Four weeks of high-intensity training in moderate, but not mild hypoxia improves performance and running economy more than normoxic training in horses.

We investigated whether horses trained in moderate and mild hypoxia demonstrate greater improvement in performance and aerobic capacity compared to horses trained in normoxia and whether the acquired training effects are maintained after 2 weeks of post-hypoxic training in normoxia. Seven untrained Thoroughbred horses completed 4 weeks (3 sessions/week) of three training protocols, consisting of 2-min cantering at 95% maximal oxygen consumption V˙O2max under two hypoxic conditions (H16, FI O2  = 16%; H18, FI O2  = 18%) and in normoxia (N21, FI O2  = 21%), followed by 2 weeks of post-hypoxic training in normoxia, using a randomized crossover study design with a 3-month washout period. Incremental treadmill tests (IET) were conducted at week 0, 4, and 6. The effects of time and groups were analyzed using mixed models. Run time at IET increased in H16 and H18 compared to N21, while speed at V˙O2max was increased significantly only in H16. V˙O2max in all groups and cardiac output at exhaustion in H16 and H18 increased after 4 weeks of training, but were not significantly different between the three groups. In all groups, run time, V˙O2max , VV˙O2max , Q˙max , and lactate threshold did not decrease after 2 weeks of post-hypoxic training in normoxia. These results suggest that 4 weeks of training in moderate (H16), but not mild (H18) hypoxia elicits greater improvements in performance and running economy than normoxic training and that these effects are maintained for 2 weeks of post-hypoxic training in normoxia.

Practical Tips on Sample Handling for Hematology, Chemistry, and Cytology Testing for Equine Patients:: Getting More Bang for your Buck.

Clinical pathology results are only as good as the quality of samples and accompanying information submitted to the diagnostic laboratory. The frustration of nondiagnostic or equivocal test results can often be avoided by taking the time to follow sample handling and submission guidelines. This article discusses preanalytical errors that commonly affect the accuracy of hematology, chemistry, and cytology testing, and offers practical tips for preventing these errors and maximizing diagnostic yield.

Coagulation Assessment in the Equine Patient.

Horses with clinical signs of unprovoked or excessive hemorrhage should be evaluated for underlying platelet defects or coagulopathies. This article provides an overview of preliminary screening and definitive tests to assess coagulation and identify hemostatic defects in horses, as well as a review of the hemostatic disorders most frequently encountered in clinical practice.

Repeatability of the hyperglycaemic clamp for assessment of ß-cell response and insulin sensitivity in horses.

BACKGROUND: The hyperglycaemic clamp has been used for measurement of insulin sensitivity (IS) but not for measurement of ß-cell response in the horse. However, the repeatability of this test has not been reported for horses. OBJECTIVES: To determine the repeatability and reliability of measures for IS and ß-cell response to glucose using the hyperglycaemic clamp in horses. STUDY DESIGN: Repeated measures, longitudinal study. METHODS: Six healthy Standardbred mares underwent a 120-min hyperglycaemic clamp on two occasions with a 10-day washout period. Indices of repeatability and reliability were calculated from measures of IS and ß-cell response to glucose derived from each hyperglycaemic clamp. RESULTS: Measures of ß-cell response to glucose, including area under the insulin response curve in the time interval 0-120 min and during steady state (60-120 min), as well as the mean insulin concentration during steady state had coefficient of variations (CV) of 9.5, 10.5 and 9.3 respectively and intraclass correlation coefficients (ICC) of 0.93, 0.93 and 0.95 respectively. The measure of glucose disposal (M) had lower CV (12.4) and ICC (0.71) compared to measure of IS (M/I-index; CV of 17.4 and ICC of 0.89). MAIN LIMITATIONS: The study was conducted in a small number of horses. CONCLUSIONS: The hyperglycaemic clamp is a diagnostic method for assessing ß-cell response to intravenous glucose with high repeatability and reliability. Insulin sensitivity can be determined with the same test but the repeatability for these measurements is lower compared to the measurements for ß-cell response.

Assessment of peripheral markers and ultrasonographic parameters in pregnant mares receiving intramuscular or intrauterine cloprostenol.

