Daniel L. Norwood

Tandem mass spectrometry: A new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism

ConclusionA method for analysis of acylcarnitines in blood at physiological concentrations has been developed. Preliminary results from umbilical cord blood and neonatal blood spotted onto Guthrie cards are encouraging. This method will detect up to at least eight inherited metabolic disorders which exhibit diagnostic acylcarnitine profiles, including medium-chain acyl-CoA dehydrogenase deficiency. The speed and simplicity of the method permit automation with existing technology, and could enable routine neonatal screening to be carried out in an efficient and cost-effective manner.

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.

Structural characterization of aquatic humic material. 2. Phenolic content and its relationship to chlorination mechanism in an isolated aquatic fulvic acid.

The complementary techniques of solid-state /sup 13/C nuclear magnetic resonance spectroscopy and chemical degradation were utilized to examine the lignin/phenolic substructure of an isolated aquatic fulvic acid capable of producing upon aqueous chlorination a number of organohalides typically found in municipal drinking water. Results indicate that while phenolic moieties are present in the fulvic acid, they account for only a minor fraction of the total carbon. A sequential chemical degradation experiment utilizing aqueous chlorine and CuO demonstrated that the lignin/phenolic substructure was attacked by the chlorine. It is concluded that while phenolic ring rupture mechanisms appear to be important in organohalide generation, other aqueous chlorination mechanisms involving aliphatic and other types of aromatic structures should also be considered. 35 references, 12 figures, 3 tables.

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.

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.

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.

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.

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.

Identification of phenylpropionylcarnitine, a new metabolite of phenylpropionic acid, in a patient with medium chain acyl-coa dehydrogenase deficiency

Following the original descriptions of patients with deficiency of medium chain acylcoenzyme A dehydrogenase (MCAD; EC 1.3.99.3) (McKusick 22274) (Kolvraa et al., 1982; Stanley et al., 1983), much interest has centred on the diagnosis of this condition. Rumsby et al. (1986) proposed oral loading with phenylpropionic acid (PPA) as an in vivo screening/diagnostic procedure which is now widely accepted for this purpose. Duran et al. (1989) have reported a number of metabolites of PPA other than phenylpropionylgtycine (PPG). This report concerns a further metabolite, phenylpropionylcarnitine (PPC), found in the urine of a child with MCAD deficiency following an oral PPA load.