An exercise-inducible metabolite that suppresses feeding and obesity.

Exercise confers protection against obesity, type 2 diabetes and other cardiometabolic diseases1-5. However, the molecular and cellular mechanisms that mediate the metabolic benefits of physical activity remain unclear6. Here we show that exercise stimulates the production of N-lactoyl-phenylalanine (Lac-Phe), a blood-borne signalling metabolite that suppresses feeding and obesity. The biosynthesis of Lac-Phe from lactate and phenylalanine occurs in CNDP2+ cells, including macrophages, monocytes and other immune and epithelial cells localized to diverse organs. In diet-induced obese mice, pharmacological-mediated increases in Lac-Phe reduces food intake without affecting movement or energy expenditure. Chronic administration of Lac-Phe decreases adiposity and body weight and improves glucose homeostasis. Conversely, genetic ablation of Lac-Phe biosynthesis in mice increases food intake and obesity following exercise training. Last, large activity-inducible increases in circulating Lac-Phe are also observed in humans and racehorses, establishing this metabolite as a molecular effector associated with physical activity across multiple activity modalities and mammalian species. These data define a conserved exercise-inducible metabolite that controls food intake and influences systemic energy balance.

Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography-High-Resolution Mass Spectrometry.

There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.

Disposition and metabolism of ethylene glycol 2-ethylhexyl ether in Sprague Dawley rats, B6C3F1/N mice, and in vitro in rat hepatocytes.

Ethylene glycol 2-ethylhexyl ether (EGEHE) is a solvent used in a variety of applications.We report disposition and metabolism of EGEHE following a single gavage or dermal administration of 50, 150 or 500 mg/kg [14C]EGEHE in rats and mice and in vitro in rat hepatocytes.EGEHE was cleared rapidly in rat hepatocytes (half-life ∼4 min) with no sex difference.EGEHE was well- and moderately absorbed following oral administration (rats: 80-96%, mice: 91-95%) and dermal application (rats: 25-37%, mice: 22-24%), respectively, and rapidly excreted in urine.[14C]EGEHE-derived radioactivity was distributed to tissues (oral: 2.3-7.2%, dermal: 0.7-2.2%) with liver and kidney containing the highest levels in both species.EGEHE was extensively metabolised with little to no parent detected in urine. The alkoxyacetic acid metabolite, which has previously been shown to mediate toxicities of other shorter-chain ethylene glycol ethers, was not detected.There were no apparent dose, species or sex differences in disposition and metabolism of EGEHE, except that the exhaled volatile compounds were greater in mice (19-20%) compared with rats (<2%).These studies address a critical gap in the scientific literature and provide data that will inform future studies designed to evaluate toxicity of EGEHE.

Detection of Methylphenidate in Equine Hair Using Liquid Chromatography-High-Resolution Mass Spectrometry.

Methylphenidate is a powerful central nervous system stimulant with a high potential for abuse in horse racing. The detection of methylphenidate use is of interest to horse racing authorities for both prior to and during competition. The use of hair as an alternative sampling matrix for equine anti-doping has increased as the number of detectable compounds has expanded. Our laboratory developed a liquid chromatography-high-resolution mass spectrometry method to detect the presence of methylphenidate in submitted samples. Briefly, hair was decontaminated, cut, and pulverized prior to liquid-liquid extraction in basic conditions before introduction to the LC-MS system. Instrumental analysis was conducted using a Thermo Q Exactive mass spectrometer using parallel reaction monitoring using a stepped collision energy to obtain sufficient product ions for qualitative identification. The method was validated and limits of quantitation, linearity, matrix effects, recovery, accuracy, and precision were determined. The method has been applied to confirm the presence of methylphenidate in official samples submitted by racing authorities.

Disposition and metabolism of 2,2'-dimorpholinodiethyl ether in sprague dawley rats and B6C3F1/N mice after oral, intravenous administration, and dermal application.

