Re-evaluation of the regulation of omeprazole in racehorses: An evidence-based approach.

Medication control and doping control have been established in horse racing to ensure the integrity of the sport and the welfare of the horses. This ensures that horses do not compete under the influence of any drugs, including omeprazole, a therapeutic medication used to treat equine gastric ulcer syndrome. In this study, pharmacokinetic data were produced in equine plasma and urine following an oral administration of 4 mg/kg of generic buffered formulation of omeprazole to six Thoroughbred horses in five daily doses to determine an appropriate screening limit and detection time in equine plasma and to assess whether the current detection time of 72 hr in equine urine would be applicable when an alternative omeprazole product is administered. Cmax of 436-2,432 ng/ml and AUC0-tau of 1,476-4,371 ng hr ml-1 were obtained for plasma and indicated, in conjunction with other published oral omeprazole studies, that an appropriate plasma screening limit would be 500 pg/ml with a detection time of 48 hr. Urine analysis showed that omeprazole could be detected for up to 25 hr above the previously established urine screening limit of 500 pg/ml and thus indicated that the detection time advice could be potentially reduced from 72 to 48 hr to allow more comprehensive treatment of gastric lesions.

Detection and pharmacokinetics of salmeterol in thoroughbred horses following inhaled administration.

Salmeterol is a man-made beta-2-adrenergic receptor agonist used to relieve bronchospasm associated with inflammatory airway disease in horses. Whilst judicious use is appropriate in horses in training, they cannot race with clinically effective concentrations of medications under the British Horseracing Authority's Rules of Racing. Salmeterol must therefore be withdrawn prior to race day and pharmacokinetic (PK) studies used to establish formal detection time advice. Salmeterol xinafoate (Serevent Evohaler® ) was administered (0.1 mg twice daily for 4.5 days) via inhalation to six horses. Urine and blood samples were taken up to 103 h postadministration. Hydrolysed samples were extracted using solid phase extraction. A sensitive Ultra high performance tandem mass spectrometry (UPLC-MS/MS) method was developed, with a Lower limit of quantification (LLOQ) for salmeterol of 10 pg/mL in both matrices. The majority of salmeterol plasma concentrations, postlast administration, were below the method LLOQ and so unusable for PK analysis. Urine PK analysis suggested a half-life consistent with duration of pharmacological effect. Average estimated urine concentration at steady-state was obtained via PK modelling and used to estimate a urine concentration of 59 ± 34 pg/mL as a marker of effective lung concentration. From this, potential detection times were calculated using a range of safety factors.

Detection and pharmacokinetics of salbutamol in thoroughbred racehorses following inhaled administration.

Salbutamol sulphate (Ventolin Evohaler) was administrated via the inhalation route to six horses at a dose of 0.5 mg every 4 h during the day for 2 days (total dose 4 mg). Urine and blood samples were taken up to 92 h postadministration. Hydrolyzed plasma and urine were extracted using solid phase extraction (SPE). A sensitive tandem mass spectrometric method was developed in this study, achieving a lower limit of quantification (LLOQ) for salbutamol of 10 pg/mL in plasma and urine. The parent drug was identified using UPLC-MS/MS. Most of the determined salbutamol plasma concentrations, post last administration, lie below the LLOQ of the method and so cannot be used for plasma PK analysis. Urine PK analysis suggests a half-life consistent with the pharmacological effect duration. An estimate of the urine average concentration at steady-state was collected by averaging the concentration measurements in the dosing period from -12 to 0 h relative to the last administered dose. The value was averaged across the six horses and used to estimate an effective urine concentration as a marker of effective lung concentration. The value estimated was 9.6 ng/mL and from this a number of detection times were calculated using a range of safety factors.

Pharmacokinetics of inorganic cobalt and a vitamin B12 supplement in the Thoroughbred horse: Differentiating cobalt abuse from supplementation.

