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Epoxyjanthitrems I&ndash, IV (1&ndash, 4) and epoxyjanthitriol (5) were isolated from seed of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Epichlo&euml, festucae var. lolii. Although structures for epoxyjanthitrems I&ndash, IV have previously been proposed in the literature, this is the first report of a full structural elucidation yielding NMR (Nuclear magnetic resonance) assignments for all five epoxyjanthitrem compounds, and additionally, it is the first isolation of epoxyjanthitriol (5). Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina (Wiseana cervinata), a common pasture pest in New Zealand. These data suggest that epoxyjanthitrems are involved in the observed effects of the AR37 endophyte on livestock and insect pests.

To determine the concentrations of fungal endophyte toxins in the milk of cows fed perennial ryegrass containing wild-type or AR37 endophyte.Groups of 10 multiparous Holstein-Friesian cows were fed wild-type (containing lolitrem B) or AR37 (containing epoxy-janthitrems) endophyte-infected perennial ryegrass (Lolium perenneL.). Animals were kept indoors and fed for 12 days. Over this period, animals were regularly assessed for ryegrass staggers and herbage intake measured. At the conclusion of the 12-day indoor-feeding period, cows were grazed on AR1 (toxin-free) pastures for a further 8 days. Daily individual milk samples and milk yields were collected over the complete 20-day period. Milk samples were analysed for endophyte toxins using HPLC methods developed during this study. Daily herbage samples were also taken and concentrations of endophyte toxins measured.Methods were successfully developed for the analysis of lolitrem B and epoxy-janthitrems in milk which allowed the concentrations of these compounds in milk to be compared with the concentrations in feed consumed by the animals. Both toxin types could be detected in milk after only 1 day of exposure to respective treatment pastures. The maximum concentration of endophyte toxins in milk was 5 ng/mL lolitrem B and 109 ng/mL epoxy-janthitrems from cows fed wild-type and AR37 endophyte-infected ryegrass pastures, respectively. Concentrations of epoxy-janthitrems present in herbage were much higher than for lolitrem B (Day 1-12 average of 14.6 and 1.8 ppm, respectively). Despite the high concentrations of epoxy-janthitrems consumed by cows fed AR37 endophyte-infected pastures no signs of ryegrass staggers were observed over the experimental period, whereas those cows fed wild-type endophyte-infected pastures all showed signs of ryegrass staggers. This is consistent with the view that epoxy-janthitrems are low potency tremorgens. At the conclusion of the toxin feeding period, endophyte toxin concentrations in milk quickly dropped to almost zero after 8 days. A comparison of the quantities of lolitrem B and epoxy-janthitrems consumed by each cow with the quantities secreted in milk showed that only very low proportions of the total amount ingested are secreted in milk (0.23% lolitrem B and 0.49% epoxy-janthitrems).Lolitrem B and epoxy-janthitrems can be detected in the milk of cows consuming wild-type and AR37 endophyte-infected perennial ryegrass, respectively. Concentrations detected were low and changed quickly in association with the amounts being consumed by the cows. Available evidence gives no indication that these compounds may pose a threat to human health.

Yessotoxins from a large-scale culture (226 L) of Protoceratium reticulatum strain CAWD129 were harvested by filtration followed by solid-phase extraction. The extract was purified by column chromatography over basic alumina and reverse-phase flash chromatography to afford pure yessotoxin (193 mg). Isolation of yessotoxin was greatly facilitated by selection of a strain which did not produce analogues that interfered with yessotoxin isolation. In addition to yessotoxin, numerous minor yessotoxins were detected by LC-MS in other fractions. From one of these, an early eluting minor analogue with the same molecular weight as yessotoxin and a similar mass spectrometric fragmentation pattern was isolated. This analogue was identified by NMR and mass spectrometry as a novel yessotoxin analogue containing a furan ring in the side chain. This finding reveals biosynthetic flexibility of the yessotoxin pathway in P. reticulatum and confirms earlier findings of production of many minor yessotoxin analogues by this alga. Production of these analogues appeared to be a constitutive trait of P. reticulatum CAWD129.

