Effects of erythropoietin on systemic hematocrit and oxygen transport in the splenectomized horse

McKeever, Kenneth H.; McNally, Beth A.; Hinchcliff, K. W.; Lehnhard, Robert A.; Poole, David C.

doi:10.1016/j.resp.2016.02.001

Cited by 6 publications

(8 citation statements)

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“…Indeed, although transferrin receptor was not detected on the surface of circulating cells, it was recently found in the exosomes (vesicles) isolated from equine blood plasma (Rout et al, 2015). Stimulation of de novo RBC production in horses was associated with an increase in heterogeneity reflected in an increase in red cell distribution width (RDW), and in most cases, with an increase in mean corpuscular volume (MCV) (Lumsden et al, 1975a,b; Radin et al, 1986; Cooper et al, 2005; Singh et al, 2007; McKeever et al, 2016). Detection of RBC life-span in horses in which stress erythropoiesis was induced by phlebotomy or erythropoietin administration, revealed a trend to faster clearance of cells produced upon stimulation of their de novo production (Lumsden et al, 1975a,b).…”

Section: Introductionmentioning

confidence: 99%

Aging Markers in Equine Red Blood Cells

et al. 2019

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Detection of hematopoietic activity in horses is a challenge due to the lack of cells carrying reticulocyte markers such as RNA remnants or CD71 in the circulation. In this study, we fractionated equine red cells according to their density and analyzed the cells forming low (L), medium (M), and high (H) density fractions for markers of aging such as membrane loss, oxidation, and alterations in the intracellular free Ca 2+ levels. Cells forming L and M fraction were highly heterogeneous in projected areas and shapes, and had higher propensity to swell in response to hypo-osmotic challenge than the cells from the H fraction. The densest cells were deprived of band 3 protein compared to the cells within L or M fraction. Furthermore, the equine red cells from the H fraction were hyper-oxidized compared to the cells within M and L fractions as follows from an increase in autofluorescence characteristic for oxidized damaged hemoglobin and from thiol oxidation as detected using monobromobimane. The lightest cells showed lower free thiol content compared to the red blood cells from the M fraction, but did not contain oxidized hemoglobin. Finally, the majority of red blood cells forming L, M, and H fraction prominently differed from each other in intracellular free Ca 2+ levels and its distribution within the cells. Based on the obtained findings, we suggest that intraerythrocytic Ca 2+ levels and its subcellular distribution, eosin-5-maleimide binding test for band 3 abundance, and autofluorescence of cells along with the changes in red blood cell indices, distribution width and creatine levels may become potential markers of regenerative erythropoiesis in horses. Validation of the power of these potential markers of red cell aging is pending.

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Section: Introductionmentioning

confidence: 99%

Aging Markers in Equine Red Blood Cells

et al. 2019

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“…In another study, McKeever et al tested and proved the hypotheses that rhEPO treatment increases systemic haematocrit, maximal O 2 uptake (VO max , by elevated perfusive and diffusive O 2 conductances) and performance in horses which had been previously splenectomized to avoid confounding effects from the mobilization of splenic red blood cell reserves.…”

Section: Peptide Hormones Growth Factors and Related Substancesmentioning

confidence: 83%

“…In a recent study referring to horse plasma samples, [35] an ELISA test served as a quick screen to detect any sample suspicious for filgrastim which could then be confirmed by LC-MS after immunoaffinity purification followed by trypsin digestion. The LOD of the LC-MS method reported to be less than 0.2 ng/mL In another study, McKeever et al [36] tested and proved the hypotheses that rhEPO treatment increases systemic haematocrit, maximal O 2 uptake (VO max , by elevated perfusive and diffusive O 2 conductances) and performance in horses which had been previously splenectomized to avoid confounding effects from the mobilization of splenic red blood cell reserves.…”

Section: Erythropoietin-receptor Agonistsmentioning

confidence: 99%

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Challenges in detecting substances for equine anti‐doping