BACKGROUND: While cobalt is an essential micronutrient for vitamin B12 synthesis in the horse, at supraphysiological concentrations, it has been shown to enhance performance in human subjects and rats, and there is evidence that its administration in high doses to horses poses a welfare threat. Animal sport regulators currently control cobalt abuse via international race day thresholds, but this work was initiated to explore means of potentially adding to application of those thresholds since cobalt may be present in physiological concentrations. OBJECTIVES: To devise a scientific basis for differentiation between presence of cobalt from bona fide supplementation and cobalt doping through the use of ratios. STUDY DESIGN: Six Thoroughbred horses were given 10 mL vitamin B12 /cobalt supplement (Hemo-15® ; Vetoquinol, Buckingham, Buckinghamshire, UK., 1.5 mg B12 , 7 mg cobalt gluconate = 983 µg total Co) as an i.v. bolus then an i.v. infusion (15 min) of 100 mg cobalt chloride (45.39 mg Co) 6 weeks later. Pre-and post-administration plasma and urine samples were analysed for cobalt and vitamin B12 . METHODS: Urine and plasma samples were analysed for vitamin B12 using an immunoassay and cobalt concentrations were measured via ICP-MS. Baseline concentrations of cobalt in urine and plasma for each horse were subtracted from their cobalt concentrations post-administration for the PK analysis. Compartmental analysis was used for the determination of plasma PK parameters for cobalt using commercially available software. RESULTS: On administration of a vitamin B12 /cobalt supplement, the ratio of cobalt to vitamin B12 in plasma rapidly increased to approximately 3 and then rapidly declined below a ratio of 1 and then back to near baseline over the next week. On administration of 100 mg cobalt chloride, the ratio initially exceeded 10 in plasma and then declined with the lower 95% confidence interval remaining above a ratio of 1 for 7 days. For two horses with extended sampling, the plasma ratio remained above one for approximately 28 days after cobalt chloride administration. The effect of the administration of the vitamin B12 /cobalt supplement on the urine ratio was transient and reached a peak value of 10 which then rapidly declined. However, a urine ratio of 10 was exceeded, with the lower 95% confidence interval remaining above a ratio of 10 for 7 days after cobalt chloride administration. For the two horses with extended sampling, the urine ratio remained above 10 for about 18 days (442 h) after cobalt chloride administration even though the absolute cobalt urine concentration had dropped below the international threshold of 100 ng/mL after 96 h. MAIN LIMITATIONS: Only one vitamin B12 /cobalt product was evaluated, a limited number of horses were included, the horses were not in full race training and the results may be specific to this population of horses. CONCLUSIONS: The results provide the basis for a potential strategy for allowing supplementation with vitamin B12 products, while controlling the misuse of high doses of cobalt, through a combination of international thresholds and ratios of cobalt to vitamin B12 , in plasma and urine.

Plasma phospholipids and fatty acid composition differ between liver biopsy-proven nonalcoholic fatty liver disease and healthy subjects.

BACKGROUND: There is growing evidence that nonalcoholic fatty liver disease (NAFLD) is associated with perturbations in liver lipid metabolism. Liver phospholipid and fatty acid composition have been shown to be altered in NAFLD. However, detailed profiles of circulating lipids in the pathogenesis of NAFLD are lacking. OBJECTIVE: Therefore, the objective of the present study was to examine circulating lipids and potential mechanisms related to hepatic gene expression between liver biopsy-proven simple steatosis (SS), nonalcoholic steatohepatitis (NASH) and healthy subjects. SUBJECTS: Plasma phospholipid and fatty acid composition were determined in 31 healthy living liver donors as healthy controls (HC), 26 patients with simple hepatic steatosis (SS) and 20 with progressive NASH. Hepatic gene expression was analyzed by Illumina microarray in a subset of 22 HC, 16 SS and 14 NASH. RESULTS: Concentrations of phosphatidylethanolamine (PE) increased relative to disease progression, HC

Development and application of a scoring system for prognostic evaluation of equine liver biopsies.

