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Effect of Dietary Starch, Fat, and Bicarbonate Content on Exercise Responses and Serum Creatine Kinase Activity in Equine Recurrent Exertional Rhabdomyolysis

Effect of Dietary Starch, Fat, and Bicarbonate Content on Exercise Responses and Serum Creatine... To determine the effect of dietary starch, bicarbonate, and fat content on metabolic responses and serum creatine kinase (CK) activity in exercising Thoroughbreds with recurrent exertional rhabdomyolysis (RER), 5 RER horses were fed 3 isocaloric diets (28.8 Mcal/d [120.5 MJ/d]) for 3 weeks in a crossover design and exercised for 30 minutes on a treadmill 5 days/wk. On the last day of each diet, an incremental standardized exercise test (SET) was performed. The starch diet contained 40% digestible energy (DE) as starch and 5% as fat; the bicarbonate-starch diet was identical but was supplemented with sodium bicarbonate (4.2% of the pellet); and the fat diet provided 7% DE as starch and 20% as fat. Serum CK activity before the SET was similar among the diets. Serum CK activity (log transformed) after submaximal exercise differed dramatically among the diets and was greatest on the bicarbonate-starch diet (6.51 1.5) and lowest on the fat diet (5.71 0.6). Appreciable differences were observed in the severity of RER among individual horses. Postexercise plasma pH, bicarbonate concentration, and lactate concentration did not differ among the diets. Resting heart rates before the SET were markedly lower on the fat diet than on the starch diet. Muscle lactate and glycogen concentrations before and after the SET did not differ markedly among the diets. A high-fat, low-starch diet results in dramatically lower postexercise CK activity in severely affected RER horses than does a low-fat, high-starch diet without measurably altering muscle lactate and glycogen concentrations. Dietary bicarbonate supplementation at the concentration administered in this study did not prevent increased serum CK activity on a high-starch diet. Key words: Exertional myopathy; Horse; Nutrition; Tying up. xertional rhabdomyolysis is a common and frustrating condition that affects many breeds of horses. Previously, it was thought to be a singular entity arising from the production of lactic acid and protons in muscle after excessive glycogenolysis in horses that were exercised after rest on a high-carbohydrate diet.1 It is now known that several distinct conditions comprise this syndrome, and lactic acidosis is not a factor in the pathophysiology of these diseases.2,3 Polysaccharide storage myopathy (PSSM) was 1st identified as a myopathy of quarter horses and, later, of draft breeds in the 1990s and is characterized by an increased clearance of glucose from the bloodstream and the accumulation of glycogen and abnormal complex polysaccharide within muscle cells.4,5 Recurrent exertional rhabdomyolysis (RER) has been identified as a common cause of pain and muscle necrosis in Thoroughbred racehorses, affecting approximately 5% of this population, and appears to constitute a heritable stress-related defect in intracellular From the Department of Clinical and Population Sciences, College of Veterinary Medicine, University of Minnesota, St Paul, MN (McKenzie, Valberg, Godden, MacLeay); Kentucky Equine Research Inc, Versailles, KY (Pagan, Geor); and the Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA (Carlson). Dr Geor is presently affiliated with R & J Veterinary Consultants Inc, Guelph, Ontario, Canada. Dr MacLeay is presently affiliated with the Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Fort Collins, CO. Previously presented at the 19th Annual Forum of the American College of Veterinary Medicine, Denver, CO, May 23–26, 2001. Reprint requests: Erica C. McKenzie, BSc, BVMS, Comparative Exercise Physiology Laboratory, 264 McElroy Hall, Oklahoma State University, Stillwater, OK 74078; e-mail: mcke0174@tc.umn.edu. Submitted October 25, 2002; Revised January 8, 2003; Accepted February 5, 2003. Copyright 2003 by the American College of Veterinary Internal Medicine 0891-6640/03/1705-0012/$3.00/0 calcium (Ca2 ) regulation.6,7 In horses with PSSM or RER, muscular pain and stiffness usually are elicited by exercise, with concurrent increases in serum creatine kinase (CK) activity. Subclinical episodes of muscular necrosis also may occur.8 Despite major advances in the understanding of the etiology and pathophysiology of these conditions, identifying consistently successful treatment and management strategies has proved challenging. A diet high in soluble carbohydrates (starch) is believed to be a predisposing factor for episodes of rhabdomyolysis in horses with PSSM or RER, but the pathophysiology and signalment associated with the 2 conditions appear to be entirely distinct.3,9 A reduction in daily grain intake is a common management strategy for both diseases. Dietary sodium bicarbonate supplementation also is frequently used in practice to manage