after an information campaign in horse journals and at meetings , 278 samples of maple tree leaves , sprouts , and seeds collected by horse owners in 2014 were sent to utrecht university ( uu ) and rikiltwageningen ur ( figs 1 , 2 , 3 ) . the samples were taken from the ground , packed in plastic , and immediately sent to the laboratory where they were stored at 20c within 24 hours of collection .
they were classified by an experienced botanist at the species level ( acer pseudoplatanus , acer platanoides , or acer campestre ) on the basis of the samples and accompanying photographs sent by owners .
sprouts were only used in this study upon positive classification by means of accompanying leaves , seeds or both . the minimum amount of material was 50 g for seeds and sprouts and 100 g for leaves .
owners were asked to complete a questionnaire with information about date of collection , address of the pasture , species of maple tree ( in their opinion ) , signalment ( name , breed and age ) of the horse(s ) , and presence of disease ( am ) in horses that were grazed in the pastures where the material was collected .
if the pasture had contained horses with am , details about the disease ( including date of onset and the final outcome ) , laboratory results and name of the veterinarian were provided .
inclusion criteria to confirm am were recumbency , dark colored urine , high serum activity of creatine kinase ( ck , either too high to measure in practice , or > 100,000 iu / l in a laboratory ) and postmortem examination , which was performed in 4 cases . exclusion criterion for am was lack of ck analysis unless postmortem examination was performed .
the geographic location of the pastures where the acer seeds , sprouts and leaves were sampled . 

a , b , c leaves of acer pseudoplatanus , acer campestre and acer platanoides .
hypoglycin a was measured using a newly validated method based on liquid chromatography coupled to tandem mass spectrometry ( lc  ms / ms).1 
  seed samples ( 50 g , as received ) were ground in a retsch mill , and leaf samples ( 50100 g ) were cryogenically milled into a powder , using liquid nitrogen .
one gram of homogenized sample material was extracted with 10 ml of milliq water ( mechanical shaker , 1 h ) .
after centrifugation ( 5000 g , 10c , 10 min ) , 10 l of the clear extract was injected into an lc  ms / ms system , acquiring 2 transitions ( m / z 142.174.1 and 142.196.0 ) .
hypoglycin a was identified based on matching retention time and ion ratio of the 2 transitions , against an analytical reference standard ( shypoglycin a2 ) dissolved in milliq water .
( residuals < 20% ) between the concentration of hypoglycin a and the msresponse was obtained in the range 0.012 mg
/ l , corresponding to 0.120 mg / kg in nondiluted extract and 102000 mg / kg for 100fold diluted extracts .
the accuracy of the method was determined as the recovery obtained after spiking known amounts of hypoglycin a to blank leaf ( 1 mg / kg ) and seed ( 10 mg / kg ) samples in 5fold dilutions .
the corresponding repeatabilities ( relative standard deviation , rsd ) were 11% and 7% , respectively .
the rsd obtained for repetitive analysis ( n = 5 ) of a homogenized seed sample containing hypoglycin a at a concentration of 194 mg / kg was 8% .
recoveries obtained for hypoglycin a in spiked seed samples ( 10 mg / kg ) concurrently analyzed with the study samples were consistent with those of the validation and in the range of 7395%
. samples with incomplete questionnaire data on relevant facts , such as address and obtainable data about the disease if present , were excluded from further statistical analysis ( 9 samples ) .
if multiple samples from 1 pasture were submitted , which happened often , the mean result of the analyses per sample type ( sprout , seed or leaf ) were counted as 1 result in further statistical analysis .
samples that could not be unambiguously assigned to an acer species ( 6 ) , samples that were not fresh ( 17 ) , and samples that contained material other than seeds , sprouts and leaves ( 10 ) also were excluded from the study .
this approach resulted in 143 usable samples , 30 from the spring and 113 from the autumn of 2014 .
the samples were grouped by whether they came from pastures grazed by healthy horses or horses with am ( nonam versus am pastures ) . mean concentrations of hypoglycin a were compared for the type of sample ( seed versus sprout versus leaf ) , season ( spring versus autumn ) and disease status for the pasture ( nonam versus am ) .
statistical analysis was performed using univariate generalized linear models with multiple comparisons , corrected with a posthoc bonferroni test.3 
  control for normal distribution was performed .
