The absorption of orally supplied beta-alanine and its effect on muscle carnosine synthesis in human vastus lateralis.

Beta-alanine in blood-plasma when administered as A) histidine dipeptides (equivalent to 40 mg . kg(-1) bwt of beta-alanine) in chicken broth, or B) 10, C) 20 and D) 40 mg . kg(-1) bwt beta-alanine (CarnoSyn, NAI, USA), peaked at 428 +/- SE 66, 47 +/- 13, 374 +/- 68 and 833 +/- 43 microM. Concentrations regained baseline at 2 h. Carnosine was not detected in plasma with A) although traces of this and anserine were found in urine. Loss of beta-alanine in urine with B) to D) was <5%. Plasma taurine was increased by beta-alanine ingestion but this did not result in any increased loss via urine. Pharmacodynamics were further investigated with 3 x B) per day given for 15 d. Dietary supplementation with I) 3.2 and II) 6.4 g . d(-1) beta-alanine (as multiple doses of 400 or 800 mg) or III) L-carnosine (isomolar to II) for 4 w resulted in significant increases in muscle carnosine estimated at 42.1, 64.2 and 65.8%.

Hair analysis as a novel investigative tool for the detection of historical drug use/misuse in the horse: a pilot study.

REASONS FOR PERFORMING STUDY: Analysis of human hair for drug residues is being used increasingly as a diagnostic tool in the investigation of drug use and abuse. Hair analysis is complementary to urine/blood testing in that it can provide an extensive historical record of drug use, is noninvasive, impersonal and can facilitate retesting. However, the technique has not been studied in horses. HYPOTHESIS: That the systemic administration of drugs in horses could be identified by the detection of drug residues in hair. OBJECTIVE: To evaluate hair analysis as a potential retrospective diagnostic test for drug administration in horses by studying the deposition of systemically administered drugs in tail hair. METHODS: Tail hairs (n = 40-50) from 4 horses with known drug histories were washed, chopped into 3-5 mm fragments and extracted overnight, in 0.1 mol/l hydrochloric acid, prior to solid-phase extraction and analysis by high-performance liquid chromatography. Horse 1, a 3-year-old Thoroughbred colt (gastric ulcer), was treated for 14 days with omeprazole; Horse 2, a 3-year-old Thoroughbred colt (anaerobic infection), was treated for 5 days with metronidazole; Horse 3, an 8-year-old Thoroughbred gelding (sinusitis), was treated for 10 days with trimethoprim/sulphadiazine; and Horse 4, a 3-year-old Thoroughbred colt (respiratory infection), was treated for 5 days with procaine benzylpenicillin. RESULTS: Omeprazole was not detected in tail hair. Metronidazole was detected in tail hair at a concentration of 0.57 ng/mg, trimethoprim and sulphadiazine at concentrations of 9.14 and 2.26 ng/mg, respectively, and procaine at a concentration of 1.66 ng/mg. CONCLUSIONS: The data presented suggest that hair analysis may become a useable technique for the retrospective detection of drug administration in horses. POTENTIAL RELEVANCE: This technique could ultimately be used as part of a prepurchase veterinary examination to identify misuse of anti-inflammatory and sedative drugs, in an in-training testing programme to identify use of anabolic agents, or to provide evidence to support post race blood or urine test results. Clearly, more extensive research will be required to evaluate the effectiveness of the technique over a much broader range of drugs.

Plasma carnosine concentration: diurnal variation and effects of age, exercise and muscle damage.

