Localization and quantification of drugs in animal tissues by use of desorption electrospray ionization mass spectrometry imaging.

Mass spectrometric imaging (MSI) has emerged as a powerful technique to obtain spatial arrangement of individual molecular ions in animal tissues. Ambient desorption electrospray ionization (DESI) technique is uniquely suited for such imaging experiments, as it can be performed on animal tissues in their native environment without prior treatments. Although MSI has become a rapid growing technique for localization of proteins, lipids, drugs, and endogenous compounds in different tissues, quantification of imaged targets has not been explored extensively. Here we present a novel MSI approach for localization and quantification of drugs in animal thin tissue sections. DESI-MSI using an Orbitrap mass analyzer in full scan mode was performed on 6 µm coronal brain sections from rats that were administered 2.5 mg/kg clozapine. Clozapine was localized and quantified in individual brain sections 45 min postdose. External calibration curves were prepared by micropipetting standards with internal standard (IS) on top of the tissues, and average response factors were calculated for the scans in which both clozapine and IS were detected. All response factors were normalized to area units. Quantifications from DESI-MSI revealed 0.2-1.2 ng of clozapine in individual brain sections, results that were further confirmed by extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis.

Identification of quinone imine containing glutathione conjugates of diclofenac in rat bile.

High-resolution accurate MS with an LTQ-Orbitrap was used to identify quinone imine metabolites derived from the 5-hydroxy (5-OH) and 4 prime-hydroxy (4'-OH) glutathione conjugates of diclofenac in rat bile. The initial quinone imine metabolites formed by oxidation of diclofenac have been postulated to be reactive intermediates potentially involved in diclofenac-mediated hepatotoxicity; while these metabolites could be formed using in vitro systems, they have never been detected in vivo. This report describes the identification of secondary quinone imine metabolites derived from 5-OH and 4'-OH diclofenac glutathione conjugates in rat bile. To verify the proposed structures, the diclofenac quinone imine GSH conjugate standards were prepared synthetically and enzymatically. The novel metabolite peaks displayed the identical retention times, accurate mass MS/MS spectra, and the fragmentation patterns as the corresponding authentic standards. The formation of these secondary quinone metabolites occurs only under conditions where bile salt homeostasis was experimentally altered. Standard practice in biliary excretion experiments using bile duct-cannulated rats includes infusion of taurocholic acid and/or other bile acids to replace those lost due to continuous collection of bile; for this experiment, the rats received no replacement bile acid infusion. High-resolution accurate mass spectrometry data and comparison with chemically and enzymatically prepared quinone imines of diclofenac glutathione conjugates support the identification of these metabolites. A mechanism for the formation of these reactive quinone imine containing glutathione conjugates of diclofenac is proposed.

Desorption electrospray ionization tissue imaging using heated nebulizing gas and high-resolution accurate mass spectra.

A new method for tissue imaging using desorption electrospray ionization (DESI) mass spectrometry is described. The technique utilizes a DESI source with a heated nebulizing gas and high-resolution accurate mass data acquired with an LTQ-Orbitrap mass spectrometer. The two-dimensional (2D) automated DESI ion source creates images using the ions that are collected under high-resolution conditions. The use of high-resolution mass detection significantly improves the image quality due to exclusion of interfering ions. The use of a heated nebulizing gas increases the signal intensity observed at lower gas pressure. The technique developed is highly compatible with soft tissue imaging due to the minimal surface destruction.

A novel bioactivation pathway for 2-[2-(2,6-dichlorophenyl)aminophenyl]ethanoic acid (diclofenac) initiated by cytochrome P450-mediated oxidative decarboxylation.

Diclofenac (2-[2-(2,6-dichlorophenyl)aminophenyl]ethanoic acid), a nonsteroidal antiinflammatory drug, undergoes bioactivation by cytochrome P450 oxidation to chemically reactive metabolites that are capable of reacting with endogenous nucleophiles such as glutathione (GSH) and proteins and that may play a role in the idiosyncratic hepatotoxicity associated with the drug. Here, we investigated the ability of diclofenac to be metabolized to 2-(2,6-dichloro-phenylamino)benzyl-S-thioether glutathione (DPAB-SG) in incubations with rat liver microsomes (RLMs) and human liver microsomes (HLMs) fortified with NADPH and GSH. Thus, after incubation of diclofenac (50 microM) with liver microsomes (1 mg protein/ml), the presence of DPAB-SG was detected in both RLM and HLM incubation extracts by liquid chromatography-tandem mass spectrometry techniques. The formation of DPAB-SG was NADPH-, concentration-, and time-dependent. Coincubation of diclofenac (10 microM) with ketoconazole (1 microM), an inhibitor of cytochrome P450 (P450) 3A4, with HLMs led to a 75% decrease in DPAB-SG formation. However, in contrast, coincubation with the P450 2C9 inhibitor sulfaphenazole (10 microM) or the P450 2D6 inhibitor quinidine (40 microM) led to a 1.9- and 1.6-fold increase in DPAB-SG production, respectively. From these data, we propose that P450 3A4 mediates the oxidative decarboxylation of diclofenac, resulting in the formation of a transient benzylic carbon-centered free radical intermediate that partitions between elimination (o-imine methide production) and recombination (alcohol formation) pathways. The benzyl alcohol intermediate, which was not analyzed for in the present studies, if formed could undergo dehydration to provide a reactive o-imine methide species. The o-imine methide intermediate then is proposed to react covalently with GSH, forming DPAB-SG.