after an information campaign in horse journals and at meetings , 278 samples of maple tree leaves , sprouts , and seeds collected by horse owners in 2014 were sent to utrecht university ( uu ) and rikiltwageningen ur ( figs 1 , 2 , 3 ) . the samples were taken from the ground , packed in plastic , and immediately sent to the laboratory where they were stored at 20c within 24 hours of collection .
they were classified by an experienced botanist at the species level ( acer pseudoplatanus , acer platanoides , or acer campestre ) on the basis of the samples and accompanying photographs sent by owners .
sprouts were only used in this study upon positive classification by means of accompanying leaves , seeds or both . the minimum amount of material was 50 g for seeds and sprouts and 100 g for leaves .
owners were asked to complete a questionnaire with information about date of collection , address of the pasture , species of maple tree ( in their opinion ) , signalment ( name , breed and age ) of the horse(s ) , and presence of disease ( am ) in horses that were grazed in the pastures where the material was collected .
if the pasture had contained horses with am , details about the disease ( including date of onset and the final outcome ) , laboratory results and name of the veterinarian were provided .
inclusion criteria to confirm am were recumbency , dark colored urine , high serum activity of creatine kinase ( ck , either too high to measure in practice , or > 100,000 iu / l in a laboratory ) and postmortem examination , which was performed in 4 cases . exclusion criterion for am was lack of ck analysis unless postmortem examination was performed .
the geographic location of the pastures where the acer seeds , sprouts and leaves were sampled . 

a , b , c leaves of acer pseudoplatanus , acer campestre and acer platanoides .
hypoglycin a was measured using a newly validated method based on liquid chromatography coupled to tandem mass spectrometry ( lc  ms / ms).1 
  seed samples ( 50 g , as received ) were ground in a retsch mill , and leaf samples ( 50100 g ) were cryogenically milled into a powder , using liquid nitrogen .
one gram of homogenized sample material was extracted with 10 ml of milliq water ( mechanical shaker , 1 h ) .
after centrifugation ( 5000 g , 10c , 10 min ) , 10 l of the clear extract was injected into an lc  ms / ms system , acquiring 2 transitions ( m / z 142.174.1 and 142.196.0 ) .
hypoglycin a was identified based on matching retention time and ion ratio of the 2 transitions , against an analytical reference standard ( shypoglycin a2 ) dissolved in milliq water .
quantification was performed by multilevel calibration . a consistent linear relationship ( residuals < 20% ) between the concentration of hypoglycin a and the msresponse was obtained in the range 0.012 mg / l , corresponding to 0.120 mg / kg in nondiluted extract and 102000 mg / kg for 100fold diluted extracts .
the accuracy of the method was determined as the recovery obtained after spiking known amounts of hypoglycin a to blank leaf ( 1 mg / kg ) and seed ( 10 mg / kg ) samples in 5fold dilutions .
the corresponding repeatabilities ( relative standard deviation , rsd ) were 11% and 7% , respectively .
the rsd obtained for repetitive analysis ( n = 5 ) of a homogenized seed sample containing hypoglycin a at a concentration of 194 mg / kg was 8% .
recoveries obtained for hypoglycin a in spiked seed samples ( 10 mg / kg ) concurrently analyzed with the study samples were consistent with those of the validation and in the range of 7395% .
samples with incomplete questionnaire data on relevant facts , such as address and obtainable data about the disease if present , were excluded from further statistical analysis ( 9 samples ) . if multiple samples from 1 pasture were submitted , which happened often , the mean result of the analyses per sample type ( sprout , seed or leaf ) were counted as 1 result in further statistical analysis .
samples that could not be unambiguously assigned to an acer species ( 6 ) , samples that were not fresh ( 17 ) , and samples that contained material other than seeds , sprouts and leaves ( 10 ) also were excluded from the study .
this approach resulted in 143 usable samples , 30 from the spring and 113 from the autumn of 2014 .
the samples were grouped by whether they came from pastures grazed by healthy horses or horses with am ( nonam versus am pastures ) .
mean concentrations of hypoglycin a were compared for the type of sample ( seed versus sprout versus leaf ) , season ( spring versus autumn ) and disease status for the pasture ( nonam versus am ) .