This study was undertaken as part of a larger investigation into carnosine metabolism and function in the Thoroughbred horse. More specifically, we wished to evaluate plasma carnosine concentration as a potential indicator of muscle carnosine status. In contrast to man, carnosine is present in equine plasma where its presence is consistent with the absence of plasma carnosinase. A significant effect of age on plasma carnosine concentration in resting Thoroughbred horses was observed. Values in horses age 3 years and older were 113-14.1 micromol/l, whereas concentrations in foals and yearlings were 3.9-8.7 micromol/l (P<0.001). Lower values in young horses may reflect lower skeletal muscle carnosine concentrations. There was no significant within-day variation in plasma carnosine concentration in fed and fasted horses (P>0.05). Intense exercise resulted in a small significant increase (P<0.05) in plasma carnosine concentration (pre-exercise, 10.3 +/- 1.0 micromol/l; postexercise, 12.4 +/- 4.4 micromol/l). Greater increases were observed (57.6-702.3 micromol/l) following onset of exercise-induced rhadomyolysis (ERS). An apparent relationship was observed between elevated plasma carnosine and increased plasma creatine kinase (CK) and aspartate transaminase (AST) activities. Plasma carnosine concentrations did not reflect the severity of the condition as determined by clinical examination. In conclusion, elevated plasma carnosine levels are observed following exercise induced muscle damage, with the greatest elevations occurring during episodes of external rhabdomylosis syndrome. Plasma carnosine measurements could provide an alternative clinical indicator of muscle damage; and in conjunction with plasma taurine measurements may be indicative of selective type 1 or type 2 muscle fibre damage. However, given the complexity of the analytical technique, its applications would probably be confined to specialist referral or research centres.

Phenobarbitone concentrations in the hair, saliva and plasma of eight epileptic dogs.

Samples of plasma, saliva and hair were collected from eight dogs receiving phenobarbitone for idiopathic epilepsy. The concentrations of phenobarbitone in hair and plasma were correlated with the daily dose rate, and the concentrations in hair were also correlated with the concentration in plasma, the dose rate of the drug over the preceding six months and the ratio of the six-month dose to body surface area. The concentration of phenobarbitone in saliva was not correlated with either the concentration in plasma or the dose rate of the drug.

Influence of oral beta-alanine and L-histidine supplementation on the carnosine content of the gluteus medius.

The aim of this work was to test the hypothesis that in vivo carnosine biosynthesis is dependent upon endogenous beta-alanine availability, by studying the effect of sustained dietary beta-alanine supplementation in the horse on the carnosine concentration in types I, IIA and IIB skeletal muscle fibres. The diets of 6 Thoroughbred horses were supplemented 3 times/day with beta-alanine (100 mg/kg bwt) and L-histidine (12.5 mg/kg bwt) for a period of 30 days. Percutaneous biopsies of the m. gluteus medius from a depth of 6 cm were taken on the days immediately before and after the supplementation period. Heparinised blood samples were collected at hourly intervals on the first and last days of supplementation, and on every sixth day during the supplementation period, 2 h after each ration. Individual muscle fibres were dissected from freeze-dried biopsies, weighed and characterised histochemically. beta-alanine, histidine and carnosine concentrations were measured in plasma. The areas under the plasma concentration-time curves (AUC) for beta-alanine and histidine were calculated as indicators of the doses absorbed. Carnosine concentrations were measured in types I, IIA and IIB muscle fibres. There was an adaptive response to sustained beta-alanine administration resulting in mean +/- s.d. beta-alanine AUC increasing significantly from 1130 +/- 612 mumol/l h (Day 1) to 2490 +/- 1416 mumol/l h (Day 30) (P < 0.05). This was probably due to increased beta-amino acid transport across the gastrointestinal lumen. There was no consistent increase in histidine AUC between Days 1 and 30, (mean +/- s.d. values being 757 +/- 447 mumol/l h Day 1[ and 1162 +/- 1084 mumol/l h Day 30[ P > 0.05[). Type IIA fibre carnosine concentrations increased from 59.9-102.6 to 76.2-112.2 mmol/kg dry weight (dw). Increases were statistically significant in 2 of the 6 horses (P < 0.05 in both instances). Type IIB fibre carnosine concentrations increased from 101.3-131.2 to 114.3-153.3 mmol/kg dw. Increases were statistically significant in 5 of the 6 horses (P < 0.05 in 3 horses, P < 0.01 in 1 horse, P < 0.005 in 1 horse). Changes in muscle carnosine concentration appeared to be influenced by beta-alanine bioavailability. Individual increases in muscle carnosine concentration were significantly correlated with individual changes in beta-alanine AUC (r2 = 0.973, P < 0.005). Increased muscle carnosine concentrations lead to increased intramuscular hydrogen ion (H+) buffering capacity.

