New approach to the direct detection of known and new diarrhoeic shellfish toxins in mussels and phytoplankton by liquid chromatography-mass spectrometry.

A new approach using combined liquid chromatography-mass spectrometry (LC-MS) with ionspray ionization is proposed for the direct detection of known and new toxins in mussels and phytoplankton. A first stage reversed-phase, negative ion mode, selected ion monitoring (SIM) LC-MS analysis was performed in order to detect DSP toxins in the same chromatographic run with a total run time of 20 min. The toxins analysed included yessotoxin (YTX), okadaic acid (OA) and four of its analogues, dinophysistoxins (i.e. DTX-1, DTX-2, DTX-2B, DTX-2C), and pectenotoxins (PTXs), involving PTX-2, two PTX-2 secoacids (PTX-2SAs), PTX-2SA, 7-epi-PTX-2SA, and AC1, the three isomeric toxins structurally related to PTX-2 recently identified in Irish phytoplankton. Positive samples can, therefore, be analyzed through reversed-phase, positive ion mode SIM LC-MS, in order to perform complete chromatographic separations of the structurally related toxins within the OA and PTX groups. Detailed toxin profiles of a number of toxic phytoplankton and shellfish, from different marine areas, were easily obtained through the new approach. PTX-2SAs and AC1 were found in phytoplankton and shellfish from Ireland as well as in Italian shellfish. Moreover, for the first time there was evidence of the presence of PTX-2 in Irish phytoplankton. YTX was present in Italian shellfish. Four isomeric OA toxins were detected in samples from Ireland with OA, DTX-2 and DTX-2B present in shellfish, and OA, DTX-2 and DTX-2C in phytoplankton. In contrast, OA was the only toxin from this group to be detected in Italian mussels.

Liquid chromatographic methods for the isolation and identification of new pectenotoxin-2 analogues from marine phytoplankton and shellfish.

Two acidic analogues of the polyether marine toxin, pectenotoxin-2 (PTX-2), responsible for diarrhetic shellfish poisoning (DSP), have been isolated from the toxic marine phytoplankton (Dinophysis acuta), collected in Irish waters. Liquid chromatography with fluorimetric detection (LC-FLD) analyses of the extracts of bulk phytoplankton samples, following derivatisation with 9-anthryldiazomethane (ADAM) or 1-bromoacetylpyrene (BAP), showed a complex toxin profile with peaks corresponding to okadaic acid (OA) and its isomers, dinophysistoxin-2 (DTX-2) and DTX-2C, as well as other unidentified lipophilic acids. LC-UV analysis showed the presence of a diene moiety in these new compounds and two acids have been isolated. LC coupled with mass spectrometry (MS) and tandem mass spectrometry (LC-MS-MS) were used to gain structural information. Through flow injection analysis (FIA)-MS, both in positive and negative ion modes, the molecular weight of 876 for both compounds was determined. Collision Induced Dissociation (CID) from each parent ion, as performed both in positive and negative ion mode, produced mass spectra which were very similar to those obtained for authentic PTX-2 (mw 858). These new compounds have been confirmed to be pectenotoxin-2 seco acids (PTX-2SAs) and they are closely related to PTX-2 except that they contain an open chain carboxylic acid rather than a lactone ring. Toxic mussels also contained these pectenotoxin-2 analogues.

Efficient isolation of the rare diarrhoeic shellfish toxin, dinophysistoxin-2, from marine phytoplankton.

The rare diarrhoeic shellfish poisoning (DSP) toxin, dinophysistoxin-2 (DTX-2), which is an okadaic acid (OA) isomer, has been isolated from a marine phytoplankton biomass that consisted mainly of Dinophysis acuta. Using a large double plankton net (length 5.9 m), bulk phytoplankton samples were collected off the south-west coast of Ireland and extracted with methanol and chloroform. Liquid chromatography coupled with ionspray mass spectrometry and tandem mass spectrometry (LC-MS, LC-MS-MS) showed the sample contained DTX-2 and OA, at a concentration of 80 pg/cell and 60 pg/cell, respectively. Flash chromatography using silica, sephadex LH20 and C18-silica, followed by preparative reversed-phase LC, separated DTX-2 from OA. The efficiency of the separation procedures was substantially improved by the use of a bioscreen to detect DSP toxins in eluate fractions and the application of a new derivatisation procedure for the chromatographic elucidation of toxin profiles with fluorimetric detection (LC-FLD). Thus, 1/1000th aliquots of eluate fractions were assayed using protein phosphatase-2A for the presence of inhibitory compounds. Positive fractions were further analysed for DSP toxins by LC-FLD following derivatisation using the hydrazine reagent, luminarine-3. The identity and purity of the free isolated DTX-2 was confirmed using flow injection analysis (FIA) and liquid chromatography (FIA-MS, LC-MS and LC-MS-MS).

Anticholinergic visual hallucinosis from atropine eye drops.

