age 12. It is recommend to not exceed the total daily dose of 4 g. Lower
maximum daily doses (e.g. 2 g) are recommended for patients who have higher risk of
15
hepatotoxicity, such as those malnourished or the patients who take enzyme-inducing
drugs or ethanol consumers (Schilling et al., 2010). There is a difference between chronic
and acute consumptions in the latter case. The effects might be the opposite: single dose of
ethanol protects against acetaminophen hepatotoxicity, but chronic ethanol administration
increases the hepatotoxicity and lethality of acetaminophen (Tredger et al., 1985; Prasad et
al., 1990; Wong et al., 1980).
Using higher doses can cause nonspecific symptoms which can start with dizziness
confusion and vomiting. It can even cause acute hepatic necrosis (Hinson, 1980;
Vermeulen et al., 1992; Prescott, 1980). Acetaminophen overdosing is the most frequent
cause of drug-induced liver failure in men (Lee, 2004). Table III shows the stages of
acetaminophen toxicity.
Table III Stages of acetaminophen toxicity according to Barile, 2004.
Stage
1
4
APAP Plasma
Concentration (mg/dl)a
≥ 150
8
≥ 75
12
≥ 35
24
≥5
Time Postingestion (h)
2
24-72
≥ 1 (at 72h)
3
72-96
≥1
4
4-14 days
≥1
16
Signs and Symptoms
Anorexia, nausea, vomiting, pallor,
diaphoresis, malaise, confusion,
hypotension, arrhythmias
Clinical improvement, right upper
quadrant pain, renal function
deterioration
Hepatic centrilobular necrosis:
jaundice, coagulopathy,
encephalopathy (coma);
reappearance of nausea and
vomiting, arrythmias, acute renal
failure, death
Resolution of hepatic dysfunction
and recovery if liver damage is
reversible
The dose of 140 mg/kg (i. e. 10 - 15g for an adult can cause hard intoxication with
serious hepatic failure and occasionally renal tubular necrosis. The hepatic failure achieves
its maximum in 3 - 4 days. The patients who used enzyme-inducing drugs
(e.g. phenobarbital, carbamazepine, phenytoin, rifampicin) are in a greater danger of
hepatic failure (even if the plasma level of acetaminophen is low). Until 4 hours after
the acetaminophen overdose it is recommended to give activated carbon to the patient, and
till 10 to 12 hours the antidote N-acetylcysteine should be used. Currently, the protocol is
the dose of 150 mg/kg of N-acetylcysteine by intravenous application followed by
12.5 mg/kg/h during 72 hours. The alternative way in acetaminophen intoxication is
the oral loading dose of 140mg/kg of N-acetylcysteine followed by 17 doses of 70 mg/kg
every 4 hours (Suchopar, 2009). N-acetylcysteine has a free thiol group that allows
the acceleration of the detoxification of toxic products of acetaminophen.
2.1.2.1.
Mechanism of toxicity
At therapeutic doses, acetaminophen is detoxified by three major pathways in
the liver. Most of the acetaminophen in the blood undergoes conjugation with glucuronic
acid and sulphates, this leads to inactive conjugates which are eliminated by urine.
Approximately 5-9% undergoes oxidative conversion by CYP 450 to the toxic metabolite
N-acetyl-p-benzoquinone imine (NAPQI). The all main CYP forms that were mentioned
previously are involved in converting acetaminophen to NAPQI. In particular, the most
active in this process are isoforms CYP3A4, CYP1A2, CYP 2D6 and CYP2E1 (Cheng,
2009; Thummel et al., 1993). The most efficient in converting acetaminophen into NAPQI
in human is CYP3A4 (Laine et al., 2009; Cheng et al., 2009).
NAPQI is a highly electrophilic metabolite and is detoxified mainly by conjugation
with glutathione (GSH) to mercapturic and cysteine conjugates (Figure 2) (Manyike et al.,
2000; James et al., 2003; Watkins et al., 2006).
