Gene delivery corrects N-acetylglutamate synthase deficiency and enables insights in the physiological impact of L-arginine activation of N-acetylglutamate synthase.

The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to urea. N-acetylglutamate synthase (NAGS) catalyzes formation of N-acetylglutamate, an essential allosteric activator of carbamylphosphate synthetase 1. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine, but the physiological significance of NAGS activation by L-arginine has been unknown. The NAGS knockout (Nags-/-) mouse is an animal model of inducible hyperammonemia, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG + Cit). We used adeno associated virus (AAV) based gene transfer to correct NAGS deficiency in the Nags-/- mice, established the dose of the vector needed to rescue Nags-/- mice from hyperammonemia and measured expression levels of Nags mRNA and NAGS protein in the livers of rescued animals. This methodology was used to investigate the effect of L-arginine on ureagenesis in vivo by treating Nags-/- mice with AAV vectors encoding either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine. The Nags-/- mice expressing E354A mNAGS were viable but had elevated plasma ammonia concentration despite similar levels of the E354A and wild-type mNAGS proteins. The corresponding mutation in human NAGS (NP_694551.1:p.E360D) that abolishes binding and activation by L-arginine was identified in a patient with NAGS deficiency. Our results show that NAGS deficiency can be rescued by gene therapy, and suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.

Effect of arginine on oligomerization and stability of N-acetylglutamate synthase.

N-acetylglutamate synthase (NAGS; E.C.2.3.1.1) catalyzes the formation of N-acetylglutamate (NAG) from acetyl coenzyme A and glutamate. In microorganisms and plants, NAG is the first intermediate of the L-arginine biosynthesis; in animals, NAG is an allosteric activator of carbamylphosphate synthetase I and III. In some bacteria bifunctional N-acetylglutamate synthase-kinase (NAGS-K) catalyzes the first two steps of L-arginine biosynthesis. L-arginine inhibits NAGS in bacteria, fungi, and plants and activates NAGS in mammals. L-arginine increased thermal stability of the NAGS-K from Maricaulis maris (MmNAGS-K) while it destabilized the NAGS-K from Xanthomonas campestris (XcNAGS-K). Analytical gel chromatography and ultracentrifugation indicated tetrameric structure of the MmMNAGS-K in the presence and absence of L-arginine and a tetramer-octamer equilibrium that shifted towards tetramers upon binding of L-arginine for the XcNAGS-K. Analytical gel chromatography of mouse NAGS (mNAGS) indicated either different oligomerization states that are in moderate to slow exchange with each other or deviation from the spherical shape of the mNAGS protein. The partition coefficient of the mNAGS increased in the presence of L-arginine suggesting smaller hydrodynamic radius due to change in either conformation or oligomerization. Different effects of L-arginine on oligomerization of NAGS may have implications for efforts to determine the three-dimensional structure of mammalian NAGS.

beta-Cyclodextrin interacts with the Alzheimer amyloid beta-A4 peptide.

Electrospray ionisation mass spectrometry has been used to show that the synthetic 40 amino acid beta-amyloid peptide (beta 1-40) interacts with the cyclic oligosaccharide beta-cyclodextrin. This interaction, presumably with the hydrophobic aromatic moieties on the peptide, has been shown to diminish substantially the neurotoxic effects of beta 1-40 in a cell line.

New mass spectrometric approaches to structural analysis in mixtures

Structural analysis of minor components in mixtures is a vital requirement in the development of any pharmaceutical compound. Mass spectrometry is uniquely able to give this kind of information on the trace amounts of material present as minor impurities in a drug substance. In this study we show that a combination of mass spectrometric analysers with different characteristics is an even more powerful approach with a higher chance of establishing a potential structure. In particular the advent of analysers capable of accurate mass measurement on small amounts of material has enabled structures to be proposed in situations where previously no real conclusions could be made. Copyright 1999 John Wiley & Sons, Ltd.

High throughput liquid chromatography/mass spectrometric analyses using a novel multiplexed electrospray interface.

A novel four- channel multiplexed electrospray liquid chromatography interface is described. This device has been used to analyse both single components and mixtures by liquid chromatography/mass spectrometry (LC/MS) as well as synthetic samples prepared by automated procedures. These data provided unambiguous molecular weight assignments to both major components and synthetic by-products in these samples. In this work particular attention has also been paid to the elimination of interchannel crosstalk. Copyright 1999 John Wiley & Sons, Ltd.

Faster compound analysis by mass spectrometry--the ToF revolution.

