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Biomedical subjects

D G Watson

Publications and source records attributed to D G Watson.

At least 19 recordsLinked to original sources
PubMed1992

Chronic lithium administration alters a prominent PKC substrate in rat hippocampus.

The therapeutic effect of lithium in the treatment of bipolar disorder exhibits a significant delay in the onset of action and a persistence of efficacy beyond abrupt discontinuation of treatment. Lithium is known to alter receptor-coupled phosphoinositide second messenger pathway in brain, resulting in indirect changes in an endogenous activator of protein kinase C (PKC). Such evidence has suggested that PKC may be involved in the mechanism of action of lithium in the brain. PKC represents a site wherein long-term regulatory changes in cell function occur through the phosphorylation of specific phosphoproteins involved in processes including neurotransmitter release and receptor activation. In studies of rats exposed to lithium, however, we have found no significant effects of chronic administration on the relative activity, subcellular distribution, or activation of PKC in hippocampus. We did find a major reduction in the in vitro PKC mediated phosphorylation of two major substrates, 83 kDa and 45 kDa, in hippocampus of rats exposed to chronic lithium and maintaining clinically relevant therapeutic levels in brain. Using immunoblot analysis we have identified a known myristoylated alanine-rich C kinase substrate (MARCKS) at 83 kDa. In vivo levels of MARCKS in hippocampus were found to be significantly reduced after chronic lithium exposure. These findings persist in animals withdrawn from lithium, but are not apparent following acute treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Determination of acidic metabolites of biogenic amines in human aqueous humour by gas chromatography--negative ion chemical ionisation mass spectrometry.

The concentrations of acidic metabolites derived from the biogenic amines o-, m-, and p-tyramines (o-, m-, and p-hydroxyphenylacetic acids), p-octopamine/p-synephrine (p-hydroxymandelic acid), and dopamine (homovanillic acid and 3,4-dihydroxyphenylacetic acid) were measured in human aqueous humour obtained from patients undergoing elective surgery for cataract removal or for trabeculectomy as a treatment for chronic open-angle glaucoma. There were no clear differences in the pattern of metabolism of neurotransmitters between the two groups. An unexpected finding was that the o-tyramine metabolite, o-hydroxyphenylacetic acid, was present in aqueous humour.

Acids↗
PubMed1991

Determination of (-)-bunolol and its metabolite, dihydro-(-)-bunolol, in human aqueous humour by gas chromatography-negative ion chemical ionisation mass spectrometry.

(-)-Bunolol (LB) was applied to the human eye in a commercially available eye drop formulation. LB and its metabolite, dihydro-(-)-bunolol (DHLB) were identified and quantified in human aqueous humour. The compounds were analysed as their trimethylsilyl-pentafluorobenzamide derivatives using gas chromatography-negative ion chemical ionisation mass spectrometry. In the case of DHLB the corresponding 2H3-labelled isotopomers were used as internal standards and LB was quantified against its methoxime derivative. Calibration curves for LB and DHLB against internal standards were linear with correlation coefficients 0.994 and 0.996, respectively. Replicate analyses of a pooled sample of aqueous humour containing LB and DHLB gave standard errors of the mean of +/- 9.8 and +/- 2.4% for the concentrations of LB and DHLB, respectively. The practical limit of detection of the method was ca. 30 pg for LB and ca. 100 pg for DHLB. The derivatization procedure was also satisfactory for the analysis of a number of other beta-blockers which are used in ophthalmological practice.

Aqueous Humor↗
PubMed1991

Biogenic amines: their occurrence, biosynthesis and metabolism in the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry.

Extraction-derivatisation techniques have been developed for the unambiguous identification of biogenic amines, and their putative amino acid precursors and metabolites (both major and minor), in single ventral thoracic nerve cords of the locust. Schistocerca gregaria, by the use of gas chromatography-negative-ion chemical ionisation mass spectrometry with selected ion monitoring. In addition the configuration of that enantiomer of p-octopamine present in the thoracid nervous system of the locust was established as R using the chiral derivatisation reagent, (-)-heptafluorobutyrylphenylalanyl chloride.