Plasma ammonia and lactate responses using incremental and constant speed exercise tests.

Plasma NH3, formed during intense exercise, results principally from the deamination of AMP in muscle. Its formation during exercise may be influenced both by the pool of fibres recruited and by changes in the intracellular environment affecting ADP homeostasis. This study compared incremental and constant speed exercise as possible protocols for the investigation of plasma NH3 accumulation with intense exercise. Six trained Thoroughbred horses, one of which had recently been operated on for recurrent laryngeal neuropathy, undertook a step-wise treadmill test with 1 min incremental steps of 6, 8, 10, 11 and 12 m/s (7.5% incline). Two and 4 weeks later horses performed a constant-speed, maximum-exercise tolerance test at 115% VO2max (7.5% incline). Blood samples from the jugular vein were drawn at 20 s intervals in all 3 tests, for plasma NH3 and lactate. There were marked differences between and within horses in their time dependant lactate and NH3 responses to exercise. Three of the 6 horses studied showed a distinct threshold for onset of plasma NH3 accumulation with incremental exercise. Distinct thresholds for the onset of NH3 accumulation were apparent also in 5 of the 6 horses during exercise at constant rate. The present study demonstrates clearly the practicality of measuring NH3 concentration curves, even during a short incremental step test which has the advantage that other measures relating to cardiovascular and respiratory functions can be measured simultaneously.

Carnosine, anserine and taurine contents in individual fibres from the middle gluteal muscle of the camel.

High muscle carnosine und anserine contents contribute significantly to intra-cellular physico-chemical buffering. Our aim was to measure carnosine, anserine and taurine contents directly in individual type I, IIA and IIB fibres from the middle gluteus muscle of the camel. Mean carnosine contents in type I, IIA and IIB were 24.6 +/- 9.2, 39.4 +/- 11.4 and 42.8 +/- 18.8 mmol kg-1 dry weight (dw), respectively. Mean anserine contents in type I, IIA and IIB fibres were 30.0 +/- 8.4, 37.3 +/- 10.1 and 34.5 +/- 9.7 mmol kg-1 dw, respectively. Mean taurine contents in type I, IIA and IIB fibres were 42.4 +/- 15.9, 20.3 +/- 12.9 and 24.7 +/- 15.9 mmol kg-1 dw, respectively. Higher carnosine contents in type II fibres emphasise the importance of carnosine to intra-muscular acid-base regulation. A specific role for taurine in type I fibres in unclear.

High-performance liquid chromatographic determination of imidazole dipeptides, histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres.

The combined solid-phase extraction (Isolute PRS columns) and reversed-phase gradient HPLC method presented provides a sensitive, reproducible and selective quantification of carnosine, balenine, homocarnosine, histidine, 1-methylhistidine and 3-methylhistidine in equine and camel muscle and individual muscle fibres. Recoveries were 91-115%. Lower limits of detection were 0.005-0.010 mmol kg-1 dry muscle. The compounds were isolated from other physiological amino acids and small peptides and resolved within a single chromatographic run of 55 min. Concentrations of these compounds in equine myocardium, diaphragm, skeletal muscle, camel muscle and individual muscle fibres of both species are presented for the first time.

High-performance liquid chromatographic determination of N-alpha-acetyl-L-carnosine in equine plasma.

