Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells.

Reactive oxygen species formed within the mammalian cell can produce 8-oxo-7,8-dihydroguanine (8-oxoG) in mRNA, which can cause base mispairing during gene expression. Here we found that administration of 8-oxoGTP in MTH1-knockdown cells results in increased 8-oxoG content in mRNA. Under this condition, an amber mutation of the reporter luciferase is suppressed. Using second-generation sequencing techniques, we found that U-to-G changes at preassigned sites of the luciferase transcript increased when 8-oxoGTP was supplied. In addition, an increased level of 8-oxoG content in RNA induced the accumulation of aggregable amyloid ß peptides in cells expressing amyloid precursor protein. Our findings indicate that 8-oxoG accumulation in mRNA can alter protein synthesis in mammalian cells. Further work is required to assess the significance of these findings under normal physiological conditions.

Thermomass Theory in the Framework of GENERIC.

Thermomass theory was developed to deal with the non-Fourier heat conduction phenomena involving the influence of heat inertia. However, its structure, derived from an analogy to fluid mechanics, requires further mathematical verification. In this paper, General Equation for Non-Equilibrium Reversible-Irreversible Coupling (GENERIC) framework, which is a geometrical and mathematical structure in nonequilibrium thermodynamics, was employed to verify the thermomass theory. At first, the thermomass theory was introduced briefly; then, the GENERIC framework was applied in the thermomass gas system with state variables, thermomass gas density ρh and thermomass momentum mh, and the time evolution equations obtained from GENERIC framework were compared with those in thermomass theory. It was demonstrated that the equations generated by GENERIC theory were the same as the continuity and momentum equations in thermomass theory with proper potentials and eta-function. Thermomass theory gives a physical interpretation to the GENERIC theory in non-Fourier heat conduction phenomena. By combining these two theories, it was found that the Hamiltonian energy in reversible process and the dissipation potential in irreversible process could be unified into one formulation, i.e., the thermomass energy. Furthermore, via the framework of GENERIC, thermomass theory could be extended to involve more state variables, such as internal source term and distortion matrix term. Numerical simulations investigated the influences of the convective term and distortion matrix term in the equations. It was found that the convective term changed the shape of thermal energy distribution and enhanced the spreading behaviors of thermal energy. The distortion matrix implies the elasticity and viscosity of the thermomass gas.

Correction to: Ten weeks of branched-chain amino acid supplementation improves select performance and immunological variables in trained cyclists.

For the author R. Mac Thompson, the first name should be R. Mac and the last name should be Thompson. On SpringerLink the name is listed correctly, but on PubMed he is listed as Mac Thompson R.

Suppression of adipocyte differentiation and lipid accumulation by stearidonic acid (SDA) in 3T3-L1 cells.

BACKGROUND: Increased consumption of omega-3 (ω-3) fatty acids found in cold-water fish and fish oil has been reported to protect against obesity. A potential mechanism may be through reduction in adipocyte differentiation. Stearidonic acid (SDA), a plant-based ω-3 fatty acid, has been targeted as a potential surrogate for fish-based fatty acids; however, its role in adipocyte differentiation is unknown. This study was designed to evaluate the effects of SDA on adipocyte differentiation in 3T3-L1 cells. METHODS: 3T3-L1 preadipocytes were differentiated in the presence of SDA or vehicle-control. Cell viability assay was conducted to determine potential toxicity of SDA. Lipid accumulation was measured by Oil Red O staining and triglyceride (TG) quantification in differentiated 3T3-L1 adipocytes. Adipocyte differentiation was evaluated by adipogenic transcription factors and lipid accumulation gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Fatty acid analysis was conducted by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). RESULTS: 3T3-L1 cells treated with SDA were viable at concentrations used for all studies. SDA treatment reduced lipid accumulation in 3T3-L1 adipocytes. This anti-adipogenic effect by SDA was a result of down-regulation of mRNA levels of the adipogenic transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBPα, C/EBPß), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol-regulatory element binding protein-1c (SREBP-1c). SDA treatment resulted in decreased expression of the lipid accumulation genes adipocyte fatty-acid binding protein (AP2), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD-1), lipoprotein lipase (LPL), glucose transporter 4 (GLUT4) and phosphoenolpyruvate carboxykinase (PEPCK). The transcriptional activity of PPARγ was found to be decreased with SDA treatment. SDA treatment led to significant EPA enrichment in 3T3-L1 adipocytes compared to vehicle-control. CONCLUSION: These results demonstrated that SDA can suppress adipocyte differentiation and lipid accumulation in 3T3-L1 cells through down-regulation of adipogenic transcription factors and genes associated with lipid accumulation. This study suggests the use of SDA as a dietary treatment for obesity.