The present study aimed to compare two methods of prostaglandin-induced abortion in mares by determining blood markers (progesterone, estradiol-17ß, alpha-fetoprotein, 13,14-dihydro-15-keto-prostaglandin-F2α (PGFM)), B-mode ultrasonographic parameters, and time until loss of fetal heartbeat. It was hypothesized that intrauterine infusion of cloprostenol results in earlier fetal compromise than intramuscular administration. Ovarian structures (number and sizes of follicles and corpora lutea area), fetal heartbeat, and fetal mobility of thirteen singleton pregnancies were assessed daily by transrectal ultrasonography until induction of pregnancy termination (60 ±â€¯2 days of gestation). Mares received 500 µg of cloprostenol intramuscularly every 12 h (IM, n = 7) or once transcervically (TC, n = 6). After initial cloprostenol administration, ultrasonographic examinations were repeated at 6-h intervals until loss of fetal heartbeat was detected. Plasma progesterone, estradiol-17ß, and alpha-fetoprotein were assessed for five days before and after pregnancy loss. In addition, plasma PGFM concentrations were assessed immediately before cloprostenol administration (0 min), and then 15, 30, and 45 min, and 1, 2, 3, 4, 6, 12 h after administration. Data were analyzed using the MIXED procedure with repeated measures in SAS. Significance was set at P < 0.05. All mares lost their pregnancies within 48 h after initial cloprostenol administration, with no difference in time to pregnancy loss. There were significant effects of time starting by 12 h post-induction of pregnancy termination but there was no time by group interaction for progesterone concentrations. Estradiol-17ß and alpha-fetoprotein concentrations were not altered upon impending abortion. Concentrations of PGFM increased significantly by 2 h after cloprostenol administration, but there were no differences between groups. No time effects or time by group interaction for fetal mobility and heartbeat was detected. Expectedly, the number and area of corpora lutea decreased significantly after cloprostenol administration with no significant differences between groups. In conclusion, intrauterine administration of cloprostenol was not different from repeated systemic administration to terminate the pregnancy. Both models for early fetal loss were equivalent for the endpoints assessed herein. The present study provides evidence that transcervical cloprostenol administration technique is repeatable in different settings and results in negligible side effects. While systemic administration results in colic-like signs and may result in severe reaction.

Assessment of equine alpha-fetoprotein levels in mares and newborn foals in the periparturient period.

Alpha-fetoprotein (AFP) is best known in human obstetrics for its association with fetal anomalies recognized in the 1970s. Although this fetal protein had been shown to be present in the sera of many mammalian species, its possible diagnostic role in the detection of abnormalities was evaluated only later, when a research laboratory published variable levels of AFP in different groups of mares with pregnancy problems (twins, conception failure, placentitis, embryonic loss), and subsequently differences were demonstrated in its serum levels between aborted and healthy mares. In this study, peri- and intrapartal AFP levels were measured in maternal serum, amniotic fluid, neonatal blood, and umbilical blood samples. The mean levels of AFP were lower in umbilical blood and amniotic fluid samples than in foal and maternal blood. Older mares had lower AFP levels correlated with their age in years. The time remaining until foaling had a significant, non-linear effect on AFP levels: an elevation could be detected in the last two weeks of pregnancy, followed by a decline after foaling. Also, AFP levels were found to be elevated in the hot summer months. There was a significant individual variation in AFP levels in the population studied.

Pharmacokinetics of intravenous lithium chloride and assessment of agreement between two methods of lithium concentration measurement in the horse.