RER horses, likely because of the false notion that lactic acid accumulation can cause rhabdomyolysis.1 Dietary fat supplementation in horses has been associated with enhanced aerobic and anaerobic performance, decreased thermal stress, decreased gut fill and water requirement, and calmer demeanor.10–13 Recently, dietary fat supplementation has been advocated for exertional rhabdomyolysis to allow exercising horses to maintain a high caloric intake without additional dietary starch.14,15 However, few standardized dietary trials to investigate the effect of fat on equine rhabdomyolysis have been performed. Many previous trials have used a variety of breeds of horses; different exercise protocols with varying degrees of fitness, exertion, and confinement; and several rations that varied in palatability as well as in type and amount of dietary fat. In these trials, success was judged subjectively by the owners and, in some instances, by the intermittent determination of serum CK activity after highly variable degrees of exertion.15,16 Exercise intensity and frequency, stall confinement, diet, and degree of fitness have been shown to influ- McKenzie et al ence the occurrence of subclinical and clinical rhabdomyolysis.3,17–20 Thus, to date, controlled dietary trials investigating the impact of dietary fat on equine rhabdomyolysis are few, and further investigation is warranted. In PSSM horses, a low-starch diet reduces glycogen accumulation in muscle and, when combined with a daily exercise regime, has been fairly successful in ameliorating the signs of rhabdomyolysis in these horses.15,16,18,21,22 A previous dietary trial in RER horses found that a high-energy, high-starch diet (28.8 Mcal/d [120.5 MJ/d]) resulted in dramatic increases in serum CK activity after treadmill exercise. Lowering the caloric intake to 21.4 Mcal/d (89.5 MJ/d) prevented marked increases in postexercise serum CK regardless of whether the calories were provided as starch or fat.3 However, to our knowledge, the effect of a high-energy, low-starch diet providing a large percentage of daily calories from fat on exercising horses with RER has not been investigated. The purpose of the current study was to assess the effects of 3 isocaloric high-energy diets (28.8 Mcal/d [120.5 MJ/d]) varying in starch, fat, and bicarbonate content on postexercise serum CK activity in Thoroughbred horses with RER undergoing treadmill exercise. The objective was to determine whether the source of calories in a high-calorie diet would affect the occurrence of episodes of muscle necrosis in RER horses and to determine the palliative effects, if any, of dietary sodium bicarbonate on the occurrence of RER. Table 1. Digestible energy (DE) and percentage of electrolyte and mineral composition of the 3 rations (hay plus pellet) on a dry matter basis. Ration Type Variable % sodium % potassium % chloride % magnesium % phosphorus % calcium % sulfur Total DE in Mcal/d (MJ/d) % DE from starch % DE from fat DE, digestible energy. Starch 0.84 1.39 1.56 0.23 0.47 0.69 0.14 28.8 (120.5) 40.0 5.0 BicarbonateStarch 1.3 1.35 1.55 0.22 0.64 0.71 0.15 28.8 (120.5) 40.0 5.0 Fat 0.80 1.54 1.47 0.27 0.42 0.97 0.19 28.8 (120.5) 7.0 20.0 incorporated into this pellet to form the bicarbonate-starch feed. Starch supplied 40% and fat 5% of the daily DE for these 2 diets. The highfat pellet was composed of 54% soy hulls, 25% rice bran,b 7% solvent extracted soybean meal, 6% allofat, 5% wheat middlings, and 2.5% pellet binder. On the fat diet, starch supplied 7% and fat 20% of the daily DE. After formulation, all diets were analyzed by a commercial laboratoryc for electrolyte and mineral concentrations and starch and fat content. Materials and Methods Horses A 3-year-old Thoroughbred stallion and 5 Thoroughbred mares (2, 6, 9, 11, and 13 years old) with RER were used. Horses were selected on the basis of criteria used in previous studies to define RER, including a history of clinical rhabdomyolysis, increases in serum CK after exercise, and an abnormally low threshold for intercostal muscle contracture in the presence of caffeine and halothane.6,23 Experimental Design A replicated 3 3 randomized design was used with 6 horses. The pelleted feeds were fed at 0.036 Mcal/kg (0.151 MJ/kg) body weight (1% of body weight), divided into 2 feedings per day. Horses were introduced to the pellet with a gradually increasing amount of the starch pellet over a 5-day period before commencement of the trial. Two horses began on the bicarbonate-starch diet, 2 horses began on the fat diet, and 2 horses began on the starch diet. After 5 days, a 6year-old mare on the starch diet was removed from the trial because of injury, and the remaining horses continued on the diets as planned. Each diet was fed for a total of 21 days. Gradually increasing amounts of the next diet to be consumed were mixed with the previous diet over a 1-week washout period to avoid sudden changes. All 3 pellets were highly palatable and were readily