Ten weeks of branched-chain amino acid supplementation improves select performance and immunological variables in trained cyclists.

We examined if supplementing trained cyclists (32 ± 2 year, 77.8 ± 2.6 kg, and 7.4 ± 1.2 year training) with 12 g/day (6 g/day L-Leucine, 2 g/day L-Isoleucine and 4 g/day L-Valine) of either branched-chain amino acids (BCAAs, n = 9) or a maltodextrin placebo (PLA, n = 9) over a 10-week training season affected select body composition, performance, and/or immune variables. Before and after the 10-week study, the following was assessed: (1) 4-h fasting blood draws; (2) dual X-ray absorptiometry body composition; (3) Wingate peak power tests; and (4) 4 km time-trials. No group × time interactions existed for total lean mass (P = 0.27) or dual-leg lean mass (P = 0.96). A significant interaction existed for body mass-normalized relative peak power (19 % increase in the BCAA group pre- to post-study, P = 0.01), and relative mean power (4 % increase in the BCAA group pre- to post-study, P = 0.01). 4 km time-trial time to completion approached a significant interaction (P = 0.08), as the BCAA group improved in this measure by 11 % pre- to post-study, though this was not significant (P = 0.15). There was a tendency for the BCAA group to present a greater post-study serum BCAA: L-Tryptophan ratio compared to the PLA group (P = 0.08). A significant interaction for neutrophil number existed (P = 0.04), as there was a significant 18 % increase within the PLA group from the pre- to post-study time point (P = 0.01). Chronic BCAA supplementation improves sprint performance variables in endurance cyclists. Additionally, given that BCAA supplementation blunted the neutrophil response to intense cycling training, BCAAs may benefit immune function during a prolonged cycling season.

Extraction, chromatographic and mass spectrometric methods for lipid analysis.

Lipids make up a diverse subset of biomolecules that are responsible for mediating a variety of structural and functional properties as well as modulating cellular functions such as trafficking, regulation of membrane proteins and subcellular compartmentalization. In particular, phospholipids are the main constituents of biological membranes and play major roles in cellular processes like transmembrane signaling and structural dynamics. The chemical and structural variety of lipids makes analysis using a single experimental approach quite challenging. Research in the field relies on the use of multiple techniques to detect and quantify components of cellular lipidomes as well as determine structural features and cellular organization. Understanding these features can allow researchers to elucidate the biochemical mechanisms by which lipid-lipid and/or lipid-protein interactions take place within the conditions of study. Herein, we provide an overview of essential methods for the examination of lipids, including extraction methods, chromatographic techniques and approaches for mass spectrometric analysis.

PHARMACOKINETIC STUDY OF ORAL ε-AMINOCAPROIC ACID IN THE NORTHERN ELEPHANT SEAL (MIROUNGA ANGUSTIROSTRIS).

ε-Aminocaproic acid (EACA) is a lysine analogue antifibrinolytic drug used to treat bleeding disorders in humans and domestic animals. Its use in zoological medicine is rare, and dosage is anecdotal. One possible application of EACA is to treat bleeding associated with prepatent Otostrongylus arteritis in Northern elephant seals ( Mirounga angustirostris ) presenting to wildlife rehabilitation centers. This study used an in vitro model of hyperfibrinolysis and a thromboelastograph-based assay to estimate the therapeutic plasma concentration of EACA in elephant seals (85 µg/ml, 95% confidence interval = 73.8-96.8 µg/ml). A concurrent pharmacokinetic study of orally administered, single-dose EACA found that doses of 75 and 100 mg/kg achieved therapeutic plasma concentrations (>85 µg/ml), but the drug was rapidly eliminated and remained in the therapeutic range for only 0.4 and 1.5 hr, respectively. Models of repeated oral dosing at 100 mg/kg every 6 hr predict that therapeutic plasma concentration will be maintained for 31.7% (7.6 hr) of a 24-hr period. More frequent dosing would be required to maintain continuous therapeutic concentrations but would be impractical in a wildlife rehabilitation setting. Further pharmacodynamic studies to evaluate the duration of action of EACA in elephant seals and a prospective, placebo-controlled study are needed to determine if EACA is effective in decreasing bleeding associated with prepatent Otostrongylus arteritis and other bleeding disorders in this species.

