BACKGROUND
ragwort  is a poisonous weed found growing on riverbanks, roadsides and pasture and is toxic to most grazing livestock.
horses are particularly sensitive to the toxic effects of ragwort and typically avoid the weed, but poisoning can occur when the animals graze on poorly maintained pastures or when feedstuff such as hay or silage is contaminated.
ragwort poisoning leads to complete liver failure, however the clinical signs can be slow to develop and may only manifest after prolonged dietary exposure.
prognosis is poor once the liver disease is advanced and current treatments are only palliative  <cit> .
the early diagnosis of ragwort poisoning is extremely difficult as many conventional biochemical and histopathological indicators of the disease are non-specific and do not discriminate ragwort poisoning from other immune, infectious or toxic diseases  <cit> .
the toxic precursors in ragwort are pyrrolizidine alkaloids  <cit> .
amongst the most prominent pyrrolizidine alkaloids found in ragwort are jacobine, erucifoline and senecionine  <cit> .
following ingestion of ragwort the pyrrolizidine alkaloids are absorbed from the gastrointestinal tract and pass to the liver where they can be rapidly metabolised by the cytochrome p <dig> system to reactive and cytotoxic pyrroles  <cit>  .
the pyrrolic metabolites bind to liver macromolecules such as dna, eliciting cell injury  <cit>  but they can also escape into the circulation where they react with the thiol groups of cysteine residues of blood proteins  <cit> .
the persistence of these adducts formed the basis of one chemical analysis that was developed over a decade ago  <cit> .
in this test, haemoglobin thioesters were treated with ethanolic silver nitrate under acidic conditions, releasing a diethoxyether form of the bound pyrrole that can be identified by thin-layer chromatography, gas chromatography or high performance liquid chromatography.
this approach has been used to confirm ragwort poisoning in horses  <cit> , cattle  <cit>  and yaks  <cit>  but has not been adopted widely.
an alternative strategy might be to directly analyse pyrrole-protein complexes in blood rather than by the recovery of the xenobiotics.
in this study an in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods.
such an approach may identify target proteins that could serve as surrogate markers of ragwort exposure.
it offers the possibility of a novel analytical strategy to improve the diagnosis of ragwort toxicity in horses and other livestock.
RESULTS
ethical considerations preclude toxicological studies that would require horses to be exposed to ragwort.
therefore, an in vitro model system was developed to determine the modifications to horse blood proteins induced by the toxic metabolites of ragwort.
commercial preparations of the major pyrrolizidine alkaloids from ragwort are expensive and therefore a structurally and chemically related pyrrolizidine alkaloid, monocrotaline, was used to optimise methodologies.
the model involved the chemical oxidation of monocrotaline to its pyrrolic derivative, dehydromonocrotaline , followed by modification of horse proteins obtained from surplus clinical blood samples taken for diagnostic purposes.
proteomic methods were then used to detect specific alterations of the equine blood proteins.
pyrrole modification of equine haemoglobin
pyrrole thioesters of haemoglobin are believed to be relatively stable  <cit>  and may remain in the bloodstream for the lifespan of the erythrocyte .
the detection of pyrrole-modified haemoglobin molecules could act as an indicator of ragwort exposure over a prolonged period.
equine haemoglobin, prepared from freshly collected blood, was incubated in vitro with varying concentrations of synthesised dhm and analysed for evidence of pyrrole modification.
1-d sds-page showed no changes in the profile of the haemoglobin following pyrrole exposure and simply separated the α- and β-chains.
this approach was unlikely to provide sufficient resolution to discriminate between modified and non-modified haemoglobin molecules if a single cysteine residue had been adducted.
the haemoglobin molecule was further analysed by intact protein mass spectrometry.
free cysteine and cysteine-containing peptides such as glutathione are readily modified by pyrrolic metabolites  <cit> .
the adduction of a pyrrole to a cysteine residue would be expected to increase the molecular weight of each haemoglobin chain by either  <dig> or  <dig> da, depending on whether the pyrrole moiety dehydrates.
intact mass analysis of the haemoglobin using electrospray ionisation-mass spectrometry  revealed two peaks at  <dig>  ±  <dig> da and  <dig>  +/-  <dig> da, which corresponded to the predicted molecular weights of the α- and β-chains respectively.