BACKGROUND: Pharmacokinetics of lithium chloride (LiCl) administered as a bolus, once i.v. have not been determined in horses. There is no point-of-care test to measure lithium (Li+ ) concentrations in horses in order to monitor therapeutic levels and avoid toxicity. OBJECTIVES: To determine the pharmacokinetics of LiCl in healthy adult horses and to compare agreement between two methods of plasma Li+ concentration measurement: spectrophotometric enzymatic assay (SEA) and inductively coupled plasma mass spectrometry (ICP-MS). STUDY DESIGN: Nonrandomised, single exposure with repeated measures over time. METHODS: Lithium chloride was administered (0.15 mmol/kg bwt) as an i.v. bolus to eight healthy adult horses. Blood samples were collected pre-administration and at multiple times until 48 h post-administration. Samples were analysed by two methods (SEA and ICP-MS) to determine plasma Li+ concentrations. Pharmacokinetics were determined based on the reference ICP-MS data. RESULTS: Adverse side effects were not observed. The SEA showed linearity, R2 = 0.9752; intraday coefficient of variation, 2.5%; and recovery, 96.3%. Both noncompartmental and compartmental analyses (traditional two-stage and nonlinear mixed-effects [NLME] modelling) were performed. Geometric mean values of noncompartmental parameters were plasma Li+ concentration at time zero, 2.19 mmol/L; terminal elimination half-life, 25.68 h; area under the plasma concentration-time curve from time zero to the limit of quantification, 550 mmol/L min; clearance, 0.273 mL/min/kg; mean residence time, 31.22 h; and volume of distribution at steady state, 511 mL/kg. Results of the traditional two-stage analysis showed good agreement with the NLME modelling approach. Bland-Altman analyses demonstrated poor agreement between the SEA and ICP-MS methods (95% limits of agreement = 0.14 ± 0.13 mmol/L). MAIN LIMITATIONS: Clinical effects of LiCl have not been investigated. CONCLUSIONS: The LiCl i.v. bolus displayed pharmacokinetics similar to those reported in other species. The SEA displayed acceptable precision but did not agree well with the reference method (ICP-MS). The Summary is available in Spanish - see Supporting Information.

Relationship between colour flow Doppler sonographic assessment of corpus luteum activity and progesterone concentrations in mares after embryo transfer.

Colour-flow Doppler sonography has been described as a means of assessing corpus luteum (CL) function rapidly, because area of luteal blood vessels correlates well with circulating progesterone (P4) concentrations [P4] in oestrous cycling mares. The aim of this study was to assess the relationships between CL size and vascularity, and circulating [P4] during early pregnancy in mares, and to determine whether luteal blood flow was a useful aid for selecting an embryo transfer recipient. Equine embryos (n=48) were recovered 8 days after ovulation and were transferred to available recipient mares as part of a commercial program with the degree of synchrony in timing of recipient ovulation ranging from 1 day before to 4 days after the donor. Immediately prior to embryo transfer (ET), maximum CL cross-section and blood vessel areas were assessed sonographically, and jugular blood was collected to measure plasma [P4]. Sonographic measurements and jugular blood collection were repeated at day 4 after ET for all mares, and again at days 11, 18 and 25 after ET in mares that were pregnant. The number of grey-scale and colour pixels within the CL was subsequently quantified using ImageJ software. The CL blood flow correlated significantly but weakly with plasma [P4] on the day of transfer and on day 4 after ET in all mares, and on days 11 and 25 after ET in pregnant mares (r=0.30-0.36). The CL area and plasma [P4] were also correlated on each day until day 11 after ET (r=0.49-0.60). The CL colour pixel area decreased significantly after day 18, whereas CL area was already decreasing by day 4 after ET. The CL area, area of blood flow, or [P4] was predictive of pregnancy. Findings in the present study suggest that both CL area and blood flow are correlated with circulating [P4] at the time of transfer and in early pregnancy. Evaluation of the CL using B-mode or CF sonography, although practical, provides no improvement in the selection of recipients or prediction of pregnancy outcomes than methods employed currently.

Analysis of metronidazole in equine plasma using liquid chromatography/tandem mass spectrometry and high-resolution accurate mass spectrometry.

RATIONALE: Treatment of racehorses with bicarbonate solutions to manage acidosis and muscle cramps prior to competition is banned in Pennsylvania (PA). Use of excess bicarbonate in horses causes diarrhea, requiring treatment with an antibiotic such as metronidazole (MTNZ). At present no method exists for detecting MTNZ in equine plasma. Thus, a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection, quantification and confirmation of MTNZ was developed. METHODS: The analyte was recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether and separated on an ACE® C18 column with its guard column. The mobile phase comprised a mixture of 5 mM ammonium formate (pH 3.5) and acetonitrile (60:40; v/v). Mass analysis was performed on an LTQ XL linear ion trap mass spectrometer in positive electrospray ionization mode while accurate mass determination was also performed in positive electrospray ionization mode using high-resolution accurate mass spectrometry (HRAMS). RESULTS: The limit of detection (LOD), limit of confirmation (LOC) and lower limit of quantification (LLOD) were 1, 2 and 50 ng/mL, respectively. The analyte in plasma was stable at -20 and -70°C for 28 days, as well as for 24 h at 20°C in the autosampler. The percentage coefficients of variation (% CV) for the intra-day and inter-day precision for the LLOQ were 5.1:3.68 and 13.21:9.95, respectively, while the intra-day accuracy was from 98.71 to 101.57% and that of the inter-day was from 88.64 to 96.6%. The matrix effect was between 9 and 24%. The precursor → product ion transition m/z 172 → 128, a retention time of 2.92 min and the accurate mass of the [M+H](+) ion of the analyte (m/z 172.0173) were used as criteria for confirmation of the presence of MTNZ in equine plasma. CONCLUSIONS: The method is highly sensitive and selective for the detection, identification and confirmation of MTNZ in equine plasma. Thus, illegal use of MTNZ in racehorses can be routinely monitored within the US State of Pennsylvania. The method is fast, sensitive, reproducible, and reliable.