The role of frataxin in doxorubicin-mediated cardiac hypertrophy.

Doxorubicin (DOX) is a highly effective anti-neoplastic agent; however, its cumulative dosing schedules are clinically limited by the development of cardiotoxicity. Previous studies have attributed the cause of DOX-mediated cardiotoxicity to mitochondrial iron accumulation and the ensuing reactive oxygen species (ROS) formation. The present study investigates the role of frataxin (FXN), a mitochondrial iron-sulfur biogenesis protein, and its role in development of DOX-mediated mitochondrial dysfunction. Athymic mice treated with DOX (5 mg/kg, 1 dose/wk with treatments, followed by 2-wk recovery) displayed left ventricular hypertrophy, as observed by impaired cardiac hemodynamic performance parameters. Furthermore, we also observed significant reduction in FXN expression in DOX-treated animals and H9C2 cardiomyoblast cell lines, resulting in increased mitochondrial iron accumulation and the ensuing ROS formation. This observation was paralleled in DOX-treated H9C2 cells by a significant reduction in the mitochondrial bioenergetics, as observed by the reduction of myocardial energy regulation. Surprisingly, similar results were observed in our FXN knockdown stable cell lines constructed by lentiviral technology using short hairpin RNA. To better understand the cardioprotective role of FXN against DOX, we constructed FXN overexpressing cardiomyoblasts, which displayed cardioprotection against mitochondrial iron accumulation, ROS formation, and reduction of mitochondrial bioenergetics. Lastly, our FXN overexpressing cardiomyoblasts were protected from DOX-mediated cardiac hypertrophy. Together, our findings reveal novel insights into the development of DOX-mediated cardiomyopathy.

Pharmacokinetics, antitumor and cardioprotective effects of liposome-encapsulated phenylaminoethyl selenide in human prostate cancer rodent models.

PURPOSE: Cardiotoxicity associated with the use of doxorubicin (DOX), and other chemotherapeutics, limits their clinical potential. This study determined the pharmacokinetics and antitumor and cardioprotective activity of free and liposome encapsulated phenyl-2-aminoethyl-selenide (PAESe). METHODS: The pharmacokinetics of free PAESe and PAESe encapsulated in liposomes (SSL-PAESe) were determined in rats using liquid chromatography tandem mass-spectrometry. The antitumor and cardioprotective effects were determined in a mouse xenograft model of human prostate (PC-3) cancer and cardiomyocytes (H9C2). RESULTS: The encapsulation of PAESe in liposomes increased the circulation half-life and area under the drug concentration time profile, and decreased total systemic clearance significantly compared to free PAESe. Free- and SSL-PAESe improved survival, decreased weight-loss and prevented cardiac hypertrophy significantly in tumor bearing and healthy mice following treatment with DOX at 5 and 12.5 mg/kg. In vitro studies revealed PAESe treatment altered formation of reactive oxygen species (ROS), cardiac hypertrophy and gene expression, i.e., atrial natriuretic peptide and myosin heavy chain complex beta, in H9C2 cells. CONCLUSIONS: Treatment with free and SSL-PAESe exhibited antitumor activity in a prostate xenograft model and mitigated DOX-mediated cardiotoxicity.

The Role of Mass Spectrometry in the Cannabis Industry.

The focus of this critical insight article is a brief overview of analytical challenges the cannabis industry faces and how analytical chemists have new opportunities to demonstrate the merits of employing mass spectrometry for the chemical analysis of cannabis and its products. The current range of cannabis products extends from recreational use to medicines, edibles, beverages, and beyond. The standards employed to assure product quality, integrity, and safety are lacking compared to those currently used by the pharmaceutical, food, and beverage industries. This manuscript overviews some of the important analytical issues that exist for the growth and harvest of the cannabis plant to the production of a wide variety of its products. Currently, the topics of interest for safety in cannabis testing where mass spectrometry can play an important role include what are currently referred to as potency, pesticides, terpenes, heavy metals, and mycotoxins from bacteria. Since each state in the USA as well as several countries has their own regulations, the analytical opportunities and challenges vary depending upon which jurisdiction a laboratory is supporting. This Critical Insight report will suggest where mass spectrometry can play an important role and provide valuable input on these topics. Graphical Abstract.

Long non-coding RNA GAS5 regulates human B lymphocytic leukaemia tumourigenesis and metastasis by sponging miR-222.