17
NHCOCH3
NHCOCH3
OC2H5
phenacetin
acetanilid
NHCOCH3
OH
acetaminophen
cytochrome P450
glucuronide
conjugation
oxidation
sulfate
conjugation
NHCOCH3
NHCOCH3
NHCOCH3
glucuronate
sulfate
O
NAPQI
glutathione
conjugation
mercapturic and
cysteine conjugates
Figure 2 Metabolic pathway of acetaminophen and its prodrugs, taken from Barile, 2004.
Glucuronidation and sulphating become saturated after an acetaminophen overdose
or by a chronic use of acetaminophen. In such case, the toxic metabolite NAPQI, formed
by CYP 450, starts to increase. When also glutathione is depleted then the NAPQI excess
is able to damage the hepatocytes (Mitchell, 1973; Prescott, 1980).
18
Malnourished patients or fasting ones are at greater risk of acetaminophen
hepatotoxicity, because they may be deficient in glutathione at baseline. In addition, even
at lower than therapeutic doses, induction of CYP450 by enzyme-inducing drugs or
chronic alcohol consumption may lead to an enhancing of NAPQI. The liver is damaged
by two mechanisms: NAPQI covalently binds to hepatic cell macromolecules and the other
mechanism is oxidative stress due to depletion of glutathione (Dahlin 1984; Schilling et al.,
2010).
The covalent binding and formation of drug-protein adducts are generally assumed
to be related to drug toxicity. The selective protein covalent binding by drug metabolites
may lead to organ toxicity (Figure 3) (Hamdan et al., 2001; Pumford et al., 1997).
Figure 3 Bioactivation of drugs to reactive metabolic intermediates, taken from Zhou, 2005.
19
The clinical consequences of drug bioactivation and covalent binding to proteins
are not completely predictable. They depend on many factors for example on administered
drugs or individual patients, etc. Theoretically, all drug-metabolizing enzymes from Phase
I and Phase II can bioactivate drugs to metabolic intermediates that may provide
a nucleophilic attack. Thus, cellular functions may be disrupted, leading to cell death or
apoptosis (Klaassen, 2001; Park et al., 2001).
There is also possibility that the protein adducts may be recognized by immune
effector cells and thereby trigger immune responses. These responses may lead to cytokine
release, autoantibodies, or mild to severe immune-mediated organ injuries (Jaeschke et al.,
2002; Kaplowitz, 2001). NAPQI reacts with many cellular proteins and non-proteins thiols
by arylation and oxidation. These reactions leading to subsequent alteration of protein
structure and function, causing dysfunction of the enzymatic systems and eventually
necrotic cell death (Bessems et al., 2001; Jaeschke et al., 2002).
20
2.2 Principles methods used
2.2.1 High performance liquid chromatography
The High performance liquid chromatography is used very often for the analytical
evaluation of drugs because it enables both qualitative and quantitative evaluation of
separated components of the mixture with a high sensitivity, selectivity and in a relatively
short time. Moreover only a very small amount of the sample is sufficient for the analysis.
The fundamental basis for HPLC consists of passing a sample in a mobile phase
through a column packed with sorbents (stationary phase). As analytes pass through
the column, they interact between the two phases, mobile and stationary, at different rates.
The difference in rates is primarily due to different polarities for the analytes. The analytes
that have low interaction with the stationary phase or high interaction with the mobile
phase will exit the column faster. Various mixtures of analytes can be analyzed by
changing the polarities of the stationary phase and the mobile phase.
The changes in the polarity of the mobile phase are another variable that can affect
the efficiency of HPLC separation. The mobile phase polarity is generally the opposite of
the stationary phase. The system allows the polarity of the mobile phase to be changed
during the course of the HPLC run. The rate at which the polarity is changed defines
the "gradient". This gradient technique helps to separate the mixtures of variously polar
analytes more precisely.
21
2.2.2 Mass spectrometry
Mass spectrometry is the method enabling the determination of molecular weight of
unknown molecules. This information is useful in determining the identity of species.
