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Biomedical subjects

D L Norwood

Publications and source records attributed to D L Norwood.

17 recordsLinked to original sources
PubMed1995

Analysis of polycyclic aromatic hydrocarbons in metered dose inhaler drug formulations by isotope dilution gas chromatography/mass spectrometry.

Organic compounds extracted into metered dose inhalers (MDIs) from the rubber components of the metering valve are of increasing interest in the development of these formulations. Polycyclic aromatic hydrocarbons (PAHs) are a class of extractable organic compounds whose source is the carbon black commonly used as a reinforcing agent in rubber. The analytical method for PAHs described in this report employs "cold filtration" to remove the suspended drug substance and excipients, and gas chromatography/mass spectrometry (GC/MS) for separation and detection of individual PAHs. After filtration, stable isotope labelled analogues of target PAHs are spiked into the drug product to act as internal standards, correcting for recovery (termed "isotope dilution GC/MS"). Validation of the method was accomplished with respect to linearity, precision, limit of detection/quantitation, selectivity and ruggedness. Application to a variety of MDI drug product formulations revealed that certain PAHs are present at the ng/inhaler level.

Gas Chromatography-Mass Spectrometry↗
PubMed1992

Rapid optimization of enzyme substrates using defined substrate mixtures.

A strategy is described for the rapid optimization of kcat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enhanced kcat/Km. High performance liquid chromatography/continuous flow fast atom bombardment mass spectrometry is used to assign structure to each peak in the chromatogram. As an example of the utility and efficiency of "substrate mapping" we describe optimization of the collagenase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (where Dnp is dinitrophenyl) at the P'1 and P'2 positions. Six different mixtures were prepared for evaluation, representing the synthesis of 128 different synthetic substrates. "Substrate mapping" has led to Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2, a substrate that possesses a 10-fold better kcat/Km than Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2.

Amino Acid Sequence↗
PubMed1991

Mitochondrial metabolism of valproic acid.

The beta-oxidation of valproic acid (2-propylpentanoic acid), an anticonvulsant drug with hepatotoxic side effects, was studied with subcellular fractions of rat liver and with purified enzymes of beta-oxidation. 2-Propyl-2-pentenoyl-CoA, a presumed intermediate in the beta-oxidation of valproic acid, was chemically synthesized and used to demonstrate that enoyl-CoA hydratase or crotonase catalyzes its hydration to 3-hydroxy-2-propylpentanoyl-CoA. The latter compound was not acted upon by soluble L-3-hydroxyacyl-CoA dehydrogenases from mitochondria or peroxisomes but was dehydrogenated by an NAD(+)-dependent dehydrogenase associated with a mitochondrial membrane fraction. The product of the dehydrogenation, presumably 3-keto-2-propylpentanoyl-CoA, was further characterized by fast bombardment mass spectrometry. 3-Keto-2-propylpentanoyl-CoA was not cleaved thiolytically by 3-ketoacyl-CoA thiolase or a mitochondrial extract but was slowly degraded, most likely by hydrolysis. The availability of 2-propylpentanoyl-CoA (valproyl-CoA) and its beta-oxidation metabolites facilitated a study of valproate metabolism in coupled rat liver mitochondria. Mitochondrial metabolites identified by high-performance liquid chromatography were 2-propylpentanoyl-CoA, 3-keto-2-propylpentanoyl-CoA, 2-propyl-2-pentenoyl- CoA, and trace amounts of 3-hydroxy-2-propylpentanoyl-CoA. It is concluded that valproic acid enters mitochondria where it is converted to 2-propylpentanoyl-CoA, dehydrogenated to 2-propyl-2-pentenoyl-CoA by 2-methyl-branched chain acyl-CoA dehydrogenase, and hydrated by enoyl-CoA hydratase to 3-hydroxy-2-propylpentanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗
PubMed1991

Biomedical applications of high-performance liquid chromatography-mass spectrometry with continuous-flow fast atom bombardment.

This report describes the application of high-performance liquid chromatography combined with continuous-flow fast atom bombardment mass spectrometry to analytical problems in the biomedical laboratory. Applications include the compound-specific detection of diagnostic acylcarnitines in human urine, the separation and analysis of acyl-coenzyme A thioesters, and qualitative studies on complex mixtures of modified peptides (dansyl and dinitrophenyl derivatives). For each of these applications standard analytical columns (3.9 mm I.D.) and 1 ml/min flow-rates were employed with post-column stream splitting (1:100) before mass spectrometry. Various mobile phase compositions and solvent gradients were employed. The addition of 1-5% glycerol to the mobile phase was shown to have little effect on the chromatography. For all compounds studied (acylcarnitines, acyl-coenzyme A thioesters, and derivatized peptides) molecular weight information was obtained and sufficient sensitivity was achieved to allow unambiguous identification of trace components in complex mixtures.

