Gloriano Moneti

The Release and Neosynthesis of Glutamic Acid Are Increased in Experimental Models of Hepatic Encephalopathy

Abstract: The effects of ammonium ions on the release of glutamic acid from the rat cerebral cortex were measured in vivo using cortical cups and a multiple ion detection technique. The neosynthesis of this amino acid from glucose was also studied in two experimental models of hepatic encephalopathy: (1) rats receiving large amounts of ammonium acetate (i.p.) and (2) rats with a surgically constructed portocaval anastomosis. Intraperitoneal administration of 8 mmol/kg of ammonium acetate increased the cortical release of glutamic acid from 9.1.0.8 to 19.2 (nmol. cm−2. min−1). Moreover, 20 min after ammonium acetate administration the rate of incorporation of 13C2, originating from [13C]glucose, into glutamic acid increased by 65%. In several brain areas of rats bearing a portocaval anastomosis and fed ad libitum for 4 weeks, the content of glutamic acid slightly increased and the rate of formation of [13C2]glutamate from [13C]glucose approximately doubled. These results indicate that ammonium ions increase the release and the formation of glutamic acid in the brain. The resulting increased concentration of this amino acid in the extracellular spaces may be one of the mechanisms of ammonia toxicity in vivo.

The excitotoxin quinolinic acid is present and unevenly distributed in the rat brain

The presence of quinolinic acid (2,3-pyridinedicarboxylic acid, QA) in the rat brain has been demonstrated using a mass-spectrometric method. Distribution studies indicate that this molecule is more concentrated in the cortex (2.1 nmol/g wet weight) than in other brain areas. Tryptophan, a possible QA precursor, administered in large doses, increases the cortical content of QA. The contrary occurs when rats are pretreated with p-chlorophenylalanine, a drug capable of decreasing brain tryptophan concentration. The neurotoxin 5,7-dihydroxytryptamine is inactive. Our findings support the idea that QA merits special attention as a potential transmitter and as an endogenous excitotoxin in brain.

ESI mass spectrometry and X-ray diffraction studies of adducts between anticancer platinum drugs and hen egg white lysozyme

The interactions of cisplatin and its analogues, transplatin, carboplatin and oxaliplatin, with hen egg white lysozyme were analysed through ESI mass spectrometry, and the resulting metallodrug-protein adducts identified; the X-ray crystal structure of the cisplatin lysozyme derivative, solved at 1.9 A resolution, reveals selective platination of imidazole Nepsilon of His15.

Modulation of Quinolinic and Kynurenic Acid Content in the Rat Brain: Effects of Endotoxins and Nicotinylalanine

: Quinolinic acid, an endogenous excitotoxin, and kynurenic acid, an antagonist of excitatory amino acid receptors, are believed to be synthesized from tryptophan after the opening of the indole ring. They were measured in the rat brain and other organs using gas chromatography‐mass spectrometry or HPLC. The enzyme indoleamine 2,3‐diox‐ygenase, capable of cleaving the indole ring of tryptophan, was induced by administering bacterial endotoxins to rats, which significantly increased the brain content of both quinolinic and kynurenic acids. Nicotinylalanine, an analogue of kynurenine, inhibited this endotoxin‐induced accumulation of quinolinic acid while potentiating the accumulation of‐kynurenic acid. The possibility of significantly increasing brain concentrations of kynurenic acid without a concomitant increase in quinolinic acid may provide a useful approach for studying the role of these electrophysiologically active tryptophan metabolites in brain function and preventing the possible toxic actions of abnormal synthesis of quinolinic acid.

The excitotoxin quinolinic acid is present in the brain of several mammals and its cortical content increases during the aging process.

The distribution of the excitotoxin quinolinic acid (QUIN) has been evaluated in the brains of rabbit, guinea pig and rat, using a mass spectrometric method. Furthermore, the cortical content of this molecule has been measured during the development and the aging of the rat. The cortex contained the highest concentration of QUIN in the three species studied. During the rat development the concentration of this molecule increased and unusually high amounts of it were found in approximately 50% of 30-month-old rats.