Acetylation↗
PubMed1991

The analysis of thiocarbamides by gas chromatography/negative-ion chemical-ionization mass spectrometry.

Thiocarbamides were converted to their di-N-pentafluorobenzyl (PFB) derivatives and analysed by gas chromatography/negative-ion chemical-ionization mass spectrometry with methane as reagent gas. The PFB derivatives of the 2-thiouracils gave mass spectra in which the ion current was carried largely by an ion arising from [M-PFB]-. The derivative was used in the determination of the uptake and metabolism of thiocarbamides by cultures of melanoma cells.

Gas Chromatography-Mass Spectrometry↗
PubMed1991

Measurement of urinary medium chain acyl glycines by gas chromatography--negative ion chemical ionization mass spectrometry.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is an inborn error of fatty acid metabolism, which is difficult to diagnose, partly because of its unpredictable clinical presentation. A specific diagnostic marker is an increased excretion of certain medium chain acyl glycines. A sensitive and specific method has been developed for the extraction, derivatization, identification and quantitation of urinary medium chain acyl glycines by gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICIMS). The following series of standard acyl glycines has been synthesized and characterized: hexanoyl, octanoyl, 3-phenylpropionyl and suberyl and their respective isotopomers (using 13C2-glycine; for use as internal standards). The range of excretion of these compounds in normal subjects has been established using this method and increased excretion of acyl glycines, particularly hexanoyl, 3-phenylpropionyl and suberyl was successfully demonstrated in three MCAD deficient subjects from one family.

Acyl-CoA Dehydrogenases↗
PubMed1991

The analysis of 5-fluorouracil in human plasma by gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICIMS) with stable isotope dilution.

5-Fluorouracil (5-FU) was extracted from plasma and converted to its di-N-ditrifluoromethylbenzyl (DTFMBz) derivative by treatment with DTFMBzBr. Under negative ion chemical ionization (NICI) conditions the derivative yielded a mass spectrum in which most of the ion current was carried by the M(-)-DTFMBz ion (m/z 355). Selected ion monitoring enabled detection of this derivative in amounts less than 1 pg, and the practical limit of detection for 5-FU extracted from plasma was ca 400 pg ml-1. 5-FU was quantified by adding a fixed amount of [15N2]5-FU to samples of plasma before extraction, and comparing the ratio of the ions of m/z 355 and 357 against a calibration curve constructed over the concentration range under investigation. The method was used to measure the variation in the concentration of 5-FU with time during continuous infusion of the drug via three, different protocols in patients with hepatic tumour.

Fluorouracil↗
PubMed1991

A mutant CHO-K1 strain with resistance to Pseudomonas exotoxin A and alphaviruses fails to cleave Sindbis virus glycoprotein PE2.

RPE.40, a mutant CHO-K1 strain selected for resistance to Pseudomonas exotoxin A, is defective in the production of infectious alphaviruses, although viruses are taken in and processed normally (J. M. Moehring and T. J. Moehring, Infect. Immun. 41:998-1009, 1983). To determine the cause of this defect, the synthesis of Sindbis virus proteins was examined. RPE.40 cells produced and glycosylated structural glycoprotein precursors PE2 and immature E1 normally. Mature E1 was formed, but PE2 was not cleaved to E2 and E3. PE2 instead was modified to a higher-molecular-weight form (PE2') in which the high-mannose oligosaccharides were processed to the complex form without proteolytic cleavage. The data suggest that the cleavage which produces E2 occurs within the trans-Golgi or in post-Golgi elements and is closely associated with the addition of sialic acid residues to the asparagine-linked oligosaccharides. RPE.40 cells make and release noninfectious Sindbis virions that contain PE2' and no detectable E2. These virions can be converted to an infectious form by treatment with trypsin. A defect in an intracellular endopeptidase activity in RPE.40 cells is postulated. Comparison of two Sindbis virus strains showed that the requirement for E2 in the virion to ensure infectivity is strain specific.

ADP Ribose Transferases↗
PubMed1991

Muscarinic receptor regulation and protein kinase C: sites for the action of chronic lithium in the hippocampus.

