Synthesis and characterization of d5 -barbarin for use in barbarin-related research.

Based on structural similarities and equine administration experiments, Barbarin, 5-phenyl-2-oxazolidinethione from Brassicaceae plants, is a possible source of equine urinary identifications of aminorex, (R,S)-5-phenyl-4,5-dihydro-1,3-oxazol-2-amine, an amphetamine-related US Drug Enforcement Administration (DEA) controlled substance considered illegal in sport horses. We now report the synthesis and certification of d5 -barbarin to facilitate research on the relationship between plant barbarin and such aminorex identifications. D5 -barbarin synthesis commenced with production of d5 -2-oxo-2-phenylacetaldehyde oxime (d5 -oxime) from d5 -acetophenone via butylnitrite in an ethoxide/ethanol solution. This d5 -oxime was then reduced with lithium aluminum hydride (LiAlH4 ) to produce the corresponding d5 -2-amino-1-phenylethan-1-ol (d5 -phenylethanolamine). Final ring closure of the d5 -phenylethanolamine was performed by the addition of carbon disulfide (CS2 ) with pyridine. The reaction product was purified by recrystallization and presented as a stable white crystalline powder. Proton NMR spectroscopy revealed a triplet at 5.88 ppm for one proton, a double doublet at 3.71 ppm for one proton, and double doublet at 4.11 ppm for one proton, confirming d5 -barbarin as the product. Further characterization by high resolution mass spectrometry supports the successful synthesis of d5 -barbarin. Purity of the recrystallized product was ascertained by High Performance Liquid Chromatography (HPLC) to be greater than 98%. Together, we have developed the synthesis and full characterization of d5 -barbarin for use as an internal standard in barbarin-related and equine forensic research.

Gabapentin, a human therapeutic medication and an environmental substance transferring at trace levels to horses: a case report.

Gabapentin, 1-(Aminomethyl)cyclohexaneacetic acid, MW 171.240, is a frequently prescribed high dose human medication that is also used recreationally. Gabapentin is orally absorbed; the dose can be 3,000 mg/day and it is excreted essentially unchanged in urine. Gabapentin is stable in the environment and routinely detected in urban wastewater. Gabapentin randomly transfers from humans to racing horses and is at times detected at pharmacologically ineffective / trace level concentrations in equine plasma and urine. In Ohio racing between January 2019 and July 2020,18 Gabapentin identifications, all less than 2 ng/ml in plasma, were reported. These identifications were ongoing because the horsemen involved were unable to pin down and therefore avoid the source of these identifications. Given that 44 ng/ml or less is an Irrelevant Plasma Concentration (IPC) of Gabapentin in horses, we proposed a 5 ng/ml plasma interim Screening Limit of Detection for Gabapentin identifications in Ohio racing, and an essentially similar 8 ng/ml plasma Screening Limit of Detection was suggested by a scientific advisor to the Ohio Horse Racing Commission. As such, an analytical Screening Limit of 8 ng /ml in plasma is an appropriate and pharmacologically conservative analytical "cut-off" or Screening Limit of Detection (SLOD) for Gabapentin in equine competitive events to avoid the calling of "positive" identifications on random unavoidable trace level identifications of this widely prescribed human therapeutic medication in equine forensic samples.

Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis.

Certain Standardbred racehorses develop recurrent exertional rhabdomyolysis (RER-STD) for unknown reasons. We compared gluteal muscle histopathology and gene/protein expression between Standardbreds with a history of, but not currently experiencing rhabdomyolysis (N = 9), and race-trained controls (N = 7). Eight RER-STD had a few mature fibers with small internalized myonuclei, one out of nine had histologic evidence of regeneration and zero out of nine degeneration. However, RER-STD versus controls had 791/13,531 differentially expressed genes (DEG). The top three gene ontology (GO) enriched pathways for upregulated DEG (N = 433) were inflammation/immune response (62 GO terms), cell proliferation (31 GO terms), and hypoxia/oxidative stress (31 GO terms). Calcium ion regulation (39 GO terms), purine nucleotide metabolism (32 GO terms), and electron transport (29 GO terms) were the top three enriched GO pathways for down-regulated DEG (N = 305). DEG regulated RYR1 and sarcoplasmic reticulum calcium stores. Differentially expressed proteins (DEP ↑N = 50, ↓N = 12) involved the sarcomere (24% of DEP), electron transport (23%), metabolism (20%), inflammation (6%), cell/oxidative stress (7%), and other (17%). DEP included ↑superoxide dismutase, ↑catalase, and DEP/DEG included several cysteine-based antioxidants. In conclusion, gluteal muscle of RER-susceptible Standardbreds is characterized by perturbation of pathways for calcium regulation, cellular/oxidative stress, inflammation, and cellular regeneration weeks after an episode of rhabdomyolysis that could represent therapeutic targets.