The introduction of the orthogonal Time of Flight (ToF) analyser has enabled ToF to be applied to continuous ionisation sources. In this technique, pulses of ions are ejected orthogonally from the main ion beam. This paper describes our development of techniques utilising the advantages of orthogonal-ToF for the analysis of small molecules especially drug substances. The fast scanning ability of the ToF enables its use with fast chromatographic procedures and can also be utilised to enable parallel detection of multiple LC columns. The medium resolution giving accurate mass measurement is utilised for faster and more reliable structural determination. These techniques are being used daily in our laboratories and some examples are shown.

Disposition of pranolium chloride in dogs, baboons and monkeys.

The general disposition of [14C]-Pranolium Chloride (SC-27761), a potential anti-arrhythmic agent, has been studied in the beagle dog, baboon and rhesus monkey. The compound was moderately absorbed from the gastro-intestinal tract of the three species at 5 mg/kg. There was appreciable inter-animal variation in the amount of absorption, and the absorption was dose-dependent in the monkey. After i.v. dosage the radioactivity was largely cleared via the kidneys. The initial elimination half-lives for Pranolium in the dog and primate were between 0.6 to 3.1 hours after i.v. dosage, but could not be determined after oral dosage. Less than 1% of the dose was localised in monkey fetal tissues, two hours after an i.v. dose was given to pregnant female rhesus monkeys, and the highest concentrations of radiolabel were detected in fetal liver. Pranolium was found to be extensively metabolised and 1-naphthol was identified as a major metabolite. Pranolium was excreted in urine both unchanged and as conjugates, but 1-naphthol was excreted largely as conjugates.

Plasma levels and urinary excretion of orally administered propantheline bromide in man.

After a single oral dose of 30 or 60 mg of propantheline bromide peak plasma levels of the drug were reached within 2 h in six healthy men. Mean peak plasma concentrations were 20.6 and 53.1 ng/ml after 30 mg and 60 mg respectively. The mean apparent absorption and elimination half-lives after 30 mg dose were 0.22 and 1.57 h respectively, and similar half-lives were found at the higher dose level. There was a dose related change in plasma levels and AUCinfinity of the drug, and some 3% to 4% of the administered dose of propantheline bromide was excreted unchanged in urine at each dose level. Comparison of the plasma levels and urinary excretion of the drug with those seen after i.v. administration in an earlier study indicated an apparently low systemic availability of orally administered propantheline bromide. There was tentative evidence of a qualitative relationship between the oral dose administered, plasma concentrations and the effects of propantheline bromide on salivary excretion.

Propantheline bromide plasma level, urinary excretion and pharmacological data in a comparison of the bioavailability of three oral formulations of Pro-Banthine.

To determine the comparative bioavailability of three oral formulations of propantheline bromide (PB) by both pharmacokinetic and pharmacodynamic parameters, six normal men received three standard Pro-Banthine 15 mg tablets, two prolonged acting (PA) Pro-Banthine 30 mg tablets or one developmental PA Pro-Banthine 45 mg capsule, in a study of balanced random crossover design. Plasma concentrations and urinary excretion of the unchanged drug were measured after each treatment using a stable isotope dilution assay. Salivary secretion rate and heart rate measurements were also made at intervals after each medication. The standard Pro-Banthine formulation was significantly more bioavailable, weight for weight, than either the developmental PA capsule (45 mg), p less than 0.05, or the two 30 mg PA tablets (60 mg), p less than 0.01, based on urinary excretion and plasma levels of PB and on salivary secretion and heart rate data. There was no evidence of significant prolonged action for the PA formulations.

Intensive nursing care of patients with a microvascular free flap after maxillofacial surgery.

This article provides an introduction to the care of patients following maxillofacial surgery, many of whom undergo the complexities of microvascular flap surgery and need careful nursing assessment in the postoperative period. A brief introduction to this surgery illustrates some of the potential reasons for admission to the intensive care unit. The nursing care is vital to maintain the survival of the flap, the details of which are discussed along with factors which potentially contribute to flap failure. Other considerations such as haemodynamic stability, postoperative complications, the importance of education and other options for postoperative management also form further points for discussion.

Rhabdomyolysis and acute renal failure in intensive care.

The aetiology and pathophysiology of the complex clinical condition rhabdomyolysis are outlined here Rhabdomyolysis can be caused by a number of factors including excessive muscle activity, direct muscle injury, ischaemia and excesses of temperature Specific nursing care strategies, focusing on the pathophysiology of the condition, are proposed The priorities of care should centre on maintaining patient safety with monitoring of cardiovascular and respiratory systems and assessment of renal function

The application of stable isotopes in biomedical research.