Quantification of the energy expenditure during training exercises in Standardbred trotters.

An appropriate energy feeding management that ensures the optimal dietary energy supply according to the energy expenditure (EE) is a crucial component for the horse's performance. The main purpose of this study was to determine the EE during four specific exercises used in the training of Standardbred trotters (promenade, jogging, parcours and interval work-outs). A total of six Standardbred geldings performed four different testing situations on a track. The intensity (expressed in percentage of the maximal velocity over 500 m, i.e. v500) and volume (distance and duration) of the testing situations were determined according to practices reported by French trainers. Promenade and jogging included only an exercise phase, whereas parcours and interval situations also included a warm-up and a recovery phase. Oxygen uptake (VO2), carbon dioxide production (VCO2) and heart rate (HR) were continuously recorded from 2 min before the beginning through to the end of the testing situations, using a portable respiratory gas analyser. Blood lactate levels and rectal temperature were determined before and immediately after the exercise phase of each testing situations. EE of the different phases (warm-up, exercise and recovery) and EE of the entire testing situations (EETOTAL) were calculated from VO2 measurements and the O2 caloric equivalent. Interval and parcours situations induced higher physiological responses than promenade and jogging situations, particularly in terms of VO2peak, VCO2peak and HRpeak. The highest blood lactate concentration (6 mmol/l) was measured after the interval exercise, and respiratory exchange ratios ⩾1 were observed only for the parcours situation. The EE of exercise phase varied from 0.49 to 1.79 kJ/min per kg for promenade and parcours situations. The EE of warm-up and recovery phases did not differ between parcours and interval situations, and was estimated at 1.04 and 0.57 kJ/min per kg BW, respectively. On average, the warm-up and the recovery phases contributed to 38% and 19% of the EETOTAL. For promenade, jogging, parcours and interval situations, EETOTAL was evaluated at 12 618, 11 119, 13 698 and 18 119 kJ, respectively.

Assessment of plasma anti-elastin antibodies for use as a diagnostic aid for chronic progressive lymphoedema in Belgian Draught Horses.

Diagnosis of chronic progressive lymphoedema (CPL) in draught horses, including the Belgian Draught Horse, is mainly based on clinical evaluation of typical lower limb lesions. A deficient perilymphatic elastic support, caused by a pathological elastin degradation in skin and subcutis, has been suggested as a contributing factor for CPL. Elastin degradation products induce the generation of anti-elastin Ab (AEAb), detectable in horse serum by ELISA. For a clinically healthy group of draught horses, a significantly lower average AEAb-level than 3 clinically affected groups (mild, moderate and severe symptoms) was demonstrated previously. To improve CPL-diagnosis, we evaluated the AEAb-ELISA as an in vitro diagnostic aid in individual horses. Test reproducibility was assessed, performing assays independently in 2 laboratories on a total of 345 horses. Possible factors associated with AEAb-levels (age, gender, pregnancy, test lab and date of blood collection) were analyzed using a mixed statistical model. Results were reproducible in both laboratories. AEAb-levels in moderately and severely affected horses were significantly higher than in healthy horses. Nevertheless, this was only demonstrated in barren mares, and, there was a very large overlap between the clinical groups. Consequently, even when a high AEAb cut-off was handled to obtain a reasonable specificity of 90%, a very low sensitivity (21%) of AEAb for CPL-diagnosis was obtained. Results on the present sample demonstrate that the described ELISA procedure is of no use as a diagnostic test for CPL in individual horses.

