Search

Laser ablation interfaced with inductively coupled plasma-mass spectrometry is described as a new method to determine the presence of phosphorylated proteins on electrophoresis gel blots. The method was applied to the phosphoprotein beta-casein with good detection levels being observed at 16 pmole. Attempts at using the technique to detect beta-casein on electrophoresis gels are also described.

Inter-individual variability in the response to numerous drugs can be traced to a number of sources. One source of variability in drug response is the variability associated with the metabolic capacity of an individual. The component of metabolic capacity that will be the focus of this article is that determined by heredity. Pharmacogenetics is frequently referred to as the study of the effects of heredity on the disposition and response to medications. This article will review the pharmacokinetic and pharmacodynamic significance of pharmacogenetics as it pertains to a select number of cardiovascular agents. The enzyme systems responsible for drug metabolism discussed in this article will be limited to the P-450IID6 and N-acetylation pathways. Given the extensive use of cardiovascular agents in clinical practice that are affected by this genetic polymorphism, it is important for the practicing pharmacist to be aware of this phenomenon and its implications. Hopefully, the knowledge gained from this article will help practicing pharmacists to appreciate the clinical significance of polymorphic drug metabolism and provide a basis for the application of this knowledge to a variety of practice settings.

Pulmonary embolus (PE) is estimated to cause 200 000 to 300 000 deaths annually. Many deaths occur in hemodynamically unstable patients and the estimated mortality for inpatients with hemodynamic instability is between 15% and 25%. The diagnosis of PE in the critically ill is often challenging because the presentation is nonspecific. Computed tomographic pulmonary angiography appears to be the most useful study for diagnosis of PE in the critically ill. For patients with renal insufficiency and contrast allergy, the ventilation perfusion scan provides an alternative. For patients too unstable to travel, echocardiography (especially transesophageal echocardiography) is another option. A positive result on lower extremity Doppler ultrasound can also aid in the decision to treat. The choice of treatment in PE depends on the estimated risk of poor outcome. The presence of hypotension is the most significant predictor of poor outcome and defines those with massive PE. Normotensive patients with evidence of right ventricular (RV) dysfunction, as assessed by echocardiography, comprise the sub-massive category and are at intermediate risk of poor outcomes. Clinically, those with sub-massive PE are difficult to distinguish from those with low-risk PE. Cardiac troponin, brain natriuretic peptide, and computed tomographic pulmonary angiography can raise the suspicion that a patient has sub-massive PE, but the echocardiogram remains the primary means of identifying RV dysfunction. The initial therapy for patients with PE is anticoagulation. Use of vasopressors, inotropes, pulmonary artery (PA) vasodilators and mechanical ventilation can stabilize critically ill patients. The recommended definitive treatment for patients with massive PE is thrombolysis (in addition to anticoagulation). In massive PE, thrombolytics reduce the risk of recurrent PE, cause rapid improvement in hemodynamics, and probably reduce mortality compared with anticoagulation alone. For patients with a contraindication to anticoagulation and thrombolytic therapy, surgical embolectomy and catheter-based therapies are options. Thrombolytic therapy in sub-massive PE results in improved pulmonary perfusion, reduced PA pressures, and a less complicated hospital course. No survival benefit has been documented, however. If one is considering the use of thrombolytic therapy in sub-massive PE, the limited documented benefit must be weighed against the increased risk of life-threatening hemorrhage. The role of surgical embolectomy and catheter-based therapies in this population is unclear. Evidence suggests that sub-massive PE is a heterogeneous group with respect to risk. It is possible that those at highest risk may benefit from thrombolysis, but existing studies do not identify subgroups within the sub-massive category. The role of inferior vena cava (IVC) filters, catheter-based interventions, and surgical embolectomy in life-threatening PE has yet to be completely defined.

To date matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) analysis has been largely concerned with mapping the distribution of known analytes in tissues. An important step in the progression of its applications is the determination of unknown variants for metabolite and protein profiling in both clinical studies and studies of disease. Principal component analysis (PCA) is a statistical approach which can be used as a means of determining latent variables in multivariate data sets. In the work reported here, PCA, in both unsupervised and supervised modes, has been used to differentiate brain regions based on their lipid composition determined by MALDI-MSI. PCA has been shown to be useful in the determination of hidden variables between spectra taken from six regions of brain tissue. It is possible to identify ions of interest from the loadings plot which are likely to be more prominent in the different regions of the brain and thus differentiating between white and grey matter. It is also possible to distinguish between the grey Cerebellar Cortex and the Hippocampal formation, due to the grey Cerebellar Cortex having a positive PC2 and the Hippocampal formation having a negative PC2 score; this is only possible in supervised PCA with this data set because with unsupervised PCA the two regions overlap.