Yessotoxin 32-O-[beta-L-arabinofuranosyl-(5'--1'')-beta-L-arabinofuranoside] (3) was isolated from extracts of Protoceratium reticulatum during a large scale isolation of yessotoxin (1). The structure was characterized by mass spectrometry and NMR spectroscopy. Di-glycoside-3, along with the corresponding mono-glycoside (2) were detected in cultures of P. reticulatum originating from Europe and New Zealand, suggesting that production of arabinosides of 1 is a normal feature of this alga. Formation of multiply charged anions and fragmentation of 3 occurred much more readily than for 1 and 2 under the LC-MS conditions used in this study.

Pectenotoxins (PTXs) accumulate in shellfish feeding on dinoflagellates of the genus Dinophysis, so that humans can be exposed to these toxins through shellfish consumption. Some PTXs are toxic to experimental animals, whereas others are of much lower toxicity. Pectenotoxin-2, the most abundant PTX from most Dinophysis spp., is rapidly metabolized by most shellfish to a mixture of pectenotoxin-2 seco acid (2) and 7-epi-pectenotoxin-2 seco acid (1). A mixture of 1 and 2 was produced during purification of an extract from in vitro enzymatic hydrolysis of pectenotoxin-2. These were separated by preparative HPLC, and the structure of 1 was confirmed by one- and two-dimensional 1H and 13C NMR spectroscopy and LC-MS3 analyses. No toxic changes were recorded in mice injected intraperitoneally with 1 or 2 at a dose of 5000 μg/kg. PTX seco acids are therefore unlikely to be of consequence to human consumers at the concentrations found in contaminated shellfish. Keywords: pectenotoxin; seco acid; PTX-2 seco acid; D...

44,55-Dihydroxyyessotoxin (1) was isolated from extracts of Protoceratium reticulatum and identified by analysis of its one- and two-dimensional NMR and mass spectra. In addition, LC–MS methods revealed the presence of compounds tentatively identified as (44-R,S)-44,55-dihydroxy-41a-homoyessotoxin (2) and (44-R,S)-44,55-dihydroxy-9-methyl-41a-homoyessotoxin (3). LC–MS analyses indicate that 1 is a constituent of P. reticulatum in New Zealand and Norway, and it was present in three species of mussels from New Zealand, Norway, and Canada.

Two analogs of yessotoxin were isolated from extracts of a culture of Protoceratium reticulatum. The structures of the analogs were identified as trihydroxylated amides of 41a-homoyessotoxin (1) and 9-methyl-41a-homoyessotoxin (2) by one- and two-dimensional 1H and 13C NMR spectroscopy and LC-MS3 analyses. Structures were further confirmed by micro-scale chemical conversions combined with LC-MS3 analyses. No toxic effects were recorded in mice injected intraperitoneally with 2 at a dose of 5000 μg/kg.

The 1,3-enone isomer ( 1 ) of heptanor-41-oxoyessotoxin ( 2 ) was isolated from extracts of Protoceratium reticulatum during large-scale production of yessotoxin ( 4 ). We found that 2 readily isomerizes to 1 in the presence of dilute ammonia and present evidence for the existence of 40- epi - 2 ( 3 ) that also isomerizes to 1 . 1 – 3 were detected by LC–MS methods both in extracts of P. reticulatum cultures and in mussels contaminated with yessotoxins. The isomerization of 2 and 3 into 1 occurs so readily that purification on basic alumina needs to be conducted carefully. No toxic effects were recorded in mice injected intraperitoneally with 1 at a dose of 5000 μg/kg.