an additional peak at  <dig>  +/-  <dig> da was due to a phe24→tyr <dig> variant of the α-chain equine haemoglobin, whilst a peak detected at  <dig>  +/-  <dig> da was attributed to the glutathionylated form of the β-chain, indicating that the cysteine residue was accessible in a significant proportion of the β-chain.
the haemoglobin was also digested with trypsin and the resultant peptides were analysed by matrix assisted laser desorption ionisation-time-of-flight-mass spectrometry .
despite 90% sequence coverage of each of the globin chains, the analysis failed to reveal any pyrrole modification of the cysteine containing peptide.
the inability to detect pyrrole adducts may be due to a combination of factors.
the pyrrole modification may be spread over the α- and β-chains of haemoglobin and it is possible that the extent of modification of haemoglobin is too low to be observed in the predominant unmodified haemoglobin pool.
it is also possible that glutathione in the erythrocytes may have protected the haemoglobin from pyrrole modification, however glutathione-pyrrole adducts were not detected in blood samples by mass spectrometric analysis.
in addition, there may be difficulties in reacting pyrroles with intact equine haemoglobin.
the α-chain cysteine residue of horse haemoglobin is situated in a relatively hydrophobic region of the protein, which is likely to render it inaccessible to pyrrole metabolites.
however, the β-chain cysteine at position  <dig> is more externally located and is more susceptible to modification  <cit> .
this conjecture was confirmed when equine haemoglobin was modified with the pyrrole under reducing and denaturing conditions and a small amount of β-chain adduction was detected by intact mass analysis using esi-ms.
pyrrole modification of equine plasma proteins
other potential protein targets of pyrrole modification in horse blood were also investigated.
as the first site of exposure to pyrroles would be plasma proteins, equine plasma was incubated with chemically synthesised dhm and the proteins analysed by 1-d sds-page.
both non-reducing and reducing gels revealed a significant alteration in the equine plasma protein profile following pyrrole exposure .
there was a shift in mobility of a number of protein bands and a protein aggregate was detected in the high molecular weight region of the resolving gel, which became more pronounced with increasing concentrations of dhm.
the aggregate was not observed in control samples where plasma was treated with a reaction mixture that was complete apart from dhm.
similarly, controls where monocrotaline or the carrier solvent, n,n-dimethylformamide , was exposed to plasma also failed to produce any changes in the protein profile.
modification was therefore caused by the presence of pyrrole in the experimental system.
dhm is capable of cross-linking proteins  <cit> .
the proteins in the high molecular weight aggregate at the top of the gel were identified using liquid chromatography-tandem mass spectrometry .
the major components of the aggregate were identified with high confidence  as serum albumin, fibrinogen, transferrin and immunoglobulin gamma  .
proteins were identified on the basis of their mowse score using the msdb database.
each band number refers to proteins identified in figure 2a.
a = mowse baseline significance score in brackets; b = for those with no percentage coverage value the database did not give this information.
the plasma proteins were identified using lc-ms/ms.
proteins were identified on the basis of their mowse score using the msdb database.
each band number refers to proteins identified in figure 2b.
a = mowse baseline significance score in brackets; b = for those with no percentage coverage value the database did not give this information.
due to the evidence of cross-linking of the major plasma proteins authentic standards of serum albumin, fibrinogen and transferrin were reacted with dhm.
as equine protein standards were not available from commercial sources bovine standards were used in their place.
fibrinogen appeared to be particularly susceptible to modification by the pyrrole, being substantially converted to a high molecular weight aggregate.
in contrast, the bovine serum albumin and transferrin standards displayed only limited reactivity with dhm and did not show the same shift in mobility corresponding to the high molecular weight aggregate.
it was possible that the presence of igg, albumin and transferrin in whole plasma was due to co-polymerisation and aggregation with the fibrinogen molecule.
since fibrinogen was reactive with dhm dose-response and time-course experiments were conducted.
the optimal concentration for pyrrole exposure was calculated at a molar ratio of dhm:protein of 10: <dig> .