N-alpha-Acetyl-L-carnosine (NAcCAR) in perchloric acid extracts of equine plasma was assayed by high-performance liquid chromatography on a 3 microns Hypersil ODS (150 x 4.6 mm I.D.) column eluted with 5 mM phosphoric acid-1 mM triethylamine, pH 2.58. NAcCAR was isolated by solid-phase extraction on Isolute PRS (propylsulphonyl) columns. The HPLC mean retention time for NAcCAR was 5.9 +/- 0.2 min. The recovery from plasma by solid-phase extraction was 93.9-99.7% and lower limit of detection in plasma was 0.18 microM. The normal NAcCAR concentration in equine plasma was 2.4 +/- 0.3 microM. The method was applied to the determination of plasma concentrations following oral and intravenous NAcCAR administration.

The pharmacokinetics or oral and intravenous allopurinol and intravenous oxypurinol in the horse.

The pharmacokinetics of oral and intravenous allopurinol was studied in five horses and compared with intravenous oxypurinol. The plasma concentration vs. time curves, following intravenous administration of 5 mg/kg, were best described by the biexponential equations Cp = 106.58e(-25.14t) + 159.93e(-10.96t) for allopurinol and Cp = 321.09e(-9.72t) + 82.39e(-0.44t) for oxypurinol, with an elimination half-life (t1/2 beta) of 0.09 h and an area under the curve (AUC) of 19.8 mumol.h/L after intravenous administration, while the t1/2 beta and AUC of oxypurinol were 1.09 h and 231 mumol.h/L, respectively. The bioavailability of allopurinol was low (14.3%), although no allopurinol was detected in the plasma of two horses after oral administration of allopurinol was equivalent to that of intravenously injected oxypurinol. The results suggest that allopurinol is rapidly metabolised in vivo and that the majority of the pharmacological activity of allopurinol in the horse may result from the action of the active metabolite, oxypurinol.

Simultaneous quantitation of oxidised and reduced glutathione in equine biological fluids by reversed-phase high-performance liquid chromatography using electrochemical detection.

A change in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) can be used to indicate oxidative stress in vivo. A rapid and highly sensitive isocratic reversed-phase high-performance liquid chromatographic method using coulometric electrochemical detection (LCEC) has been developed to simultaneously detect GSH and GSSG in equine biological fluids. Perchloric acid was used to extract GSH and GSSG from equine plasma and haemolysates, and methanol was used to deproteinise bronchoalveolar lavage fluid samples. Injection of extracts onto a Hypersil ODS HPLC column produced well resolved peaks corresponding to GSH and GSSG. The concentrations of GSH and GSSG found in equine haemolysates were similar to those previously found in humans and laboratory animals, although, to the authors' knowledge, previous attempts to measure GSH and GSSG in bronchoalveolar lavage fluid using LCEC have been unsuccessful. This method can be used to measure the GSH redox ratio in biological fluids during physiological conditions that may induce oxidative stress, such as exercise and disease.

Physiological, metabolic and biochemical responses of horses competing in the speed and endurance phase of a CCI*****3-day-event.

The present study was undertaken to investigate physiological, metabolic, haematological and biochemical changes in horses competing in the Speed and Endurance test of a Concours Complet International (CCI)*****3-day-event held under FEI rules. A total of 28 horses competing in the Burghley Horse Trials Speed and Endurance test were selected to be monitored: 11 horses in 1993 and 17 horses in 1994. Of the 28 horses selected, 17 completed the Speed and Endurance test and went on to complete the showjumping test. Mean +/- s.d. shade temperature and relative humidity, black globe temperature and wind speed were 13 +/- 1 and 20 +/- 2 degrees C, 54 +/- 3 and 55 +/- 10%, 17 +/- 2 and 29 +/- 4 degrees C and 2.7 +/- 0.7 and 1.2 +/- 0.3 m/s, for 1993 and 1994, respectively. Mean heart rate during Phases A, B and D was not significantly different between years, but mean heart rate during Phase C and X was significantly higher in 1994. Mean (+/- s.d.) heart rate on Phase B and D for all horses in both 1993 and 1994 was 198 +/- 8 and 188 +/- 11 beats/min, respectively. Mean heart rate during Phase D showed a poor correlation with mean speed (r = 0.412). Total mean (+/- s.d.) weight loss from the start of Phase A to the end of Phase D was 15.5 +/- 6.1 kg in 1993 and 16.5 +/- 5 kg in 1994 and did not differ significantly between years. Following 14-18 h completion of Phase D, mean bodyweight was not significantly different from that at the start of Phase A in either year. Mean rectal temperature at the end of Phase D was 41 +/- 0.6 degrees C and 41.1 +/- 0.6 degrees C in 1993 and 1994, respectively (P > 0.05). Both the lowest (39.7 degrees C) and highest (41.8 degrees C) rectal temperatures were recorded at the end of Phase D in 1994. Plasma lactate concentrations at the end of Phase D were 8.5-38.5 mmol/l. The highest lactate concentration also coincided with the highest plasma glucose concentration (11.4 mmol/l) as well as the joint fastest time in either year, although overall lactate showed only weak correlations with mean speed on Phase D (r = 0.12, 1993; r = 0.58, 1994). While the Speed and Endurance test at CCI*****level run in a temperate climate presents a considerable challenge to the fitness and ability of the horses competing, the metabolic and physiological changes are not extreme. The majority of horses that finish the test appear to undergo a rapid and considerable degree of recovery and are able to present sound at the final inspection, take part in the showjumping test and complete the competition.

