Brain extracellular levels of the putative antipsychotic CI-1007 and its effects on striatal and nucleus accumbens dopamine overflow in the awake rat.

The compound CI-1007 (R-(+)-1,2,3,6-tetrahydro-4-phenyl-1 [(3-phenyl-3-cyclohexen-1-yl)methyl]pyridine maleate) has been identified as a partial dopamine agonist and putative antipsychotic in in-vitro and in-vivo neurochemical, neurophysiological and behavioural tests. By use of microdialysis in conjunction with high-performance liquid chromatography (HPLC) with electrochemical detection, the effects of the drug on brain dopamine release, previously observed in anaesthetized animals, were shown to occur in awake animals also. Detection of peripherally administered CI-1007 in the brain, i.e. evidence of the drug's ability to penetrate the blood-brain barrier, was achieved by use of in-vivo brain microdialysis in awake, freely moving rats and capillary HPLC in combination with tandem mass spectrometry (HPLC/MS/MS). Intravenous administration of CI-1007 (40 mg kg-1) significantly inhibits dopamine release in the nucleus accumbens, a region associated with dopamine hyperactivity in schizophrenia, while having a non-significant impact on the striatal dopamine neurotransmission which is critical to regular motor function. The differential neurochemical profile of the drug indicates its potential usefulness in treating positive disease symptoms and implies that its extrapyramidal side effects are lower than those of typical antipsychotics.

Evaluation of the uncertainty of molecular mass measurement by electrospray ionization mass spectrometry.

Electrospray ionization (ES) is a novel method used in mass spectrometry (MS) for producing gas-phase ions from substances in solutions. Common practices for molecular mass estimation from ES spectra summarize the spectrum as a single peak giving no estimate of uncertainty or treat each peak as an independent molecular mass measurement. ES-MS data analysis showed that each peak in an ES spectrum does not always provide an independent measure of molecular mass. Underestimation of measurement uncertainty is a possible result. An elementary time series method, the Yule-Walker equations, was applied to molecular mass estimation from ES data.

Conodipine-M, a novel phospholipase A2 isolated from the venom of the marine snail Conus magus.

We describe the purification and first biochemical characterization of an enzymatic activity in venom from the marine snail Conus magus. This enzyme, named conodipine-M, is a novel phospholipase A2 with a molecular mass of 13.6 kDa and is comprised of two polypeptide chains linked by one or more disulfide bonds. The amino acid sequence of conodipine-M shows little if any homology to other previously sequenced phospholipase A2 enzymes (PLA2s). Conodipine-M thus represents a new group of PLA2s. This is remarkable, since conodipine-M displays a number of properties that are similar to those of previously characterized 14-kDa PLA2s. The enzyme shows little, if any, phospholipase A1, diacyglycerol lipase, triacylglycerol lipase, or lysophospholipase activities. Conodipine-M hydrolyzes the sn-2 ester of various preparations of phospholipid only in the presence of calcium and with specific activities that are comparable to those of well known 14-kDa snake venom and pancreatic PLA2s. The Conus enzyme binds tightly to vesicles of the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol and catalyzes the hydrolysis of this substrate in a processive fashion. Conodipine-M does not significantly discriminate against phospholipids with unsaturated versus saturated fatty acids at the sn-2 position or with different polar head groups. Linoleoyl amide and a phospholipid analog containing an alkylphosphono group at the sn-2 position are potent inhibitors of conodipine-M. We suggest that the functional resemblance of conodipine-M to other PLA2s might be explained by the utilization of similar catalytic residues.

Aqueous stability of human epidermal growth factor 1-48.