consumed by all horses. Training To establish a similar plane of fitness, horses were exercised 5 days/ wk for a total of 5 weeks on a high-speed treadmill before the trial. Horses performed alternating intervals of walk (1.9 m/s), trot (4.0 m/s), and canter (7.0 m/s). Daily exercise gradually was increased to a maximum of 30 minutes/d. On the Wednesday of the last 2 training weeks, the final canter interval was replaced by a 2-minute gallop (11 m/s) with the treadmill inclined to a 6% slope. Throughout the study, all horses were confined to a stall when they were not exercising on the treadmill and were rested on the Saturday and Sunday of every week. Daily Exercise Regime Daily exercise throughout the trial consisted of alternating 2-minute intervals of walk, trot, and canter for a total of 30 minutes, 5 days/ wk. On the Wednesday of the 1st 2 weeks on each diet, the last canter interval was replaced by a 2-minute gallop (11 m/s) on a 6% slope, designed to achieve a heart rate of 200 beats/min. During the week, when diet changeovers were occurring, the horses continued to undergo the same daily exercise routine, but postexercise CK activity was not measured. Horses did not gallop on the Wednesday during the 3rd week of each diet when the standardized exercise test (SET) was to be performed. Diets All horses were fed grass hay at 0.022 Mcal/kg (0.092 MJ/kg) body weight per day (1.2% of body weight). Three isocaloric pelleted feeds were designed that, in combination with hay, provided 28.8 Mcal (120.5 MJ) in digestible energy (DE) per day (Table 1). All 3 pellets were supplemented with additional dicalcium phosphate, sodium chloride, and a combined vitamin, electrolyte, and mineral supplementa to meet National Research Council recommendations.24 The pellet of the starch and bicarbonate-starch diets was composed of approximately 38% ground corn, 32% wheat middlings, 15% oats, 10% soy meal and hulls, and 4% molasses. Sodium bicarbonate at 4.2% dry matter was Daily Sample Collection Immediately postexercise throughout the trial, jugular venous blood was collected into lithium heparin blood gas syringes for the measurement of plasma pH, bicarbonate (HCO3 ), ionized calcium (Ca2 ), sodium (Na ), and potassium (K ) concentrations. On the last 5 exercise days of each diet, venous blood gas samples also were obtained Diet and Equine Exertional Rhabdomyolysis Table 2. Mean ( SD) preexercise plasma acid-base values and electrolyte concentrations in 5 Thoroughbreds with recurrent exertional rhabdomyolysis (RER) consuming 3 diets varying in starch, bicarbonate, and fat content. Variable Blood pH Bicarbonate (mmol/L) Total CO2 (mmol/L) Base excess Ionized calcium (mmol/L) Sodium (mmol/L) Potassium (mmol/L) N 7.40 30.4 31.9 5.6 1.53 129 4.2 Starch 5 (n 30) 0.02 1.95 2.03 1.79 0.10 3.35 0.67 Bicarbonate-Starch N 5 (n 30) 7.40 30.8 32.3 6.1 1.53 129 4.0 0.02 1.84 1.93 1.60 0.08 3.43 0.51 A A A A A A A Fat 5 (n 30) A B B B A A B AB AB AB A A A N, number of horses; n, total number of samples; RER, recurrent exertional rhabdomyolysis. a Differing letters indicate significant differences attributable to diet among horses. immediately before exercise. Four hours after daily exercise, blood was obtained for the measurement of serum CK activity. compound symmetry correlation structure, and repeated on subject horse. The main effects that were offered to the models used in the analysis included diet (starch, bicarbonate-starch, or fat), day (Monday–Friday), and a diet day interaction term. A preliminary review of descriptive statistics by the Proc Univariate showed that raw serum CK data were not normally distributed (skewness statistic value for all measures, 10.6; mean skewness value by diet, 5.0 [range, 3.4–7.7]; mean skewness by horse, 2.9 [range, 1.6–4.9]). All other blood parameters measured were normally distributed. Because normality is one of the 3 assumptions required for the results of repeated-measures analysis of variance to be valid, serum CK data were transformed to the natural logarithm (ln) before statistical analysis. Significance was set at P .05. Further analysis of variance (Proc Mixed in SAS26) included horse as a fixed effect in the model statement as well as a horse diet interaction term. As a final alternative approach to transforming the CK data, a nonparametric approach to analysis of the raw CK data also was completed26,f by the Wilcoxon statistic, after categorizing the CK data into one of 6 categories: (1) 404 U/L, (2) 404– 999 U/L, (3) 1,000–4,999 U/L, (4) 5,000–99,999 U/L, (5) 10,000– 999,999 U/L, and (6) 100,000 U/L. Standardized Exercise Test On the final Thursday (horses 1–3) or Friday (horses 4–6) of each diet, a near-maximal SET was performed by each horse. Before the SET, concurrent urine and serum samples were obtained from all horses to calculate the urinary fractional excretion of Ca2 , phosphorus (P), magnesium (Mg2 ), Na , K , and chloride (Cl ). The SET consisted of a 12-minute step test. Initially, horses