A mass spectrometer determines the mass of a molecule by measuring the mass-tocharge ratio (m/z) of its ion. Ions are generated by inducing either the loss or gain of
a charge from neutral species. Once formed, ions are electro-statically directed into mass
analyzer where they are separated according to m/z and finally detected. The result of
molecular ionization, ion separation, and ion detection is a spectrum that can provide
molecular mass and even structural information.
A mass spectrum will usually be presented as a vertical bar graph, in which each bar
represents an ion having a specific mass-to-charge ratio (m/z) and the length of the bar
indicates the relative abundance of the ion. Most of the ions formed in a mass spectrometer
have a single charge, so the m/z value is equivalent to mass itself. Modern mass
spectrometers easily distinguish ions differing by only a single atomic mass unit (amu),
and thus provide completely accurate values for the molecular mass of a compound.
The highest-mass ion in a spectrum is normally considered to be the molecular ion, and
lower-mass ions are fragments from the molecular ion, assuming the sample is a single
pure compound.
22
3. The aim of the study
The aims of this study are:
a) confirmation that NAPQI is the toxic intermediate of acetaminophen which
attacks free thiol groups.
b) in vitro preparation of purified NAPQI-peptide conjugate by use of HPLC with
gradient
elution
and
identification
of
the
product
by
liquid
chromatography/mass spectrometry (LC/MS) system.
This study is the initiation of the further research of polyclonal antibodies.
The antibodies will be used in further experimental studies in vitro/ in vivo (details are not
specified due to protection of unpublished data).
23
4. Practical part
4.1 Materials
4.1.1 Chemicals
Acetaminophen
Orion; Helsinki, Finland
Acetonitrile
J.T. Baker; St.Louis MO, USA
Dipotassium salt of ethylenediaminetetraacetic acid (K2- EDTA)
Fluka 03660, Chemika; Switzerland
Formic acid
Merk KGaA; Darmstadt, Germany
Magnesium chloride
Sigma-Aldrich chemicals; Helsinki, Finland
NADPH
Sigma-Aldrich chemicals; Helsinki, Finland
Potassium phosphate
Sigma-Aldrich chemicals; Helsinki, Finland
Synthetic peptide composed of seven amino acids (gly-tyr-pro-cys-pro-his-pro)
GL Biochem; Sanghai, China
Tris hydrochloride buffer
MP Biomedicals; France
24
4.1.2 Equipment
Incubator 1000
Heidolph Instruments GmbH & Co. KG;
Germany
Cenrifuge Multifuge 3 L-R
Heraeus; Germany
Centrifuge Avanti J-30 I
Beckman Coulter; USA
Centrifuge Sorvall WX 90
Thermo Fisher Scientific Inc.; USA
Homogenisator Potter-Elvehjem
Pyrex; USA
Evaporating centrifuge RC 10-10
Jouan, France
HPLC
Shimadzu; Japan
Column Supelco 150 cm x 4 mm, 5µm C 18
Detector SPD-10AVP
Fraction collector FRC-10A
Preparative LC pump unit LC-8A
System controller SCL-10AVP
The LC/MS system
Finnigan, USA
Surveyor separations module
Ion trap LTQ
Column X-terra 150mm x 2.5; 3.5µ C8
25
4.2 Animals and animal liver microsomes
DBA/2N/Kuo Wistar rats were received from the National Laboratory Animal
Centre, Kuopio University. Six rats were pretreated with dexamethasone (100 mg/kg)
which was given i.p. in 0.5 ml oil suspension for three days. The animals had unrestricted
access to water and standard chow (Lactamin R36, Lactamin AB, Södertälje, Sweden).
In the in vitro experiments, liver microsomes and the cytosolic fraction were prepared from
livers of animals (described in next paragraph) (Lang et al. 1981). The Ethics Committee
for Animal Experiments, University of Kuopio approved these experiments.
26
4.3 Methods
4.3.1 Isolation of microsomes
Animals were killed by decapitation and their liver were removed and cut into
the pieces. The pieces of liver were put into the tubes and buffer pH 7.4 containing 0.1 M
Tris-HCl and 1mM K2-EDTA was added. 4 ml of buffer were equal to 1 gram of the liver
of a rat. Mixture was homogenized 10 times with Potter-Elvehjem homogenizer.