REASONS FOR PERFORMING STUDY: The study was designed to investigate the prognostic value of liver biopsy during investigation of cases of suspected liver disease in mature horses. HYPOTHESIS: That liver biopsy is the most prognostically useful technique in common usage in the investigation of suspected liver disease. METHODS: This study examined the prognostic value of liver biopsy during the investigation of suspected liver disease in 73 mature horses. Histopathological variables comprising fibrosis, irreversible cytopathology, inflammatory infiltration, haemosiderin accumulation and biliary hyperplasia were found to be significant predictors of nonsurvival and were used to formulate a weighted biopsy score representing a prognostically useful broad comparative index of histopathological severity. Minimum and maximum possible scores were 0 and 14 points, respectively. RESULTS: Retrospective application of the biopsy scoring system to the study population indicated that horses with scores of 0 or 1 were equally likely to survive to 6 months with a combined mortality of 4%. Horses with biopsy scores between 2 and 6 had a combined mortality of 33% and were at a 12-fold increased risk of nonsurvival within 6 months (hazard ratio = 12.04, 95% CI 134-107.81, P = 0.026) compared to horses with a biopsy score of 0. Horses with biopsy scores between 7 and 14 had a combined mortality of 86% and were at a 46-fold increased risk of nonsurvival (hazard ratio = 46.01, 95% CI 5.92-357.5, P < 0.001) compared to horses with biopsy score 0. CONCLUSIONS: Application of the biopsy scoring system to histopathological findings in liver biopsy specimens was very useful in predicting survival of cases of suspected liver disease. POTENTIAL RELEVANCE: Application of the findings in this study may not be directly applicable to other case populations. However, liver biopsy appears to be the most valuable prognostic technique employed during the investigation of suspected cases of liver disease in mature horses.

Retrospective analysis of historical, clinical, ultrasonographic, serum biochemical and haematological data in prognostic evaluation of equine liver disease.

REASONS FOR PERFORMING STUDY: Results of noninvasive tests of liver disease do not always correlate with the degree of hepatic disease nor outcome of the case. OBJECTIVE: To investigate the prognostic value of data collected using noninvasive tests during the investigation of cases of suspected liver disease in mature horses. HYPOTHESIS: Much of the data gathered during the investigation of suspected hepatopathy cases offers little prognostic guidance and interpretation of such data can be misleading. METHODS: The results from a range of common and noninvasive diagnostic techniques applied in 116 mature horses with suspected liver disease, were assessed for their ability to predict survival within a 6 month period. RESULTS: A significantly poorer prognosis was found in association with clinical signs suggestive of liver disease, presence of hepatic encephalopathy, ultrasonographic abnormalities, increased serum globulins, increased total bile acids (TBA), increased alkaline phosphatase (AP), increased gamma-glutamyl transferase (gammaGT), erythrocytosis, leucocytosis, low serum albumin and low serum urea. Additional significant novel findings of interest included an association between increased plasma fibrinogen and low serum creatinine concentrations with nonsurvival in cases of liver disease, an association between raised serum concentrations of AP and gammaGT with biliary hyperplasia and also an association between hepatic fibrosis, haemosiderosis and biliary hyperplasia with ultrasonographically detected hepatic abnormalities. CONCLUSIONS: The most useful noninvasive prognostic test in cases of suspected liver disease in mature horses is the severity of clinical signs. Other data may be of some limited prognostic value. POTENTIAL RELEVANCE: Application of the findings in this study may not be directly applicable to other case populations. However, the findings should at least be considered when prognosis is based on similar criteria.

Cold-pressed flaxseed oil reverses age-associated depression in a primary cell-mediated adaptive immune response in the mouse.

The objective of this investigation was to determine the influence of flaxseed oil on responses representative of primary humoral and cell-mediated adaptive immune competence in immunosenescent mice. Male and female C57BL/6J mice, 85 weeks old, were randomized between two complete purified diets differing only in oil source (cold-pressed safflower or flaxseed). After 8 weeks, humoral competence was assessed in six mice per group as the serum haemagglutinin titre to sheep red blood cells (SRBC) and cell-mediated competence was assessed, in an additional six mice per group, as the delayed hypersensitivity response to SRBC. A zero-time control group (88 weeks old) and a young adult positive control group (12 weeks old) were each tested similarly (six per immune response), revealing age-related depression in both antibody and cell-mediated competence at 88 weeks of age. After the 8-week experimental period, the antibody response of the two test groups of geriatric mice remained below the young adult level (P=0.04) and the cell-mediated response of the safflower oil group also continued to exhibit age-related depression (20 % of young adult level, P=0.0002). By contrast, the anti-SRBC delayed hypersensitivity response of the flaxseed group no longer differed from the response of the young adults but exceeded that of the safflower and zero-time control senescent groups (P=0.0002). Depression in primary cell-mediated competence, the most outstanding aspect of immunosenescence, can be addressed by means of a dietary source of 18 : 3n-3 without longer-chain PUFA.