walked at 1.9 m/s for 2 minutes. The treadmill then was inclined to a 6% slope, with 2 additional minutes of walking. Horses subsequently performed 2-minute increments of trot (4.5 m/s) and canter (7 m/s) and two 2-minute gallop intervals at 10 and then 11 m/s, gauged to achieve a peak heart rate of approximately 200 beats/min. Heart rate was recorded as the horses stood on the treadmill before commencement of the SET, during the last 15 seconds of each speed increment during the SET, and 5 minutes after the SET with the Equistat heart rate monitor (model HR8AE).d Serial blood samples for measurement of blood lactate concentrations were drawn through a preplaced jugular catheter at the same intervals as for heart rate measurement. Before and 4 hours after the SET, a serum sample was obtained for analysis of serum CK activity. Immediately before and after the SET, samples of the middle gluteal muscle were obtained 15 cm along a straight line from the top of the tuber coxa to the point of the tail through a single incision at a depth of 8 cm with a modified Bergstrom needle. Muscle samples for measurement of muscle lactate and glycogen concentrations were immediately frozen in liquid nitrogen and stored at 80 C for later analysis. Results Five of the 6 horses completed the trial successfully. One mare required 2 days of rest while consuming the starch diet because of postexercise stiffness and muscle cramping on the second Monday of the starch diet (CK, 109,650 U/L). Body weight did not differ markedly among the diets throughout the study (starch, 496.8 75.9 kg; bicarbonatestarch, 493.6 82.5 kg; and fat, 494.1 74.7 kg). Analysis of Samples Blood gas samples were stored on ice and analyzed within 30 minutes with a blood gas analyzer. Serum concentrations of Ca2 , P, Mg2 , Na , K , and Cl as well as serum CK activity were measured on an automated chemistry analyzer. Urine concentrations of Na , K , Ca2 , P, and Mg2 were determined by emission spectrometry, and urine Cl and creatinine concentrations were measured on an automated chemistry analyzer after preparation by a method described in a previously published study.23 Plasma lactate concentrations were determined with an automated lactate analyzer. Muscle for glycogen and lactate analysis was freeze-dried and dissected free of blood, fat, and connective tissue. Samples for glycogen analysis were boiled for 2 hours in 1 M HCl to produce glucose residues. Glucose residues and lactate concentrations were determined by fluorometric analysis according to the methods of Lowry and Passonneau (1973).25 Preexercise Plasma Acid-Base Status and Electrolyte Concentrations At rest, consumption of the bicarbonate-starch diet resulted in significantly mild increases in plasma HCO3 and total carbon dioxide concentrations and plasma base excess compared to the fat diet (Table 2). The plasma K concentration was markedly greater in horses consuming the fat diet than in horses consuming the other 2 diets. Postexercise Plasma Acid-Base Status, Electrolytes, and Minerals Exercise resulted in an increase in the plasma K concentration and decreases in plasma base excess, ionized Ca2 , total carbon dioxide, and HCO3 concentrations on all diets compared to preexercise values (Tables 2, 3). No differences were observed among any diets in post- Statistical Analysis Serum results were analyzed by repeated-measures analysis of variance26,e by means of a maximum likelihood estimation, specifying a McKenzie et al Table 3. Mean ( SD) and postexercise plasma acid-base values and electrolyte concentrations in 5 Thoroughbreds with RER consuming 3 diets varying in starch, bicarbonate, and fat content performing submaximal exercise or submaximal exercise including a 2-minute gallop on a 6% incline. Submaximal Exercise Variable Blood pH Bicarbonate (mmol/L) Total CO2 (mmol/L) Base excess Ionized calcium (mmol/L) Sodium (mmol/L) Potassium (mmol/L) Raw serum CK (U/L) ln(CK) N Starch 5 (n 55) 0.03 A 3.2 A 3.2 A 3.2 A 0.07 A 3.5 A 0.7 A 14,954 A 1.28 A Submaximal Exercise plus Gallop Interval N Fat 5 (n 7.41 29.2 30.6 5.0 1.46 129 5.3 387 5.71 54) 0.04 A 3.1 A 3.2 A 3.2 A 0.09 B 3.1 B 1.0 A 403 A 0.63 C N Starch 5 (n 10) 7.35 24.4 25.6 0.01 1.40 129 5.9 877 6.07 0.05 A 5.5 A 5.7 A 5.4 A 0.05 A 3.2 A 1.0 A 1,535 AB 1.06 B N Bicarbonate 5 (n 10) 7.35 23.9 25.1 0.35 1.38 130 5.5 2,560 6.64 0.06 A 5.4 A 5.6 A 5.4 A 0.05 A 3.5 A 1.1 A 4,063 A 1.69 A N Fat 5 (n 7.34 23.1 24.3 1.51 1.40 129 5.6 574 5.93 10) 0.05 A 4.4 A 4.5 A 4.5 A 0.07 A 2.5 A 1.3 A 680 B 0.87 B Bicarbonate 5 (n 54) 7.40 30.0 30.3 4.7 1.41 129 5.3 0.04 A 3.3 A 3.4 A 3.3 A 0.08 A 3.3 B 0.9 A 6,186 A 1.50 B 7.40 28.8 30.1 4.6 1.41 127 5.5 3,064 6.24 2,618 6.51 N, number of horses; n, total number of samples; RER, recurrent exertional rhabdomyolysis; CK, creatine kinase; ln, natural logarithm. a Differing letters indicate significant differences in parameters between diets in horses undergoing submaximal exercise or submaximal exercise plus a 