Effects of two training protocols on Angiotensin I-converting enzyme (ACE) activity in horses

Reasons for performing study: Studies in man have shown a correlation between Angiotensin I‐converting enzyme (ACE) genetic polymorphisms, ACE activity in the blood and superior athletic performance in sports requiring endurance. It has been hypothesised that the same correlation occurs in horses. There is no information in the literature concerning the effects of training on ACE activity in equine plasma. Hypothesis: Exercise training influences the activity of circulating ACE and the response observed is dependent on the exercise protocol. Methods: Thirteen horses of mixed breeds were randomly allocated 2 different training protocols to be carried out for a period of 15 weeks. Blood samples were collected from each horse before the beginning of training to determine baseline values. Subsequent sampling took place every 15 days throughout the training phase and for 8 weeks of paddock rest. Angiotensin I‐converting enzyme activity was determined by automated spectrophotometry. Results: Training for 15 weeks significantly increased plasma ACE activity, irrespective of training protocol. Differences observed in ACE activity pattern between the 2 training protocols were not statistically significant. Increase in ACE activity peaked with maximum workload. As soon as training was interrupted, ACE levels significantly decreased. Conclusions and discussion: Exercise training affects levels of ACE activity in equine plasma. The mechanism for this is not yet elucidated, but cardiovascular adaptation to exercise and blood pressure changes might be involved in this regulation. Potential relevance: Exercise training produced a gradual increase in enzymatic activity and might warrant the use of ACE as a tool for fitness monitoring. Angiotensin I‐converting enzyme enzymatic activity in the plasma might be directly correlated to a change in genetic expression and that variability must be taken into account when evaluating results from horses undergoing a physical training programme.     

Dose-dependent inhibition of angiotensin converting enzyme by enalapril in cats

Although heart failure in cats is treated with angiotensin converting enzyme (ACE) inhibitors, data on the effects of different doses of enalapril on hemodynamics and the inhibition of ACE activity have not been published. To evaluate the effect of enalapril, 0.25, 0.5, or 1.0 mg/kg was given once (s.i.d., p.o.) or twice (b.i.d., p.o.) a day, and plasma ACE activity, indirect blood pressure, and heart rate were measured. Plasma ACE activity and blood pressure fell dose-dependently. There was a biphasic effect on blood pressure with twice daily administration. Enalapril 0.25 mg/kg b.i.d. inhibited plasma ACE activity by 40% after 24 hr, which was almost the same as the effect of 0.5 and 1.0 mg/kg s.i.d., and 0.5 and 1.0 mg/kg b.i.d., while 0.25 mg/kg s.i.d. inhibited it by 23%. Thus, enalapril with a daily dose exceeding 0.5 mg/kg may provide similar efficacy of ACE inhibition in cats.     

Assessment of three variations of the 1,9-dimethylmethylene blue assay for measurement of sulfated glycosaminoglycan concentrations in equine synovial fluid

OBJECTIVE: To determine whether 3 variations of the 1,9-dimethylmethylene blue (DMMB) assay yield comparable results when measuring sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF). SAMPLE POPULATION: 25 samples of SF collected from affected joints of 13 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses. PROCEDURE: Sulfated glycosaminoglycan concentrations were measured by the direct spectrophotometric (ie, Farndale), microplate, and indirect DMMB assays in samples of SF collected from normal and affected joints and in samples digested with nucleases, papain, and hyaluronidase. RESULTS: All 3 assays reacted similarly to standard solutions of sGAGs and digestion of SF samples with nucleases, papain, and hyaluronidase. Nucleic acids were not important interfering substances, and papain and hyaluronidase could not be used interchangeably to digest SE All 3 assays proved to have satisfactory precision (SD < 10%), but each DMMB assay resulted in significantly different measures of sGAG in equine SF. CONCLUSIONS AND CLINICAL RELEVANCE: Samples of SF should be digested with papain or hyaluronidase prior to measurement via DMMB assay. Researchers currently are unable to compare clinical information when variations of the DMMB assay are used, because each DMMB assay yields substantially different sGAG concentrations in SF. Of the 3 assays examined here, we recommend use of the direct spectrophotometric DMMB assay.     