Accumulating evidence has shown that lncRNA GAS5 is a novel tumour-promoting RNA that contributes to tumour progression by sponging miRNAs. However, the detailed role of lncRNA GAS5 in B lymphocytic leukaemia is still unclear. A qRT-PCR assay was used to examine the levels of lncRNA GAS5 and miR-222 in leukomonocytes of patients with B lymphocytic leukaemia and in healthy donors. Raji cells were transfected with GAS5 overexpression or shRNA-GAS5 plasmids for 48⁢h, and cell proliferation was assessed by the CCK-8 assay, while apoptosis and cell cycle progression were assessed using flow cytometry. The Transwell assay was applied to detect the invasion of Raji cells with GAS5 overexpression or knockdown. The dual luciferase reporter assay and regression curve were conducted to evaluate the binding interaction between lncRNA GAS5 and miR-222. The results showed that the expression of lncRNA GAS5 was decreased in B lymphocytic leukaemia patients compared with the healthy group, and the levels of lncRNA GAS5 in B lymphocytic leukaemia cell lines were significantly higher than those in the normal B cell line, whereas the levels of miR-222 were increased in B lymphocytic leukaemia patients compared with the healthy group. Moreover, cell culture experiments indicated that lncRNA GAS5 overexpression decreased B lymphocytic leukaemia cell proliferation, promoted B lymphocytic leukaemia cell apoptosis, arrested B lymphocytic leukaemia cells in the G1 phase of the cell cycle, and inhibited B lymphocytic leukaemia cell invasion. Finally, the luciferase reporter assay showed a direct target interaction between lncRNA GAS5 and miR-222. The regression analysis showed a negative correlation between the levels of lncRNA GAS5 and miR-222. Thus, our data suggested that lncRNA GAS5 could effectively sponge miR-222 to modulate human B lymphocytic leukaemia cell tumourigenesis and metastasis. This work advances our understanding of the clinical significance of lncRNA GAS5 from the perspective of lncRNA-miRNA regulation.

Eupatilin inhibits microglia activation and attenuates brain injury in intracerebral hemorrhage.

Inflammation serves a critical role in the pathophysiology of intracerebral hemorrhage (ICH)-induced brain injury. Eupatilin, a pharmacologically active flavone derived from Artemisia sp., has been reported to have antioxidant, anti-inflammatory, anti-allergic and antitumor activities. However, the effect of eupatilin in ICH has not been well studied. The aim of the present study was to investigate the effect of eupatilin on ICH-induced microglial inflammation. The MTT and Transwell migration assay results revealed that eupatilin significantly inhibited microglial migration. It also decreased the production of inflammatory cytokines in erythrocyte lysis-induced BV2 cells, as well as the level of intracellular reactive oxygen species. The anti-inflammatory mechanism of eupatilin was also investigated using ELISAs and western blotting and the results demonstrated that eupatilin was able to inhibit erythrocyte lysis-induced NF-κB activation in BV2 cells. Taken together, the results of the present study suggest that eupatilin serves neurological protective effects via inhibiting microglial inflammation, providing an experimental basis for the use of eupatilin as a therapeutic target for ICH.

The effects of interleukin 2 and rAd-p53 as a treatment for glioblastoma.

Interleukin 2 (IL-2) is an anti-cancer cytokine that stimulates T cell propagation, triggering innate and adaptive immunity. IL-2 has been used for cancer therapy and has achieved curative effects. Recombinant adenovirus p53 injection (rAd­p53) is a gene therapeutic agent that may improve the prognosis of patients with glioblastoma (GBM). In the present study, the effect of combined IL­2 and rAd­p53 treatment was studied. The ability of IL­2 to stimulate immunoregulation and the ability of p53 to induce apoptosis for GBM was researched in the GBM tumor model. In addition, the activity of IL­2 was analyzed. The antitumor potential of IL­2 and rAd­p53 was studied using xenograph mice carrying GBM cells. Tumor­specific CD4+ and CD8+ T cells were also analyzed in the GBM­bearing models. The results demonstrated that IL­2 and rAd­p53 not only stimulated tumor­specific cytotoxic T­lymphocyte responses and increased regulatory CD4+ and cytotoxic CD8+ T cell proliferation, however additionally increased expression of apoptosis­associated genes. The treatment with IL­2 and rAd­p53 resulted in tumor regression and prolonged the survival of glioma­bearing mice. Taken together, a combination of IL­2 and rAd­p53 treatment combines the effects of immunotherapy and oncolytic therapy and may be a comprehensive therapeutic schedule for clinical application in future cancer therapies.