Regulation of muscarinic receptor activity in critical regions of the limbic system may represent a site for the therapeutic action of lithium. Muscarinic receptors in the hippocampus are coupled to phosphoinositide (PI) hydrolysis and the generation of the second messengers inositol (1,4,5) trisphosphate [Ins (1,4,5)P3] as well as diacylglycerol (DAG), which can directly activate protein kinase C (PKC). Since lithium may affect the regeneration of critical receptor-coupled pools of phosphatidylinositol 4,5-bisphosphate, studies in our laboratory have investigated the effects of chronic lithium on the regulation of the muscarinic receptor response. We have recently demonstrated that, following chronic administration of atropine in control and chronic lithium animals, there is an up-regulation of muscarinic receptor binding sites in the hippocampus; however, a concomitant sensitization of the carbachol-stimulated PI response is observed only in control animals. We have so far detected no effects of either in vitro or in vivo lithium on muscarinic receptor interactions with G proteins. However, we have observed a reduction in the in vitro phosphorylation of a major PKC phosphoprotein substrate in the rat hippocampus, following chronic lithium treatment. This effect may be related to long-lasting changes in regulation of receptor activity.

Animals↗
PubMed1990

Identification and quantitation of phenylalanine, tyrosine and dihydroxyphenylalanine in the thoracic nervous system of the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry.

Phenylalanine, tyrosine and dihydroxyphenylalanine (DOPA) were identified unambiguously and quantitatively determined in single ventral thoracic nerve cords from the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry. Deuterium-labelled analogues of each compound were added to a single ventral thoracic nerve cord in hydrochloric acid; the tissue was homogenised and the suspension centrifuged. The remaining hydrochloric acid was eliminated azeotropically by repeated additions of acetonitrile followed by evaporation under a stream of nitrogen and the resultant residue derivatised by reaction with hexafluoroisopropanol and pentafluoropropionic anhydride. Under negative-ion chemical ionisation conditions, the hexafluoroisopropanol-pentafluoropropionyl derivatives produced characteristic ions which were sufficiently abundant to be suitable for selected-ion monitoring. This method is highly specific and gave a limit of detection below the nanogram level. The amounts of phenylalanine, tyrosine and DOPA in a single ventral thoracic nerve cord were, respectively, 194 +/- 81, 347 +/- 88 and 11 +/- 11 ng per tissue.

Animals↗
PubMed1990

Identification and quantitation of N-acetyl metabolites of biogenic amines in the thoracic nervous system of the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry.

The N-acetylated metabolites of p-tyramine, p-octopamine and dopamine were identified unambiguously and quantitatively determined in a single ventral thoracic nerve cord of the locust, Schistocerca gregaria, by gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-NICIMS). Deuterium-labelled analogues of each compound were added to a single ventral thoracic nerve cord in acetonitrile: the tissue was homogenised and the suspension centrifuged. The solvent was removed from the supernatant and the resultant residue was derivatised with trifluoroacetic anhydride. Under negative-ion chemical ionisation conditions, the trifluoroacetyl derivatives produced ions which were sufficiently abundant to be suitable for selected-ion monitoring. This method is highly specific and gave a limit of detection below the picogram levels. N-Acetyl-5-hydroxytryptamine was determined using a previously published GC-NICIMS technique [S.P. Markey, R.W. Colburn and J.N. Johannessen, Biomed. Mass Spectrom., 7 (1981) 301]. The concentrations of N-acetyltyramine, N-acetyloctopamine, N-acetyldopamine and N-acetyl-5-hydroxytryptamine in locust thoracic nerve cords were, respectively, 1.86 +/- 0.71, 1.13 +/- 0.34, 6.77 +/- 8.48 and 0.07 +/- 0.02 ng per tissue.

Acetic Anhydrides↗
PubMed1990

Dichlorotetrafluoroacetone as a derivatisation reagent in the analysis of mandelic acids in human urine.