Metoprolol is a lipophilic, cardioselective beta-adrenergic blocking agent commercially available as a racemic compound. A normal phase high-performance liquid chromatographic method was developed to directly determine individual enantiomeric concentrations of metoprolol in human serum. Separation of the enantiomers was accomplished by a cellulose-tris(3,5-dimethylphenylcarbamate) chiral stationary phase. Metoprolol enantiomers were detected by means of fluorescence with excitation and emission wavelengths of 275 and 315 nm, respectively. Standard curves were linear over the concentration range 12.5-400 ng/ml for each enantiomer. Within-day coefficient of variation was less than 15% at all concentrations and the between-day coefficient of variation ranged from 4.1 to 11.2%. The limit of detection was determined to be 5 ng/ml for each enantiomer and the stereoselective resolution (alpha) of R- and S-metoprolol was 3.08. The assay was employed to determine enantiomeric serum concentrations of metoprolol in healthy male volunteers.

Despite recent improvements to in situ proteolysis strategies, a higher efficiency is still needed to increase both the number of peptides detected and the associated ion intensity, leading to a complete and reliable set of biomarkers for diagnostic or prognostic purposes. In the study presented here, an extract of a systematic study is illustrated investigating a range of surfactants assisting trypsin proteolytic activity. Method development was trialled on fingermarks; this specimen results from a transfer of sweat from an individual's fingertip to a surface upon contact. As sweat carries a plethora of biomolecules, including peptides and proteins, fingermarks are, potentially, a very valuable specimen for non-invasive prognostic or diagnostic screening. A recent study has demonstrated the opportunity to quickly detect peptides and small proteins in fingermarks using Matrix Assisted Laser Desorption Ionization Mass Spectrometry Profiling (MALDI MSP). However, intact detection bears low sensitivity and does not allow species identification; therefore, a shotgun proteomic approach was employed involving in situ proteolysis. Data demonstrate that in fingermarks, further improvements to the existing method can be achieved using MEGA-8 as surfactant in higher percentages as well as combinations of different detergents. Also, for the first time, Rapigest SF, normally used in solution digestions, has been shown to successfully work also for in situ proteolysis.

The coupling of the techniques, high-performance liquid chromatography (HPLC), orthogonal acceleration time-of-flight mass spectrometry (OATOF-MS) and inductively coupled plasma mass spectrometry (ICP-MS) provides a very powerful method for identifying and quantifying the products of bradykinin metabolism. In this study, we were able to identify the major metabolites of bradykinin degradation reported in the literature. In addition, a new bradykinin metabolite corresponding to bradykinin 5,9 fragment (BK-(5,9)-fragment) was identified as a product of neutral endopeptidase (NEP) activity. This finding establishes that NEP cleaves bradykinin simultaneously at the positions 4–5 and 7–8. We also demonstrate the equivalent participation of NEP and angiotensin-converting enzyme (ACE) within the rat lung tissue membranes (RLTM) in bradykinin degradation, suggesting its suitability as a model for the assay of dual ACE/NEP inhibitors. On the contrary, in rat kidney brush border membranes (KBBM), ACE is not significantly involved in bradykinin metabolism, with NEP being the major enzyme. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.

The potential for developing improved procedures for phosphate measurement through combinations of gel electrophoresis and quadrupole-based ICP mass spectrometry utilising (47)PO(+) is investigated. Laser ablation of gels offers a rapid and direct quantitation route, but is subject to high blanks due to P impurities in gels and associated reagents; nevertheless optimisation of laser sampling afforded improved method sensitivity (limit of detection 0.09 microg g(-1)). Implementation of whole gel elution (WGE) with FI-ICP-MS (conventional solution nebulisation) following gel electrophoresis permitted quantitation at the sub microg l(-1) level, and microcolumn processing (activated alumina) was effective at rejecting phosphate contamination. The potential for S-induced molecular ion interference at mass 47 was demonstrated.

Both LC and capillary LC (CapLC) have been successfully interfaced with inductively coupled plasma mass spectrometry (ICP-MS). Gradients of acetonitrile and aqueous based solvents have been employed to separate several compounds of pharmaceutical interest. This paper will describe four application areas in the pharmaceutical industry, and examples will be shown where CapLC, LC and gel electrophoresis via laser ablation have been coupled with ICP-MS. The four areas highlighted in this paper are: (1) the use of derivatisation reactions to “make the invisible visible”. Methods involving derivatisations with copper and iron will be described that can be used for the analysis of amines and carboxylic acids by ICP-MS. (2) The profiling of metal ion content (in particular bromine) in biological samples such as human plasma, this study will focus on the metabolism of bromine-labelled peptides (e.g. substance P). (3) The analysis of materials derived from single, solid-phase beads used in combinatorial chemistry, and (4) also discussed will be our findings from investigations into the use of laser ablation ICP-MS on the determination of protein phosphorylation on electrophoresis gel blots.