Sporadic worldwide "clusters" of feed driven Zilpaterol identifications in racing horses: a review and analysis.

Zilpaterol is a ß2-adrenergic agonist medication approved in certain countries as a cattle feed additive to improve carcass quality. Trace amounts of Zilpaterol can transfer to horse feed, yielding equine urinary "identifications" of Zilpaterol. These "identifications" occur because Zilpaterol is highly bioavailable in horses, resistant to biotransformation and excreted as unchanged Zilpaterol in urine, where it has a 5 day or so terminal half-life.In horses, urinary steady-state concentrations are reached 25 days (5 half-lives) after exposure to contaminated feed. Zilpaterol readily presents in horse urine, yielding clusters of feed related Zilpaterol identifications in racehorses. The first cluster, April 2013, involved 48 racehorses in California; the second cluster, July 2013, involved 15 to 80 racehorses in Hong Kong. The third cluster, March 2019, involved 24 racehorses in Mauritius; this cluster traced to South African feedstuffs, triggering an alert concerning possible Zilpaterol feed contamination in South African racing. The fourth cluster, September/October 2020 involved 18 or so identifications in French racing, reported by the French Laboratories des Courses Hippiques, (LCH), and in July 2021, a fifth cluster of 10 Zilpaterol identifications in South Africa.The regulatory approach to these identifications has been to alert horsemen and feed companies and penalties against horsemen are generally not implemented. Additionally, given their minimal exposure to Zilpaterol, there is little likelihood of Zilpaterol effects on racing performance or adverse health effects for exposed horses.The driving factor in these events is that Zilpaterol is dissolved in molasses for incorporation into cattle feed. Inadvertent incorporation of Zilpaterol containing molasses into horse feed was the source of the California and Hong Kong Zilpaterol identifications. A second factor in the 2019 Mauritius and 2020 French identifications was the sensitivity of testing for Zilpaterol in Mauritius and France, with the French laboratory reportedly testing at a "more sensitive level for Zilpaterol". As of January 1st, 2021, the new FEI Atypical Finding (ATF) policy specifies Zilpaterol as a substance to be treated as an Atypical Finding (ATF), allowing consideration of inadvertent feed contamination in the regulatory evaluation of Zilpaterol identifications.

Pathways of calcium regulation, electron transport, and mitochondrial protein translation are molecular signatures of susceptibility to recurrent exertional rhabdomyolysis in Thoroughbred racehorses.

Recurrent exertional rhabdomyolysis (RER) is a chronic muscle disorder of unknown etiology in racehorses. A potential role of intramuscular calcium (Ca2+) dysregulation in RER has led to the use of dantrolene to prevent episodes of rhabdomyolysis. We examined differentially expressed proteins (DEP) and gene transcripts (DEG) in gluteal muscle of Thoroughbred race-trained mares after exercise among three groups of 5 horses each; 1) horses susceptible to, but not currently experiencing rhabdomyolysis, 2) healthy horses with no history of RER (control), 3) RER-susceptible horses treated with dantrolene pre-exercise (RER-D). Tandem mass tag LC/MS/MS quantitative proteomics and RNA-seq analysis (FDR <0.05) was followed by gene ontology (GO) and semantic similarity of enrichment terms. Of the 375 proteins expressed, 125 were DEP in RER-susceptible versus control, with 52 ↑DEP mainly involving Ca2+ regulation (N = 11) (e.g. RYR1, calmodulin, calsequestrin, calpain), protein degradation (N = 6), antioxidants (N = 4), plasma membranes (N = 3), glyco(geno)lysis (N = 3) and 21 DEP being blood-borne. ↓DEP (N = 73) were largely mitochondrial (N = 45) impacting the electron transport system (28), enzymes (6), heat shock proteins (4), and contractile proteins (12) including Ca2+ binding proteins. There were 812 DEG in RER-susceptible versus control involving the electron transfer system, the mitochondrial transcription/translational response and notably the pro-apoptotic Ca2+-activated mitochondrial membrane transition pore (SLC25A27, BAX, ATP5 subunits). Upregulated mitochondrial DEG frequently had downregulation of their encoded DEP with semantic similarities highlighting signaling mechanisms regulating mitochondrial protein translation. RER-susceptible horses treated with dantrolene, which slows sarcoplasmic reticulum Ca2+ release, showed no DEG compared to control horses. We conclude that RER-susceptibility is associated with alterations in proteins, genes and pathways impacting myoplasmic Ca2+ regulation, the mitochondrion and protein degradation with opposing effects on mitochondrial transcriptional/translational responses and mitochondrial protein content. RER could potentially arise from excessive sarcoplasmic reticulum Ca2+ release and subsequent mitochondrial buffering of excessive myoplasmic Ca2+.