Modulation of the tissue reninangiotensin-aldosterone system in dogs with chronic mild regurgitation through the mitral valve

OBJECTIVE: To investigate whether the tissue and plasma renin-angiotensin-aldosterone system (RAAS) is activated in dogs with mild regurgitation through the mitral valve and determine the contribution of chymase and angiotensin-converting enzyme (ACE) to the activation of the RAAS and potential production of angiotensin II during the chronic stage of mild mitral valve regurgitation. ANIMALS: 5 Beagles with experimentally induced mild mitral valve regurgitation and 6 clinically normal (control) Beagles. PROCEDURES: Tissue ACE and chymase-like activities and plasma RAAS were measured and the RAAS evaluated approximately 1,000 days after experimental induction of mitral valve regurgitation in the 5 dogs. RESULTS: Dogs with experimentally induced mitral valve regurgitation did not have clinical signs of the condition, although echocardiography revealed substantial eccentric hyper- trophy. On the basis of these findings, dogs with mitral valve regurgitation were classified as International Small Animal Cardiac Health Council class Ib. Plasma activity of renin and plasma concentrations of angiotensin I, angiotensin II, and aldosterone were not significantly different between dogs with mitral valve regurgitation and clinically normal dogs. Tissue ACE activity was significantly increased and chymase-like activity significantly decreased in dogs with mitral valve regurgitation, compared with values in clinically normal dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The tissue RAAS was modulated without changes in the plasma RAAS in dogs with mild mitral valve regurgitation during the chronic stage of the condition. An ACE-dependent pathway may be a major route for production of angiotensin II during this stage of the condition.     

Development of a solid-phase assay for measurement of sulfated glycosaminoglycan concentrations in equine synovial fluid

OBJECTIVE: To develop a new 1,9-dimethylmethylene blue (DMMB) assay for measurement of sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF) by use of membrane technology and to compare the assay's ability to measure sGAG concentrations with that of 2 other established DMMB assays. SAMPLE POPULATION: 25 samples of SF collected from affected joints of 14 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses. PROCEDURE: A solid-phase DMMB assay was developed to measure sGAG concentrations in SE Results for the assay were then compared with results obtained by use of the direct spectrophotometric method (ie, Famdale method) and microplate DMMB assay. RESULTS: The solid-phase assay and direct spectrophotometric assay measured the same sGAG concentrations in identical equine SF, but those concentrations differed significantly from results obtained by use of the microplate DMMB assay. All other aspects of the solid-phase DMMB assay were comparable to both the direct spectrophotometric and microplate DMMB assays. CONCLUSIONS AND CLINICAL RELEVANCE: The new solid-phase assay can be used interchangeably with the direct spectrophotometric method to measure sGAG concentrations in equine SF samples, but it cannot be interchanged with the microplate DMMB assay. Results can be rapidly obtained with the solid-phase assay. Also, the solid-phase assay can detect nanogram quantities of sGAGs in SF, circumvent the problem of premature precipitation of sGAG-dye complexes, and provide quantitative or qualitative results. The solid-phase assay may replace other DMMB assays for measuring sGAG concentrations in SF obtained from horses.     

Effects of acute exercise on angiotensin I-converting enzyme (ACE) activity in horses

Angiotensin I-converting enzyme (ACE) level measurement in blood samples is an important tool in human medicine for the detection, treatment and control of diseases such as sarcoidosis and hypertension. Recently ACE has been advocated as being correlated to athletic aptitude in human athletes and a genetic polymorphism has been shown to be responsible for the enzymatic levels in the circulation. The objective of this research was to evaluate the effects of acute exercise in horses in order to increase the understanding of a possible correlation between ACE levels in plasma and performance in equine athletes. A standardised exercise test (SET) to fatigue was conducted on 8 horses and repeated venous blood collections carried out for ACE activity measurements before, during and after the SET. Our results show an increase in ACE activity up to fatigue and a return to baseline values at 30 min post exercise.     