Dichlorotetrafluoroacetone has been used to prepare 4-substituted 2-bis (chlorodifluoromethyl)-1,3-dioxolan-5-one derivatives of mandelic acids which were found to be suitable for the analysis of these compounds by gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-NICIMS). The high specificity of the derivatising agent facilitates the identification and quantitation of small amounts of mandelic acids in complex biological matrices. The derivatisation procedure was used to determine the concentrations of m- and p-hydroxymandelic acids and vanillylmandelic acid in to the urine of normal subjects. The method may also have application in the determination of isomeric phenylethylene glycols, the corresponding products of reductive metabolism of biogenic amines.

Acetone↗
PubMed1990

Analysis of biogenic amines and their metabolites in biological tissues and fluids by gas chromatography-negative ion chemical ionization mass spectrometry (GC-NICIMS).

GC-NICIMS has been employed in the analysis of biogenic amines and their metabolites in human urine and human, bovine and porcine aqueous and vitreous humour. Several new chemical derivatization procedures have been developed in order to analyse these compounds. Concentrations of octopamines and synephrines were determined in urine from treated and untreated hypertensive subjects and normotensive individuals; there were no significant differences in concentrations of these metabolites between these groups. Human urine contained several dihydroxy-phenylethylamines which have not been reported as natural metabolites before and also 5- and 6-hydroxydopamine in relatively large amounts. Aqueous and vitreous humour contained very low quantities of noradrenaline, tyramine and dopamine but measurements were inconsistent because sometimes the levels were below the limits of detection. Metabolites of a number of biogenic amines were readily detected in aqueous and vitreous humour.

Animals↗
PubMed1990

The pharmacokinetics of 5-fluorouracil administered by arterial infusion in advanced colorectal hepatic metastases.

The pharmacokinetics of 5-fluorouracil (5FU) following its administration via the hepatic artery in conjunction with biodegradable albumin microspheres and angiotensin II have been studied. Peripheral venous concentrations of 5FU are lower and plasma clearance values higher following intrahepatic arterial administration compared with a similar dose administered by intravenous infusion over both 2 h and 24 h. For the 2 h drug infusions, plasma 5FU concentrations following co-treatment with angiotensin II and microspheres via the hepatic artery were intermediate between those of arterial and venous infusions of 5FU alone. There was a trend towards the peak plasma drug concentrations and the area under the plasma concentration-time curve (AUC) being significantly lower following co-treatment with angiotensin II and microspheres compared with intra-arterial and intravenous infusions of 5FU over 24 h. Co-administration of 5FU, angiotensin II and microspheres via the hepatic artery may reduce drug exposure in the systemic compartment and therefore may increase the therapeutic ratio of 5FU administration via the hepatic artery.

Colorectal Neoplasms↗
PubMed1990

Penetration of synthetic corticosteroids into human aqueous humour.

The penetration of prednisolone acetate (1%) and fluorometholone alcohol (0.1%) into human aqueous humour following topical application was determined using the very sensitive and specific technique of Gas Chromatography with Mass Spectrometry (GCMS). Prednisolone acetate afforded peak mean concentrations of 669.9 ng/ml within two hours and levels of 28.6 ng/ml in aqueous humour were detected almost 24 hours post application. The peak aqueous humour level of fluorometholone was 5.1 ng/ml. The results are compared and contrasted with the absorption of dexamethasone alcohol (0.1%), betamethasone sodium phosphate (0.1%) and prednisolone sodium phosphate (0.5%) into human aqueous humour.

Aged↗
PubMed1990

Penetration of topically applied betamethasone sodium phosphate into human aqueous humour.

Gas Chromatography combined with Negative Chemical Ionisation Mass Spectrometry (GCMS) was used to determine the absorption of topically applied beta-methasone sodium phosphate into the aqueous humour of human subjects undergoing routine intraocular surgery. The Betamethasone concentration was greatest in the interval 91-120 minutes following topical administration (mean peak concentration = 7.7 ng/ml). At twelve hours post instillation the mean concentration of Betamethasone was 2.5 ng/ml and detectable levels were recorded in the aqueous humour 24 hours after application (mean concentration 0.4 ng/ml).

Administration, Topical↗