Aptamer based peptide enrichment for quantitative analysis of gonadotropin-releasing hormone by LC-MS/MS.

Over recent years threats to racing have expanded to include naturally occurring biological molecules, such as peptides and proteins, and their synthetic analogues. Traditionally, antibodies have been used to enable detection of these compounds as they allow purification and concentration of the analyte of interest. The rapid expansion of peptide-based therapeutics necessitates a similarly rapid development of suitable antibodies or other means of enrichment. Potential alternative enrichment strategies include the use of aptamers, which offer the significant advantage of chemical synthesis once the nucleic acid sequence is known. A method was developed for the enrichment, detection and quantitation of gonadotropin-releasing hormone (GnRH) in equine urine using aptamer-based enrichment and LC-MS/MS. The method achieved comparable limits of detection (1 pg/mL) and quantification (2.5 pg/mL) to previously published antibody-based enrichment methods. The intra- and inter-assay precision achieved was less than 10% at both 5 and 20 pg/mL, and displayed a working dynamic range of 2.5-100 pg/mL. Significant matrix enhancement (170 ± 8%) and low analytical recovery (29 ± 15%) was observed, although the use of an isotopically heavy labelled GnRH peptide, GnRH (Pro(13)C5,(15)N), as the internal standard provides compensation for these parameters. Within the current limits of detection GnRH was detectable up to 1h post administration in urine and identification of a urinary catabolite extended this detection window to 4h. Based on the results of this preliminary investigation we propose the use of aptamers as a viable alternative to antibodies in the enrichment of peptide targets from equine urine.

Biosecurity and vaccination strategies to minimise the effect of an equine influenza outbreak on racing and breeding.

Three biosecurity and relief-and-recovery initiatives adopted by the NSW horse racing industries reduced the economic and social disruption caused by the disease and subsequent movement controls during the 2007 Australian equine influenza (EI) incursion. The first was the creation of biosecure horse training and racing precincts around the Sydney area to permit racing to continue with healthy horses. Infection was excluded for 3-5 weeks and race meetings were conducted safely during this period. The second was a vaccination program of racehorses at these and other precincts to maintain an ongoing healthy pool of racehorses. Vaccination commenced too late to enable viable racing to continue in Sydney in the short term, but assisted in managing an early return to racing throughout the state before EI-free status had been regained. The third was the establishment of approved quarantine stations to facilitate the movement of racing and breeding horses out of high-risk regions. The difficulties in establishing and managing these initiatives in the face of the EI incursion are discussed.

The metabolism of norethandrolone in the horse: characterization of 16-, 20- and 21-oxygenated metabolites by gas chromatography/mass spectrometry.

After oral administration to a thoroughbred gelding, the anabolic steroid norethandrolone was converted into a complex mixture of oxygenated metabolites. These metabolites were extracted from the urine, deconjugated by methanolysis and converted to their O-methyloxime trimethylsilyl derivatives. Gas chromatographic/mass spectrometric analysis indicated the major metabolites to be 19-norpregnane-3,16,17-triols, 19-norpregnane-3,17,20-triols and 3,17-dihydroxy-19-norpregnan-21-oic acids. Some minor metabolites were also detected.

Metabolism of methandrostenolone in the horse: a gas chromatographic-mass spectrometric investigation of phase I and phase II metabolism.

The phase I and phase II metabolism of the anabolic steroid methandrostenolone was investigated following oral administration to a standardbred gelding. In the phase I study, metabolites were isolated from the urine by solid-phase extraction, deconjugated by acid catalysed methanolysis and converted to their O-methyloxime trimethylsilyl derivatives. GC-MS analysis indicated the major metabolic processes to be sequential reduction of the A-ring and hydroxylation at C6 and C16. In the phase II study, unconjugated, beta-glucuronidated and sulfated metabolites were fractionated and deconjugated using a combination of liquid-liquid extraction, enzyme hydrolysis, solid-phase extraction and acid catalysed methanolysis. Derivatization followed by GC-MS analysis revealed extensive conjugation to both glucuronic and sulfuric acids, with only a small proportion of metabolites occurring in unconjugated form.