The specialty amine catalyst 2,2'-dimorpholinodiethyl ether (DMDEE) is a high-production volume chemical used in the production of flexible foam, high-resilient molded foam, and in coatings and adhesives. The disposition and metabolism of [14C]DMDEE (20 or 200 mg/kg) were determined in male ane female rats and mice after oral and intravenous administration and dermal application. In male and female rats, following a single oral administration, [14C]DMDEE was well-absorbed and excreted rapidly and extensively via urine (75-93%) and some in feces (∼4-8%). The total radioactivity in tissues at 24 h and 72 h (males only) following oral administration was 8-10% and ∼4%, respectively, suggesting considerable tissue distribution. A moderate amount of the total tissue radioactivity in kidney and liver were unextractable suggesting covalent binding of [14C]DMDEE-derived products in tissue macromolecules. Absorption following a single dermal application in rats was significant (∼64%) with a similar disposition pattern to oral. The oral and dermal disposition of [14C]DMDEE in male and female mice was similar to rats. Urinary products of DMDEE identified were oxidative metabolism of the morpholine ring. Coadministration of DMDEE with nitrite in rats didn't produce the rodent carcinogen, N-nitrosomorpholine.

N6-Formyllysine as a Biomarker of Formaldehyde Exposure: Formation and Loss of N6-Formyllysine in Nasal Epithelium in Long-Term, Low-Dose Inhalation Studies in Rats.

Exposure to both endogenous and exogenous formaldehyde has been established to be carcinogenic, likely by virtue of forming nucleic acid and proteins adducts such as N6-formyllysine. To better assess N6-formyllysine as a biomarker of formaldehyde exposure, we studied accumulation of N6-formyllysine adducts in tissues of rats exposed by inhalation to 2 ppm [13C2H2]-formaldehyde for 7, 14, 21, and 28 days (6 h/day) and investigated adduct loss over a 7-day postexposure period using liquid chromatography-coupled tandem mass spectrometry. Our results showed formation of exogenous adducts in nasal epithelium and to some extent in trachea but not in distant tissues of lung, bone marrow, or white blood cells, with a 2-fold increase over endogenous N6-formyllysine over a 3-week exposure period. Postexposure analyses indicated a biexponential decay of N6-formyllysine in proteins extracted from different cellular compartments, with half-lives of ∼25 and ∼182 h for the fast and slow phases, respectively, in cytoplasmic proteins. These results parallel the behavior of DNA adducts and DNA-protein cross-links, with protein adducts cleared faster than DNA-protein cross-links, and point to the potential utility of N6-formyllysine protein adducts as biomarkers of formaldehyde.

Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5α-dihydroprogesterone.

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.

Pleiotropic effect of the proton pump inhibitor esomeprazole leading to suppression of lung inflammation and fibrosis.

BACKGROUND: The beneficial outcome associated with the use of proton pump inhibitors (PPIs) in idiopathic pulmonary fibrosis (IPF) has been reported in retrospective studies. To date, no prospective study has been conducted to confirm these outcomes. In addition, the potential mechanism by which PPIs improve measures of lung function and/or transplant-free survival in IPF has not been elucidated. METHODS: Here, we used biochemical, cell biological and preclinical studies to evaluate regulation of markers associated with inflammation and fibrosis. In our in vitro studies, we exposed primary lung fibroblasts, epithelial and endothelial cells to ionizing radiation or bleomycin; stimuli typically used to induce inflammation and fibrosis. In addition, we cultured lung fibroblasts from IPF patients and studied the effect of esomeprazole on collagen release. Our preclinical study tested efficacy of esomeprazole in a rat model of bleomycin-induced lung injury. Furthermore, we performed retrospective analysis of interstitial lung disease (ILD) databases to examine the effect of PPIs on transplant-free survival. RESULTS: The cell culture studies revealed that esomeprazole controls inflammation by suppressing the expression of pro-inflammatory molecules including vascular cell adhesion molecule-1, inducible nitric oxide synthase, tumor necrosis factor-alpha (TNF-α) and interleukins (IL-1ß and IL-6). The antioxidant effect is associated with strong induction of the stress-inducible cytoprotective protein heme oxygenase-1 (HO1) and the antifibrotic effect is associated with potent inhibition of fibroblast proliferation as well as downregulation of profibrotic proteins including receptors for transforming growth factor ß (TGFß), fibronectin and matrix metalloproteinases (MMPs). Furthermore, esomeprazole showed robust effect in mitigating the inflammatory and fibrotic responses in a murine model of acute lung injury. Finally, retrospective analysis of two ILD databases was performed to assess the effect of PPIs on transplant-free survival in IPF patients. Intriguingly, this data demonstrated that IPF patients on PPIs had prolonged survival over controls (median survival of 3.4 vs 2 years). CONCLUSIONS: Overall, these data indicate the possibility that PPIs may have protective function in IPF by directly modulating the disease process and suggest that they may have other clinical utility in the treatment of extra-intestinal diseases characterized by inflammatory and/or fibrotic phases.