Homogenate was transferred into the centrifuge tubes. The tubes were centrifuged for
20 min (10,000 g, 4°C) by Avanti centrifuge. The supernatant was taken into the new
centrifuge and it was centrifuged for 1 hour (10,000 g, 4°C) by Sorvall centrifuge.
The supernatant was taken of the tubes and frozen. Pellet was homogenized with a PotterElvehjem homogenizer using a mixture where 1ml of the buffer was equal to 1g of
the liver. Microsomes were frozen at -80 °C in Eppendorf tubes and then used for
incubation. The ice was used in every step of the process.
4.3.2 Incubation of NAPQI-peptide conjugate
Mixture of compounds for incubation contained 200 mM potassium phosphate
buffer pH 7.4; 5 mM MgCl2; and 1 mM NADPH. The enzyme source was dexamethasoneinduced rat liver microsomal fraction. The amount of microsomal protein was 3.7 mg/ml.
The concentration of acetaminophen was 11 mM. The concentration of the peptide (glytyr-pro-cys-pro-his-pro) was 176 µg/ml and after first identifications was increased to 400
µg/ml. The reaction was conducted at 37 oC in the incubator for two hours with shaking.
Then the reaction was terminated by addition of equal amount of acetonitrile (ACN). The
test tubes were centrifuged for 20 min 2000 x g by centrifuge Multifuge 3 L-R. The
supernatant was put into the Eppendorf tubes and evaporated 1 h in evaporating centrifuge
at room temperature.
The samples were separated and collected with HPLC and measured with LC/MS
spectrometer.
27
4.3.3 The HPLC separation
The NAPQI-peptide conjugate was collected with the HPLC equipped with
the fraction collector. The column was Supelco C18, 150 x 4 mm, 5µm. Mobile phase
consisted of A: 0.1% formic acid and B: 90% acetonitrile (in 0.1% formic acid). The flow
rate was 0.8 ml/min.
After first identifications by mass spectrometry, the injection volume, gradient and
the run time were changed as follows: The starting injection volume was 100 µl which
after the first identifications by mass spectrometry increased to 200 µl. The original 65 min
of the process run decreased to 55 minutes. During the gradient elution the proportion of
the 90 % acetonitrile was linearly increased (Table IV).
Table IV shows concentration of the 90% acetonitrile during a certain periods of time.
I concentrations of acetonitrile were changed after the first set of samples to II.
Time
0,05
I ACN (%)
2
II ACN (%)
2
30
40
35
100
40
45
50
55
60
65
40
100
100
2
2
0
100
2
2
0
28
4.3.4 Specific details of LC/MS analysis for detection
NAPQI-peptide conjugate
Peptide was adducted with an excess of acetaminophen in CYP enzyme incubation
conditions in vitro, and the adduct was separated with HPLC and analyzed by LC/MS. Full
scan, the MS/MS spectra and exact molecular weight spectra were acquired. The relative
chromatographic peak areas of substrate-peptide were used for calculation of the results;
the peak areas of the double charged peaks were also calculated. The typical fragment ions
of the product ion spectra of NAPQI-peptide conjugate confirmed proved that correct was
monitored.
The LC/MS system was equipped with a pump separations module coupled to
a Finnigan LTQ ion trap mass spectrometer with an electrospray ionization (ESI) source.
The LC separation was carried out with an X-terra C8 3.5µ 2.5 x 150 mm column at room
temperature. The mobile phase consisted of A: 0.1 % formic acid and B: 90 % acetonitrile
(in 0.1 % formic acid). The flow rate was 200 µl/min. Samples were injected by an
autosampler. The injection volume was 10 µl. In gradient elution, the proportion of 90 %
acetonitrile was linearly increased from 2 to 50 % in 12 min and then returned to 2 %.
The run time was 18 min. The flow was diverted to waste for the first 2 min and after
9 minutes was diverted to waste again for the remaining time of the run.