2-minute gallop interval. exercise acid-base parameters in horses undergoing daily submaximal exercise (Table 3). The postexercise plasmaionized Ca2 concentration was significantly increased on the fat diet compared to the other 2 diets. A slightly but significantly lower postexercise plasma Na concentration was observed with the starch diet than with the other diets after submaximal exercise. The inclusion of a 2-minute gallop interval to the exercise routine resulted in a decrease in plasma pH and base excess as well as in plasma concentrations of HCO3 and total carbon dioxide compared to nongallop days (Table 3). Higher plasma concentrations of Na and K were observed after exercise on gallop days than on other days of the week. No significant difference was observed among the diets with regard to acid-base parameters or plasma electrolyte concentrations on gallop days (Table 3). Four-Hour Postexercise Serum CK The mare that was consuming the high-starch diet and dropped from the trial after 5 days of exercise had a mean CK of 383 U/L for the 5-day period (range, 285–948 U/L). For the 5 horses that completed the study, the raw mean postexercise serum CK activity after daily submaximal exercise was within the normal range (57–404 U/L) when they consumed the high-fat diet but was 7.9 times higher when they consumed the starch and bicarbonate-starch diets (Table 3). Repeated-measures analysis of variance identified a significant effect of both diet and day on postsubmaximal exercise values for the ln(CK). No significant interaction was observed between the terms describing diet and day. Subsequent contrast analysis showed that a significant difference in the ln(CK) existed among all 3 diets, with the postexercise ln(CK) being highest on the bicarbonate-starch diet and lowest on the fat diet (Fig 1). The statistical power to detect a treatment difference with the respective means, standard deviations, and sample sizes for the ln(CK) (1tailed test, .05) was .36 between the starch and bicarbonate-starch diets, .98 between the bicarbonate-starch and fat diets, and .82 between the starch and fat diets. Nonparametric testing of the untransformed CK data provided identical inferences (data not shown). Postgallop serum CK activity (ln) was significantly greater in horses consuming the bicarbonate-starch diet than in horses consuming the other diets (Table 3). Within each diet, no significant difference in serum CK activity (raw means or ln) was ob- Fig 1. Scatterplot of 4-hour postexercise serum creatine kinase (CK) activity in 5 horses with recurrent exertional rhabdomyolysis (RER) consuming 3 diets that varied in starch, fat, and bicarbonate content. Horizontal bars indicate median serum CK activity for each diet. Differing superscript letters indicate significant differences attributable to diet in mean natural logarithm (ln) CK activity 4 hours postexercise. N, number of plasma samples; N 65 (starch diet); N 64 (bicarbonate-starch and fat diets). Diet and Equine Exertional Rhabdomyolysis Day-of-Week Effect Across diets, a significant effect of day of the week on serum CK activity was observed on the analysis of logtransformed postexercise serum CK data. Serum CK activity (ln) on Monday was significantly higher than on Tuesday (P .06) as well as on the remaining days of the week (P .05). Individual statistical examination of the diets revealed that on the bicarbonate-starch diet, the ln(CK) on Monday was significantly higher than on Wednesday, Thursday, and Friday (Table 4). In horses consuming the fat diet, the ln(CK) on Monday was different from that on all other days except for Wednesday, when horses galloped. Fig 2. Scatterplot of 4-hour postexercise serum creatine kinase (CK) activity for 5 individual horses (TO, MG, MT, NA, and ST) with recurrent exertional rhabdomyolysis (RER) consuming 3 diets that varied in starch (triangles), fat (circles), and bicarbonate content (squares). Horizontal bars indicate median serum CK activity for each horse. Standardized Exercise Test served between 30 minutes of submaximal exercise compared to 28 minutes of submaximal exercise plus a 2-minute gallop interval. Subsequent analysis of variance indicated that a significant horse diet interaction existed; therefore, analysis was repeated after stratifying the data by horse. For stratified analysis, a significant effect of diet on ln(CK) values for horses 1 (NA), 3 (TO), and 4 (MA) was observed, with values being the lowest for the fat diet. There was a tendency for an effect of diet on the ln(CK) for horse 5 (MT, P .1) and no effect of diet on the ln(CK) for horse 2 (ST, P .3). Significant individual variation was observed in postexercise serum CK activity (Fig 2). For example, horse TO had the highest postexercise serum CK activity of all the horses when consuming the starch diet (mean CK, 12,014 U/L) and bicarbonate-starch diet (mean CK, 9,803 U/L) but had lower postexercise serum CK activity when consuming the high-fat diet (mean CK, 958 