Detection of doping peptides and basic drugs in equine urine using liquid chromatography-mass spectrometry.

The abuse of prohibited agents including peptides and basic small-molecule drugs is an area of great concern in horseracing due to their high potential to act as doping agents. These compound classes include agents such as growth hormone-releasing peptides, peptide analgesics, beta-2-adrenergic receptor agonists, and quaternary ammonium drugs that can be challenging to detect and regulate because of their chemical properties and potential rapid elimination following administration. The use of highly sensitive and selective analytical techniques such as liquid chromatography-mass spectrometry (LC-MS) is necessary to provide coverage of these substances and their potential metabolites. This study describes the development and validation of methodology capable of the detection of over 50 different peptide-based doping agents, related secretagogues, quaternary ammonium drugs, and other challenging small molecules in equine urine following solid-phase extraction using a mixed mode weak cation exchange sorbent. Following sample extraction, the compounds were analyzed using LC-MS with chromatographic separation via a reverse phase gradient and detection via selective reaction monitoring following introduction to a triple-stage quadrupole mass spectrometer using positive mode electrospray ionization. Validation parameters including limits of detection and quantitation, accuracy, precision, linear range, recovery, stability, and matrix effects were determined. Briefly, the limits of detection for most compounds were in the sub-ng/mL ranges with adequate precision and accuracy sufficient for an initial testing procedure. Stability studies indicated that most compounds were sufficiently stable to allow for effective screening using conditions commonly utilized in drug testing laboratories.

Dosimetry of N6-formyllysine adducts following [¹³C²H2]-formaldehyde exposures in rats.

With formaldehyde as the major source of endogenous N6-formyllysine protein adducts, we quantified endogenous and exogenous N6-formyllysine in the nasal epithelium of rats exposed by inhalation to 0.7, 2, 5.8, and 9.1 ppm [¹³C²H2]-formaldehyde using liquid chromatography-coupled tandem mass spectrometry. Exogenous N6-formyllysine was detected in the nasal epithelium, with concentration-dependent formation in total as well as fractionated (cytoplasmic, membrane, nuclear) proteins, but was not detected in the lung, liver, or bone marrow. Endogenous adducts dominated at all exposure conditions, with a 6 h 9.1 ppm formaldehyde exposure resulting in one-third of the total load of N6-formyllysine being derived from exogenous sources. The results parallel previous studies of formaldehyde-induced DNA adducts.

Formaldehyde carcinogenicity research: 30 years and counting for mode of action, epidemiology, and cancer risk assessment.

Formaldehyde is a widely used high production chemical that is also released as a byproduct of combustion, off-gassing of various building products, and as a fixative for pathologists and embalmers. What is not often realized is that formaldehyde is also produced as a normal physiologic chemical in all living cells. In 1980, chronic inhalation of high concentrations of formaldehyde was shown to be carcinogenic, inducing a high incidence of nasal squamous cell carcinomas in rats. Some epidemiologic studies have also found increased numbers of nasopharyngeal carcinoma and leukemia in humans exposed to formaldehyde that resulted in formaldehyde being considered a Known Human Carcinogen. This article reviews the data for rodent and human carcinogenicity, early Mode of Action studies, more recent molecular studies of both endogenous and exogenous DNA adducts, and epigenetic studies. It goes on to demonstrate the power of these research studies to provide critical data to improve our ability to develop science-based cancer risk assessments, instead of default approaches. The complexity of constant physiologic exposure to a known carcinogen requires that new ways of thinking be incorporated into determinations of cancer risk assessment for formaldehyde, other endogenous carcinogens, and the role of background endogenous DNA damage and mutagenesis.