The MS was operated in positive ESI mode. Nitrogen was used as sheath gas at
the flow of 90 arbitrary units. The spray voltage was set at 4.5 kV and the capillary
temperature was 225 º C. Mass range was 140.00 - 2000.00. Full scan, MS/MS spectra and
the exact molecular weight spectra were acquired. Within day reproducibility was with
acetaminophen standard (50 µM) 11 % RSD (n=4). Between day reproducibility was 19 %
RSD (n=10).
29
4.4 Results
Analysis of NAPQI-peptide conjugation formation represents an indirect way to
monitor the bioactivation of acetaminophen in vitro. Peptide was adducted with
acetaminophen in the incubation with liver microsomes and NADPH which produce
NAPQI-peptide conjugate (Figure 4). This adduct was collected with the HPLC.
Figure 4 Formula of NAPQI-peptide conjugate. It contains N-acetyl-p-benzoquinone imine
(red contour) binding to cysteine in a chain of amino acids (gly-tyr-pro-cys-pro-his-pro).
30
4.4.1 Separation of NAPQI-peptide conjugate with
the HPLC
The individual fractions were collected by HPLC with fractional collector. Figure 5
shows the first chromatogram. The quantitative characteristics are the areas under
the chromatographic peaks. These fractions were collected and then identified by LC/MS
analysis. There is also gradient elution curve which shows the proportion of the 90 %
acetonitrile (Table IV). Some of the conditions were changed (Figure 6) after the first
identifications as described in the Methods part.
Figure 5 Chromatogram with first set of samples of NAPQI-peptide conjugate. Collected fractions are
green and blue areas under the peaks. Injection volume was 100 µl and the concentration of the peptide (glytyr-pro-cys-pro-his-pro) was 176 µg/ml.
31
Figure 6 Chromatogram of NAPQI-peptide conjugate. Injection volume was increased to 200 µl, it
improved the visibility and separation of individual fractions, and concentration of the peptide (gly-tyr-procys-pro-his-pro) was increased to 400 µg/ml.
32
4.4.2 LC/MS analysis of the NAPQI-peptide conjugate
The adduct (Figure 7) was analyzed by LC/MS. Full scan, the MS/MS spectra and
exact molecular weight spectra were acquired.
862.35
318.14
350.1
221.09
699.2
193.09
804.31
776.31
Figure 7 Formula of NAPQI-peptide conjugate with intimated typical fragment ions e.g.-193.1 a2, 318.1
b3, 350.2 y3 , 699.2 y5 , 776.4 a6, 804.3 b6 , 822.3 b +H2O 6 and 862.5 y6.
33
4.4.2.1 The peptide spectra
Figure 8 shows the peptide (gly-tyr-pro-cys-pro-his-pro) spectrum which was
performed like a standard for measurement NAPQI-peptide conjugate. NAPQI is bound to
cysteine.
C:\Xcalibur\...\071009jelkats am ples 42
10/8/2009 12:57:47 AM
RT: 0.00 - 18.07 SM: 7B
NL: 1.15E6
RT: 5.56
AA: 55852637
100
Bas e Peak F: ITMS + c
ESI Full m s 2
[email protected] [
210.00-1000.00] MS
ICIS
071009jelkats am ples 42
90
80
Relative Abundance
70
60
50
40
30
20
10
0
0
1
2
3
4
5
6
7
8
9
Tim e (m in)
10
11
12
13
14
15
16
17
18
071009jelkats am ples 42 #867 RT: 5.56 AV: 1 NL: 1.23E6
F: ITMS + c ESI Full m s 2 [email protected] [ 210.00-1000.00]
655.33
50
45
40
550.29
Relative Abundance
35
30
25
20
15
10
532.35
435.20
350.22
752.34
637.32
235.11
673.41
5
274.29
320.32
250
300
404.38
455.21
400
450
386.34
0
350
478.33
568.38
518.27
500
550
621.28
713.41
600
m /z
650
700
750
800
850
900
950
1000
Figure 8 Peptide spectrum. Top panel: The relative chromatographic peak area of peptide (m/z 770.00)
and the lower panel shows its fragment ions e.g. 193.1 a2, 221.1 b2, 350.2 y3, 550.3 y5, 655.3 b6, 627.3 a6,
673.4 b + H2O 6 and 713.4 y6.