Risk factors associated with musculoskeletal injuries in Australian thoroughbred racehorses.

Risk factors for musculoskeletal injury in racing Thoroughbreds were investigated in a case-control study conducted at racetracks administered by the Australian Jockey Club. Univariable analysis of 137 cases from the official Veterinary Surgeon's reports and an equal number of randomly selected controls from the Australian Race Results identified field size, barrier position and class of race as being significantly associated with breakdown (P < 0.05). Multiple logistic regression was then used to investigate the effect of each putative risk factor whilst controlling for all others. Horses at greater risk were older, started from a wider barrier position, ran at the same distance as their previous race and raced in the highest class of race. There was no significant difference between tracks or significant association with track condition. The incidence of fatalities in the study population was less than that reported in the UK and USA.

Effects of sodium bicarbonate and sodium chloride on the elimination of etorphine in equine urine.

The combination of large doses of sodium bicarbonate and the potent narcotic, etorphine, has reportedly been given to racehorses in attempts to improve their performance and also to "mask" the presence of etorphine in urine samples. The increased urinary output and pH associated with sodium bicarbonate (approximately 500 g) administration may reduce the urinary concentration of etorphine, making it more difficult to detect. Our experiment was designed to examine the effects of this combination. Six Thoroughbred horses were used in a latin-square design with three horse pairs and three treatments consisting of the following: etorphine (20 micrograms), etorphine (20 micrograms) plus sodium bicarbonate (1.0 g/kg), and etorphine (20 micrograms) plus sodium chloride (0.7 g/kg). Sodium chloride was used to distinguish between the urinary alkalinizing effects of sodium bicarbonate and the diuretic effects associated with the large electrolyte load. Venous blood and urine samples were collected prior to and for 24 h post-treatment. Sodium bicarbonate produced a significant metabolic alkalosis and an increase in urine pH. Both sodium bicarbonate and sodium chloride produced a profound diuresis. After sodium bicarbonate and sodium chloride treatments, the urinary concentration of etorphine, measured by radioimmunoassay (RIA), was reduced and in some cases could not be detected. Extraction of the urine samples, prior to RIA analysis, increased the sensitivity of the assay and in most cases gave a positive result. We conclude that the coadministration of etorphine and sodium bicarbonate or sodium chloride can make the detection of etorphine more difficult because of the dilutional effects associated with the administration of a large electrolyte load.

Effects of sodium bicarbonate on cardiorespiratory measurements and exercise capacity in thoroughbred horses.

We examined the effects of sodium bicarbonate in 6 Thoroughbred horses during submaximal and maximal treadmill exercise. Cardiorespiratory function was assessed together with the effect on exercise capacity by determining the run time to fatigue at maximal intensities. To discriminate between sodium bicarbonate's alkalinising effects and the fluid shifts that could result from the high osmotic load, we administered an equimolar solution of sodium chloride as a control. The horses were given sodium bicarbonate (1 g/kg bwt) or an equivalent number of moles of sodium chloride by nasogastric tube. Arterial blood samples were collected before exercise and 5 h after treatment, resulting in mean standard bicarbonate values of 39.6 mmol/l in horses treated with sodium bicarbonate compared with 24.2 mmol/l in horses that received saline. The horses were exercised on a treadmill at 40, 60 and 80% of their VO2max for 4, 2 and 2 mins respectively. The horses were walked for 3 mins and accelerated rapidly to a speed approximately equivalent to 110% VO2max and run until fatigued. The horses ran for 170 +/- 20 secs (mean +/- sem) after administration of sodium bicarbonate compared with 128 +/- 13 secs after receiving sodium chloride (P < 0.02). At rest and throughout submaximal and maximal exercise, the bicarbonate-treated horses had significantly lower arterial oxygen tensions and higher arterial carbon dioxide tensions. There were no differences in cardiac output, heart rate, oxygen uptake or carbon dioxide production between the saline and bicarbonate treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Gas chromatographic mass-specific investigation of dextromoramide (Palfium) metabolism in the horse.

