E. L. Esmans

Liquid chromatography–mass spectrometry in nucleoside, nucleotide and modified nucleotide characterization

Abstract Macromolecules such as deoxyribonucleic acid (DNA) and ribonucleic acid as well as their constituents play an important role in all kinds of biochemical reactions in nature. Hence their isolation and identification plays a major role in biochemical analysis. Mass spectrometry (MS) has gained an important position in this field because of the development of soft ionization techniques such as fast atom bombardment (FAB), liquid secondary mass spectrometry, thermospray ionization (TSP), the atmospheric pressure ionization techniques, electrospray ionization and atmospheric pressure chemical ionization and matrix assisted laser desorption. Because of their polar nature, mixtures of nucleosides, nucleotides and oligonucleotides are wel separated by liquid chromatography (LC) and electrophoretic techniques. Therefore it is not surprising to note that a lot of effort has been put into the development of LC–MS methods for the analysis of these compounds. In this review, covering the period 1990–1996, the LC–MS analysis of nucleobases, nucleosides, nucleotides, oligonucleotides and DNA adducts by TSP, continuous flow FAB and electrospray MS is discussed.

Detection of Estrogen DNA-Adducts in Human Breast Tumor Tissue and Healthy Tissue by Combined Nano LC-Nano ES Tandem Mass Spectrometry

For the first time estrogen DNA-adducts were identified in DNA human breast tumor tissue using nano-LC coupled to nano-Electrospray Tandem Mass Spectrometry. Normal breast tissue was analyzed analogously. The data obtained in the five breast tumor and five adjacent normal tissue samples were compared qualitatively, but no straightforward difference was observed. Prior to LC-MS analysis the DNA was enzymatically hydrolyzed to a nucleoside pool. The DNA-hydrolysates were directly injected onto a column switching system developed for on-line sample clean-up and subsequent analysis of the DNA-adducts. In four patients using Premarin, DNA-adducts of 4-hydroxy-equilenin (4OHEN) were detected. All except three samples contained DNA-adducts from 4-hydroxy-estradiol or 4-hydroxy-estrone. Also DNA isolated from eight alcohol fixed and paraffin embedded breast tumor tissue showed the presence of different estrogen DNA-adducts. Worthwhile mentioning is the presence of adducts responding to m/z 570 > m/z 454 transition. This is a well-known SRM-transition indicative for the presence of the 2′-deoxyguanosine (dGuo) adduct of Benzo[a]pyrene.

Evaluation of the toxicological effects of perfluorooctane sulfonic acid in the common carp (Cyprinus carpio).

In the present study we evaluated the toxicological effects of a scarcely documented environmental pollutant, perfluorooctane sulfonic acid (PFOS), on selected biochemical endpoints in the common carp, Cyprinus carpio. Juvenile organisms were exposed to PFOS through a single intraperitoneal injection (liver concentrations ranging from 16 to 864 ng/g after 5 days of exposure) and after 1 and 5 days effects were assessed in liver and serum of the exposed organisms. The investigation of the hepatotoxicity of PFOS included the determination of the peroxisome proliferating potential (peroxisomal palmitoyl CoA oxidase and catalase activity) and the compounds influence on the average DNA basepair length (ABPL) by agarose gel electrophoresis. Total antioxidant activity (TAA), cholesterol and triglyceride levels were monitored in the serum. After 1 day of exposure the ABPL was significantly increased in the 270 and 864 ng/g treatment groups. After 5 days of exposure significant increases relative to the control were observed for the 16, 270 and 864 ng/g treatment groups. Enzyme leakage from the liver was investigated by measurement of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in the serum. At 561, 670 and 864 ng/g PFOS a significant increase in serum ALT activity became apparent after 5 days of exposure with values ranging from 159 to 407% relative to the control. For serum AST activity a significant increase for the 864 ng/g treatment group was observed with a value of 112% relative to the control. Determination of the polymorphonuclear leukocyte migration into liver tissue as assessed through myeloperoxidase (MPO) activity in liver, was used as an indicator for inflammation. It appeared that inflammation was not involved in the observed membranous enzyme leakage for the 561, 670 and 864 ng/g PFOS treatment groups. The results of this study suggest that PFOS induces inflammation-independent enzyme leakage through liver cell membranes that might be related to cell necrosis. Furthermore, results show that PFOS does not significantly affects serum antioxidant levels nor does it clearly induce peroxisome proliferation in carp. This study also points out that PFOS might interfere with homeostasis of the DNA metabolism. The results of these biochemical analyses were used to perform an initial hazard assessment study indicating that PFOS levels observed in tissues of wildlife populations could induce a clear rise in serum transaminase levels indicative for disruption of hepatocyte membrane integrity.