34
4.4.2.2 The exact molecular weight of NAPQI-peptide
conjugate
The precise molecular weight was determined by liquid chromatography (Figure 9)
and compared with theoretical values.
C:\Xcalibur\...\101209jelkats am ples 08
12/10/2009 3:57:14 PM
101209jelkats am ples 08 #634 RT: 6.09 AV: 1 NL: 5.34E3
F: ITMS + p ESI Z m s [ 914.00-924.00]
919.36
100
90
80
Relative Abundance
70
60
50
920.27
40
30
921.36
20
10
0
914.0
914.91 915.27
914.27
914.5
915.0
915.5
915.91
917.36
916.36
916.0
916.5
917.0
917.5
922.36
917.82 918.27
918.0
918.5
923.27
919.0
m /z
919.5
920.0
920.5
921.0
921.5
922.0
922.5
923.0
923.5
923.82
924.0
101209jelkats am ples 08 #635 RT: 6.09 AV: 1 NL: 3.57E4
F: ITMS + p ESI Z m s [ 455.00-465.00]
460.27
100
90
80
Relative Abundance
70
60
50
40
30
461.18
20
461.64
10
0
455.0
455.73 456.09 456.45
455.5
456.0
456.5
456.91
457.0
457.45
458.00
458.45
457.5
458.0
458.5
459.45
459.0
459.5
462.09
460.0
m /z
460.5
461.0
461.5
462.0
462.73 463.00
462.5
463.0
463.55
463.5
464.00 464.36
464.0
464.5
465.0
Figure 9 The exact molecular weight of NAPQI-peptide conjugate. It was 919.36 and a theoretical
value was 919.88. Also in bottom panel: The double charged peak (m/z 460.18) of NAPQI-peptide adduct
peak m/z 460.27 was detected.
35
4.4.2.3 Mass spectra of NAPQI-peptide conjugate
Mass
spectra
of
NAPQI-peptide
conjugate
(Figure
10).
The
relative
chromatographic peak areas of NAPQI-peptide (m/z 919.88) and its double charged peak
(m/z 460.18) were observed. The product ion spectra of NAPQI-peptide resulted in typical
fragment ions m/z 193.12, 350.10, 699.30, 776.52, 804.38, 822.28 for example, which was
compared with theoretical values (Table V). These theoretical numbers were acquired by
ProteinProspector program - MS-Product (Table VI, VII, VIII).
All the collected fractions by HPLC separation were analyzed in order to find the
NAPQI-peptide conjugate.
C:\Xcalibur\...\071009jelkats am ples 48
10/8/2009 2:54:20 AM
R T : 0.00 - 18.09 SM : 7B
Relativ e Abundanc e
80
60
RT: 14.07
A A : 3903
40
20
0
N L: 5.63E4
B ase P eak F : ITM S + c
ESI F ull ms 2
[email protected] [
125.00-1000.00] M S ICIS
071009jelkatsamples 48
RT: 6.38
A A : 3349696
100
Relative Abundance
N L: 1.17E2
B ase P eak F : ITM S + c
ESI F ull ms 2
[email protected] [
250.00-1500.00] M S ICIS
071009jelkatsamples 48
RT : 6.37
A A : 5490
100
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9
T im e (m in)
10
11
12
13
14
15
16
17
18
071009jelkatsam ples48 # 955 RT: 6.37 A V: 1 NL: 1.29E2
F : ITM S + c ESI F ull ms2 [email protected] [ 250.00-1500.00]
699.43
50
804.32
45
Relativ e Abundanc e
40
35
622.47
602.44
30
25
873.18
776.52
20
736.38
584.45
15
875.78
571.40
350.10
10
5
322.09
0
250
300
758.99
650.43
401.55
507.39
361.08
350
837.01
489.34
400
450
901.10
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
m /z
071009jelkatsam ples48 # 939 RT: 6.27 A V: 1 NL: 8.09E4
F : ITM S + c ESI F ull ms2 [email protected] [ 125.00-1000.00]
350.36
24
22
Relative Abundance
20
18
16
14
699.30
12
10
8
6
700.35
4
2
193.12
136.03
0
150
166.99
221.02
194.24
200
222.12 245.90
250
293.11
300
318.28
351.33
350
450.93
389.32 413.67
400
469.27 498.73
450
500
539.31 570.28 584.21 602.26
550
600
670.41
650
785.30 804.38
701.02
700
750
800
850
900
950
1000
m /z
Figure 10 Mass spectrum of NAPQI-peptide conjugate. The top panel shows High performance liquid
chromatography chromatogram and the bottom panel typical fragment ions of compound. The relative
chromatographic peak areas of NAPQI - peptide ( m/z 919.88 ) and its double charged peak ( m/z 460.18 ).