Comparison among the euglycemic-hyperinsulinemic clamp, insulin-modified frequently sampled intravenous glucose tolerance test, and oral glucose tolerance test for assessment of insulin sensitivity in healthy Standardbreds.

OBJECTIVE: To compare, in horses, estimates of insulin sensitivity obtained from minimal model analysis (MMA) of a frequently sampled IV glucose tolerance test (FSIGTT) with estimates from the euglycemic-hyperinsulinemic clamp (EHC) and to evaluate the validity of surrogate estimates of insulin sensitivity derived from an oral glucose tolerance test (OGTT). ANIMALS: 18 mature Standardbreds (mean ± SD body weight, 428.9 ± 35.9 kg; mean ± SD body condition score, 4.4 ± 1.0 [on a scale of 1 to 9]). PROCEDURES: All horses underwent at least 2 of the 3 procedures (EHC [n = 15], insulin-modified FSIGTT [18], and OGTT [18]) within a 10-day time frame to evaluate insulin sensitivity. RESULTS: Insulin sensitivity variables derived from the EHC and FSIGTT were strongly correlated (r = 0.88). When standardized to the same units of measure, these measures were still strongly correlated (r = 0.86) but were not equivalent. Area under the curve, peak insulin concentration, insulin concentration at 120 minutes, and 2 calculated indices from glucose and insulin data from the OGTT were significantly correlated with the EHC- and FSIGTT-derived estimates of insulin sensitivity. CONCLUSIONS AND CLINICAL RELEVANCE: In healthy Standardbreds with moderate body condition score, insulin sensitivities from the EHC and FSIGTT were strongly correlated but not equivalent. Estimates derived from an OGTT also may be useful to estimate insulin sensitivity.

Assessment of serum levels of allergen-specific immunoglobulin E in different seasons and breeds in healthy horses.

The present study was designed to asses specific IgE towards environment allergens in 42 healthy horses. Determination of this immunoglobulin in serum serve as diagnostic tools in allergic diseases to improve efficacy of the treatment and proper allergen selection to specific immunotherapy. Serum levels of allergen specific IgE were measured with equine monoclonal antibody, using 15 individual and 5 mix allergens in North European Panel. The study revealed season dependent increased levels of allergen specific IgE in normal horses. It is noteworthy that healthy horses show high percentage of positive reactions, most commonly towards to domestic mites D. farinae (80%), D. pteronyssinus (35.71%) and storage mites T. putrenscentiae (42.86%), Acarus siro (40.48%). These allergens play an important role in equine, canine and feline atopic dermatitis. We also demonstrated high IgE levels in the group of horse specific insect allergens. Tabanus sp. (35.71%), Culicoides sp. (28.57%) and Simulium sp. (26.19%) were the most frequent insect positive reaction allergens. No positive reactions in all groups of allergens were found in winter season, low and merely detectable levels of antibodies have been found relating to D. farianae and T. putrescentiae allergen. We observed elevated mould-IgE levels in horses that live in stables, while outdoor living horses showed very low levels. Amongst all positive reactions we observed only weak and moderate reactions but no strong positive reactions were found. No significant differences were observed between three breeds of horses with the exception of moulds and D. pteronyssinus allergens.

Fast and sensitive analysis of dermorphin and HYP6-dermorphin in equine plasma using liquid chromatography tandem mass spectrometry.

Dermorphin and HYP(6) -dermorphin are hepta-peptides and natural opioids originally isolated from the skin of South American frogs. They are more potent than morphine but less likely to produce drug tolerance and addiction. These properties make them ideal candidates for the doping of racehorses to enhance performance during competition. Dermorphin was recently classified as a Class I drug by Racing Commissioners International (RCI), indicating that it is a banned substance in equine athletes. To enforce this ban, a fast and sensitive method was developed for dermorphin and HYP(6)-dermorphin analysis in equine plasma. Equine plasma (2 ml) was extracted on a mixed mode cation exchange solid-phase column. After extraction, dermorphin and HYP(6)-dermorphin were separated and detected using a liquid chromatography (LC) triple quadrupole linear ion trap mass spectrometry in positive multiple-reaction-monitoring (MRM) mode. Each analysis was 3.5 min. Four MRM transitions were used for identification of each compound. The extraction procedure was efficient and the limits of detection (LOD) were 2 pg/ml and 10 pg/ml for dermorphin and HYP(6)-dermorphin, respectively. The method has good selectivity and precision. Results of stability studies showed that both analytes were stable at low temperature. This is the first report of dermorphin and HYP(6)-dermorphin analysis in equine plasma, which could be adopted as a regular screening or confirmation method for controlling the abuse of these compounds in equine sports.