Ecto-nucleotide pyrophosphatase modulates the purinoceptor-mediated signal transduction and is inhibited by purinoceptor antagonists.

The effect of ecto‐nucleotide pyrophosphatase (ecto‐NPPase; EC 3.6.1.9) on the ATP‐ and ADP‐mediated receptor activation was studied in rat C6 glioma cells. The P2‐purinoceptor antagonists pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC50=12±3 μM) of the latter enzyme. 4,4′‐diisothiocyanatostilbene‐2,2′ disulfonic acid (DIDS), 5′‐phosphoadenosine 3′‐phosphate (PAP) and suramin were less potent inhibitors with an IC50 of 22±4, 36±7 and 72±11 μM respectively. P1‐purinoceptor antagonists CGS 15943, cyclo‐pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto‐NPPase. ATP‐ and ADP‐mediated P2Y1‐like receptor activation inhibited the (−)‐isoproterenol‐induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y‐antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto‐NPPase. We conclude that ecto‐NPPase has a modulatory effect on purinoceptor‐mediated signalling in C6 glioma cell cultures.

New α-pyrones from Iboza riparia

Abstract The structures for umuravumbolide, 5,6-dihydro-6-(3-acetoxy-1-heptenyl)-2-pyrone, a new α-pyrone from Iboza riparia (Labiatae) and its corresponding deacylated product have been established. Deacetylboronolide was also isolated and identified by different spectroscopic techniques.

Acetogenins from root bark of Uvaria narum

Abstract Three new acetogenins, isodesacetyluvaricin, narumicins I and II were isolated from hexane and ethyl acetate extracts of the root bark of Uvaria narum , in addition to the known compounds glutinone, glutinol, taraxerol, β-sitosterol and benzyl benzoate.

Squamocin-28-one and panalicin, two acetogenins from Uvaria narum

Abstract Squamocin-28-one and panalicin, two new acetogenins containing bis-tetrahydrofuran rings were isolated and characterized in addition to a known, but stereochemically undefined, compound (squamocin) from the root bark of Uvaria narum . The relative configuration of the bis-THF part in squamocin is discussed.

Fast Atom Bombardment and Tandem Mass Spectrometry for the Identification of Nucleoside Adducts with Phenyl Glycidyl Ether

Abstract The present study deals with the use of fast atom bombardment (FAB) in combination with constant neutral loss (CNL) scanning, high resolution mass spectrometry and tandem mass spectrometry (MS-MS) with collisionally activated decomposition (CAD), as complementary methods for the identification and structural analysis of phenyl glycidyl ether-nucleoside adducts. Selective detection of the parent ions of the modified nucleosides at the 1–10 ng level has been achieved by suitably designed CNL scans. The elemental composition of the adducts has been determined by accurate mass measurements. CAD-MS has been carried out on the [M + H]+ and [M - H]− ions to derive structural data on the size and nature of the base, sugar and alkyl substituent. In some cases, information on the alkylation site has been obtained, which is very useful for distinguishing isomeric adducts.

Evaluation of liquid chromatography—thermospray mass spectrometry in the determination of some phenylglycidyl ether-2′-deoxynucleoside adducts

Abstract The adducts formed between 2′-deoxyadenosine (dAdo), 2′-deoxycytidine (dCyd) and 2′-deoxyuridine (dUrd) and phenyl glycidyl ether (PGE) were analysed by HPLC and LC—thermospray (TSP)-MS. Good results were obtained on a 10 RP Select B column (12.5 cm × 4 mm I.D.) using 0.1 M NH4OAcCH3OH at a flow-rate of 0.8 ml/min. The mass spectra of the 2′-deoxynucleoside—PGE adducts, obtained under LC-TSP-MS conditions were all characterized by the presence of the protonated molecule [MH]+ and [BH + H]+ ions. The PGE-dCyd adduct underwent hydrolytic deamination to the corresponding PGE-dUrd adduct. There was an indication that this process of hydrolytic deamination also took place in the TSP interface. Localization of the alkylation site was possible in the PGE-dUrd adduct by the presence of an RDA rearrangement leading to a fragment ion at m/z 194. Preliminary sensitivity studies on PGE-dUrd showed a detection limit of 500 pg (signal-to-noise ratio = 2) in multiple ion monitoring at m/z 263 and 379.

Urinary modified nucleosides as tumor markers

Abstract Extracts of urinary nucleosides have been sequentially purified and examined by mass spectrometric analysis. Seventeen modified nucleosides have been unequivocally identified and a further five provisionally identified. While several nucleosides were found only in a small number of extracts, the occurrence and levels of others were found to correlate with the tumour type and stage.