Characterization of the pharmacokinetic and pharmacodynamic properties of the angiotensin-converting enzyme inhibitor, enalapril, in horses

The pharmacokinetics of enalapril (0.5 mg/kg i.v.) and the pharmacodynamics of enalapril (0.5 mg/kg PO) in 5 mares were investigated. After single i.v. dosing, concentrations of enalapril and enalaprilat, its active metabolite, were measured. Two weeks later, enalapril was administered by nasogastric tube. Potassium, creatinine, blood urea nitrogen (BUN), enalapril, and enalaprilat concentrations and angiotensin converting enzyme (ACE) activity were measured in serum. In addition, heart rate, blood pressure, digital venous blood gases, and lactate were measured. Two weeks later, enalapril was again administered by nasogastric tube. To mimic activation of the renin-angiotensin-aldosterone system, angiotensin I (0.5 microg/kg) was administered at fixed intervals, followed by blood-pressure and heart-rate measurement. The elimination half lives of enalapril and enalaprilat were 0.59 and 1.25 hours, respectively, after i.v. administration. After PO administration, enalapril and enalaprilat were not detectable in serum. There was a tendency (P = .0625) toward a decrease in ACE activity 45-120 minutes after enalapril administration, but ACE activity suppression was never > 16%. There was a tendency (P = .0625) toward a decrease in mean arterial pressure (MAP) 6-8 hours after enalapril administration. Serum concentrations of potassium, creatinine, and BUN and digital venous blood gases and lactate concentrations did not change. In response to angiotensin I, there was a tendency (P = .0625) toward a decrease in the MAP response 4-24 hours after enalapril administration. Single-dose enalapril at 0.5 mg/kg PO did not demonstrate significant availability, pharmacodynamic effect, or substantial suppression of ACE activity.     

Analytic validation and comparison of three commercial immunoassays for measurement of plasma atrial/A-type natriuretic peptide concentration in horses

Measurement of atrial/A-type natriuretic peptide (ANP) concentrations may be of use for assessment of cardiac disease, and reliable data on the analytic performance of available assays are needed. To assess the suitability for clinical use of commercially available ANP assays, intra-assay and inter-assay coefficient of variation and dilution parallelism were calculated for three immunoassays (RIA_Pen, RIA_Phoen, and an ELISA_Pen) using blood samples from healthy and diseased horses to cover a wide range of ANP concentrations. Further, agreement between assays was assessed using linear regression and Bland–Altman analyses. For all assays, precision was moderate but acceptable and dilution parallelism was good. All assays showed analytic performance similar to other immunoassays used in veterinary medicine. However, the results from the three assays were poorly comparable. Our study highlights the need for an optimised species-specific assay for equine samples.     

Influence of chemotactic agents on the locomotion of equine polymorphonuclear and mononuclear leucocytes

Subpopulations of equine leucocytes, polymorphonuclear and mononuclear cells, were separated from whole blood on a discontinuous Percoll gradient and used in studies of chemokinesis and chemotaxis. Polymorphonuclear cells responded to the chemo-attractant properties of zymosan-activated plasma in Boyden chamber and agarose microdroplet assays but they responded only slightly (Boyden chamber) or not at all (agarose microdroplet) to the peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). Equine mononuclear cell movement was increased by FMLP in both assay systems and these cells also responded to zymosan activated plasma in the Boyden chamber assay but not in the agarose microdroplet. It is concluded that factors controlling equine polymorphonuclear and mononuclear cell movements into inflammatory exudates may differ.     

Evaluation of commercially available assays for the measurement of equine insulin

Determining circulating equine insulin concentrations is becoming increasingly important in equine clinical practice and research. Most available assays are optimized for human medicine, but there is strong equine cross-reactivity because of the highly conserved nature of insulin. To identify an accurate and reliable assay for equine insulin, 6 commercial immunoassays were evaluated for precision, accuracy, and specificity. Only 1 assay initially reached the requisite standard: Mercodia Equine Insulin Enzyme-linked Immunosorbent assay (ELISA). Plasma matrix interferences were identified when the provided assay buffer was used with the Siemens Count-a-Coat Insulin radioimmunoassay (RIA) but not when charcoal-stripped equine plasma was used as the diluent. This modified RIA and the Mercodia Equine Insulin ELISA were evaluated further by directly examining accuracy by comparing their results for 18 equine plasma samples with values obtained using liquid chromatography and high-resolution/high-accuracy mass spectrometry (LC-MS). Compared with LC-MS measurements, the modified Siemens Insulin RIA rendered a moderate Lin's concordance coefficient (ρ_c) of 0.41, whereas the Mercodia Equine Insulin ELISA rendered a very poor ρ_c of 0.06. This suggests that the Siemens Insulin RIA is appropriate to use for routine evaluations when LC-MS is not available.     