Correlation of serum IgG concentration in foals and refractometry index of the dam's pre- and post-parturient colostrums: an assessment for failure of passive transfer in foals.

The object of this study was to evaluate the usefulness of measuring the differences in the values of the serum total protein (DVSTP) concentration of foals and the refractometry index (DVRI) of the milk of dams before and after nursing of the colostrum for assessing failure of passive transfer (FPT) in foals. Serum samples from 31 foals were collected before the first nursing and other 1 to 6 times between 4 and 24 hr after birth. Paired colostrum and milk samples were collected from 14 of their dams at the same time. Serum samples were analyzed for IgG concentration using a single radial immunodiffusion (SRID) test (98 samples) and total protein concentration using a temperature-compensating refractometer (98 samples). Colostrum and milk samples were analyzed for refractometry index (RI) using a Brix refractometer (71 samples). DVSTP concentration and DVRI were significantly correlated with serum IgG concentration. The negative predictive values (NPVs) of DVSTP concentration for detecting serum IgG concentrations<400 mg/dl and<800 mg/dl were 98.2% and 91.3% when the cutoff value is set to 0.4 mg/dl and 0.8 mg/dl, respectively. Furthermore, the NPVs of DVRI for detecting serum IgG concentrations<400 mg/dl and<800 mg/dl were 97.3% and 96.3% when the cutoff value is set to 6% and 10%, respectively. The results suggest that measurement of DVRI is useful in assessing FPT as an initial "stall-side" screening test, because it is easy, inexpensive to perform and allows for rapid interpretation.

Pharmacokinetic assessment of ketanserin in the horse.

The purpose of this study was to determine the pharmacokinetics (PK) of the 5-HT(2A) receptor antagonist ketanserin in healthy adult horses, and to develop a computational model that could be used to optimize dosing. Plasma concentrations of ketanserin were determined using liquid chromatography with mass spectrometry after single and multiple intravenous administration in the horse. A two-compartment linear pharmacokinetic model described the plasma concentration-time profile of ketanserin after single and multiple doses in healthy horses; the terminal half-life was 11.5 h; steady-state volume of distribution was 10.5 L/kg; AUC was 115 ng · h/mL; and clearance was 0.87 L/h/kg. Model simulations followed by the examination in three healthy horses suggest 0.3 mg/kg q.8 h exhibited linear PK and produced consistent systemic blood concentrations of ketanserin above 3 ng/mL.

Clinical assessment of blood glucose homeostasis in horses: comparison of a continuous glucose monitoring system with a combined intravenous glucose and insulin test protocol.

BACKGROUND: The combined glucose-insulin test (CGIT) is helpful for evaluating insulin sensitivity. A continuous glucose monitoring system (CGMS) reports changes in interstitial glucose concentrations as they occur in the blood. Use of the CGMS minimizes animal contact and may be useful when performing a CGIT. HYPOTHESIS: Results obtained using a CGMS are useful for the evaluation of glucose responses during the evaluation of insulin sensitivity in equids. ANIMALS: Seven mature, obese ponies. METHODS: Ponies were equipped with CGMS for determination of interstitial glucose concentrations. Glucose (150 mg/kg, i.v.) and insulin (0.1 U/kg, i.v.) were administered and blood glucose concentrations determined at (minutes after time zero) 1, 5, 15, 25, 35, 45, 60, 75, 90, 105, and 120 with a hand-held glucometer. Blood chemistry results were compared with simultaneously obtained results using CGMS. RESULTS: Concordance coefficients determined for comparison of blood glucose concentrations determined by a hand-held glucometer and those determined by CGMS after the zero time point were 0.623, 0.764, 0.834, 0.854, and 0.818 (for delays of 0, 5, 10, 15, and 20 minutes, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Interstitial glucose concentrations obtained by the CGMS compared favorably to blood glucose concentrations. CGMS may be useful for assessment of glucose dynamics in the CGIT.

Assessment of glucose disposal with the hyperglycaemic clamp technique during low intensity exercise in Warmblood horses.