lower concentrations of the pyrrole failed to completely modify the protein, whereas high concentrations resulted in instantaneous and complete modification.
figure  <dig> shows 1-d sds-page analysis of pyrrole-treated fibrinogen over a  <dig> min incubation period.
the density of the fibrinogen bands, and in particular the α-chain, diminished over time, suggesting that the protein is gradually modified in a way which prevents it from entering the gel and migrating to its usual position.
this correlates well with the appearance of high molecular weight aggregates.
all of the cysteine residues in fibrinogen maintain the tertiary structure of the protein through disulphide bonds.
in principle these oxidised cysteines residues would not be available to react with an alkylating agent such as pyrrole, and modification of amino acid residues other than cysteine may have occurred.
tentative identifications of pyrrole modified peptides were made from maldi-tof-ms analysis of in-gel and in-solution tryptic digests of the fibrinogen aggregate, however, additional mass spectrometric analysis using lc-ms/ms was unable to confirm the site pyrrole modification.
this may occur due to the fact that the covalent linkage between the pyrrole and amino acid residues may not be stable to conditions of analysis by mass spectrometry  <cit> .
the formation of specific pyrrole derived fragment ions can vary depending on the amino acid adducted and the surrounding peptide sequence.
pyrrole modification also disrupts the normal peptide fragmentation  <cit>  complicating the ability to obtain sequence information from modified peptides.
western blotting of pyrrole modified plasma proteins
in order to further investigate the response of plasma proteins with dhm, western blots were performed using antibodies to equine fibrinogen.
after exposure to dhm, the fibrinogen α-chain band was no longer observed.
western blots using antibodies to equine serum albumin and transferrin indicated that albumin was modified to a lesser extent following dhm exposure, whereas the transferrin demonstrated no reactivity with pyrrole.
a dose-response study revealed that the fibrinogen aggregation is initiated when an approximate sixty-fold molar excess of pyrrole is added to plasma .
CONCLUSIONS
although ragwort poisoning is recognised as a threat to equidae, the true extent of this threat cannot be assessed in the absence of a definitive and specific test for exposure to the toxin.
at present, clinical tests are focused on a more generalised manifestation of hepatic damage.
the poisoning is chronic and accumulative, and the emergence of symptoms may post date the exposure to the plant by weeks or even months.
there is therefore a pressing need for a test that can assess the history of exposure.
the most successful test used previously was based on qualitative assessment of adducts of the pyrrole with blood proteins  <cit> .
accordingly, an in vitro model system, not requiring deliberate exposure of horses to pyrrolizidine alkaloids, was developed to explore the specificity of adduction.
perhaps surprisingly, the highly abundant haemoglobin chains were not modified, which might reflect lack of availability of reactive side chains, or the inability of the pyrrole to survive for long enough in plasma to cross the erythrocyte membrane.
plasma proteins might therefore be targets for pyrrole modification, and the discovery that fibrinogen was modified is the first step towards such a specific test.
it is not known whether a protein-based analysis is more sensitive than a test based on measurement of pyrrole residues after release from proteins, or indeed, is sensitive enough for a routine analysis.
however, the mass-shifted modification of fibrinogen chains is easily detected by western blotting, and could be readily performed in many laboratories, requiring minimal investment in equipment and using a commercially available antiserum.
because the test is based on a mass shift, a simple elisa cannot be substituted, as this would not discriminate between the unmodified and modified fibrinogen chains.
deployment of this simple assessment may be of greatest value in identifying and establishing a pool of cases that can then be used to develop even more sensitive test, based on specific peptide modification or on direct detection of pyrrole moieties in a blood sample.
such a screening test may enable veterinarians to identify ragwort exposure and would therefore be an invaluable diagnostic tool with substantial welfare benefits.