Rapid assay of topiramate in dried blood spots by a new liquid chromatography-tandem mass spectrometric method

Topiramate (TPM) is a new antiepiletic drug with efficacy in several types of seizures. Therapeutic drug monitoring of TPM is essential for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to determinate the TPM levels during the treatment. Advantages of DBS include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. Performance comparison between this method and the commercially available fluorescence-polarization immunoassay (FPIA) was made. The analysis was performed in selected reaction monitoring (SRM) mode. The calibration curve in matrix using D(12)-topiramate was linear in the concentration range of 0.0166-1.66mg/L (0.5-50mg/L in DBS) of topiramate with correlation coefficient value of 0.9985. In the concentration range of 0.5-50mg/L, the coefficients of variation in DBS were in the range 2.13-11.85% and the accuracy ranged from 93.93% to 110.67%. There was no significant differences between the concentrations (ranging 0.5-50mg/L) measured both FPIA on venous samples and LC-MS/MS assay on simultaneous DBS samples. The sensitivity and specificity of tandem mass spectrometry allow now high throughput topiramate analysis (the improvement was three times in comparison with FPIA). This new assay has favourable characteristics being highly precise and accurate. FPIA also proved to be precise and accurate, but is not always suitable for the sample collection in neonates in whom obtaining larger blood samples is not convenient or possible.

Inhibition of Nicotinamide Phosphoribosyltransferase: CELLULAR BIOENERGETICS REVEALS A MITOCHONDRIAL INSENSITIVE NAD POOL*

The NAD rescue pathway consists of two enzymatic steps operated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide mononucleotide adenylyltransferases. Recently, the potent Nampt inhibitor FK866 has been identified and evaluated in clinical trials against cancer. Yet, how Nampt inhibition affects NAD contents and bioenergetics is in part obscure. It is also unknown whether NAD rescue takes place in mitochondria, and FK866 alters NAD homeostasis within the organelle. Here, we show that FK866-dependent reduction of the NAD contents is paralleled by a concomitant increase of ATP in various cell types, in keeping with ATP utilization for NAD resynthesis. We also show that poly- and mono(ADP-ribose) transferases rather than Sirt-1 are responsible for NAD depletion in HeLa cells exposed to FK866. Mass spectrometry reveals that the drug distributes in the cytosolic and mitochondrial compartment. However, the cytoplasmic but not the mitochondrial NAD pool is reduced upon acute or chronic exposure to the drug. Accordingly, Nampt does not localize within the organelles and their bioenergetics is not affected by the drug. In the mouse, FK866-dependent reduction of NAD contents in various organs is prevented by inhibitors of poly(ADP-ribose) polymerases or the NAD precursor kynurenine. For the first time, our data indicate that mitochondria lack the canonical NAD rescue pathway, broadening current understanding of cellular bioenergetics.

Primary Aromatic Amines from Side-Stream Cigarette Smoke are Common Contaminants of Indoor Air

A very sensitive mass-spectrometry method has been developed for the analysis of aromatic amines in tobacco smoke and in indoor air. Cigarettes were smoked with a smoking machine; the anwies from the smoke were trapped in a 5% HCl water solution containing internal standards and detected by gas chromatography/mass spectrometry in the selected-ion-monitoring (SIM) mode. The amines measured were the following: aniline. 2-toluidine, 3-toluidine, 4-toluidine, 2-ethylaniline, 3-ethylaniline, 4-ethylaniline, 2,3-dimethylaniline, 2,4-dimethylaniline, 2,5-dimethylaniline, 2,6-dimethylaniline, 1-naphthylamine, 2-naphthylamine, 2-methyl-1-naphthylamine, 2-aminobiphenyl,3-aminobiphenyl and 4-aminobiphenyl. We analyzed nine brands of cigarettes sold commercially in Italy (Gauloise, Nazionali, Marlboro, Camel, MS, MS mild and MS lights), with and without filter. Main-stream smoke contained a lower amount of aromatic amines than side-stream smoke: the total level of these amines in main-stream smoke ranged from 200 to 1300 nglcigarette, whereas the level of aromatic amines in side-stream smoke varied from 20,000 to 30,000 nglcigarette. The smoke of black-tobacco cigarettes had higher levels of aromatic amines compared to light-tobacco cigarettes and the filters significantly reduced aromatic amines in main-stream smoke. We also determined the levels of aromatic amines in ambient air, offices and houses. Some aromatic amines (aniline and toluidine) were detected in ambient air, as well as in rooms of non-smokers. Most measurements showed a considerable contamination of aromatic amines derived from side-stream smoke, which was detected also in parts of the buildings in which smoking was not allowed.