We sought to identify certain clinical characteristics associated with a poor clinical outcome in patients acutely infected with Influenza A.We performed a retrospective chart review of inpatients with acute Influenza A infection comparing a poor outcome group (POG; n=27), defined as patients who died and/or developed respiratory failure or shock, with a usual outcome group (UOG; n=105).Compared with the UOG, the POG had a significantly greater percentage of patients with: a history of chronic obstructive pulmonary disease (41% vs 20%), coronary artery disease (63% vs 38%), congestive heart failure (44% vs 23%), transient ischemic attack (TIA) or stroke (44% vs 21%), chronic renal insufficiency (22% vs 8%) and dialysis (11% vs 1%). Shortness of breath as a chief complaint (74% vs 44%), lower initial oxygen saturations (0.86 vs 0.92), as well as higher mean respiratory rates (28/minute vs 22/minute) occurred more frequently in the POG. The POG also had a greater frequency of CHF/ vascular congestion (26% vs 8%), and interstitial involvement (22% vs 6%) on admission chest roentgenogram. Independent predictors of poor outcome identified by multivariate analysis included low oxygen saturation on admission, history of TIA or stroke, and history of dialysis.The presence of certain comorbidities as well as clinical and radiographic evidence of respiratory compromise on admission may be helpful in identifying high-risk patients acutely infected with Influenza A.

Publisher Summary This chapter describes production of novel derivatives of a gastrin antagonist (GW1) using biotransformation. The idea of using micro-organisms to carry out biotransformations has proved to be a powerful aid to drug metabolism. The use of micro-organisms for metabolite production offers various advantages over more traditional approaches, such as (1) the use of radiochemicals can often be avoided, (2) micro-organisms are readily amenable to scale up supporting the production of gram quantities of known or putative mammalian metabolites, and (3) the demand for animal studies is reduced which is important both from an economic and a humanitarian standpoint. Sixty-five organisms are screened for possible biotransformation of GW1. Twenty showed significant (> 60%) substrate utilization. Further analysis shows that four organisms (all Streptomycetes) are worthy of scale-up based on both the diversity and the levels of compounds produced. Isolated yields of compound are typically 0.2–4 mg from each organism processed.

A high-performance liquid chromatography (HPLC) assay for the simultaneous quantitation of dextromethorphan and its O-demethylated metabolite dextrorphan from urine is described. A cyano analytical column was used with a mobile phase consisting of MeOH 16%, acetonitrile 3%, and triethylamine 0.06% at pH 2.8 and a flow rate of 1.0 ml/min. Betaxolol was used as the internal standard. Standard curves from 50 ng/ml to 10,000 ng/ml (dextrorphan), and from 50 ng/ml to 8,000 ng/ml (dextromethorphan) were developed. The peaks eluted at 7.8 min (dextrorphan), 12.2 min (betaxolol), and 17.8 min (dextromethorphan). The coefficients of variance ranged from 1.3 to 4.5% at 250 ng/ml and 0.9 to 2.5% at 5,000 ng/ml. This assay was used to determine dextromethorphan/dextrorphan molar ratios in healthy male volunteers for the purpose of determining phenotype status for the P450IID6 isozyme.

During the screening of fungi for inhibitors of squalene synthase, Phoma sp. C2932 was found to produce three structurally related novel inhibitors. These compounds, designated the squalestatins, exhibited potent activity against both mammalian (rat liver) and fungal (Candida albicans) squalene synthase. Furthermore, they also had broad spectrum in vitro antifungal activity.

A number of previous studies of the involvement of 2-methyl-6-phytyl-1,4-benzoquinol in the biosynthesis of α-tocopherol have failed to take account of the fact that this quinol and its quinone have very similar chromatographic properties to those of 2-methyl-3-phytyl-1,4-benzoquinol and 2-methyl-3-phytyl-1,4-benzoquinone respectively. It has now been shown that the two quinones can be separated from each other either by multidevelopment TLC or by HPLC and that the claims made earlier with regard to the biosynthesis and metabolism of 2-methyl-6-phytyl-1,4-benzoquinol in chloroplasts are correct. In particular, it has been established that this quinol is the only methyl phytylbenzoquinol formed from homogentisate and phytyl pyrophosphate in chloroplast preparations. It has also been shown for the first time that lettuce chloroplasts are able to synthesize 3 H-labelled α- and γ-tocopherols from [ methylene - 3 H] homogentisate.

Inoculation of stem or leaf tissue of Coleostephus myconis with the fungus Botrytis cinerea induced the biosynthesis of an antifungal spiroketalenol ether. With the aid of 1 H and 13 C NMR, IR and mass spectroscopy, the antifungal compound was identified as ( E ) -7-(2,4)-hexadiynyliden-1,6 -dioxaspiro -[4.4]nona-2,8-dien-4-ol, which we name mycosinol. This is the first report of a spiroketalenol ether as a phytoalexin and the first report of a phytoalexin from a member of the tribe Anthemideae of the Compositae.