A comparison of the capacity of six cold-pressed plant oils to support development of acquired immune competence in the weanling mouse: superiority of low-linoleic-acid oils.

The objective of this investigation was to compare, at several levels of intake, the capacity of diverse cold-pressed plant oils to support development of acquired immune competence assessed in vivo in the weanling mouse. Safflower, maize, soyabean, rapeseed, flaxseed and olive oils were selected to represent widely differing 18 : 1n-9, 18 : 2n-6 and 18 : 3n-3 contents, and each oil was fed at three dietary levels (40, 80 and 160 g/kg) as the exclusive source of fat. C57BL/6J mice, ten males and ten females, had free access to each diet for 28 d beginning at 19 d of age. The primary serum haemagglutinin response to sheep red blood cells and the primary cutaneous delayed hypersensitivity response to dinitrochlorobenzene were used to assess humoral and cell-mediated competence respectively, on day 28. A zero-time control group, assessed immunologically at 19 d of age, was also included (n 32). Independently of dietary oil level, flaxseed, rapeseed, olive and soyabean oils supported development of a more vigorous antibody response than safflower (a useful point of reference, being rich in 18 : 2n-6 but low in 18 : 1n-9 and 18 : 3n-3), whereas only flaxseed oil supported development of cell-mediated responsiveness exceeding that of safflower-fed mice. Independently of oil type, development of both immunological indices correlated negatively with intake of 18 : 2n-6, and development of humoral competence varied inversely with dietary oil level. A low content of 18 : 2n-6, perhaps less than 20 g/100 g fatty acids, appears important to the capacity of a plant oil to support development of acquired immune competence in the young.

Interleukin-10 concentration determined by sandwich enzyme-linked immunosorbent assay is unrepresentative of bioactivity in murine blood.

Two experiments were performed, each using six male and six female C57BL/6J mice collectively ranging from 4 wk to 17 mo of age. Blood was obtained following CO2 anesthesia, and the IL-10 concentration of each serum sample was determined both by sandwich enzyme-linked immunosorbent assay (ELISA) and by bioassay. In the first experiment, mean serum IL-10 immunoactivity was 9.3 pg/ml while the mean bioactivity was 700 times greater, i.e., 6.5 ng/ml. However, the bioassay required sample dilution, which might have released bound cytokine that the ELISA could also detect. In the second experiment, therefore, the ELISA was applied to samples diluted to 20% as for the bioassay. Nevertheless, the immunoassay continued to detect only a small fraction of the serum IL-10 identified by the bioassay (mean values: 32.4 pg/ml vs. 2.6 ng/ml). Although currently the preferred method, the sandwich ELISA is inappropriate for quantification of blood IL-10 concentrations. Moreover, studies of the actions of IL-10 are needed at the concentrations revealed in the blood by bioassay and currently considered supraphysiological.

T cells with a quiescent phenotype (CD45RA+) are overabundant in the blood and involuted lymphoid tissues in wasting protein and energy deficiencies.