Dextromoramide (Palfium) was given by intravenous injection to a Thoroughbred horse at a dosage of 20 mg and urine was collected 2, 4, 6 and 8 h after drug administration. Enzymatic hydrolysis of the urine followed by solvent extraction gave a residue which was back-extracted into 0.1 M sulphuric acid. After basification to pH 9 and solvent extraction, the resulting residue was submitted to gas chromatographic-mass spectrometric analysis. Both electron-impact and ammonia chemical-ionization mass spectra were recorded and, based on the observed fragmentation patterns, the principal metabolites in horse urine were shown to be 2,2-diphenyl-3-methyl-4-morpholinobutyramide (compound 2) and the product of hydroxylation of one phenyl ring in dextromoramide (compound 3), respectively. The electron-impact mass spectra of compounds 2 and 3, and of their derivatisation products from oncolumn methylation in the gas chromatograph, are reported.

An evaluation of an oral glucose-glycine-electrolyte solution for the treatment of experimentally induced dehydration in the horse.

Five standardbred geldings were given 1 mg/kg bodyweight of frusemide by intramuscular injection to induce mild dehydration. After food and water deprivation overnight, the mean weight loss was 24.4 +/- 1.8 kg (5.5 per cent of bodyweight). The horses were then given an equivalent volume of an oral glucose-glycine-electrolyte solution by stomach tube. No more than 10 litres was given every 30 minutes until the calculated bodyweight loss had been replaced. Measurements made before, during and after the fluid administration included bodyweight, arterial blood haematocrit, PCO2, pH, standard bicarbonate, base excess and plasma concentrations of sodium, potassium, chloride, total protein, glucose, urea and creatinine. The final measurement was taken eight hours after the last dose of fluid and no food or water was offered to the horses during this time. Administration of the solution caused a rapid correction of the frusemide-induced dehydration and metabolic alkalosis. Absorption of the fluid from the gastrointestinal tract appeared to be very rapid because by 30 minutes after the last dose of the solution, plasma protein values were not significantly different from those before administration of frusemide. Plasma glucose concentrations became significantly increased for up to three hours after the fluid was given and an increase in creatinine and urea concentrations, which was observed after the administration of frusemide, was still evident at eight hours. The glucose-glycine-electrolyte solution was well retained, there being a mean bodyweight loss of 2.8 kg at three hours and 6.2 kg at eight hours after the last dose of fluid.

Congenital malformation of the large colon causing colic in a horse.

An abnormal mesocolic attachment which resulted in a stellate malformation of the left colon adjacent to the pelvic flexure was suspected to be the cause of intermittent episodes of colic in a horse. Resection and side-to-side anastomosis of the large colon at the level of the sternal and diaphragmatic flexures was performed and the horse made an uneventful recovery from surgery. Only minor serum biochemical changes were observed in the initial postoperative period. The abnormal mesocolic attachment was probably a congenital anomaly.

Surgical treatment of paraphimosis in a pony.

Circumcision (or reefing operation) was performed on an aged pony stallion to remove excessive granulation tissue involving the preputial integument following an injury and subsequent paraphimosis. Postoperative swelling of the penis and prepuce was reduced daily with gentle massage. Initially, an improvised suspensory was used to support the penis postoperatively. The pony was able to voluntarily retract its penis six months following surgery.

The treatment of transection of the extensor carpi radialis muscle as a result of a lacerating wound in a horse.

A 16 month old filly was presented with the complaint of a severe laceration to the right foreleg with resultant transection of the extensor carpi radialis. Normal principles of wound treatment were followed and a bandage and splint were used for support and immobilization. The return to partial function of the damaged extensor carpi radialis was evidenced by resolution of the wound and an improvement in the patient's gait four months after the time of injury.