Validation of the Lactate Plus Lactate Meter in the Horse and Its Use in a Conditioning Program

The equine industry has a need for a convenient, rapid, and reliable method of measuring blood lactate concentrations ([LA]). We hypothesized that the handheld Lactate Plus lactate meter (LPlus), developed and tested for use in humans, would provide dependable results when used in horses undergoing an exercise conditioning program and that horse's fitness would improve following individualized conditioning based on each horse's velocity at which [LA] = 4 mmol/L (V_LA4) was reached. Five adult horses underwent a 4-week training program that consisted of 3 exercise bouts/wk. Horses were subjected to an incremental step standardized exercise test (SET) before starting (SET-1) and after the completion of the program (SET-2). Blood samples were collected before each increase in speed until [LA] reached ≥4 mmol/L, and then the SET was terminated. The [LA] sample range in our study was 0–8 mmol/L. Blood was analyzed at the time of collection using a calibrated LPlus, and plasma was collected for [LA] determination using the lactate dehydrogenase–based enzymatic colorimetric method. Although the LPlus tended to significantly underestimate [LA] by 0.39 mmol/L (P < .001), the LPlus proved to be a dependable device for use in horses based on good correlation with the biochemical analysis (r = 0.978) and Bland–Altman limits of agreement and 95% confidence intervals. All horses showed an increase in V_LA4 from SET-1 to SET-2, consistent with improved fitness following our 3 exercise bout/wk training protocol. The LPlus can reliably be used in horses to determine [LA] ranging from 0–8 mmol/L. When determining serial [LA], analytical techniques should not be used interchangeably.     

Use of nutraceuticals in the stallion: Effects on semen quality and preservation

Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from different sources, vitamins and antioxidants, carnitine and botanical extracts. Different nutraceuticals have been highlighted to improve stallion fertility by providing optimal levels of antioxidants, with the most promising results obtained by the combination of PUFAs and antioxidants that resulted to be essential for the maintenance of normal reproductive functions and the reduction of cryodamage in cooled and frozen equine semen.     

Comparison of Pulsed Wave and Color Doppler Myocardial Velocity Imaging in Healthy Dogs

Background: Tissue velocity imaging (TVI) is increasingly used in small animal cardiology. Tissue velocity of the myocardial wall can be measured by pulsed wave (PW) or color Doppler (CD) imaging methods. Currently, the same reference ranges are used for PW TVI and CD TVI methods. However, if and how both methods correlate, and whether they can be used interchangeably, have not been assessed in small animals. Objectives: To compare the results of PW TVI and CD TVI measurements. Animals: Seventy‐one healthy dogs. Methods: Longitudinal myocardial velocity profiles were recorded from the 4‐chamber left apical view. Peak maximal systolic (S), early (E), and late diastolic (A) velocities were measured off‐line in a blinded fashion in the septal and lateral left ventricular wall by PW TVI and CD TVI. Differences between peak PW TVI and CD TVI waves were analyzed by a paired t‐test. Regression analysis and Bland‐Altman difference plots also were used to assess agreement between methods. Results: There was a significant correlation between PW TVI and CD TVI (P < .001). However, S, E, and A waves measured by PW TVI were significantly higher than the CD TVI values (P < .001). Peak systolic and diastolic PW velocities were approximately 2.20 cm/s higher than corresponding mean CD TVI velocities. Conclusions and Clinical Importance: PW TVI measurements are significantly higher compared with CD TVI measurements. Theses differences are clinically relevant. These methods should not be used interchangeably, and different reference ranges for PW TVI and CD TVI should be used.     