REASONS FOR PERFORMING STUDY: The quantity of glucose disposal during exercise (walk and trot) compared to rest by use of the hyperglycaemic clamp technique has not been reported previously and has relevance to nutritional requirements. HYPOTHESIS: Exercise (walk and trot) significantly increases glucose disposal compared to rest. METHODS: Seven healthy Dutch Warmblood mares, all in dioestrus, mean ± s.d. age 11.6 ± 2.4 years and weighing 569 ± 40 kg were fasted for 12 h prior to a hyperglycaemic clamp at rest (maintaining a steady state of the blood glucose concentration during 30 min), walk (10 min, 1.5 m/s), trot (20 min, 4.4 m/s), walk (10 min, 1.5 m/s) and rest again (maintaining a steady state during 30 min). Plasma glucose concentrations were measured every 5 min. The mean rate of glucose disposal was calculated by corrections for glucose loss via the glucose space and urine. A one-way ANOVA with post hoc Bonferroni was performed. RESULTS: The mean ± s.d. rate of glucose disposal was 15.0 ± 2.1 at first rest, 25.1 ± 6.2 at first walk, 37.4 ± 9.1 at trot, 33.0 ± 13.1 at second walk and 18.7 ± 4.6 µmol/kg bwt/min at second rest. Values at trot and at second walk differed significantly from values at first rest, whereas values at both rests were similar as well as at first rest and at first walk. CONCLUSIONS: Mean rate of glucose disposal of Warmblood horses increased 2.5 times during trot compared to basal. POTENTIAL RELEVANCE: The hyperglycaemic clamp technique is an attractive nonisotope method to assess the rate of glucose disposal in exercising horses.

Safety pharmacology, toxicology and pharmacokinetic assessment of human Gc globulin (vitamin D binding protein).

Gc globulin is an important protein of the plasma actin-scavenger system. As such, it has been shown to bind free actin and prevent hypercoagulation and shock in patients with massive actin release resulting from severe tissue injuries. Treatment of such patients with Gc globulin could therefore potentially be life-saving. This article presents pre-clinical toxicology experiments conducted on purified plasma-derived human Gc globulin. The Gc globulin formulation was shown to be stable for at least 4 years with full retention of actin-binding capacity. In vitro studies did not reveal activation of the kallikrein system or the complement system and cellular studies showed no toxic effects on a variety of human cell lines. In vivo studies showed no acute toxic effects in mice, rats or guinea pigs upon intravenous infusion. A 14-day local tolerance study in rabbits showed no adverse effects, and 14-day toxicity studies in rats and horses did not show any unwanted reactions. In a 14-day toxicology study in beagle dogs, formation of antibodies was seen and in the end of the study period, three out of four dogs showed clinical immunological reactions, which could be ascribed to the formation of antibodies. The half-life, T, for human Gc globulin was 12 hr in rats, 16 hr in horses and 30 hr in dogs. The safety profile of plasma-derived Gc globulin is concluded to be consistent to that required for use in man.

Fabrication of an implantable fine needle-type glucose sensor using gamma-polyglutamic acid.

Implantable fine needle-type glucose sensors with an outer diameter of less that 0.2 mm were fabricated using a low-cost and non-animal origin polyamide, gamma-polyglutamic acid (PGA) as a glucose oxidase (GOx) immobilizing material. Two types of PGA, gamma-polyglutamic acid (PGAH) and gamma-polyglutamic acid sodium salt (PGANa), were employed to prepare GOx immobilized film by the covalent attachment of GOx using water-soluble carbodiimide (EDC). Nafion/cellulose acetate composite film and polyurethane/polydimethylsiloxane composite film were employed as a permselective inner film and a biocompatible outer film, respectively. The procedure of enzyme-immobilized film fabrication affected the stability of the sensor; that is, GOx immobilized film prepared by pouring a mixture solution of GOx and EDC on a PGA precoated surface showed higher sensor stability than that prepared by pouring a mixture solution of GOx, PGA and EDC. Although, obvious differences in the sensor properties were not observed between the use of PGANa and PGAH, the electrode prepared with PGAH had a lower swelling degree. The glucose sensors prepared with both PGANa and PGAH were practically not affected by the existence of electroactive compounds, such as uric acid, and provided long-term stability for approximately 5 weeks. These sensors also showed good performance in horse serum.