The objective of this investigation was to determine the influence of distinct forms of acute weight loss on the expression of the quiescence marker, CD45RA, by T cells in several lymphoid compartments including the blood. Male and female weanling mice, CBA/J and C57BL/6J strains, were allocated to the following groups: ad libitum intake of a complete purified diet; restricted intake of the complete diet; and ad libitum intake of an isocaloric low-protein diet. The restricted intake protocol produced weight loss through energy deficiency (marasmic-type malnutrition), whereas the low-protein diet caused wasting through inadequate protein nitrogen and induced a condition mimicking incipient kwashiorkor. In one experiment, weanling mice of both strains were maintained for 14 days according to each of these dietary protocols and, in a supplementary study, C57BL/6J weanlings consumed either the complete diet or the low-protein diet ad libitum for 21 days. Zero-time control groups (19-days old and 23-days old in C57BL/6J and CBA/J strains, respectively) were included in the first experiment to control for ontogeny-related change. Expression of CD45RA was assessed by two-colour flow cytometry in CD4+ and CD8+ T cells from the spleen, mesenteric lymph nodes and blood. Within 14 days, energy-restricted mice exhibited a high percentage of CD4+ T cells expressing CD45RA in all three lymphoid compartments in both mouse strains (an average of 50% CD45RA+ versus 9% in well-nourished controls), and a similar outcome was apparent in the CD8+ subset (93% CD45RA+ versus 63%). Mice fed the low-protein diet required up to 21 days to exhibit the same imbalance within the CD4+ T-cell subset (33% CD45RA+ versus 4% in well-nourished controls). A shift toward a quiescent phenotype occurs throughout the peripheral T-cell system in acute wasting disease. Consequently, the quiescence-activation phenotype of blood T cells reflects the same index in secondary lymphoid organs in such pathologies. Naive-type quiescence among T cells is implicated as a component of depressed adaptive immunocompetence in the advanced stages of diverse forms of acute weight loss.

The proportion of CD45RA(+)CD62L(+) (quiescent-phenotype) T cells within the CD8(+) subset increases in advanced weight loss in the protein- or energy-deficient weanling mouse.

Male and female C57BL/6J mice, initially 19 d old, had free access to a complete purified diet, were fed this diet in restricted daily quantities, or had free access to a low-protein diet. Three separate studies were conducted with feeding periods of 14, 9 or 6 d (n = 7-8 per dietary group and feeding period; 6 d: restricted intake and age-matched controls only). A zero-time control group (19 d old) was included in each study. Malnourished mice lost approximately 2% of initial body weight daily. Naïve-phenotype (quiescent) CD8(+) T cells of the blood, spleen and mesenteric lymph nodes were identified on the basis of surface coexpression of CD45RA and CD62L. Relative to age-matched controls, the percentage of naïve-phenotype CD8(+) T cells was high in energy-restricted groups after 9 d and 14 d of weight loss and in the protein-restricted groups after 14 d (P < or = 0.05). No ontogenetic change was apparent (age-matched vs. zero-time control). Other studies have demonstrated depression in cell-mediated immune competence in both malnutrition models within the first week of weight loss. An overabundance of quiescent-phenotype T cells within the involuted CD8(+) compartment may contribute to established immune depression but not to its initiation in weight loss pathologies.

The CD45RA+ (quiescent) cellular phenotype is overabundant relative to the CD45RA- phenotype within the involuted splenic T cell population of weanling mice subjected to wasting protein-energy malnutrition.

The objective of this investigation was to determine whether an imbalance between naive- and memory-phenotype cells occurs within CD4+ and/or CD8+ splenic T cell subsets in models of protein-energy malnutrition (PEM) which produce wasting disease (loss of approximately 1.6% of body weight per day for 14 d) and profound depression in thymus-dependent immunity. Male and female weanling mice of disparate inbred strains, CBA/J and C57BL/6J, were allocated to the following groups: zero-time control (23 d old and 19 d old, respectively), ad libitum intake of a complete purified diet (19% crude protein, 17 kJ/g gross energy), restricted intake of the complete diet, and (C57BL/6J, only) ad libitum intake of an isocaloric low protein diet (0.6% crude protein). Surface expression of isoforms of CD45, a component of the T cell receptor complex, as well as of the accessory molecule, CD2, were assessed by flow cytometry of splenic mononuclear cell suspensions. Both major T cell subsets in the malnourished groups contained a significantly higher proportion of cells expressing the surface marker, CD45RA, than was found in the spleen cells of the control groups. CD45RA+ (naive-phenotype) T cells represent the extreme of quiescence and stringent activation requirements among thymic lymphocytes. The results provide the first clear evidence of a T cell subset imbalance in PEM which is consistent with depression in acquired immunity and which occurs, apart from antigenic challenge, in a site wherein immune responses take place. The T cell receptor complex may emerge as a focal point of the depressive influence of PEM on the competence of thymic lymphocytes.