Acyl Coenzyme A↗
PubMed1990

Combined high-performance liquid chromatographic-continuous-flow fast atom bombardment mass spectrometric analysis of acylcoenzyme A compounds.

A high-performance liquid chromatographic method for the analysis of coenzyme A thioesters which employs continuous-flow fast atom bombardment mass spectrometric detection is presented. The chromatographic system utilizes gradient elution with reversed-phase conditions using ammonium acetate-acetonitrile from both standard analytical (3.9 mm I.D.) and microbore (1 mm I.D.) columns. Applications to coenzyme A thioesters of various acyl group chain length (C2-C18) and functionality (-COOH, -OH, -C = C-) are described. The system is also applied to an in vitro enzyme reaction (crotonase) to directly follow the disappearance of substrate and appearance of product. The mass spectrometry of coenzyme A thioesters, their chromatographic behavior, system stability, and sensitivity of detection are discussed.

Acyl Coenzyme A↗
PubMed1990

Quantitative assay of free and total carnitine using tandem mass spectrometry.

A new, specific method for isotope dilution assay of total and free carnitine in urine has been developed. The method utilizes fast atom bombardment ionization with tandem mass spectrometry and requires minimal sample preparation. It compared well with radioenzymatic assay in terms of specificity, precision and accuracy, but was much more convenient in terms of analysis time and sample throughput. The new method is also applicable to the determination of free and short-chain total carnitine in plasma.

Carnitine↗
PubMed1990

Dipyridamole-cisplatin potentiation: enhanced in vivo cytotoxicity in xenograft models of human testicular and bladder cancers.

The antitumor efficacy and host toxicity of dipyridamole (DP), methotrexate (MTX) and cisplatin (CDDP) alone and combined were evaluated in a nude mouse supported human bladder cancer model. Single agent post treatment tumor volume growth ratio [TGR] values of DP, MTX and CDDP were 97%, 65% and 49% of control. While the MTX/DP combination produced only mild cytotoxic enhancement, CDDP/DP and CDDP/MTX/DP reduced TGR to 20% and 17%, respectively. A second multi-dose evaluation of CDDP/DP using human testicular carcinoma in this model also showed a CDDP dose-dependent response with achievable complete tumor regression. Host toxicity was not substantially increased by DP. DP would appear to be effective in vivo as a chemosensitizer of CDDP; it may enhance the therapeutic efficacy of CDDP in a variety of tumors.

Animals↗
PubMed1990

2,4-Dienoyl-coenzyme A reductase deficiency: a possible new disorder of fatty acid oxidation.

Several inherited disorders of fatty acid beta-oxidation have been described that relate mainly to saturated precursors. This study is the first report of an enzyme defect related only to unsaturated fatty acid oxidation and provides the first in vivo evidence that fat oxidation in humans proceeds by the reductase-dependent pathway. The patient was a black female, presenting in the neonatal period with persistent hypotonia. Biochemical studies revealed hyperlysinemia, hypocarnitinemia, normal organic acid profile, and an unusual acylcarnitine species in both urine and blood. The new metabolite was positively identified by mass spectrometry as 2-trans,4-cis-decadienoylcarnitine, derived from incomplete oxidation of linoleic acid. In spite of dietary therapy, the patient died of respiratory acidosis at four months of age. Samples of liver and muscle from the autopsy were assayed for 2,4-dienoyl-coenzyme A reductase activity. Using the substrate 2-trans,4-cis-decadienoylcoenzyme A, the reductase activity was 40% of the control value in liver and only 17% of that found in normal muscle. It is suggested that unsaturated substrates should be used for in vitro testing to cover the full range of potential beta-oxidation defects and that acylcarnitine species identification be used for in vivo detection of this disorder.

Fatty Acid Desaturases↗
PubMed1990

Carnitine homeostasis in the organic acidurias.

The detection of acylcarnitines by FAB-MS in human physiological fluids has added another valuable diagnostic tool for the recognition of specific metabolic diseases. The newly developed technique of FAB-MS/MS, which embodies the principles of tandem mass spectrometry, affords a quantum leap in specificity, enabling the detection and quantification of acylcarnitines in concentrations well within the physiological ranges in urine, blood and tissue. The therapeutic use of L-carnitine appears to offer protection against the harmful consequences of catabolism. A reduction in the number of clinical episodes requiring hospitalization has been observed in most cases. When such occurrences have necessitated intravenous fluids, the addition of L-carnitine to the IV infusate has led to dramatic reduction in recovery times. It has also been shown that there is no discernable toxicity associated with carnitine therapy, even under high-dose IV conditions.