Variation in the concentration of long chain free fatty acids in equine plasma over 24 hours.

The primary aim of this study was to examine the within-day variation in the concentration of total and individual long chain free fatty acids (C > 14) in normally fed horses. Plasma samples were collected over a 24 h period from 12 resting horses during three separate sessions (six horses in the first session and three in the second and third). Samples were analysed for individual long chain free fatty acids (FFA) and glucose. During normal feeding, the predominant FFA in plasma were palmitic (C16:0), linoleic (C18:2), oleic (C18:1), stearic (C18:0) and linolenic (C18:3). Together these acids constituted over 90% of the total concentration. Other FFA present were myristic (C14:0) and palmitoleic (C16:1) both of which constituted < 5% of the total concentration. Ten out of the 12 horses sampled exhibited an early morning increase in FFA (P < 0.001) localized around 0700 h and which was independent of feeding. The mean concentration of total FFA increased 4.5 fold (range 2.0-8.5) between 0400 and 1000 h. The predominant FFA showed the largest increase.

Taurine content and distribution in equine skeletal muscle.

Taurine (TAU) is found in large but variable amounts in the skeletal muscles of many species. It has been reported that slow twitch muscles in the rat exhibit higher TAU levels than fast twitch muscles. Variation in muscle taurine content may be attributable to differences in the fibre type composition of different muscles. TAU content (mmol kg-1 dry muscle) and percentage type-1, type-2A, and type-2B fibre section area (f.s.a.) were measured in muscle samples taken from up to six sites in the middle gluteal muscle of four horses and one pony at post mortem and in biopsy samples taken from twenty Thoroughbred horses in race training. TAU was positively correlated to type-1 f.s.a. (r = 0.94, p < 0.001) in both post mortem samples and biopsies from horses in race-training. Multiple linear regression analysis was used to estimate the TAU content of individual fibre types when present at 100%. TAU is almost exclusively localized in type-1 fibres. The TAU content of type-1 and type-2A fibres was estimated to be 45.4 mmol kg-1 d.m. and 4.5 mmol kg-1 d.m. respectively in the post mortem horses, and 32.4 mmol kg-1 d.m. and 7.9 mmol kg-1 d.m. respectively in the horses in training. TAU was estimated to be absent from type-2B fibres in both horse groups.

Estimation of the carnosine content of different fibre types in the middle gluteal muscle of the thoroughbred horse.