Synthesis and characterization of barbarin, a possible source of unexplained aminorex identifications in forensic science.

Aminorex is a US DEA Schedule 1 controlled substance occasionally detected in racing horses. A number of aminorex identifications in sport horses were thought to have been caused by exposure to plant sources of aminorex. Glucobarbarin, found in plants of the Brassicaceae family, has been suggested as a potential proximate chemical source by being metabolized in the plant or the horse to aminorex. In Brassicaceae, glucobarbarin is hydrolyzed by myrosinase to yield barbarin, which serves as an insect repellant and/or attractant and is structurally related to aminorex. The synthesis, purification, and characterization of barbarin is now reported for use as a reference standard in aminorex related research concerning equine urinary identifications of aminorex and also for possible use in equine administration experiments. Synthesis of barbarin was performed via ring closure between phenylethanolamine and carbon disulfide in tetrahydrofuran with the catalyst pyridine under reflux. The reaction yielded a white crystalline substance that was purified and chemically characterized as barbarin for use as a Certified Reference Standard or for studies related to equine aminorex identification.

RAPID COMMUNICATION: Differential skeletal muscle mitochondrial characteristics of weanling racing-bred horses1.

Responses of equine skeletal muscle characteristics to growth and training have been shown to differ between breeds. These differential responses may arise in part because muscle fiber type and mitochondrial density differ between breeds, even in untrained racing-bred horses. However, it is not known when these breed-specific differences manifest. To test the hypothesis that weanling Standardbreds (SB) and Thoroughbreds (TB) would have higher mitochondrial measures than Quarter Horses (QH), gluteus medius samples were collected from SB (mean ± SD; 6.2 ± 1.0 mo; n = 10), TB (6.1 ± 0.5 mo; n = 12), and QH (7.4 ± 0.6 mo; n = 10). Citrate synthase (CS) and cytochrome c oxidase (CCO) activities were assessed as markers of mitochondrial density and function, respectively. Mitochondrial oxidative (P) and electron transport system (E) capacities were assessed by high-resolution respirometry (HRR). Data for CCO and HRR are expressed as integrated (per mg protein and per mg tissue wet weight, respectively) and intrinsic (per unit CS). Data were analyzed using PROC MIXED in SAS v 9.4 with breed as a fixed effect. Mitochondrial density (CS) was higher for SB and TB than QH (P ≤ 0.0007). Mitochondrial function (integrated and intrinsic CCO) was higher in TB and QH than SB (P ≤ 0.01). Integrated CCO was also higher in TB than QH (P < 0.0001). However, SB had higher integrated maximum P (PCI+II) and E (ECI+II) than QH (P ≤ 0.02) and greater integrated and intrinsic complex II-supported E (ECII) than both QH and TB (P ≤ 0.02), whereas TB exhibited higher integrated P with complex I substrates (PCI) than SB and QH (P ≤ 0.003) and higher integrated PCI+II and ECI+II than QH (P ≤ 0.02). In agreement, TB and QH had higher contribution of complex I (CI) to max E than SB (P ≤ 0.001), whereas SB had higher contribution of CII than QH and TB (P ≤ 0.002). Despite having higher mitochondrial density than QH and TB, SB showed lower CCO activity and differences in contribution of complexes to oxidative and electron transport system capacities. Breed differences in mitochondrial parameters are present early in life and should be considered when developing feeding, training, medication, and management practices.