Exploring metallodrug-protein interactions by mass spectrometry: comparisons between platinum coordination complexes and an organometallic ruthenium compound.

Electrospray ionisation mass spectrometry was used to analyse the reactions of metal compounds with mixtures of selected proteins. Three representative medicinally relevant compounds, cisplatin, transplatin and the organometallic ruthenium compound RAPTA-C, were reacted with a pool of three proteins, ubiquitin, cytochrome c and superoxide dismutase, and the reaction products were analysed using high-resolution mass spectrometry. Highly informative electrospray ionisation mass spectra were acquired following careful optimisation of the experimental conditions. The formation of metal–protein adducts was clearly observed for the three proteins. In addition, valuable information was obtained on the nature of the protein-bound metallofragments, on their distribution among the three different proteins and on the binding kinetics. The platinum compounds were less reactive and considerably less selective in protein binding than RAPTA-C, which showed a high affinity towards ubiquitin and cytochrome c, but not superoxide dismutase. In addition, competition studies between cisplatin and RAPTA-C showed that the two metallodrugs have affinities for the same amino acid residues on protein binding.

Exploring Metallodrug–Protein Interactions by ESI Mass Spectrometry: The Reaction of Anticancer Platinum Drugs with Horse Heart Cytochrome c

Since DNA is commonly believed to be the primary target for platinum metallodrugs, researchers’ interest has mainly focused on the characterisation of platinum–nucleic acid adducts while devoting much less attention to platinum–protein adducts. However, protein-bound platinum fragments probably represent truly active anticancer species—rather than mere drug-inactivation products—provided that metal transfer among distinct binding sites is kinetically allowed. Moreover, platination of specific side chains, which can affect the function of biologically crucial proteins or enzymes through the formation of tight coordinative bonds, might play a relevant role in the overall mechanism and toxicity of platinum drugs. The state of the art of platinum–protein interactions is described in a few articles and reviews; in any case, this issue warrants further experimental work. Thanks to the latest improvements, electrospray ionisation mass spectrometry (ESI-MS) today represents a very powerful method for exploring metallodrug–protein interactions. Owing to the introduction of “soft” ionisation methods, it is possible to transfer the intact metal–protein adduct—whole, in the gas phase—to determine its molecular mass with high accuracy and, thus, obtain its full molecular characterisation. However, much work is still required for the optimisation and the standardisation of experimental ESI-MS procedures directed at these systems. A great variability in ESI-MS responses is generally found in the current literature that depends on many factors, such as the nature of the protein, the nature of the metal, the specific solution conditions, the nature of the metalbound ligands, pH and the kind of buffer. Apparently, the intrinsic “fragility” of the metal–protein coordination bonds represents a major obstacle, often leading to extensive bond cleavage during ionisation and to loss of chemical information. Some pioneering ESI-MS studies of platinum–protein interactions were reported a few years ago by Gibson and co-workers, who used either ubiquitin or myoglobin as model proteins. A number of platinum–protein adducts were identified and characterised in detail. Afterwards, a few additional ESI-MS studies of various metallodrug–protein adducts were reported by other research groups. Cytochrome c is a small electron-carrier heme protein, localised in the mitochondria, that plays a crucial role in apoptotic pathways. Cytochrome c is also known to be an excellent ESI-MS probe and has been the subject of a number of investigations. This led us to choose cytochrome c as the model protein for our study. The following classical platinum drugs were selected: cisplatin, transplatin, carboplatin and oxaliplatin (Scheme 1).