Author Correction: Soy protein supplementation is not androgenic or estrogenic in college-aged men when combined with resistance exercise training.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Soy protein supplementation is not androgenic or estrogenic in college-aged men when combined with resistance exercise training.

It is currently unclear as to whether sex hormones are significantly affected by soy or whey protein consumption. Additionally, estrogenic signaling may be potentiated via soy protein supplementation due to the presence of phytoestrogenic isoflavones. Limited also evidence suggests that whey protein supplementation may increase androgenic signaling. Therefore, the purpose of this study was to examine the effects of soy protein concentrate (SPC), whey protein concentrate (WPC), or placebo (PLA) supplementation on serum sex hormones, androgen signaling markers in muscle tissue, and estrogen signaling markers in subcutaneous (SQ) adipose tissue of previously untrained, college-aged men (n = 47, 20 ± 1 yrs) that resistance trained for 12 weeks. Fasting serum total testosterone increased pre- to post-training, but more so in subjects consuming WPC (p < 0.05), whereas serum 17ß-estradiol remained unaltered. SQ estrogen receptor alpha (ERα) protein expression and hormone-sensitive lipase mRNA increased with training regardless of supplementation. Muscle androgen receptor (AR) mRNA increased while ornithine decarboxylase mRNA (a gene target indicative of androgen signaling) decreased with training regardless of supplementation (p < 0.05). No significant interactions of supplement and time were observed for adipose tissue ERα/ß protein levels, muscle tissue AR protein levels, or mRNAs in either tissue indicative of altered estrogenic or androgenic activity. Interestingly, WPC had the largest effect on increasing type II muscle fiber cross sectional area values (Cohen's d = 1.30), whereas SPC had the largest effect on increasing this metric in type I fibers (Cohen's d = 0.84). These data suggest that, while isoflavones were detected in SPC, chronic WPC or SPC supplementation did not appreciably affect biomarkers related to muscle androgenic signaling or SQ estrogenic signaling. The noted fiber type-specific responses to WPC and SPC supplementation warrant future research.

Overexpression of GRP75 inhibits inflammation in a rat model of intracerebral hemorrhage.

Glucose­regulated protein 75 (GRP75) is a member of the heat shock protein 70 family and previous studies have demonstrated that GRP75 is involved in diseases of the central nervous system. However, the biological function of GRP75 in intracerebral hemorrhage (ICH) remains to be clarified. Thus, the aim of the present study was to evaluate the effects of GRP75 in a rat model of ICH. Western blotting was used to detect the protein expression of GRP75, active caspase­3, Bax, Bcl­2, p­Akt and Akt in brain tissues following ICH. The levels of tumor necrosis factor­α (TNF­α) and interleukin (IL)­1ß were evaluated using ELISA assay. Expression of GRP75 mRNA and protein was demonstrated to be reduced in the brain tissues of rats with ICH compared with sham­operated rats. In addition, overexpression of GRP75 in brain tissues with ICH significantly inhibited the production of the inflammatory cytokines TNF­α and IL-1ß and increased Bcl­2/decreased Bax levels compared with ICH alone. Furthermore, overexpression of GRP75 in brain tissues with ICH resulted in significantly increased phosphorylation of Akt compared with ICH alone. Therefore, the present study demonstrated, for the first time to the best of our knowledge, significantly reduced GRP75 expression in brain tissues following ICH, and that overexpression of GRP75 inhibits inflammation and potentially inhibits neuronal apoptosis in a rat model of ICH. GRP75 may, therefore, represent a promising target in the treatment of ICH.

Effect of aminocaproic acid on clot strength and clot lysis of canine blood determined by use of an in vitro model of hyperfibrinolysis.