Rapid Communication: Differential skeletal muscle mitochondrial characteristics of weanling racing-bred horses.

Responses of equine skeletal muscle characteristics to growth and training have been shown to differ between breeds. These differential responses may arise in part because muscle fiber type and mitochondrial density differ between breeds, even in untrained racing-bred horses. However, it is not known when these breed-specific differences manifest. To test the hypothesis that weanling Standardbreds (SB) and Thoroughbreds (TB) would have higher mitochondrial measures than Quarter Horses (QH), gluteus medius samples were collected from SB (mean ± SD; 6.2 ± 1.0 mo; n = 10), TB (6.1 ± 0.5 mo; n = 12), and QH (7.4 ± 0.6 mo; n = 10). Citrate synthase (CS) and cytochrome c oxidase (CCO) activities were assessed as markers of mitochondrial density and function, respectively. Mitochondrial oxidative (P) and electron transport system (E) capacities were assessed by high-resolution respirometry (HRR). Data for CCO and HRR are expressed as integrated (per mg protein and per mg tissue wet weight, respectively) and intrinsic (per unit CS). Data were analyzed using PROC MIXED in SAS v 9.4 with breed as a fixed effect. Mitochondrial density (CS) was higher for SB and TB than QH (P ≤ 0.0007). Mitochondrial function (integrated and intrinsic CCO) was higher in TB and QH than SB (P ≤ 0.01). Integrated CCO was also higher in TB than QH (P < 0.0001). However, SB had higher integrated maximum P (PCI+II) and E (ECI+II) than QH (P ≤ 0.02) and greater integrated and intrinsic complex II-supported E (ECII) than both QH and TB (P ≤ 0.02), while TB exhibited higher integrated P with complex I substrates (PCI) than SB and QH (P ≤ 0.003) and higher integrated PCI+II and ECI+II than QH (P ≤ 0.02). In agreement, TB and QH had higher contribution of complex I (CI) to max E than SB (P ≤ 0.001), while SB had higher contribution of CII than QH and TB (P ≤ 0.002). Despite having higher mitochondrial density than QH and TB, SB showed lower CCO activity and differences in contribution of complexes to oxidative and electron transport system capacities. Breed differences in mitochondrial parameters are present early in life and should be considered when developing feeding, training, medication, and management practices.

Coding sequences of sarcoplasmic reticulum calcium ATPase regulatory peptides and expression of calcium regulatory genes in recurrent exertional rhabdomyolysis.

BACKGROUND: Sarcolipin (SLN), myoregulin (MRLN), and dwarf open reading frame (DWORF) are transmembrane regulators of the sarcoplasmic reticulum calcium transporting ATPase (SERCA) that we hypothesized played a role in recurrent exertional rhabdomyolysis (RER). OBJECTIVES: Compare coding sequences of SLN, MRLN, DWORF across species and between RER and control horses. Compare expression of muscle Ca2+ regulatory genes between RER and control horses. ANIMALS: Twenty Thoroughbreds (TB), 5 Standardbreds (STD), 6 Quarter Horses (QH) with RER and 39 breed-matched controls. METHODS: Sanger sequencing of SERCA regulatory genes with comparison of amino acid (AA) sequences among control, RER horses, human, mouse, and rabbit reference genomes. In RER and control gluteal muscle, quantitative real-time polymerase chain reaction of SERCA regulatory peptides, the calcium release channel (RYR1), and its accessory proteins calsequestrin (CASQ1), and calstabin (FKBP1A). RESULTS: The SLN gene was the highest expressed horse SERCA regulatory gene with a uniquely truncated AA sequence (29 versus 31) versus other species. Coding sequences of SLN, MRLN, and DWORF were identical in RER and control horses. A sex-by-phenotype effect occurred with lower CASQ1 expression in RER males versus control males (P < .001) and RER females (P = .05) and higher FKBP1A (P = .01) expression in RER males versus control males. CONCLUSIONS AND CLINICAL IMPORTANCE: The SLN gene encodes a uniquely truncated peptide in the horse versus other species. Variants in the coding sequence of SLN, MLRN, or DWORF were not associated with RER. Males with RER have differential gene expression that could reflect adaptations to stabilize RYR1.

Aminorex identified in horse urine following consumption of Barbarea vulgaris; a preliminary report.