Use of LC-MS/MS and stable isotopes to differentiate hydroxymethyl and methyl DNA adducts from formaldehyde and nitrosodimethylamine.

Formaldehyde is a known human and animal carcinogen that forms DNA adducts, and causes mutations. While there is widespread exposure to formaldehyde in the environment, formaldehyde is also an essential biochemical in all living cells. The presence of both endogenous and exogenous sources of formaldehyde makes it difficult to develop exposure-specific DNA biomarkers. Furthermore, chemicals such as nitrosodimethylamine form one mole of formaldehyde for every mole of methylating agent, raising questions about potential cocarcinogenesis. Formaldehyde-induced hydroxymethyl DNA adducts are not stable and need to be reduced to stable methyl adducts for detection, which adds another layer of complexity to identifying the origins of these adducts. In this study, highly sensitive mass spectrometry methods and isotope labeled compounds were used to differentiate between endogenous and exogenous hydroxymethyl and methyl DNA adducts. We demonstrate that N(2)-hydroxymethyl-dG is the primary DNA adduct formed in cells following formaldehyde exposure. In addition, we show that alkylating agents induce methyl adducts at N(2)-dG and N(6)-dA positions, which are identical to the reduced forms of hydroxymethyl adducts arising from formaldehyde. The use of highly sensitive LC-MS/MS and isotope labeled compounds for exposure solves these challenges and provides mechanistic insights on the formation and role of these DNA adducts.

Detection and confirmation of zilpaterol in equine hair using liquid chromatography-mass spectrometry.

Zilpaterol is a ß2 -adrenergic agonist and a repartitioning agent that has a high potential for abuse in equine performance athletes. Analysis of zilpaterol in hair is an alternative sampling matrix that extends detection time periods beyond those found in urine or blood samples. Our laboratory has been screening for zilpaterol in hair for many years and recently detected and confirmed its presence in official samples. Accordingly, a liquid chromatography-mass spectrometry method was developed and validated to detect and confirm zilpaterol in equine hair. Briefly, equine hair was decontaminated, cut, and pulverized prior to disruption and liquid-liquid extraction in basic conditions. Following extraction, the sample was introduced to an Agilent 1260 HPLC and zilpaterol was separated using a reverse phase gradient with a total run time of 12.5 min. Following chromatographic separation, zilpaterol and its corresponding stable isotope labeled internal standard were introduced via positive mode electrospray ionization to a Thermo Q-Exactive Plus mass spectrometer and spectra collected using parallel reaction monitoring. The methodology was validated using in-house criteria including characterization of accuracy, precision, recovery, linear range, matrix effects, limit of detection, and limit of quantitation, and the method was found to be fit-for-purpose to confirm the presence of zilpaterol in equine hair. This methodology has been used to detect and confirm the presence of zilpaterol from out-of-competition hair samples submitted by regional racing authorities.

Investigation of the Role of Healthy and Sick Equids in the COVID-19 Pandemic through Serological and Molecular Testing.

More and more studies are reporting on the natural transmission of SARS-CoV-2 between humans with COVID-19 and their companion animals (dogs and cats). While horses are apparently susceptible to SARS-CoV-2 infection based on the homology between the human and the equine ACE-2 receptor, no clinical or subclinical infection has yet been reported in the equine species. To investigate the possible clinical role of SARS-CoV-2 in equids, nasal secretions from 667 horses with acute onset of fever and respiratory signs were tested for the presence of SARS-CoV-2 by qPCR. The samples were collected from January to December of 2020 and submitted to a commercial molecular diagnostic laboratory for the detection of common respiratory pathogens (equine influenza virus, equine herpesvirus-1/-4, equine rhinitis A and B virus, Streptococcus equi subspecies equi). An additional 633 serum samples were tested for antibodies to SARS-CoV-2 using an ELISA targeting the receptor-binding domain of the spike protein. The serum samples were collected from a cohort of 587 healthy racing Thoroughbreds in California after track personnel tested qPCR-positive for SARS-CoV-2. While 241/667 (36%) equids with fever and respiratory signs tested qPCR-positive for at least one of the common respiratory pathogens, not a single horse tested qPCR-positive for SARS-CoV-2. Amongst the racing Thoroughbreds, 35/587 (5.9%) horses had detectable antibodies to SARS-CoV-2. Similar to dogs and cats, horses do not seem to develop clinical SARS-CoV-2 infection. However, horses can act as incidental hosts and experience silent infection following spillover from humans with COVID-19. SARS-CoV-2-infected humans should avoid close contact with equids during the time of their illness.