 ragwort is a poisonous weed found growing on riverbanks, roadsides and pasture and is toxic to most grazing livestock. horses are particularly sensitive to the toxic effects of ragwort and typically avoid the weed, but poisoning can occur when the animals graze on poorly maintained pastures or when feedstuff such as hay or silage is contaminated. ragwort poisoning leads to complete liver failure, however the clinical signs can be slow to develop and may only manifest after prolonged dietary exposure. prognosis is poor once the liver disease is advanced and current treatments are only palliative. the early diagnosis of ragwort poisoning is extremely difficult as many conventional biochemical and histopathological indicators of the disease are non-specific and do not discriminate ragwort poisoning from other immune, infectious or toxic diseases. the toxic precursors in ragwort are pyrrolizidine alkaloids. amongst the most prominent pyrrolizidine alkaloids found in ragwort are jacobine, erucifoline and senecionine. following ingestion of ragwort the pyrrolizidine alkaloids are absorbed from the gastrointestinal tract and pass to the liver where they can be rapidly metabolised by the cytochrome p system to reactive and cytotoxic pyrroles. the pyrrolic metabolites bind to liver macromolecules such as dna, eliciting cell injury but they can also escape into the circulation where they react with the thiol groups of cysteine residues of blood proteins. the persistence of these adducts formed the basis of one chemical analysis that was developed over a decade ago. in this test, haemoglobin thioesters were treated with ethanolic silver nitrate under acidic conditions, releasing a diethoxyether form of the bound pyrrole that can be identified by thin-layer chromatography, gas chromatography or high performance liquid chromatography. this approach has been used to confirm ragwort poisoning in horses, cattle and yaks but has not been adopted widely. an alternative strategy might be to directly analyse pyrrole-protein complexes in blood rather than by the recovery of the xenobiotics. in this study an in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods. such an approach may identify target proteins that could serve as surrogate markers of ragwort exposure. it offers the possibility of a novel analytical strategy to improve the diagnosis of ragwort toxicity in horses and other livestock.  ethical considerations preclude toxicological studies that would require horses to be exposed to ragwort. therefore, an in vitro model system was developed to determine the modifications to horse blood proteins induced by the toxic metabolites of ragwort. commercial preparations of the major pyrrolizidine alkaloids from ragwort are expensive and therefore a structurally and chemically related pyrrolizidine alkaloid, monocrotaline, was used to optimise methodologies. the model involved the chemical oxidation of monocrotaline to its pyrrolic derivative, dehydromonocrotaline, followed by modification of horse proteins obtained from surplus clinical blood samples taken for diagnostic purposes. proteomic methods were then used to detect specific alterations of the equine blood proteins. pyrrole modification of equine haemoglobin pyrrole thioesters of haemoglobin are believed to be relatively stable and may remain in the bloodstream for the lifespan of the erythrocyte. the detection of pyrrole-modified haemoglobin molecules could act as an indicator of ragwort exposure over a prolonged period. equine haemoglobin, prepared from freshly collected blood, was incubated in vitro with varying concentrations of synthesised dhm and analysed for evidence of pyrrole modification. 1-d sds-page showed no changes in the profile of the haemoglobin following pyrrole exposure and simply separated the α- and β-chains. this approach was unlikely to provide sufficient resolution to discriminate between modified and non-modified haemoglobin molecules if a single cysteine residue had been adducted. the haemoglobin molecule was further analysed by intact protein mass spectrometry. free cysteine and cysteine-containing peptides such as glutathione are readily modified by pyrrolic metabolites. the adduction of a pyrrole to a cysteine residue would be expected to increase the molecular weight of each haemoglobin chain by either or da, depending on whether the pyrrole moiety dehydrates. intact mass analysis of the haemoglobin using electrospray ionisation-mass spectrometry revealed two peaks at ± da and +/- da, which corresponded to the predicted molecular weights of the α- and β-chains respectively. an additional peak at +/- da was due to a phe24→tyr variant of the α-chain equine haemoglobin, whilst a peak detected at +/- da was attributed to the glutathionylated form of the β-chain, indicating that the cysteine residue was accessible in a significant proportion of the β-chain. the haemoglobin was also digested with trypsin and the resultant peptides were analysed by matrix assisted laser desorption ionisation-time-of-flight-mass spectrometry. despite 90% sequence coverage of each of the globin chains, the analysis failed to reveal any pyrrole modification of the cysteine containing peptide. the inability to detect pyrrole adducts may be due to a combination of factors. the