The product ion spectra of NAPQI-peptide resulted in typical fragment ions 193.1 a2, 318.2 b3, 350.1 y3 ,
699.3 y5 , 776.5 a6, 804.4 b6 , 822.3 b +H2O 6 and 862.5 y6.
36
Table V Comparison measured and theoretical fragment ions of the N- terminal and the C-terminal.
Substance
N-acetyl-p-benzoquinone imine (NAPQI)
Adduct
+ peptide (gly-tyr-pro-cys-pro-his-pro)
Mass
919.88
Measured mass
919.36
Teoretical
fragment 350.18 y3
ions C-terminal y-ions
602.24 y4
699.29 y5
862.36 y6
Measured
fragment 350.10
ions C-terminal y-ions
602.20
699.30
862.49
Theoretical fragment
ions N-terminal a,b and 193.09 a2
b+ H2O - ions
318.14 b3
776.31 a6
804.31 b6
822.32 b
+H2O 6
Measured
fragment
ions N-teminal a,b and 193.12
b+ H2O - ions
318.26
776.52
804.38
822.28
Table VI Main Sequence Ions.
b
y
---
1
G
7
---
221.0921
2
Y
6
862.3552
318.1448
3
P
5
699.2919
570.2017
4
u
4
602.2391
667.2545
5
P
3
350.1823
804.3134
6
H
2
253.1295
---
7
P
1
116.0706
37
Table VII All Sequence Ions
N-terminal
a
193.0972 290.1499 542.2068 639.2595 776.3185
b
221.0921 318.1448 570.2017 667.2545 804.3134
b+H2O
1
G
7
-
---
---
---
---
822.3239
2
Y
6
3
P
5
4
u
4
5
P
3
6
H
2
7
P
1
C-terminal
y
862.3552 699.2919 602.2391 350.1823 253.1295 116.0706
Table VIII Theoretical Peak table
70.0651
P 138.0662 H 318.1448 b3 639.2595 a5 822.3239 b6+H2O
110.0713 H 193.0972 a2 350.1823 y3 667.2545 b5 862.3552
y6
116.0706 y1 221.0921 b2 542.2068 a4 699.2919 y5 919.3767
MH
126.0550 P 253.1295 y2 570.2017 b4 776.3185 a6
136.0757 Y 290.1499 a3 602.2391 y4 804.3134 b6
38
5. Discussion
Acetaminophen was first marketed in the United States in 1953 and since eighth
decade of 20th century has been one of the most widely used analgesic. On the other hand,
it is also one of the most widely used hepatotoxic drugs in the world. Acetaminophen is
safe at therapeutic doses, but causes liver failure in overdoses. According to several studies
the toxicity causes reactive metabolite NAPQI (Albano et al., 1985; Dahlin et al., 1984).
Indeed, in this study, the formation of toxic metabolite NAPQI was confirmed with rat
liver microsomes induced by three day administrations of 100 mg/kg of dexamethasone.