Comparison of paired serum and lithium heparin plasma samples for the measurement of serum amyloid A in horses using an automated turbidimetric immunoassay

This study aimed to evaluate whether equine serum amyloid A (SAA) concentrations could be reliably measured in plasma with a turbidimetric immunoassay previously validated for equine SAA concentrations in serum. Paired serum and lithium-heparin samples obtained from 40 horses were evaluated. No difference was found in SAA concentrations between serum and plasma using a paired t test (P = 0.48). The correlation between paired samples was 0.97 (Spearman’s rank P < 0.0001; 95% confidence interval 0.95–0.99). Passing-Bablok regression analyses revealed no differences between paired samples. Bland–Altman plots revealed a positive bias in plasma compared to serum but the difference was not considered clinically significant. The results indicate that lithium-heparin plasma samples are suitable for measurement of equine SAA using this method. Use of either serum or plasma allows for greater flexibility when it comes to sample collection although care should be taken when comparing data between measurements from different sample types.     

Investigation of the Role of Androstenedione-19-oic Acid in the Presence of 19-Norandrostenedione in Intact Male Horse Plasma Using Liquid Chromatography–Tandem Mass Spectrometry

A liquid chromatography–tandem mass spectrometry method was developed to confirm the presence of androstenedione-19-oic acid in intact male equine plasma and to show the source of 19-norandrostenedione in equine plasma. Androstenedione-19-oic acid was recovered from acidified plasma by liquid–liquid extraction using methyl tert-butyl ether and separated on an Ace 5 C₈ column. A triple quadrupole mass spectrometer was used to detect the analytes in negative electrospray ionization mode. Limits of detection, quantification, and confirmation of the method were 0.1, 0.5, and 1.0 ng/mL, respectively. The linear dynamic range of quantification was 0.5–50 ng/mL. The presence of androstenedione-19-oic acid was confirmed in all plasma samples obtained from intact male horses but not those from gelded and female horses; the average concentration was 3.1 ± 1.6 ng/mL, suggesting androstenedione-19-oic acid is an endogenous compound only in intact male horse plasma samples. The conversion of androstenedione-19-oic acid to 19-norandrostenedione in equine plasma was demonstrated by spiking androstenedione-19-oic acid into blank plasma and monitoring the generation of 19-norandrostenedione and its increase in concentration during storage. Results indicated that androstenedione-19-oic acid was readily converted into 19-norandrostenedione; the higher the storage temperature, the faster the conversion. The conversion was not affected by the types of plasma samples collected from gelded and female horses or by anticoagulants used in blood collection to harvest plasma. Compared with other matrices such as water, methanol, and phosphate-buffered saline, the conversion of androstenedione-19-oic to 19-norandrostenedione in equine plasma was faster, suggesting that there is an unknown factor(s) in equine plasma that enhances the conversion.     

Evaluation of a new handheld point-of-care blood gas analyser using 100 equine blood samples

Objectives

To determine whether the Enterprise point-of-care blood analysis system (EPOC) produces results in agreement with two other blood gas analysers in regular clinical use (i-STAT and Radiometer ABL77) and to investigate the precision of the new machine when used with equine whole blood.

Association between circulating angiotensin-converting enzyme and exercise-induced pulmonary haemorrhage in Thoroughbred racehorses

Exercise-induced pulmonary haemorrhage has an impact on racehorse performance. Although endoscopic diagnosis (with or without the aid of bronchoalveolar lavage) is considered to be the standard diagnostic method for this condition, the use of biomarkers that could aid in quantifying risk and severity of the condition would represent an advance in equine sport medicine. This preliminary research investigated the use of angiotensin-converting enzyme (ACE) activity in plasma of racehorses and demonstrated that ACE activity is increased in horses with higher degrees of haemorrhage and is a promising biomarker for EIPH in racehorses.     