Identification of a novel glutathione conjugate of diclofenac by LTQ-Orbitrap.

High resolution accurate MS with an LTQ-Orbitrap identified two novel metabolites of diclofenac in rat bile and rat and human hepatocyte incubations: a benzyl-S-glutathione conjugate and 2-(2,6-dichlorophenylamino) benzoic acid. A mechanism for the bioactivation of diclofenac involving decarboxylation is proposed.

Identification of a novel mitochondrial protein ("mitoNEET") cross-linked specifically by a thiazolidinedione photoprobe.

Thiazolidinediones address underlying causes of type 2 diabetes, although their mechanism of action is not clearly understood. The compounds are thought to function as direct activators of the nuclear receptor PPARgamma (peroxisome proliferator-activated receptor-gamma), although pioglitazone, the weaker agonist of the two thiazolidinediones now in clinical use, seems to have more useful effects on circulating lipids. We have used tritiated pioglitazone and a photoaffinity cross-linker to identify a novel binding site in mitochondria. A saturable binding site for [3H]pioglitazone was solubilized from the membranes with CHAPS and migrated as a large complex by size exclusion chromatography. The binding correlated with a <17-kDa protein (m17), marked by a photoaffinity cross-linker, in both subcellular location and selectivity of competition by analogs. The protein was isolated and identified by mass spectrometry analysis and NH2-terminal sequencing. Three synthetic peptides with potential antigenic properties were synthesized from the predicted nontransmembrane sequence to generate antibodies in rabbits. Western blots show that this protein, which we have termed "mitoNEET," is located in the mitochondrial fraction of rodent brain, liver, and skeletal muscle, showing the identical subcellular location and migration on SDS-PAGE as the protein cross-linked specifically by the thiazolidinedione photoprobe. The protein exists in low levels in preadipocytes, and expression increases exponentially in differentiated adipocytes. The synthetic protein bound to solid phase associated with a complex of solubilized mitochondrial proteins, including the trifunctional beta-oxidation protein. It is possible that thiazolidinedione modification of the function of the mitochondrial target may contribute to lipid lowering and/or antidiabetic actions.

Studies on the chemical reactivity of diclofenac acyl glucuronide with glutathione: identification of diclofenac-S-acyl-glutathione in rat bile.

Diclofenac, a nonsteroidal anti-inflammatory drug, is metabolized to a reactive acyl glucuronide that has been proposed to mediate toxic adverse drug reactions associated with its use. In the present study, we examined the ability of diclofenac acyl glucuronide (D-1-O-G) to transacylate glutathione (GSH) in vitro in buffer and in vivo in rats. Thus, in vitro reactions of D-1-O-G (100 microM) with GSH (10 mM) at pH 7.4 and 37 degrees C showed a linear time-dependent formation of diclofenac-S-acyl-glutathione (D-SG, 3 microM/h) through 60 min of incubation, reaching a maximum of 3.7 microM after 2 h of incubation. The major reaction that occurred was acyl migration of D-1-O-G (t1/2, 54 min) to less reactive isomers. The D-SG thioester product was shown to be unstable by degrading primarily to 1-(2,6-dichlorophenyl)indolin-2-one and by hydrolysis to diclofenac. After administration of diclofenac to rats (200 mg/kg), bile was collected and analyzed for D-SG by liquid chromatography-tandem mass spectrometry. Results indicated the presence of D-SG, which was confirmed by coelution with synthetic standard and by its tandem mass spectrum. When the reactivity of D-SG (100 microM) was compared with D-1-O-G (100 microM) in vitro in reactions with N-acetylcysteine (NAC, 10 mM), results showed the quantitative reaction of D-SG with NAC after 30 min of incubation, whereas only approximately 1% of D-1-O-G reacted to form diclofenac-S-acyl-NAC at the same time point. Results from these studies indicate that GSH reacts with D-1-O-G in vitro, and presumably in vivo, to form D-SG, and that the product D-SG thioester is chemically more reactive in transacylation-type reactions than the D-1-O-G metabolite.

Cross-linking in the living cell locates the site of action of oxazolidinone antibiotics.

Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.