U/L). In comparison, 2 horses (NA and ST) had serum CK activity 1,000 U/L on all diets; therefore, diet had little effect regarding abnormal increases in postexercise serum CK activity for these 2 horses. Pre-SET fractional excretion values of Cl , K , Ca2 , Mg2 , and P did not differ significantly among the diets and were within previously published normal ranges for horses.27–30 The pre-SET fractional excretion of Na was significantly increased in horses consuming the bicarbonate-starch diet (0.55 0.49%) compared to that of horses consuming the starch diet (0.05 0.03%) and the fat diet (0.07 0.07%). Diet had no significant impact on postSET plasma acid-base parameters or plasma electrolyte concentrations (data not shown). The resting heart rate (beats per minute) measured in horses standing on the treadmill before each SET was significantly lower in horses consuming the fat diet (36 5.4 beats/min) than in horses consuming the starch diet (43 2.7 beats/min). The pre-SET heart rate in horses consuming the bicarbonate-starch diet was not different from that in horses consuming the other 2 diets (40 2.9 beats/min). No significant dietary effect on maximal heart rate was observed during the SET or on the mean 5-minute post-SET heart rate (Table 5). Diet had no effect on resting, peak, or 5-minute post-SET plasma lactate concentrations and no effect on pre-SET or post-SET serum CK activity or the magnitude of change in CK activity with the SET. Table 4. Mean 4-hour postexercise serum creatine kinase activity (CK U/L) in 5 Thoroughbreds with RER consuming 3 diets that vary in starch, bicarbonate, and fat content undergoing daily exercise.a Starch Day Monday Tuesday Wednesday Thursday Friday CK (U/L) N 5, n 65 8,301 28,096 (173–109,650) 1,611 3,784 (173–14,907) 787 1,309 (173–5,173) 724 1,109 (211–3,816) 717 1,018 (184–3,356) ln(CK) N 5, n 65 6.59 6.23 6.02 6.05 6.05 1.73 1.29 0.99 0.91 0.96 Ab A A A A Bicarbonate-Starch CK (U/L) N 5, n 64 4,255 9,478 (152–36,060) 2,891 6,390 (160–24,740) 1,935 3,404 (142–12,840) 1,857 3,167 (164–10,200) 1,475 2,710 (165–9,024) ln(CK) N 5, n 64 6.78 6.54 6.48 6.37 6.36 1.74 1.61 1.47 1.55 1.31 A A B B B Fat CK (U/L) N 5, n 64 546 648 (186–2,688) 368 359 (147–1,483) 502 567 (160–2,285) 298 210 (158–775) 296 166 (151–632) N ln(CK) 5, n 64 0.74 0.64 0.77 0.55 0.50 A B A B B N, number of horses; n, total number of samples; CK, creatine kinase; RER, recurrent exertional rhabdomyolysis; ln, natural logarithm. a CK values are presented as raw and log-transformed (ln) mean ( SD). The range of CK values is presented in brackets. b Differing letters indicate significant differences in ln(CK) between Monday and other days of the week within each diet. McKenzie et al Table 5. Mean ( SD) parameters before and immediately after the standardized exercise test (SET) in Thoroughbred horses with recurrent exertional rhabdomyolysis consuming 3 diets varying in starch, bicarbonate, and fat content. Variablea Peak SET HR Post-SET HR Preplasma lactate Peak plasma lactate Postplasma lactate Pre-SET CK (U/L) Post-SET CK Premuscle glycogen Postmuscle glycogen muscle glycogen Premuscle lactate Postmuscle lactate muscle lactate N Starch 5, n 15 17.2 A 8.3 A 0.18 A 2.53 A 3.29 A 324 A 275 A 57.1 A 67.3 A 32.9 A 33.5 A 65.0 A 33.0 A Bicarbonate-Starch N 5, n 15 205 80 0.74 8.22 7.12 296 409 676.7 597.1 79.5 114.8 149.0 34.2 12.8 A 3.4 A 0.22 A 3.39 A 3.75 A 176 A 285 A 72.0 A 100.6 A 48.6 A 40.6 A 69.7 A 32.6 A N 199 77 0.78 6.56 5.23 222 293 692.5 570.6 121.9 112.6 161.6 49.0 Fat 5, n 11.7 A 10.9 A 0.15 A 3.40 A 3.37 A 35 A 90 A 147.0 A 146.7 A 126.1 A 12.42A 18.1 A 27.7 A SET, standardized exercise test; HR, heart rate in beats per minute; CK, serum creatine kinase activity; N, number of horses; n, total number of samples. a difference between preexercise and postexercise muscle glycogen and lactate values. Plasma lactate is measured as mmol/L and serum CK as U/L. Muscle glycogen and lactate concentrations are measured as mmol/kg dry weight. b Letters indicate statistical comparison in parameters between diets. Muscle Glycogen and Lactate Muscle glycogen concentration did not differ significantly among the diets before or after the SET (Table 5). No significant difference in pre-SET and post-SET muscle lactate concentrations was found among the diets. Diet did not significantly affect the magnitude of change in muscle glycogen and lactate concentrations with exercise (Table 5). Discussion In the present study, we showed that when RER-susceptible horses are fed a diet providing 40% of the DE in the form of starch, marked subclinical and clinical rhabdomyolysis may occur during exercise. The horses used in the present study have a well-defined form of RER that appears to involve an inherited defect in IM Ca2 regulation.6,31 An individual horse effect was noted, and for 1 stallion, no increases of CK were recorded regardless of diet. Equine diet trials such as these often are limited in the number of horses tested because