pyrrole modification may be spread over the α- and β-chains of haemoglobin and it is possible that the extent of modification of haemoglobin is too low to be observed in the predominant unmodified haemoglobin pool. it is also possible that glutathione in the erythrocytes may have protected the haemoglobin from pyrrole modification, however glutathione-pyrrole adducts were not detected in blood samples by mass spectrometric analysis. in addition, there may be difficulties in reacting pyrroles with intact equine haemoglobin. the α-chain cysteine residue of horse haemoglobin is situated in a relatively hydrophobic region of the protein, which is likely to render it inaccessible to pyrrole metabolites. however, the β-chain cysteine at position is more externally located and is more susceptible to modification. this conjecture was confirmed when equine haemoglobin was modified with the pyrrole under reducing and denaturing conditions and a small amount of β-chain adduction was detected by intact mass analysis using esi-ms. pyrrole modification of equine plasma proteins other potential protein targets of pyrrole modification in horse blood were also investigated. as the first site of exposure to pyrroles would be plasma proteins, equine plasma was incubated with chemically synthesised dhm and the proteins analysed by 1-d sds-page. both non-reducing and reducing gels revealed a significant alteration in the equine plasma protein profile following pyrrole exposure. there was a shift in mobility of a number of protein bands and a protein aggregate was detected in the high molecular weight region of the resolving gel, which became more pronounced with increasing concentrations of dhm. the aggregate was not observed in control samples where plasma was treated with a reaction mixture that was complete apart from dhm. similarly, controls where monocrotaline or the carrier solvent, n,n-dimethylformamide, was exposed to plasma also failed to produce any changes in the protein profile. modification was therefore caused by the presence of pyrrole in the experimental system. dhm is capable of cross-linking proteins. the proteins in the high molecular weight aggregate at the top of the gel were identified using liquid chromatography-tandem mass spectrometry. the major components of the aggregate were identified with high confidence as serum albumin, fibrinogen, transferrin and immunoglobulin gamma. proteins were identified on the basis of their mowse score using the msdb database. each band number refers to proteins identified in figure 2a. a = mowse baseline significance score in brackets; b = for those with no percentage coverage value the database did not give this information. the plasma proteins were identified using lc-ms/ms. proteins were identified on the basis of their mowse score using the msdb database. each band number refers to proteins identified in figure 2b. a = mowse baseline significance score in brackets; b = for those with no percentage coverage value the database did not give this information. due to the evidence of cross-linking of the major plasma proteins authentic standards of serum albumin, fibrinogen and transferrin were reacted with dhm. as equine protein standards were not available from commercial sources bovine standards were used in their place. fibrinogen appeared to be particularly susceptible to modification by the pyrrole, being substantially converted to a high molecular weight aggregate. in contrast, the bovine serum albumin and transferrin standards displayed only limited reactivity with dhm and did not show the same shift in mobility corresponding to the high molecular weight aggregate. it was possible that the presence of igg, albumin and transferrin in whole plasma was due to co-polymerisation and aggregation with the fibrinogen molecule. since fibrinogen was reactive with dhm dose-response and time-course experiments were conducted. the optimal concentration for pyrrole exposure was calculated at a molar ratio of dhm:protein of 10:. lower concentrations of the pyrrole failed to completely modify the protein, whereas high concentrations resulted in instantaneous and complete modification. figure shows 1-d sds-page analysis of pyrrole-treated fibrinogen over a min incubation period. the density of the fibrinogen bands, and in particular the α-chain, diminished over time, suggesting that the protein is gradually modified in a way which prevents it from entering the gel and migrating to its usual position. this correlates well with the appearance of high molecular weight aggregates. all of the cysteine residues in fibrinogen maintain the tertiary structure of the protein through disulphide bonds. in principle these oxidised cysteines residues would not be available to react with an alkylating agent such as pyrrole, and modification of amino acid residues other than cysteine may have occurred. tentative identifications of pyrrole modified peptides were made from maldi-tof-ms analysis of in-gel and in-solution tryptic digests of the fibrinogen aggregate, however, additional mass spectrometric analysis using lc-ms/ms was unable to confirm the site pyrrole modification. this may occur due to the fact that the covalent linkage between the pyrrole and amino acid residues may not be stable to conditions