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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. 7 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. 8 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. 9 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. 14 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. 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