Human epidermal growth factor 1-48 (hEGF 1-48, Des(49-53)hEGF) is a single chain polypeptide (48 amino acids; 3 disulfide bonds; 5445 Da) possessing a broad spectrum of biologic activity including the stimulation of cell proliferation and tissue growth. In this study, three primary aqueous degradation products of hEGF 1-48 were isolated using isocratic, reverse phase/ion-pair HPLC. The degradation products were characterized using amino acid sequencing, electrospray ionization mass spectrometry, isoelectric focusing, and degradation kinetics. Results indicate that hEGF 1-48 degrades via oxidation (Met21), deamidation (Asn1), and succinimide formation (Asp11). The relative contribution of each degradation pathway to the overall stability of hEGF 1-48 changes as a function of solution pH and storage condition. Succinimide formation at Asp11 is favored at pH < 6 in which aspartic acid is present mostly in its protonated form. Deamidation of Asn1 is favored at pH > 6. The relative contribution of Met21 oxidation is increased with decreasing temperature, storage as a frozen solution (-20 degrees C), and exposure to fluorescent light.

Reversed-phase capillary high-performance liquid chromatography with on-line UV, fluorescence and electrospray ionization mass spectrometric detection in the analysis of peptides and proteins.

Analysis of peptide mixtures by reversed-phase capillary HPLC with gradient elution using three detectors in series: UV (214 nm), fluorescence (lambda exc. = 280 nm, lambda emiss. = 356 nm), and electrospray ionization mass spectrometry (ES-MS) is reported. The chromatographic integrity of the system and the detection limits were evaluated. The effect of the mass spectrometer's acquisition rate on the total ion current (TIC) profile was also examined. The utility of fluorescence monitoring with UV and ES-MS detection was demonstrated in the analysis of proteolytic digests of proteins. The native fluorescence character of tryptophan-containing peptides provides selectivity in peptide mapping, while monitoring UV absorption at 214 nm affords detection of the peptide bond. Three tryptophan-containing tryptic peptides of bovine serum albumin were immediately located by fluorescence among many UV peaks and ES-MS provided molecular masses allowing the peptides to be identified.

Control of exogenous factors affecting plasma homovanillic acid concentration.

Measurements of plasma homovanillic acid (pHVA) concentrations appear to be a valid research strategy in psychiatric disorders in which a central dopamine (DA) abnormality has been implicated. This study provides guidance about the control of some of the exogenous factors affecting pHVA concentrations. Fasting for 14 hours eliminates the dietary effects on pHVA in healthy human subjects. Changing position, walking for 30 minutes, or smoking two cigarettes has no effect on pHVA concentrations.

Mixture analysis by triple-quadrupole mass spectrometry: metabolic profiling of urinary carboxylic acids.

In this new method for profiling urinary carboxylic acids, lyophilized urine samples are analyzed directly with a triple-quadrupole mass spectrometer. Extraction, derivatization, and lengthy gas-chromatographic separation procedures are obviated by this approach. Total sample-preparation, instrument, and data-analysis time per sample is about 15 min. More than 100 different organic acids can be detected in a typical urine sample. All components of the solid urine residue are volatilized into the ion source of the mass spectrometer and converted to (M-1)- ions by reaction with OH- under chemical ionization conditions. Quadrupole 1 is set to transmit ions of a particular m/z ratio, which in turn collide with molecular nitrogen in quadrupole 2 and dissociate to smaller charged fragments. Carboxylic acid (M-1)- ions uniquely lose either CO2 (44 atomic mass units) or both H2O and CO2 (62 atomic mass units). Quadrupole 3 is set to pass only ions that have lost either 44 or 62 atomic mass units. Accordingly, the instrument specifically detects carboxylic acids in the urine matrix and no other components. Direct analysis of polyethylene glycols and their acidic metabolites in urine and serum of a burn patient treated with an antimicrobial cream having a polyethylene glycol base is also discribed.

Sequence analysis of polypeptides by collision activated dissociation on a triple quadrupole mass spectrometer.

A new approach to the direct sequencing of oligopeptides in complex mixtures is described. Mixtures of [2Ho]/[2H3]-N-acetylated and N,O-permethylated peptides are analyzed by collision activate dissociation on a triple quadrupole mass spectrometer using isobutane chemical ionization. Analysis of the collision activated dissociation spectra enables peptide sequences to be deduced. Use of electron capture negative chemical ionization for the sequence analysis of neuropeptides at the picomole level is also described.