We have developed a simple and effective method for isolating pectenotoxin-2 (PTX-2) from Dinophysis cells collected from a natural bloom. A two-step extraction procedure followed by two column chromatography steps produced PTX-2 in high purity suitable for use as an analytical standard and for toxicological studies. Incubation of purified PTX-2 with the supernatant from ultracentrifuged blue (Mytilus edulis) or Greenshell (Perna canaliculus) mussel hepatopancreas homogenate caused rapid conversion to pectenotoxin-2 seco acid (PTX-2 SA). Purification of PTX-2 SA was achieved by solvent extraction followed by column chromatography. PTX-2 and PTX-2 SA were fully characterized by LC-MS and NMR, and full (1)H and (13)C NMR assignments were obtained. Okadaic acid C(8)-diol ester was isolated during the purification of PTX-2, and its identity confirmed by NMR and LC-MS analyses. Pectenotoxin-2 seco acid methyl ester, identified by LC-MS, was also produced during the hydrolytic procedure due to the presence of methanol. PTX-2 was acutely toxic to mice by i.p. injection (LD(50)=219 microg/kg) but no effects were seen with PTX-2 SA at 5000 microg/kg. Neither PTX-2 nor PTX-2 SA was overtly toxic to mice by the oral route at doses up to 5000 microg/kg. No diarrhea was observed in mice dosed with either compound, suggesting that pectenotoxins do not belong in the diarrhetic shellfish poison group.

Lolitrem A, the second most abundant minor lolitrem in endophyte-infected perennial ryegrass, has been isolated and its structure determined by mass spectrometry and one- and two-dimensional NMR spectroscopy. Lolitrem A, which other workers have suggested to be 7α-hydroxylolitrem B, was found to be an inseparable mixture of the 44R and 44S isomers of 44,45-epoxylolitrem C. An assignment of the methyl group proton and carbon resonances of lolitrem B is also reported. The intensity and time course of the tremorgenic activity of lolitrem A in mice were identical to that of lolitrem B.

A cis-isomer of a C8-diol ester of okadaic acid (1) was isolated during large-scale purification of pectenotoxins (PTXs) from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compound was identified by NMR spectroscopic and liquid chromatography-mass spectrometry (LC-MS) studies, and is the first reported cis-isomer of an okadaic acid C8-diol-ester identified in Dinophysis. The more abundant trans-C8-diol ester of okadaic acid (2) isolated from the same Dinophysis extract was rapidly hydrolyzed to okadaic acid in vitro by the supernatant from green-lipped mussel hepatopancreas.

Two novel pectenotoxins (PTXs), PTX-13 and -14, were isolated from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compounds were identified as oxidized analogues of PTX-2 by NMR spectroscopic and LC-MS studies. PTX-13 (32R-hydroxyPTX-2) corresponds to the unidentified analogue PTX-11x reported by [Suzuki et al., 2003. Liquid chromatography-mass spectrometry of spiroketal stereoisomers of pectenotoxins and the analysis of novel pectenotoxin isomers in the toxic dinoflagellate Dinophysis acuta from New Zealand. J. Chromatogr. A 992, 141-150]. PTX-13 underwent slow deuteration at the 13beta-position during NMR analysis. PTX-14 corresponds to the 32,36-dehydration product of PTX-13, and may be an artifact.

Preparative HPLC purification of a side-fraction obtained during purification of 44,55-dihydroxyyessotoxin (6) afforded fractions containing previously unidentified yessotoxin analogues. Careful analysis of these fractions by HPLC-UV, LC–MS3, and NMR spectroscopy, revealed the identities of some of these analogues as 45-hydroxy-46,47-dinoryessotoxin (1), 44-oxo-45,46,47-trinoryessotoxin (2) and 9-methyl-42,43,44,45,46,47,55-heptanor-38-en-41-oxoyessotoxin (5). Numerous other analogues were present but could only be characterized by HPLC-UV and LC–MS3 due to their low abundance. The HPLC-UV and LC–MS3 data confirm the presence of large numbers of yessotoxin analogues, some of which may be oxidative degradation products, in extracts of Protoceratium reticulatum. Compound-1 is the first 46,47-dinoryessotoxin to be identified.