NAPQI targets free thiol group, in particular, of glutathione, mainly because of its
physiological redundance over other molecules containing free thiol groups (Soglia et al.,
2004). Glutathione, or free thiol group, were mimicked by a synthetic peptide containing
cystein with a free thiol group in this study.
The dexamethasone-induced rat liver microsomes were used as the enzyme source,
because they have the most effective bioactivation capacity due to the inductive effect on
CYP 3A4 (10 to 20 times higher in comparison to control microsomes). CYP 3A4
metabolizes more than 60% of the clinically prescribed drugs and has the major role in
catalysing acetaminophen oxidation to NAPQI (Laine et al., 2009).
Our synthetic peptide, composed of seven amino acids (gly-tyr-pro-cys-pro-hispro), was established for this study. Proteins are major targets of reactive electrophiles due
to their diverse nucleophilic side chains. Cysteine, as mentioned above, is particularly
important because its thiol group has high reactivity toward electrophiles and oxidants.
Modification of thiol groups on functional protein sensors is thought to be a primary
mechanism of initiating signalling reactions associated with cell toxicity and adaptive
responses to electrophiles (Kemp et al., 2008). There are also molecules of proline which
can help that the peptide and afterwards also the adduct would not break up during
the incubation by the enzymes.
39
This method is analogous with the methods used for NAPQI-GSH conjugates
(Zheng et al., 2007; Soglia et al., 2006; Masubuchi et al., 2007) and it is based on
the knowledge that the hepatotoxicity of many known drugs is mediated by reactive
metabolites that covalently bind to proteins. By use of this LC/MS technique the selective
and sensitive identification NAPQI-peptide conjugate could have been realized. Indeed, in
this project a good yield of specific NAPQI-peptide conjugate was produced, purified,
identified and collected by HPLC and LC/MS technique.
During this project there were no changes in principle. After the first identifications
was the process run lowered by 10 minutes and the injection volume increased 2 times
during the separation by HPLC. These arrangements were made to speed up the collection
and also improved visibility and separation of individual fractions. Change, for acquired
bigger amount of adduct, was the increase of the concentration of the peptide (gly-tyr-procys-pro-his-pro) in the incubation mixture. It was originally 176 µg/ml and after the first
identifications the concentration of the peptide increased up to 400 µg/ml.
Pathophysiologically, redundant NAPQI reacts firstly with glutathione, when
glutathione is depleted, it may react with diverse nucleophilic groups on the hepatocyte
membranes. And therefore, this thesis is a part of larger research aimed at the treatment of
acetaminophen overdoses. This study is the initiation of the acquiring of polyclonal
antibodies. The antibodies will be used in further experimental studies in vitro/in vivo.
The detailed mechanism cannot be elucidated in the present, due to security of yet
unpublished data.
40
6. Conclusion
NAPQI was confirmed as the toxic intermediate of acetaminophen metabolism,
which bind free thiol groups and is therefore responsible for liver injury. NAPQI was
bound on a synthetic peptide to form a peptide conjugate, which was separated and
purified by HPLC. After the first identifications the injection volume, gradient, the run
time and proportion of substrates for incubation were changed. These changes led to
achievement of bigger amount of the adduct in a shorter period of time. The adduct was
identified by liquid chromatography/mass spectrometry system. This adduct will be then
processed to produce polyclonal antibodies by hen eggs which will be used for further
diagnostic use.
41
7. List of abbreviations
ACN
acetonitrile
COX
cyclooxygenase
CYP
cytochrome P450
Cys
cysteine
Gly
glycine
GSH
glutathione
His
histidine
HPLC
high performance liquid chromatography
K2 - EDTA
dipotassium salt dyhydrate of ethylenediaminetetraacetic acid
LC
liquid chromatography
MgCl2
magnesium chloride
MS
mass spectrometry
NADPH
nicotinamide adenine dinucleotide phosphate
NAPQI
N-acetyl-p-benzoquinone imine
NSAIDs
nonsteroidal anti-inflammatory drugs
Pro
proline
Tris - HCl
Tris hydrochloride
Tyr
tyrosine
42
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