of analysis by mass spectrometry. the formation of specific pyrrole derived fragment ions can vary depending on the amino acid adducted and the surrounding peptide sequence. pyrrole modification also disrupts the normal peptide fragmentation complicating the ability to obtain sequence information from modified peptides. western blotting of pyrrole modified plasma proteins in order to further investigate the response of plasma proteins with dhm, western blots were performed using antibodies to equine fibrinogen. after exposure to dhm, the fibrinogen α-chain band was no longer observed. western blots using antibodies to equine serum albumin and transferrin indicated that albumin was modified to a lesser extent following dhm exposure, whereas the transferrin demonstrated no reactivity with pyrrole. a dose-response study revealed that the fibrinogen aggregation is initiated when an approximate sixty-fold molar excess of pyrrole is added to plasma.  although ragwort poisoning is recognised as a threat to equidae, the true extent of this threat cannot be assessed in the absence of a definitive and specific test for exposure to the toxin. at present, clinical tests are focused on a more generalised manifestation of hepatic damage. the poisoning is chronic and accumulative, and the emergence of symptoms may post date the exposure to the plant by weeks or even months. there is therefore a pressing need for a test that can assess the history of exposure. the most successful test used previously was based on qualitative assessment of adducts of the pyrrole with blood proteins. accordingly, an in vitro model system, not requiring deliberate exposure of horses to pyrrolizidine alkaloids, was developed to explore the specificity of adduction. perhaps surprisingly, the highly abundant haemoglobin chains were not modified, which might reflect lack of availability of reactive side chains, or the inability of the pyrrole to survive for long enough in plasma to cross the erythrocyte membrane. plasma proteins might therefore be targets for pyrrole modification, and the discovery that fibrinogen was modified is the first step towards such a specific test. it is not known whether a protein-based analysis is more sensitive than a test based on measurement of pyrrole residues after release from proteins, or indeed, is sensitive enough for a routine analysis. however, the mass-shifted modification of fibrinogen chains is easily detected by western blotting, and could be readily performed in many laboratories, requiring minimal investment in equipment and using a commercially available antiserum. because the test is based on a mass shift, a simple elisa cannot be substituted, as this would not discriminate between the unmodified and modified fibrinogen chains. deployment of this simple assessment may be of greatest value in identifying and establishing a pool of cases that can then be used to develop even more sensitive test, based on specific peptide modification or on direct detection of pyrrole moieties in a blood sample. such a screening test may enable veterinarians to identify ragwort exposure and would therefore be an invaluable diagnostic tool with substantial welfare benefits. 
in this study a non-invasive in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods. the principal toxins of ragwort are the pyrrolizidine alkaloids that are rapidly metabolised to highly reactive and cytotoxic pyrroles, which can escape into the circulation and bind to proteins. one dimensional gel electrophoresis revealed a significant alteration in the equine plasma protein profile following pyrrole exposure and the formation of a high molecular weight protein aggregate. using mass spectrometry and confirmation by western blotting the major components of this aggregate were identified as fibrinogen, serum albumin and transferrin. this model has the potential to form the basis of a novel proteomic strategy aimed at identifying surrogate protein biomarkers of ragwort exposure in horses and other livestock. the high molecular weight aggregate may result from extensive inter- and intra-molecular cross-linking of fibrinogen with the pyrrole.
 ingestion of the poisonous weed ragwort by horses leads to irreversible liver damage. the principal toxins of ragwort are the pyrrolizidine alkaloids that are rapidly metabolised to highly reactive and cytotoxic pyrroles, which can escape into the circulation and bind to proteins. in this study a non-invasive in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods.  one dimensional gel electrophoresis revealed a significant alteration in the equine plasma protein profile following pyrrole exposure and the formation of a high molecular weight protein aggregate. using mass spectrometry and confirmation by western blotting the major components of this aggregate were identified as fibrinogen, serum albumin and transferrin.  these findings demonstrate that pyrrolic metabolites can modify equine plasma proteins. the high molecular weight aggregate may result from extensive inter- and intra-molecular cross-linking of fibrinogen with the pyrrole. this model has the potential to form the basis of a novel proteomic strategy aimed at identifying surrogate protein biomarkers of ragwort exposure in horses and other livestock. 