Many of the ways in which isotopes are used in biomedical research are reviewed. The use of stable isotopes in stable isotope dilution assays, for metabolite identification and in pharmacokinetic studies, is discussed and relevant examples are given to illustrate the various points made. Isotope effects and their implications for future drug design are considered. Some of the toxicity problems associated with the use of stable isotopes are also considered. Finally, a brief subjective view of possible future advances is made.

Rapid analysis of a peptide by fast-atom bombardment mass spectrometry after polyacrylamide gel electrophoresis.

Gel electrophoresis has been a powerful technique for the separation of peptides and proteins for many years. After electrophoresis separation on a polyacrylamide gel, the peptide bradykinin was localized using Coomassie Blue as a staining dye. Excess dye was removed by washing the gel with water. For mass spectrometric analysis, bands containing the peptide were crushed, extracted with acetic acid and the eluent applied to the fast-atom bombardment probe. Under these conditions the protonated molecule of bradykinin was clearly observed. Also apparent were sequence ions at about the same intensity observed from authentic bradykinin.

The replacement of trideuteromethyl by methyl groups when using methane as a reagent gas in chemical ionization mass spectrometry.

The methane chemical ionization spectra of a number of tertiary and quaternary amines with a N-trideuteromethyl group show substitution of this group by methyl. The isotopic purity of the original amines was shown to be greater than 99% using ammonia as a reagent gas.

Pyrolysis-gas chromatography-mass spectrometry of a series of bile acid sequestrants.

Pyrolysis of a series of polymers based on polystyrene and used as bile acid sequestrants produced characteristic mixtures of compounds which were analysed by gas chromatography-mass spectrometry. The nature of the substituent groups was clearly apparent while the polymer backbone gave rise to representative styrenes. The reproducibility of the results was examined by experimenting with the temperature of pyrolysis. It was found that at low temperatures very little fragmentation of the polystyrene backbone occurred but the substituents were still released in high yield. The orientation of the various substituted styrenes generated by pyrolysis was confirmed by the use of gas chromatography with infrared and mass spectrometric detection.

The use of negative ion thermospray liquid chromatography/tandem mass spectrometry for the determination of bile acids and their glycine conjugates.

A study was carried out on the negative ion thermospray liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry of a group of bile acids and their glycine conjugates. The liquid chromatographic/tandem mass spectrometric experiments were performed using low-energy collisions on a triple-quadrupole mass spectrometer. It was found that relatively high collision energies and collision gas pressures were required to produce fragmentation and that some unusual fragments were produced.

A coordinated approach towards the molecular weight determination of peptides by gel electrophoresis and fast-atom bombardment mass spectrometry.

The molecular weight of peptides can be determined by fast-atom bombardment mass spectrometry (FAB-MS) following polyacrylamide gel electrophoresis (PAGE). Initial results combining the fast and efficient separation of peptides by PAGE and the unambiguous determination of molecular weights by FAB-MS have been demonstrated for the three peptides bradykinin, neurotensin and gramicidin S. This method has also been applied to the determination of the molecular weights of two fragments from the tryptic digest of horse-heart cytochrome c.

Structural studies of alfalfa roots infected with nodulation mutants of Rhizobium meliloti.

Alfalfa roots infected with four nodulation defective (Nod-) mutants of Rhizobium meliloti which were generated by transposon Tn5 mutagenesis were examined by light and electron microscopy. In one class of Nod- mutants, which we can nonreactive, the bacteria did not induce root hair curling or penetrate host cells. In a second class of Nod- mutants, which we call reactive, the bacteria induced some root hair curling and entered root epidermal cells, although no infection threads were formed. In addition, reactive Nod- mutants induced extensive root hair proliferation and hypertrophied roots. This study presents the details of the phenotype of the association between each mutant strain and alfalfa roots.

The use of tandem mass spectrometry for the differentiation of bile acid isomers and for the identification of bile acids in biological extracts.

Tandem mass spectrometric (MS/MS) techniques hae been widely used for the differentiation of isomeric compounds, since their spectra may show differences sufficient to distinguish between them. There are several different ways by which the MS/MS data can be obtained depending on the energies of the ions and the collisions. In this paper MS/MS spectra have been obtained for a group of isomeric bile acids using: 1, low-energy ions and low-energy collisions in a triple quadrupole mass spectrometer by liquid chromatography/MS/MS; 2, high-energy ions and low-energy collisions in a hybrid mass spectrometer by fast-atom bombardment MS/MS. Liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) has also been used to identify the bile acids present in biological matrices such as bile extracts.