OBJECTIVE To determine pharmacodynamic and pharmacokinetic profiles of aminocaproic acid (ACA) by use of a thromboelastography (TEG)-based in vitro model of hyperfibrinolysis and high-performance liquid chromatography-mass spectrometry. ANIMALS 5 healthy adult dogs. PROCEDURES A single dose of injectable ACA (20, 50, or 100 mg/kg) or an ACA tablet (approximately 100 mg/kg) was administered orally. Blood samples were collected at 0, 15, 30, 45, 60, 90, 120, and 240 minutes after ACA administration for pharmacokinetic analysis. Samples were obtained at 0, 60, and 240 minutes for pharmacodynamic analysis by use of a TEG model of hyperfibrinolysis. RESULTS No adverse effects were detected. In the hyperfibrinolysis model, after all doses, a significantly higher TEG maximum amplitude (clot strength), compared with baseline, was detected at 60 and 240 minutes. Additionally, the percentage of fibrinolysis was reduced from the baseline value at 60 and 240 minutes, with the greatest reduction at 60 minutes. At 240 minutes, there was significantly less fibrinolysis for the 100 mg/kg dose than the 20 mg/kg dose. Maximum plasma ACA concentration was dose dependent. There was no significant difference in pharmacokinetic parameters between 100 mg/kg formulations. CONCLUSIONS AND CLINICAL RELEVANCE In an in vitro model of hyperfibrinolysis, ACA inhibited fibrinolysis at all doses tested. At 240 minutes after administration, the 100 mg/kg dose inhibited fibrinolysis more effectively than did the 20 mg/kg dose. Thus, ACA may be useful for in vivo prevention of fibrinolysis in dogs. IMPACT FOR HUMAN MEDICINE These data may improve research models of hyperfibrinolytic diseases.

Pharmacokinetic and pharmacodynamic evaluation of oral rivaroxaban in healthy adult cats.

OBJECTIVES: To determine the pharmacodynamics and pharmacokinetics of rivaroxaban (RVX), in healthy cats and to evaluate the clinicopathologic effects of various plasma RVX concentrations within target therapeutic ranges established for people. DESIGN: Prospective randomized cross-over study performed between July 2013 and November 2014. SETTING: Veterinary university teaching hospital. ANIMALS: Six healthy adult domestic shorthair cats (3 males, 3 females). INTERVENTIONS: Cats were treated with oral RVX at single, fixed doses (1.25, 2.5, 5 mg PO), q 12 h for 3 days (1.25 mg); q 24 h for 7 days (2.5 mg); and q 24 h for 28 days (1.25 mg). Blood samples were collected for complete blood count, blood chemistry, and RVX anticoagulant activity based on prolongation of dilute prothrombin time, activated partial thromboplastin time (aPTT), activated Factor X (FXa) inhibition (anti-Xa activity [aXa]) and high-pressure liquid chromatography tandem mass spectrometry determination of drug concentration. MEASUREMENTS AND MAIN RESULTS: Treated cats had no signs of hemorrhage or clinicopathologic off-target adverse effects. There were dose-dependent prolongations of coagulation times and increase in aXa, with peak effect at 3 hours postadministration. There was a direct correlation between plasma RVX concentration and dilute prothrombin time and aXa. Coagulation parameters returned to baseline by 24 hours after the last dose. CONCLUSIONS: Oral RVX was well tolerated by healthy cats with predictable pharmacokinetics and anticoagulant effects. Clinical studies of RVX are warranted in cats with heart disease.

Pharmacokinetic evaluation of immediate- and extended-release formulations of levetiracetam in dogs.

OBJECTIVE: To compare the pharmacokinetics of various formulations of levetiracetam after oral administration of a single dose to healthy dogs. ANIMALS: 6 neurologically normal mixed-breed dogs. PROCEDURES: A crossover study design was used. Blood samples for serum harvest were collected from each dog before and at various points after oral administration of one 500-mg tablet of each of 2 generic extended-release (ER) formulations, 1 brand-name ER formulation, or 1 brand-name immediate-release (IR) formulation of levetiracetam. Serum samples were analyzed to determine pharmacokinetic properties of each formulation by means of ultra-high-performance liquid chromatography with tandem mass spectrometry. RESULTS: No dogs had clinically important adverse effects for any formulation of levetiracetam. All ER formulations had a significantly lower maximum serum drug concentration and longer time to achieve that concentration than did the IR formulation. Half-lives and elimination rate constants did not differ significantly among formulations. Values for area under the drug concentration-versus-time curve did not differ significantly between ER formulations and the IR formulation; however, 1 generic ER formulation had a significantly lower area under the curve than did other ER formulations. CONCLUSIONS AND CLINICAL RELEVANCE: All ER formulations of levetiracetam had similar pharmacokinetic properties in healthy dogs, with some exceptions. Studies will be needed to evaluate the clinical efficacy of the various formulations; however, findings suggested that twice-daily administration of ER formulations may be efficacious in the treatment of seizures in dogs.