statistical analysis was performed using univariate generalized linear models with multiple comparisons , corrected with a posthoc bonferroni test.3 
  control for normal distribution was performed . a p value of .05 was set as statistically significant .
in total , 143 of the collected samples ( 93 seed samples , 14 sprout samples , and 36 leave samples ) from a total of 81 locations were included ( fig 1 ) . none of the acer platanoides ( n = 11 ) and acer campestre ( n = 13 ) samples ( 29 samples of seeds , 11 of leaves and 1 of sprouts ) contained detectable concentrations of hypoglycin a. in contrast , most acer pseudoplatanus leaves , seeds , and sprouts contained the toxin . in 2 samples of seeds and 6 samples of leaves ,
no hypoglycin a was found ( ie , concentration below limit of quantification [ 1 mg / kg for leaves and 10 mg / kg for seeds ] ) .
the factor season was not statistically significant ( p = .841 ) and was excluded from further statistical analysis .
the mean  sd concentrations of hypoglycin a in the samples are presented in table 1 .
hypoglycin a concentrations differed significantly among the sample types ( seeds and sprouts , p < .001 ; seeds and leaves , p < .001 ; sprouts and leaves , p = .000 ) .
concentrations were significantly higher in seeds from the am group than in seeds from the nonam group ( seeds , 856  677 versus 456  358 mg / kg ; p = .039 ) . in sprouts , ( 1365  795 mg /
kg versus 1097  310 , p = .493 ) and leaves ( 31  49 versus 37  34 mg / kg , p = .819 ) this was not the case and when values for all seeds , sprouts , and leaves were combined the difference between nonam and am was not significant either ( p = .127 ) ; ( table 1 ) .
the concentration ( mg / kg ) of hypoglycin a in samples of acer pseudoplatanus taken from pastures or paddocks grazed by healthy horses and horses with atypical myopathy ( am ) concentration significantly different between pastures used by healthy and am horses ( p = 0.039 ) .
there were 16 horses ( providing 29 samples ) with am of which 11 died and 5 survived .
there was no relationship between the concentration of hypoglycin a in the samples and outcome of the disease ( p = .250 ) .
, acer negundo ( box elder tree ) and acer pseudoplatanus ( sycamore maple ) have been associated with am.15 , 17 
  the species acer campestre ( hedge maple ) and acer platanoides ( norway maple ) did not contain detectable hypoglycin a concentrations , and thus it is probably safe , in terms of am , to grow these species in or around pastures and paddocks . in 1973 , semiquantitative measurements ( notdetected , trace , weak , moderate , and strong ) also indicated that these tree species were free of hypoglycin a.18 
  the most common maple tree in the netherlands is acer pseudoplatanus .
we found that the leaves , sprouts , and seeds contained measurable concentrations of hypoglycin a. although hypoglycin a concentrations were significantly lower in seeds collected from pastures grazed by healthy horses than in samples from pastures grazed by am horses , concentrations were too variable to allow predictions to be made as to whether or not the presence of an individual tree would be a threat to horses grazing in its vicinity .
our results are consistent with a study that found hypoglycin a concentrations in seeds to be highly variable among trees on the same or different farms.17 hypoglycin a concentrations in our study were similar to those previously reported.17 in the previous study , a mean concentration of 690 mg / kg was found with variation between 40 and 2810 mg / kg and in our study a mean of 856 mg / kg with variation between 25 and 3683 mg / kg was found
. the question arises as to why not all horses grazing pastures containing or surrounded by acer pseudoplatanus develop am .
the most likely explanation might relate to differences in the amount of seeds , sprouts , and leaves ingested , other components of the diet , and the sensitivity of the individual horse .
the availability of seeds , leaves , and sprouts largely is determined by the season and weather , with changes in wind and temperature loosening the attachment of leaves and seeds.6 , 10 , 15 , 19 indeed , inclement weather or high winds have been reported to precede clinical signs of am.6 , 12 , 15 strong winds can disperse leaves and seeds over a long distance , perhaps depositing them in pastures that do not contain acer trees .
all of these factors could explain why acer trees are not always found in the paddock or pastures grazed by horses with am.8 
  our results suggest that , per kilogram , sprouts are most dangerous to horses , followed by seeds , with leaves being potentially least harmful .