Contractile function in vitro of slow-twitch skeletal muscle from weanling mice subjected to wasting malnutrition.

Our hypothesis was that malnutrition sufficient to produce weight loss in weanling mice would decrease the ability of slow-twitch skeletal muscle to develop and maintain force. We isolated muscles from 3 groups (n = 5) of weanling C57BL/6J mice of both sexes (i) mice at 19 days of age serving as zero-time or baseline controls (CONT) (ii) mice fed for the next 14 days with a low-protein diet that produces features of incipient kwashiorkor (LPD) and (iii) mice fed for the next 14 days with a complete diet (NORM). Muscles were also obtained from 5 adult mice 7-9 months of age (MAT). We stimulated the soleus at 50 Hz for 500 ms at 0.6 tetanic contractions per min (tet x min(-1)), 6 tet x min(-1), and 30 tet x min(-1) in Krebs-Henseleit bicarbonate buffer at 27 degrees C gassed with 95% O2 and 5% CO2. The initial developed force (mN x mm(-2)) at 0.6 tet x min(-1) did not differ across groups (CONT 211.7 +/- 16.0, LPD 274.2 +/- 41.6, NORM 246.8 +/- 38.0, MAT 210.8 +/- 10.6). The fatigue rate (mN x mm(-2) x min(-1)) at 6 tet x min(-1) was significantly slower in muscles from CONT (0.6 +/- 0.3) and LPD (0.6 +/- 0.4) than in NORM (2.4 +/- 0.6) and MAT (2.3 +/- 0.2). At 30 tet x min(-1), the fatigue rate (mN x mm(-2) x min(-1)) did not differ across groups (CONT 2.4 +/- 0.5, LPD 2.7 +/- 0.5, NORM 2.5 +/- 0.4, MAT 2.0 +/- 0.2). After stimulation at 6 tet x min(-1) and 30 tet x min(-1), only muscles from CONT and LPD recovered to 100%. Because muscles from LPD mice developed equal force, fatigued less, and recovered from fatigue to a greater extent than muscles from NORM mice, we rejected the hypothesis. The function of the tissue remaining in the muscles from LPD mice approximated that of muscles from mice at 19 days of age rather than muscles from either mice of the same age fed a complete diet or adult mice.

Overabundance of CD45RA(+) (quiescent-phenotype) cells within the involuted CD4(+) T-cell population follows initiation of immune depression in energy-deficient weanling mice and reflects involution exclusive to the CD45RA(-) subset.

Previous studies have identified an overabundance of quiescent-phenotype (CD45RA(+)) CD4(+) T cells throughout the lymphoid system of weanling mice at an advanced stage of food intake restriction mimicking marasmus. The objective of this investigation was to determine the timing of this phenomenon relative to the development of depression in cell-mediated immune competence. Two experiments were conducted in which male and female weanling C57BL/6J mice, initially 19 d of age, either were permitted free access to a complete purified diet or were subjected to restricted intake of this diet, producing loss of 1.5-2% of initial body weight daily. In the first experiment, feeding periods of 3, 6, 9, 12 and 14 d were examined, and a zero-time control group (19 d old) was also included. Expression of CD45RA was assessed by flow cytometry in CD4(+) T cells from the blood, spleen and mesenteric lymph nodes. Despite reduction in CD4(+) T-cell numbers, evident in all three lymphoid compartments of the malnourished mice by d 6, energy-restricted mice maintained the numbers of CD4(+)CD45RA(+) T cells at the level found in the zero-time control group. Consequently, the malnourished group exhibited a high percentage of CD4(+) T cells expressing CD45RA by d 9 in the blood and mesenteric nodes and by d 12 in the spleen. In the second study, malnourished and age-matched control groups were sensitized to sheep red blood cells on d 3 and energy-restricted mice exhibited depression in the delayed hypersensitivity response to this antigen when assessed on d 9 after challenge 24 h previously. Energy deficiency pathology includes a shift toward CD4(+) T cell quiescence that may contribute to ongoing immunodepression without being involved in its initiation. Remarkably, this imbalance develops because involution of the CD4(+) subset in the energy-deficient mice is confined to the CD45RA(-) population.