A 37-year-old man with type I diabetes mellitus and chronic renal failure presented to the emergency department complaining of hallucinations. He was 5 days postoperative for left pars plana vitrectomy and intra-ocular lens implantation and had been taking ophthalmic atropine, tobramycin and prednisolone. He had presented 5 months earlier, on the same ophthalmic medications, with postoperative hallucinations after a right pars plana vitrectomy. Visual hallucinations are a major side effect of anticholinergic poisoning. Ophthalmic instillation of atropine has been documented to cause many central nervous sytstem symptoms, including hallucinations.

Isolation of a new okadaic acid analogue from phytoplankton implicated in diarrhetic shellfish poisoning.

A new analogue of okadaic acid (OA), the toxin mainly responsible for diarrhetic shellfish-poisoning (DSP) phenomena in Europe, has been isolated from toxic phytoplankton (Dinophysis acuta) collected in Irish waters. Fluorimetric LC analyses of the extracts of bulk phytoplankton samples using derivatisation with 9-anthryldiazomethane (ADAM) showed a complex toxin profile, with peaks corresponding to OA and dinophysistoxin-2 (DTX-2) as well as a third unidentified compound. This minor unidentified component was isolated by chromatographic techniques such as normal-phase chromatography, gel permeation on Sephadex, solid-phase extraction and reversed-phase separations. Ionspray mass spectrometry (MS) was used for structural investigation on this compound due to the very small amount of isolated material. Flow injection analysis (FIA)-MS of the isolated compound gave positive-ion mass spectrum dominated by the protonated molecule, [M + H]+, at signal m/z 805, whereas the deprotonated molecule [M - H]- was observed in the negative-ion spectrum at signal m/z 803, thus indicating the molecular weight of 804 for the new toxin, the same as OA and its known isomers, DTX-2 and DTX-2B. Collision-induced dissociation (CID) as obtained by positive and negative tandem mass spectrometry (MS-MS) showed a fragmentation pattern for the new compound which was very similar to that of OA, DTX-2 and DTX-2B. Ionspray microLC-MS of a mixture containing the compound under investigation together with OA analogues showed the compound eluted after OA, DTX-2, DTX-2B and before DTX-1. All the chromatographic and mass spectrometric data indicated the compound to be another OA isomer and it was therefore coded DTX-2C. To the best of our knowledge this is the first report on the isolation of a new compound related to DSP toxins from natural communities of toxic phytoplankton.

Isolation of dinophysistoxin-2 and the high-performance liquid chromatographic analysis of diarrhetic shellfish toxins using derivatisation with 1-bromoacetylpyrene.

The rare diarrhetic shellfish toxin, dinophysistoxin-2 (DTX-2), was isolated from the digestive glands of mussels (Mytilus edulis). This was achieved by chromatography on silica and Sephadex LH-20 followed by reversed-phase solid phase extraction and semi-preparative high-performance liquid chromatography (HPLC) with an Ultremex C18 column. Using 1-bromoacetylpyrene (BAP), as a precolumn derivatisation reagent, the diarrhetic shellfish toxins, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and DTX-2, were determined by HPLC with fluorimetric detection. Derivatisation using BAP was compared with 9-anthryldiazomethane (ADAM) and, although the latter exhibited a four-fold better sensitivity, the BAP method gave fewer artefact peaks from reagent decomposition. The limits of detection of OA and DTX-2 were 0.4 ng on-column using BAP, which permits this method to be used for the regulatory control of these toxins in shellfish.

Local cytokine production in a murine model of Escherichia coli pyelonephritis.

Cytokines may play an important role in the regulation of host defense against local bacterial infections. We have evaluated the local production of cytokines in a BALB/c mouse model of Escherichia coli pyelonephritis. Kidneys, draining lymph nodes, and spleens, were harvested at specific time intervals after bladder inoculation with E. coli corresponding to the stages of renal infection, infiltration, and bacterial clearance seen in this model. The presence of messenger RNA for specific cytokines (interleukins 1 through 6, chemotactic factors, granulocyte and granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF alpha) and beta, IFN gamma, transforming growth factor (TGF beta), and cytokine synthesis inhibitory factor (CSIF)/IL-10) was determined by polymerase chain reaction (PCR) amplification of reverse transcribed RNA. We have demonstrated mRNA encoding IL-1, IL-6, G-CSF, GM-CSF, TNF alpha, H400 (a protein homologous to a family of chemotactic factors and identical to MIP-1 beta), and CSIF/IL-10 in the kidney at 12 h and 1, 2, and 3 d after bacterial challenge. No signal was seen in normal animals or in mice after 5 d. This pattern of cytokine expression was observed only in renal tissues suggesting a localized response. IL-6 was present in the urine at 4 h with rapid resolution to baseline levels by 24 to 48 h. In contrast, IL-6 was not usually detectable in the serum. TNF alpha was not detectable in the serum or urine during the course of the infection. By immunohistochemical staining of kidney sections we have shown that IL-6 is produced predominantly by mesangial cells rather than by the inflammatory infiltrate. This study provides additional evidence utilizing novel techniques that specific cytokines are produced locally in response to bacterial infections. The time course of production demonstrated in this model supports the important role of cytokines in natural host resistance to local infection.