To study the urinary disposition of orally administered sporidesmins A and D in sheep and identify factors influencing their kinetics, particularly the influence of breeding for resistance and susceptibility to sporidesmin, the mycotoxin responsible for the hepatogenous photosensitisation, facial eczema.A competitive ELISA was used to monitor urinary output of immunoreactive metabolites after the intraruminal administration, to female Romney sheep, of either sporidesmin A or sporidesmin D, the nontoxic analogue. Preliminary characterisation of metabolites was carried out using HPLC with fractions monitored by ELISA.Maximum urinary excretion rates of immunoreactive metabolites occurred 2-8 h after dosing with sporidesmin D and 15-30 h after dosing with sporidesmin A. Sporidesmin D caused no liver injury, as detected by changes in serum enzyme activity, while the liver injury caused by sporidesmin A was greatest for the sheep with the highest cumulative output of metabolite. When sporidesmin D was administered in two separate doses to sheep bred for either resistance or susceptibility to facial eczema, the variability of metabolic output between sheep within groups was much less after the second dose. The mean urinary metabolite excretion was greater for the susceptible than the resistant sheep but the difference was not significant. Potentiation (caused by pre-administration of small doses of sporidesmin A) resulted in a more severe reaction to the dosed sporidesmin A. Urinary output of metabolite was less in the potentiated than in the unpotentiated sheep. When resistant and susceptible sheep were dosed with sporidesmin A after potentiation there was no difference between them in their cumulative totals or excretion rates of immunoreactive metabolites. However, the volume of urine produced by the susceptible sheep was lower and less variable than the resistant sheep and consequently the concentration of their urinary metabolites was higher. Preliminary ELISA examination of HPLC-fractionated urine from a sheep dosed with sporidesmin A indicated the presence of several metabolites of sporidesmin.Sporidesmin A and metabolites are rapidly excreted in urine but not as rapidly as sporidesmin D and its metabolites. Only minor differences between sheep bred for resistance and susceptibility were seen. Potentiation caused a more severe reaction to sporidesmin A and less urinary excretion of the sporidesmin and its metabolites.This work is part of a programme with the aim of identifying FE-resistant animals without the need for sporidesmin dosing.

The lolitrem neurotoxins, potent tremorgenic toxins isolated from perennial ryegrass, were examined using high-performance liquid chromatography with stop-flow UV spectral characterization. Comparison with some known indoles and indolic tremorgenic mycotoxins, together with chemically reduced lolitrem B, the major lolitrem neurotoxin, established the central indole chromophore of the lolitrems. The stop-flow UV spectral characterization was useful for identification of lolitrem B in ryegrass plant and seed extracts.

A rapid, sensitive and quantitative method, based on high-performance liquid chromatography with fluorescence detection, is described for the determination of the neurotoxin lolitrem B in perennial ryegrass, in the ppm to sub-ppm range. The method, which requires a minimal clean-up step prior to chromatographic analysis, is suitable for the routine analysis of large numbers of ryegrass samples, and is currently being used in New Zealand to study the livestock disorder ryegrass staggers. The method is suitable for determining lolitrem B in the whole plant, the seed, and dissected plant components.

Extract Sir:- Recently, two potent neurotoxins named lolitrem A and lolitrem B were isolated from herbage collected from pastures on which the livestock disorder ryegrass staggers (RGS) occurred. et al.(1) We now wish to report that samples of perennial rygrass (Loliun yeretzrze L.) seed obtained from several locations in New Zealand contained potent neurotoxins which include lolitrem A and lolitrem B. Further, when pellets made from lolitrem neurotoxin-containing seed were fed to sheep, symptoms indistinguishable from those of severe, natural RGS were induced in four sheep, and mild symptoms in three sheep.

The lolitrems, tremorgenic neurotoxins from perennial ryegrass, are implicated in ryegrass staggers disorder in livestock; the assignment of structure (1) to the major neurotoxin, lolitrem B, is based on its spectroscopic properties, particularly a detailed structure of its high-field 1H and 13C n.m.r. spectra, as well as chemical evidence.