Fragkaki

Kioukia‐Fougia

Kiousi

et al. 2017

Drug Testing and Analysis

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The artificial increase of the physical capability of horses using drugs is well known in racing and other equine sports. Both illicit and therapeutic substances are regarded as prohibited substances in competition in most countries. Some countries make distinctions for a few, specific drugs which are, however, allowed for use in other countries. The primary objective in the case of doping control is the detection of any trace of drug exposure, either parent drug or any of its metabolites, using the most powerful analytical methods which are generally based on chromatographic/mass spectrometric techniques. Of major concern in horseracing is the absence of a single organization regulating the anti-doping framework; instead of this, individual racing authorities provide rules and regulations often resulting in variations in the applied doping control programmes of different countries. The aim of this paper is to review the recent literature (approximately from 2012 to mid-2016) to highlight the numerous and diverse challenges faced in doping control of racing and equestrian sports, including the detection of designer drugs (anabolic steroids or stimulants) and of other emerging prohibited substances, such as peptides and noble gases in horse urine and plasma. Moreover, the application of 'omics' techniques (especially of metabolomics) deserves attention for establishing possible fingerprints of drug abuse as well as the evolution of instrumental analysis resulting a powerful ally in the fight against doping in equine sports.

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“…5 The abuse of rHuEPO and its analogs promotes an increase in a splenectomized horse's systemic hematocrit and maximal O 2 uptake. 6 This is a major concern for regulators in the horse racing industry because, similar to many other banished substances, the use of such agents during competition breaches the rule of competition and threatens animal welfare. Horse EPO doping is also associated with health problems that may occur due to production of anti-rHuEPO antibodies that can block endogenous EPO action.…”

Section: Introductionmentioning

confidence: 99%

Detection of ESAs in equine urine and blood by SAR‐PAGE

Cavalcanti

Teixeira

Levy

et al. 2019

Drug Testing and Analysis

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Erythropoiesis‐stimulating agents (ESAs) have been used in horses for doping purposes to increase the performance of these animals in endurance sports. Currently, enzyme‐linked immunosorbent assay (ELISA) and mass spectrometry methods are used to detect ESA abuse in equines. However, the sarcosyl polyacrylamide gel‐electrophoresis (SAR‐PAGE) technique could also be used, since its application in human doping control is well established and has proven to be more sensitive. In this work, the SAR‐PAGE method was used to detect recombinant human erythropoietin (rHuEPO), novel erythropoiesis stimulating protein (NESP), continuous erythropoietin receptor activator (CERA), and fusion protein of erythropoietin with human immunoglobulin heavy chain Fc region (EPO‐Fc) in horse blood and urine. The purification technique for human blood using MAIIA kits worked well for horse samples. The major challenge was horse urine immunopurification, which proved difficult due to filter clogging, but heating and cooling of the horse urine followed by filtration in 30‐kDa molecular weight cut‐off filters solved this problem. The limits of detection (LODs) of 1.3, 1.6, 6.6, and 13.3 pg/mL for rHuEPO, NESP, CERA, and EPO‐Fc, respectively, obtained in spiked urine and 40, 100, 80, and 400 pg/mL for rHuEPO, NESP, CERA, and EPO‐Fc, respectively, acquired in spiked blood are lower than the LODs reported in the literature using liquid chromatography–mass spectrometry (LC–MS) methods. In addition, the presence of ESAs was detected up to 9 days after the administration of microdoses of Hemax (rHuEPO), NESP, and CERA in horse blood and urine. SAR‐PAGE may be implemented in the routine analysis of horse doping control laboratories for screening and confirmation of ESA abuse, mainly due to its high sensitivity for both matrices compared to published mass spectrometric methods.

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Effects of erythropoietin on systemic hematocrit and oxygen transport in the splenectomized horse

Cited by 6 publications

References 59 publications

Aging Markers in Equine Red Blood Cells

Aging Markers in Equine Red Blood Cells

Challenges in detecting substances for equine anti‐doping

Detection of ESAs in equine urine and blood by SAR‐PAGE

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