of the expense and availability of suitable horses, and this limitation may affect the ability to achieve statistical significance for observed biologic differences. The power calculations performed with data from the ln(CK) in this study indicated that sufficient power was present to detect differences between the starch and fat diets and the bicarbonate-starch and fat diets. Rhabdomyolysis in RER horses may be affected by a combination of the total number of calories fed as well as by the starch composition of the diet. For example, in a previous crossover trial with some of the same RER horses, the postexercise serum CK activity was not increased (383 237 U/L) when a 21.4-Mcal/d (89.5 MJ/d) sweet feed diet was fed compared to a 28.8-Mcal/d (120.5 MJ/d) sweet feed diet (CK, 839 243 U/L).3 In the present study, the pelleted 28.8-Mcal/d (120.5 MJ/d) starch diet resulted in a mean postexercise serum CK 3,000 U/L. High-calorie starch diets containing corn, oats, and wheat middlings may be a critical factor in triggering muscular necrosis with exercise in susceptible Thoroughbreds. These findings become particularly important given that approximately 5% of Thoroughbred racehorses suffer from exertional rhabdomyolysis and that large amounts of sweet feed are commonly fed to racing Thoroughbreds.7 It is of great importance, therefore, that by replacing starch with a specifically designed fat ration, a high-calorie intake can be provided to racehorses without producing rhabdomyolysis. The high-fat diet used in the present study had to be specially formulated because it was not possible to provide a palatable 28.8-Mcal/d (120.5 MJ/d) low-starch, high-fat ration by feeding rice bran alone or by adding corn oil, which would require supplementation with approximately 800 mL of oil per day. This amount of supplementation is likely to be unpalatable to many horses.15 By providing 7% of the DE in starch and 20% of the DE from the fat found in rice bran and tallow, rhabdomyolysis was ameliorated in the horses of our study that developed high serum CK activities on the starch diet (Fig 2). It is unclear, however, whether the source of fat had any effect on the results of the present study. In addition, whether a high-fat diet provides a passive beneficial effect by the exclusion of dietary starch or offers unidentified specific protective effects directly from high amounts of dietary fat, as has been suggested, is not known.15,22 Previously, a minimum of 3 months of consuming a high-fat diet has been suggested to be required for metabolic adaptation and clinical improvement in signs of exertional rhabdomyolysis.15,22 However, in the current study, a rapid impact was observed, with dramatic reduction in or normalization of postexercise serum CK activity in all RER horses within the 1st week of consuming a high-fat diet, even in the most severely affected horse of the study. It seems unlikely that important changes in muscle metabolic processes are responsible for the palliative effects of a highfat, low-starch diet on RER because, in normal horses, a Diet and Equine Exertional Rhabdomyolysis minimum of 3–4 weeks are required to show metabolic adaptations to a high-fat diet, including alterations in muscle glycogen concentrations.32,33 In the present study, the high-fat or high-starch diets did not affect resting muscle lactate or muscle glycogen concentrations and did not create a significant difference in glycogen usage rates, in agreement with a previous dietary trial in RER horses.34 In contrast, 3 weeks of feeding rice bran as a dietary fat supplement to horses with PSSM resulted in a marked decrease in their abnormally high resting muscle glycogen concentrations.18 Thus, it appears unlikely that the addition of a fat supplement to RER horses provides a specific beneficial effect via alterations in muscle lactate or glycogen metabolism during exercise. Dietary fat supplementation has been reported to have a calming effect, whereas consumption of a high-starch diet is associated with excitable behavior.13,35 Recent epidemiologic studies have identified stress and anxiety as important predisposing factors in Thoroughbreds with exertional rhabdomyolysis.7 In the current study, horses consuming the fat diet subjectively were much calmer and easier to work with and had lower resting heart rates than the increased reactiveness to unexpected stimuli, misbehavior on the treadmill, and higher resting heart rates observed when RER horses consumed a high-starch diet. This finding is consistent with the lower resting heart rates and PCVs documented in RER horses consuming a fat-supplemented diet compared to a high-starch diet in a previous study.34 Thus, for high-calorie diets, one of the beneficial effects of replacing starch with fat for RER horses may be a modification of nervousness and excitability, which may make them less predisposed to rhabdomyolysis with exercise. Whether this beneficial effect is a reflection of decreased psychologic