Daan Noort

Quantitative analysis of O-isopropyl methylphosphonic acid in serum samples of Japanese citizens allegedly exposed to sarin: estimation of internal dosage

Abstract A convenient and rapid micro-anion exchange liquid chromatography (LC) tandem electrospray mass spectrometry (MS) procedure was developed for quantitative analysis in serum of O-isopropyl methylphosphonic acid (IMPA), the hydrolysis product of the nerve agent sarin. The mass spectrometric procedure involves negative or positive ion electrospray ionization and multiple reaction monitoring (MRM) detection. The method could be successfully applied to the analysis of serum samples from victims of the Tokyo subway attack and of an earlier incident at Matsumoto, Japan. IMPA levels ranging from 2 to 135 ng/ml were found. High levels of IMPA appear to correlate with low levels of residual butyrylcholinesterase activity in the samples and vice versa. Based on our analyses, the internal and exposure doses of the victims were estimated. In several cases, the doses appeared to be substantially higher than the assumed lethal doses in man.

Immunomagnetic Separation and Quantification of Butyrylcholinesterase Nerve Agent Adducts in Human Serum

A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.

Liquid chromatography/tandem mass spectrometry detection of covalent binding of acetaminophen to human serum albumin

Covalent binding of reactive electrophilic intermediates to proteins is considered to play an important role in the processes leading to adverse drug reactions and idiosyncratic drug reactions. Consequently, both for the discovery and the development of new drugs, there is a great interest in sensitive methodologies that enable the detection of covalent binding of drugs and drug candidates in vivo. In this work, we present a strategy for the generation and analysis of drug adducts to human serum albumin. Our methodology is based on the isolation of albumin from blood, its digestion to peptides by pronase E, and the sensitive detection of adducts to the characteristic cysteine-proline-phenylalanine (CPF) tripeptide by liquid chromatography/tandem mass spectrometry. We chose acetaminophen (APAP) as a model compound because this drug is known to induce covalent binding to proteins when bioactivated by cytochromes P450 to its reactive N-acetyl-p-benzoquinoneimine metabolite. First, by microsomal incubations of APAP in presence of CPF and/or intact albumin, in vitro reference adducts were generated to determine the mass spectrometric characteristics of the expected CPF adducts and to confirm their formation on pronase E digestion of the alkylated protein. When applying this methodology to albumin isolated from blood of patients exposed to APAP, we were indeed able to detect the corresponding CPF adducts. Therefore, this strategy could be seen as a potential biomonitoring tool to detect in vivo reactive intermediates of drugs and drug candidates, e.g., in the preclinical and clinical development phase.

Covalent binding of nitrogen mustards to the cysteine-34 residue in human serum albumin

Abstract. Covalent binding of various clinically important nitrogen mustards to the cysteine-34 residue of human serum albumin, in vitro and in vivo, is demonstrated. A rapid method for detection of these adducts is presented, based on liquid chromatography-tandem mass spectrometry analysis of the adducted tripeptide Cys*-Pro-Phe after digestion of the protein with Pronase.

Evaluation of the antibacterial spectrum of drosocin analogues.

Drosocin is a 19‐mer, cationic antimicrobial peptide from Drosophila melanogaster. The aim of the study was to examine the antibacterial spectrum of unglycosylated drosocin analogues. Furthermore, the amino acid sequence of DnaK, drosocins intracellular target, from susceptible species was aligned and studied for sequence homology. From this a panel of 31 bacterial strains, including Salmonella strains with truncated lipopolysaccharide structures, was tested for susceptibility towards three drosocin analogues. Available bacterial DnaK amino acid sequences were retrieved from the ExPASy proteomics server of the Swiss Institute of Bioinformatics studied for sequence homology. Seventeen of the 31 strains tested were susceptible for the drosocin analogues. Minimal inhibitory concentration values against mainly Gram‐negative bacteria ranged from 3.1 to 100 μm. With the exception of Micrococcus luteus and Xanthomonas campestris all drosocin analogue‐susceptible strains were Enterobacteriaceae showing a high DnaK amino acid sequence homology.

Development of an automated on-line pepsin digestion-liquid chromatography-tandem mass spectrometry configuration for the rapid analysis of protein adducts of chemical warfare agents

Rapid monitoring and retrospective verification are key issues in protection against and non-proliferation of chemical warfare agents (CWA). Such monitoring and verification are adequately accomplished by the analysis of persistent protein adducts of these agents. Liquid chromatography-mass spectrometry (LC-MS) is the tool of choice in the analysis of such protein adducts, but the overall experimental procedure is quite elaborate. Therefore, an automated on-line pepsin digestion-LC-MS configuration has been developed for the rapid determination of CWA protein adducts. The utility of this configuration is demonstrated by the analysis of specific adducts of sarin and sulfur mustard to human butyryl cholinesterase and human serum albumin, respectively.

An adamantyl amino acid containing gramicidin S analogue with broad spectrum antibacterial activity and reduced hemolytic activity

The cyclic cationic antimicrobial peptide gramicidin S (GS) is an effective topical antibacterial agent that is toxic for human red blood cells (hemolysis). Herein, we present a series of amphiphilic derivatives of GS with either two or four positive charges and characteristics ranging between very polar and very hydrophobic. Screening of this series of peptide derivatives identified a compound that combines effective antibacterial activity with virtually no toxicity within the same concentration range. This peptide acts against both Gram-negative and Gram-positive bacteria, including several MRSA strains, and represents an interesting lead for the development of a broadly applicable antibiotic.

Diagnosis and dosimetry of exposure to sulfur mustard: development of a standard operating procedure for mass spectrometric analysis of haemoglobin adducts: exploratory research on albumin and keratin adducts

Experiments were carried out to develop a standard operating procedure for analysis of sulfur mustard adducts to the N‐terminal valine in haemoglobin and to explore adduct formation with albumin and keratin.

Structural Study of the Complex Stereoselectivity of Human Butyrylcholinesterase for the Neurotoxic V-Agents.

Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the PS adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VXR-(+) and VXS-(−) solutions lead to the formation of the PS and PR adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VXR-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VXS-(−). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with PR enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the PS enantiomer and ages.

Exploring the conformational and biological versatility of β-turn-modified gramicidin S by using sugar amino acid homologues that vary in ring size.

Monobenzylated sugar amino acids (SAAs) that differ in ether ring size (containing an oxetane, furanoid, and pyranoid ring) were synthesized and incorporated in one of the β-turn regions of the cyclo-decapeptide gramicidin S (GS). CD, NMR spectroscopy, modeling, and X-ray diffraction reveal that the ring size of the incorporated SAA moieties determines the spatial positioning of their cis-oriented carboxyl and aminomethyl substituents, thereby subtly influencing the amide linkages with the adjacent amino acids in the sequence. Unlike GS itself, the conformational behavior of the SAA-containing peptides is solvent dependent. The derivative containing the pyranoid SAA is slightly less hydrophobic and displays a diminished haemolytic activity, but has similar antimicrobial properties as GS.