BACKGROUND: Aminorex, (RS)-5- Phenyl-4,5-dihydro-1,3-oxazol-2-amine, is an amphetamine-like anorectic and in the United States a Drug Enforcement Administration [DEA] Schedule 1 controlled substance. Aminorex in horse urine is usually present as a metabolite of Levamisole, an equine anthelmintic and immune stimulant. Recently, Aminorex identifications have been reported in horse urine with no history or evidence of Levamisole administration. Analysis of the urine samples suggested a botanical source, directing attention to the Brassicaceae plant family, with their contained GlucoBarbarin and Barbarin as possible sources of Aminorex. Since horsepersons face up to a 1 year suspension and a $10,000.00 fine for an Aminorex identification, the existence of natural sources of Aminorex precursors in equine feedstuffs is of importance to both individual horsepersons and the industry worldwide. RESULTS: Testing the hypothesis that Brassicaceae plants could give rise to Aminorex identifications in equine urine we botanically identified and harvested flowering Kentucky Barbarea vulgaris, ("Yellow Rocket") in May 2018 in Kentucky and administered the plant orally to two horses. Analysis of post-administration urine samples yielded Aminorex, showing that consumption of Kentucky Barbarea vulgaris can give rise to Aminorex identifications in equine urine. CONCLUSIONS: Aminorex has been identified in post administration urine samples from horses fed freshly harvested flowering Kentucky Barbarea vulgaris, colloquially "Yellow Rocket". These identifications are consistent with occasional low concentration identifications of Aminorex in equine samples submitted for drug testing. The source of these Aminorex identifications is believed to be the chemically related Barbarin, found as its precursor GlucoBarbarin in Kentucky Barbarea vulgaris and related Brassicaceae plants worldwide.

A cluster of trace-concentration methamphetamine identifications in racehorses associated with a methamphetamine-contaminated horse trailer: A report and analysis.

Three low concentration methamphetamine "positive" tests were linked to use of a methamphetamine-contaminated trailer to transport the affected horses. This incident establishes methamphetamine as a human-use substance that can inadvertently enter the environment of racing horses, resulting in urinary methamphetamine "positives;" an interim regulatory cut-off of 15 ng/mL for methamphetamine in post-race urine is proposed.

Identifications de concentrations de méthamphétamine à l'état de traces chez des chevaux de course associées à une remorque contaminée : rapport et analyse. Trois tests «positifs¼ de faibles concentrations de méthamphétamine ont été associés à l'utilisation d'une remorque contaminée par les méthamphétamines qui était utilisée pour transporter les chevaux affectés. Cet incident établit la méthamphétamine comme une substance à utilisation humaine qui peut pénétrer par inadvertance dans le milieu des chevaux de course, entainant ainsi des tests d'urine «positifs¼; un niveau intérimaire réglementaire de 15 ng/mL pour les méthamphétamines est proposé pour les tests d'urine après la course.(Traduit par Isabelle Vallières).

Field efficacy of ivermectin plus praziquantel oral paste against naturally acquired gastrointestinal nematodes and cestodes of horses in North America and Europe.

The efficacy of an oral formulation of ivermectin plus praziquantel in the reduction of nematode and cestode egg counts in horses was assessed in 273 horses under field conditions at 15 sites in North America (n = 6) and Europe (n = 9). Horses were confirmed by fecal examination to have natural infections of strongyles (100%) and tapeworms (76%). Replicates of four horses were formed at each site, and in each replicate three animals received ivermectin (0.2 mg/kg body weight) plus praziquantel (1 mg/kg body weight) oral paste and one animal remained untreated or received vehicle paste. Fecal samples were collected for fecal nematode and cestode egg counting before and 7, 8, 9, 14, 15, and 16 days after treatment. Horses treated with ivermectin plus praziquantel oral paste had significantly (P <.01) lower posttreatment strongylid and cestode egg counts (reductions of 98% or more) than controls. Combined site analyses revealed that 95% or 96% of the horses positive for cestode eggs before treatment that were treated with ivermectin plus praziquantel were negative for cestode eggs at each posttreatment fecal examination. No adverse reactions attributable to ivermectin plus praziquantel oral paste treatments were observed. The results of the studies demonstrated that ivermectin plus praziquantel paste was highly effective in reducing egg shedding by gastrointestinal nematodes and cestodes, and no adverse reactions were observed in horses treated under field conditions.