1. Skeletal muscle samples were obtained by needle biopsy from one of two depths of the m. gluteus medius in a group of twenty race-trained thoroughbred horses. 2. The content of carnosine was determined in each muscle sample, part of which was used for histochemical analysis. Fibres were classified as type I, type IIA or type IIB on the basis of the pH dependent lability of the myosin ATPase reaction. 3. Muscle samples with a higher type II fibre section area (FSA) have a higher carnosine content than those with a higher type I FSA. 4. Multiple linear regression analysis was used to estimate the mean carnosine content of individual fibre types. The results estimated a mean carnosine content in type I fibres of 54 mmol (kg dry muscle (DM))-1, in type IIA fibres 85 mmol (kg DM)-1 and in type IIB fibres 180 mmol (kg DM)-1. 5. Based on the estimated values of single fibre carnosine content, there was close concordance between the estimated and the measured carnosine content of mixed fibre samples. 6. It would appear from this and other studies that carnosine has an important role as a physico-chemical buffer in equine middle gluteal muscle and that this is greatest in type IIB fibres, where it may account for up to 50% of physico-chemical buffering of H+ produced by muscle in the pH range 7.1-6.5.

Determination of carnosine and other biogenic imidazoles in equine plasma by isocratic reversed-phase ion-pair high-performance liquid chromatography.

The isocratic reversed-phase ion-pair high-performance liquid chromatographic technique presented provides a sensitive, rapid and reproducible analytical method for the selective determination of carnosine and other biogenic imidazoles in equine plasma. Plasma was deproteinized with 5-sulphosalicylic acid and the compounds of interest were isolated by sorbent extraction on Bond Elut PRS cartridges. Recoveries were 97-105% and the lowest limits of detection were 58.3-80.1 nM. All compounds of interest were well resolved within a maximum retention time of 9.2 min. The mean equine plasma carnosine level determined by this method was 11.31 microM. Comparative determinations were made in canine and human plasma. Carnosine was not detected in human plasma. Concentrations of imidazole in canine plasma are reported here for the first time.

Carnosine and anserine concentrations in the quadriceps femoris muscle of healthy humans.

The content of anserine and carnosine in the lateral portion of the quadriceps femoris muscle of 50 healthy, human subjects has been studied. Anserine was undetectable in all muscle samples examined. Muscle carnosine values for the group conformed to a normal distribution with a mean (SD) value of 20.0 (4.7) mmol.kg-1 of dry muscle mass. The concentration of carnosine was significantly higher in the muscle of male subjects (21.3, 4.2 mmol.kg-1 dry mass) than in females of a similar age and training status (17.5, 4.8 mmol.kg-1 dry mass) (P less than 0.005). The test-retest reliability of measures was determined on a subgroup of 17 subjects. No significant difference in mean carnosine concentration was found between the two trials [21.5 (4.0) and 22.0 (5.2) mmol.kg-1 dry muscle mass; P greater than 0.05]. The importance of carnosine as a physicochemical buffer within human muscle was examined by calculating its buffering ability over the physiological pH range. From the range of carnosine concentrations observed (7.2-30.7 mmol.kg-1 dry muscle mass), it was estimated that the dipeptide could buffer between 2.4 and 10.1 mmol H+.kg-1 dry mass over the physiological pH range 7.1-6.5, contributing, on average, approximately 7% to the total muscle buffering. This suggests that in humans, in contrast to many other species, carnosine is of only limited importance in preventing the reduction in pH observed during high intensity exercise.

Reverse-phase ion-pairing high-performance liquid chromatography of phosphocreatine, creatine and creatinine in equine muscle.

A simple, robust and reproducible analytical method for the determination of phosphocreatine (PCr), creatine (Cr) and creatinine (Cn) in equine skeletal muscle is presented. The technique used isocratic reverse-phase ion-pairing high-performance liquid chromatography. Neutralized perchloric acid extracts of equine muscle biopsies were analysed and the values obtained were compared with determinations from an established enzymic procedure. Good resolution of all three metabolites was achieved within a retention time of less than 11 min. Linearity for each metabolite within the concentration range in the samples was demonstrated. Peak purity was specifically addressed. The abolition of each creatine in a pooled extract by enzymic incubation showed no underlying peaks. It was concluded that peaks were free of co-eluents which would otherwise lead to an overestimation of PCr, Cr and Cn concentrations.