data show that there are more cases of am in autumn than in spring.8 because sprouts are more common in spring and seeds are more common in autumn , it would appear that horses eat more seeds than sprouts either because more seeds are available or because they prefer seeds to sprouts .
however , whether horses eat seeds or sprouts is to a large extent determined by the availability of other feedstuff.20 in general , spring pasture contains more and better grass than autumn pasture , and thus horses may have less reason to eat other feedstuff.20 
  the finding that the concentration of hypoglycin a is high in sprouts suggests that horse owners should be alert to the presence of acer sprouts in pastures .
owners should be advised to mow areas of pasture with maple sprouts and to remove the mown material .
a factor that needs further investigation is tree stress.20 , 21 tree stress or abiotic stress may increase the hypoglycin a concentration in seeds .
several owners who sent in samples did not return the questionnaire or did not complete it fully .
moreover , if there was doubt ( after consultation with the owner 's veterinarian ) about whether a horse suffered from am or not , data for samples collected from that horse 's paddock or pasture were not used .
results of the samples of 1 type ( seed , sprout , or leaf ) originating from 1 pasture were pooled and the mean concentration of these was treated as 1 sample .
because toxin concentrations are highly variable among trees on the same farm , it would have been better to have taken more samples of all materials .
the seeds were fresh frozen and not desiccated before freezing and changes in toxin concentrations as a result of artificial freezing could have occurred in the spring samples in the same fashion as occurs naturally in autumn . ideally all cases would have been confirmed by analyzing blood or urine for madd or toxin or toxin metabolites . because doing so was not always possible , the described inclusion criteria were used to make the diagnosis . in conclusion , it is important to know the tree species when giving advice regarding the toxicity of maple trees and the potential risk for horses , because some acer species contain hypoglycin a and others do not .
unfortunately , the amount of hypoglycin a in seeds is so variable that a reliable prediction of the occurrence of am can not be made for individual farms .
therefore , it is not worthwhile to measure hypoglycin concentrations in individual seed samples in order to predict am risk .
however , in all cases in which no hypoglycin a was found , no clinical signs of am were observed .
thus , the most important recommendation is that owners should prevent horses from eating seeds , sprouts , leaves , or any combination of these from acer pseudoplatanus ( sycamore maple ) , whereas the seeds of acer campestre ( hedge maple ) and acer platanoides ( norway maple ) appear to be safe for horses .
steps to prevent the ingestion of toxin include moving the horse to a safer pasture , decreasing the size of the pasture ( away from the trees ) , blowing away seeds and leaves , or mowing and removing sprouts .
 backgroundatypical myopathy ( am ) in horses is caused by the plant toxin hypoglycin a , which in europe typically is found in the sycamore maple tree ( acer pseudoplatanus ) . 
 owners are concerned about whether their horses are in danger if they graze near maple trees.hypothesis/objectivesto measure hypoglycin a in the most common maple tree species in the netherlands , and to determine whether concentration of toxin is a predictor of am in horses.methodsa total of 278 samples of maple tree leaves , sprouts , and seeds were classified by species . 
 mean concentrations of hypoglycin a were compared for the type of sample , the season and the occurrence of am in the pasture ( nonam versus am ) . 
 statistical analysis was performed using generalized a linear model ( spps22).resultsalmost all acer pseudoplatanus samples contained hypoglycin a , with concentrations differing significantly among sources ( p < .001 ) . 
 concentrations were significantly higher in seeds from the am group than in seeds from the nonam group ( 856  677 and 456  358 mg / kg , respectively ; p = .039 ) . in sprouts and leaves this was not the case . 
 acer platanoides and acer campestre samples did not contain detectable concentrations of hypoglycin a.conclusions and clinical importance 
 acer platanoides and campestre seem to be safe around paddocks and pastures , whereas almost all acer pseudoplatanus samples contained hypoglycin a. in all am cases , acer pseudoplatanus was found . despite significantly higher concentration of hypoglycin a in seeds of pastures where am has occurred , individual prediction of am can not be made by measuring these concentrations because of the high standard deviation . 
Materials and Methods
Collection of Leaves, Seeds, and Sprouts
Chemical Analysis
Selection and Grouping of Samples
Statistical Analysis
Results
Discussion
Supporting information