Carnitine↗
PubMed1989

Application of fast atom bombardment with tandem mass spectrometry and liquid chromatography/mass spectrometry to the analysis of acylcarnitines in human urine, blood, and tissue.

Using a precursor-ion scan function on a triple quadrupole mass spectrometer, acylcarnitines were detected in the target matrices at or below concentrations of 1 nmol per gram by fast atom bombardment mass spectrometry. Acylcarnitine profiles from patients with known metabolic disorders were consistent with previously acquired data. Putative acylcarnitine signals were confirmed in one case by administration of stable isotope-labeled carnitine, which equilibrated rapidly with the endogenous pool. The addition of a continuous flow system enabled rapid sequential analysis without operator intervention, indicating the potential for automation of the analytical procedure. Incorporation of a micro-LC column enabled on-line liquid chromatographic/mass spectrometric analysis of selected patient samples. Large-scale screening and quantitative analysis of urine or blood for diagnostic acylcarnitines are now practicable.

Acylation↗
PubMed1988

Application of continuous-flow liquid chromatography/fast-atom bombardment mass spectrometry to the analysis of diagnostic acylcarnitines in human urine.

Acylcarnitine profiles of diagnostic value were generated from the equivalent of 0.1 microL of raw urine using the continuous-flow liquid chromatography/fast-atom bombardment mass spectrometry (LC/FAB-MS) interface for sample introduction. Further analysis was accomplished by gradient LC/MS using a commercially available packed fused-silica microbore column.

Carnitine↗
PubMed1988

Estimates of radiation dose and health risks to the United States population following the Chernobyl nuclear plant accident.

Estimates of both individual and collective doses received by the United States population following the Chernobyl accident have been made by using the data obtained from the U.S. Environmental Protection Agency's Environmental Radiation Ambient Monitoring System. Radionuclides associated with the debris first were measured in precipitation and surface air particulates at Portland, OR and Olympia, WA on 5 May 1986. Iodine-131 was the most consistently measured nuclide in all media, although several Cs and Ru isotopes also were observed. Strontium and any actinides notably were absent from the samples at the lower level of detection. The highest calculated individual-organ dose due to intake during May and June 1986 was 0.52 mSv to the infant thyroid in the state of Washington. This was predominantly (98%) from the ingestion of milk. The maximum U.S. collective dose equivalent to any organ was calculated to be 3,300 person-Sv to the thyroid. Risk estimates project three excess lung cancer deaths and an additional four deaths due to cancers of thyroid, breast and leukemia in the U.S. population over the next 45 y from exposure during the May-June 1986 interval. The only long-lived radionuclide measured in milk samples following the accident was 137Cs. We estimate 20 excess fatalities from the ingestion of 137Cs in milk during all subsequent years, with six of these due to lung cancer and the majority of the remainder distributed approximately equally among cancers of the thyroid, breast, liver and leukemia. A total of 100 excess fatalities from all dietary components was estimated. Because of the uncertainty of risk estimates from data such as those available for this study, all calculated values carry a range of uncertainty from a minimum of one-half the calculated value to a maximum of two times the calculated value. The estimated excess fatalities given above may be compared with corresponding projected cancer mortality from all other causes: 41,000 fatalities from thyroid cancer and 3,800,000 fatalities from lung cancer are estimated to occur within the U.S. population during the next 45 y.

Accidents↗
PubMed1982

Reaction products of aquatic humic substances with chlorine.

A major concern of the chlorination of aquatic humic materials is the ubiquitous production of trihalomethanes. A large number of other chlorinated organic compounds, however, have been shown to be formed by chlorine's reaction with humic substances. In this study, humic material was concentrated from a coastal North Carolina lake and chlorinated at a chlorine to carbon mole ratio of 1.5 at pH 12. A high pH was necessary for complete dissolution of the humic material and for production of adequate quantities of oxidation and chlorination products for extraction, separation and mass spectrometric identification. After concentration in ether, samples were methylated, separated with a 50-m OV-17 glass capillary column or a 25 m SP-2100 fused-silica column and identified. A Hewlett-Packard 5710A gas chromatograph interfaced to a VG Micromass 7070F double-focusing mass spectrometer was used. Low resolution, accurate mass measurements were made with a combined EI-Cl source. The ability to do low resolution, accurate mass measurements made possible a rapid scan function necessary for capillary column gas chromatography. Accurate mass measurements allowed increased confidence in the identification of compounds, most of which are not available as standards. The products identified in these studies were chlorinated aliphatic straight-chain acids dominated by di- and trichloroacetic acid and the chlorinated dicarboxylic acids: succinic, fumaric and maleic acids. Chlorinated and unchlorinated aliphatic mono- and dicarboxylic acids and unchlorinated polycarboxylic aromatic acids comprise the remaining bulk of the compounds identified.

Chemical Phenomena↗