Determination of N2-hydroxymethyl-dG adducts in the nasal epithelium and bone marrow of nonhuman primates following 13CD2-formaldehyde inhalation exposure.

The presence of endogenous and exogenous N(2)-hydroxymethyl-dG adducts in DNA from the nasal mucosa and bone marrow of cynomolgus macaques exposed to 1.9 and 6.1 ppm of [(13)CD(2)]-formaldehyde for 6 h a day for 2 consecutive days was investigated using a highly sensitive nano-UPLC-MS/MS method with a limit of detection of 20 amol. Both exogenous and endogenous adducts were readily detected and quantified in the nasal tissues of both exposure groups, with an exposure dependent increase in exogenous adducts observed. In contrast, only endogenous adducts were detectable in the bone marrow, even though ∼10 times more DNA was analyzed.

Evaluation of dried blood spots as an alternative sample matrix for equine antidoping analysis.

Controlling the abuse of prohibited substances such as anabolic steroids, selective androgen receptor modulators, ß-adrenoceptor agonists, and blood doping agents is of great interest to racing authorities. The use of dried blood spots (DBS) as an alternative sampling approach may be a feasible approach for controlling the use of these agents. To assess the feasibility of using DBS in equine blood, an 11-min liquid chromatography-mass spectrometry method was developed on a triple quadrupole mass spectrometer following extraction from Whatman 903 DBS cards. A total of 50 compounds across multiple compound classes were detectable with reproducible results. The stability was assessed with good results after almost 3 months of storage at ambient temperatures. These results suggest that the use of DBS may be a feasible alternative sampling approach in equine drug testing.

Simultaneous quantification of vitamin E and vitamin E metabolites in equine plasma and serum using LC-MS/MS.

Vitamin E deficiencies can impact normal growth and development in humans and animals, and assessment of circulating levels of vitamin E and its metabolites may be an important endpoint for evaluation. Development of a sensitive method to detect and quantify low concentrations of vitamin E and metabolites in biological specimens allows for a proper diagnosis for patients and animals that are deficient. We developed a method to simultaneously extract, detect, and quantify the vitamin E compounds alpha-tocopherol (α-TP), gamma-tocopherol (γ-TP), alpha-tocotrienol (α-TT), and gamma-tocotrienol (γ-TT), and the corresponding metabolites formed after ß-oxidation of α-TP and γ-TP, alpha-carboxymethylbutyl hydroxychroman (α-CMBHC) and alpha- or gamma-carboxyethyl hydroxychroman (α- or γ-CEHC), respectively, from equine plasma and serum. Quantification was achieved through liquid chromatography-tandem mass spectrometry. We applied a 96-well high-throughput format using a Phenomenex Phree plate to analyze plasma and serum. Compounds were separated by using a Waters ACQUITY UPLC BEH C18 column with a reverse-phase gradient. The limits of detection for the metabolites and vitamin E compounds were 8-330 pg/mL. To validate the method, intra-day and inter-day accuracy and precision were evaluated along with limits of detection and quantification. The method was then applied to determine concentrations of these analytes in plasma and serum of horses. Alpha-TP levels were 3-6 µg/mL of matrix; the metabolites were found at much lower levels, 0.2-1.0 ng/mL of matrix.

Ibuprofen inhibits key genes involved in androgen production in theca-interstitial cells.