Comparison of enzyme-linked immunosorbent assay and immunofluorescence test for determination of anti-Encephalitozoon cuniculi antibodies in sera from rabbits with different clinical and histopathological presentations

Encephalitozoon cuniculi is a microsporidian that infects rabbits resulting in a varied pathology of the nervous, renal, and ocular tissues. Serology based studies using different methods have been conducted indicating a high seroprevalence worldwide in pet rabbits. While qualitative results are not especially helpful in the diagnosis of this infection, quantitative titers can have increased predictive value. In the current study, results of enzyme-linked immunosorbent assay (ELISA) and immunofluorescence test methods were compared using banked samples from rabbits with confirmed infection and clinically normal rabbits. Pearson's analysis found good and significant correlation (R = 0.56, P = 0.0002). Bland Altman analysis demonstrated the methods were similar but not equivalent. The sensitivity ranged from 0.79 to 0.84 and the specificity was 1.00 for both assays. Anti-E. cuniculi IgM titers were also determined by ELISA to be an independent measure from IgG (R = 0.31, P = 0.06) with a sensitivity of 0.24 and specificity of 1.00. In total, these results confirm an agreement in the detection of IgG antibody by two different methods conducted at laboratories in the United States and Austria that are involved in research and clinical diagnostic testing of this agent.     

The effect of enalapril on furosemide‐activated renin–angiotensin–aldosterone system in healthy dogs

Studies in our laboratory have revealed that furosemide‐induced RAAS activation, evaluated via the urine aldosterone‐to‐creatinine ratio (UAldo:C), was not attenuated by the coadministration of benazepril, while enalapril successfully suppressed amlodipine‐induced urinary aldosterone excretion. This study was designed to evaluate the efficacy of enalapril in suppressing ACE activity and furosemide‐induced circulating RAAS activation. Failure to do so would suggest that this failure may be a drug class effect. We hypothesized that enalapril would suppress ACE activity and furosemide‐induced circulating RAAS activation. Sixteen healthy hound dogs. The effect of furosemide (2 mg/kg PO, q12 h; Group F) and furosemide plus enalapril (0.5 mg/kg PO, q12 h; Group FE) on circulating RAAS was determined by plasma ACE activity, 4–6 h post‐treatment, and urinary A:C on days ?1, ?2, 1, 4, and 7. There was a significant increase in the average urine aldosterone‐to‐creatinine ratio (UAldo:C) after administration of furosemide (P < 0.05). Enalapril inhibited ACE activity (P < 0.0001) but did not significantly reduce aldosterone excretion. A significant (P < 0.05) increase in the UAldo:C was maintained for the 7 days of the study in both groups. Enalapril decreased plasma ACE activity; however, it did not suppress furosemide‐induced RAAS activation, as determined by the UAldo:C. While enalapril blunts ACE activity, the absence of circulating RAAS suppression may be due to angiotensin II reactivation, alternative RAAS pathways, and furosemide overriding concurrent ACE inhibition, all indicating the existence of aldosterone breakthrough (ABT). Along with similar findings with benazepril, it appears that failure to suppress aldosterone suppression with furosemide stimulation may be a drug class effect. The discrepancy between the current data and the documented benefits of enalapril likely reflects the efficacy of this ACE inhibitor in suppressing tissue RAAS, variable population responsiveness to ACE‐inhibition, and/or providing additional survival benefits, possibly through as yet unknown mechanisms.     

The effect of an angiotensin-converting enzyme inhibitor on water and electrolyte balance in water-restricted sheep

The importance of angiotensin II in the regulation of water and electrolyte balance in sheep is questionable. In this trial the effects of an angiotensin-converting enzyme (ACE) inhibitor were quantified in sheep on restricted water intake. Comparing the phase of water restriction only with that of water restriction plus ACE inhibition, significant increases were observed during the latter phase in urine volume, sodium and potassium excretion via the urine, sodium concentration in the plasma and osmolar clearance. Urine osmolarity decreased with inhibition of angiotensin II formation while variables such as water, sodium and potassium loss via the faeces were unaffected. Most of the renal effects of ACE inhibition, except the increase in urinary potassium excretion, were explicable in terms of the established functions of angiotensin II. Furthermore, results of this trial indicate that angiotensin II has no significant effect on the intestine in regulating water and electrolyte excretion via the faeces.