OBJECTIVE: To study the effects of ibuprofen on androgen production, gene expression, and cell viability in rat theca-interstitial cells exposed to the proinflammatory stimuli interleukin-1ß (IL-1ß) and lipopolysaccharide (LPS). DESIGN: Animal study. SETTING: University-based research laboratory. PATIENTS/ANIMALS: Theca-interstitial cells were isolated from 30 day old female Sprague Dawley rats. INTERVENTIONS: Theca cells were cultured with pro-inflammatory media containing IL-1ß and LPS and compared with cells cultured in control media. MAIN OUTCOME MEASURES: Androstenedione quantification was performed on conditioned cell culture medium using liquid chromatography-mass spectrometry. Theca cell viability was assessed using PrestoBlue cell viability assay. The gene expression of Cyp17a1, Cyp11a1, and Hsd3b was analyzed using quantitative polymerase chain reaction. RESULTS: Both proinflammatory stimuli IL-1ß and LPS increased androstenedione concentration in cell culture medium, and these effects were mitigated with ibuprofen. Both inflammatory agents in addition increased the expression of key genes involved in androgen synthesis: Cyp17a1, Cyp11a1, and Hsd3b; the addition of ibuprofen to the culture medium inhibited these effects. Theca cell viability increased with IL-1ß and LPS. Ibuprofen inhibited the IL-1ß-mediated increase in cell viability but did not reverse the effects of LPS. CONCLUSIONS: In conclusion, our findings support the hypothesis that many of the alterations induced by inflammatory stimuli in theca-interstitial cells are abrogated by the addition of ibuprofen.

Determination of vitamin E and its metabolites in equine urine using liquid chromatography-mass spectrometry.

Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) is a hereditary, deteriorating central nervous disease in horses. Currently, the only way to confirm eNAD/EDM is through a postmortem histological evaluation of the central nervous system. Vitamin E, specifically the isoform alpha-tocopherol (α-TP), is known to protect eNAD/EDM susceptible horses from developing the clinical phenotype. While vitamin E is an essential nutrient in the diet of horses, there are no diagnostic tests able to quantitate vitamin E and its metabolites in urine. An ultra-performance liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry (UPLC-APCI-MS/MS) method was developed and validated following acidic hydrolysis and solid phase extraction to quantitate vitamin E and its metabolites in equine urine. A blank control horse urine matrix was used and spiked with different concentrations of analytes to form a standard curve using either alpha-tocopherol-d6 or chlorpropamide as the internal standard. Inter-day and intra-day statistics were performed to evaluate the method for accuracy (90% to 116%) and precision (0.75% to 14%). Matrix effects, percent recovery, and stability were also assessed. The method successfully analyzed alpha-carboxyethyl hydroxychroman (α-CEHC), alpha-carboxymethylbutyl hydroxychromans (α-CMBHC), gamma-carboxyethyl hydroxychroman γ-CEHC, and α-TP concentrations in urine to determine a baseline levels of analytes in healthy horses, and can be used to determine concentrations of vitamin E metabolites in equine urine allowing for its evaluation as a diagnostic approach in the treatment of eNAD/EDM.

Disposition and metabolism of 2',2'"-Dithiobisbenzanilide in rodents following intravenous and oral administration and dermal application.

2',2'''-Dithiobisbenzanilide (DTBBA) is a high-production-volume chemical used as a peptizing agent for rubber. The disposition and metabolism of [14C]DTBBA were determined in male and female rats and mice following oral (4, 40, or 400 mg/kg) and intravenous (IV) (4 mg/kg) administration and dermal application (0.4 or 4 mg/kg). [14C]DTBBA was well absorbed following oral administration (> 60%) and dermal application (∼40-50%) in rats and mice. Following oral administration, the majority of radioactivity was excreted in urine (29 - 70%) and feces (16 - 45%). Unlike rats, mice excreted ∼1-5% of the dose as exhaled CO2. The residual radioactivity in tissues was <1% in both species and sexes. The pattern of disposition following IV administration in male rats was similar to that following oral. When [14C]DTBBA was administered via IV to rats, a significant portion of the dose was recovered in bile (∼13%) suggesting that at least a portion of the dose recovered in feces following oral administration was likely the absorbed dose. The profiles of urine from rats and mice were similar and consisted of four major metabolites and three minor metabolites. The predominant metabolite in urine was the S-glucuronide of the thiol/sulfide cleavage product N-(2-mercaptophenyl)benzamide, which accounted for more than 50% of radioactivity in the radiochromatogram.