stress and anxiety in the calmer fat-fed horse or a result of beneficial influences on unidentified physiologic and neurohormonal mechanisms is currently not known. Dietary trials in normal horses have found high-fat diets to reduce preexercise and postexercise serum cortisol concentrations.36,37 Unfortunately, plasma cortisol concentrations were not measured in the current study. Another potential mechanism by which a high-fat diet may ameliorate rhabdomyolysis in RER horses is the modification of muscle membrane fatty acid composition with resultant alterations in inflammatory mediator production.38,39 Rice bran constituted a substantial portion of the high-fat diet in the current study and has a high oil content (18–22%), which includes 38.4% oleic acid (n-9 series), 34.4% linoleic acid (n-6 series), and 2.2% -linolenic acid (n-3 series).40 The ingestion of polyunsaturated fatty acids (PUFAs) of the (n-3) series has been associated with a modification of the inflammatory response in several species, including horses, after several weeks of intake.38,41 However, the high (n-6) : (n-3) PUFA ratio of rice bran and the rapid amelioration of rhabdomyolysis that was observed in this study shortly after horses commenced consuming the high-fat diet suggest that alteration of muscle membrane lipid composition is not an important reason for the beneficial effect of this diet. Other speculative modes by which a high-fat diet may affect exertional rhabdomyolysis include alteration of substrate usage during exercise and enhancement of muscular oxidative capacity.42,43 Measurements of fatty acid oxidation were not recorded in this study. Oleic acid has been shown in vitro to alter the threshold for halothane-induced Ca2 release from isolated human and porcine sarcoplasmic reticulum.44 RER horses also have altered muscle contracture thresholds in response to halothane, and another potential mechanism for the beneficial effect of fat diets in RER horses is modulation of IM Ca2 regulation.6 Alterations in electrolyte balance, such as those characterized by low fractional excretions of Na and K and high fractional excretions of P, have previously been implicated in the development of exertional rhabdomyolysis.45 The fractional excretion of electrolytes in the present study was within the normal range across the diets for all RER horses and did not appear to contribute to the differences in postexercise serum CK activity among the diets. In fact, horses consuming the bicarbonate-starch diet had a high fractional excretion of Na , with serum CK activity much higher than the normal reference range. Dietary supplementation with sodium bicarbonate at the concentration provided in this study appeared to have no beneficial effect on RER. These findings do not support the antiquated belief that lactic acidosis is the cause of clinical signs of rhabdomyolysis.46 One of the effects of the fat diet was to increase the plasmaionized Ca2 concentration after submaximal exercise, but it did not alter the magnitude of change in ionized Ca2 concentration with exercise. Although RER in Thoroughbreds appears to be due to an inherent defect in intracellular Ca2 regulation in the myocyte, it is not known whether plasma-ionized Ca2 concentrations are capable of influencing intracellular Ca2 concentrations.6 Serum CK activity in the current study was consistently higher on Mondays than on other days of the week. Anecdotally, subclinical and clinical rhabdomyolysis with return to exercise after short periods of rest has long been known to occur and also has been documented.1,20 That this observation occurred even in horses consuming the fat diet confirms that exercise after a period of rest is a predisposing factor for muscle damage. Whether the detrimental effects of exercise after rest are exerted independently from the detrimental effects of a high-starch diet is not known. Serum CK activity decreases over several weeks in PSSM horses, provided constant turnout, and remains abnormally high if the horses are confined to a stall.47 The successful management of RER horses also may require feeding a high-fat, low-starch diet accompanied by an appropriate exercise regimen that does not allow excessive periods of rest or stall confinement. Previous epidemiologic studies have suggested that holding back fit RER horses at a slower pace during exercise also predisposes them to rhabdomyolysis.7 Results of the current study indicate that in RER Thoroughbreds requiring a high caloric intake, a specially formulated low-starch, high-fat diet is a convenient and practical means to rapidly and dramatically reduce exercise-induced rhabdomyolysis as assessed by increases in serum CK activity. Additional research is necessary to determine the mechanism by which diet affects rhabdomyolysis. Footnotes Combined vitamin, electrolyte, and mineral supplement (KER 5X premix is a blend of essential vitamins and trace elements that was McKenzie et al 16. Valentine BA, Reynolds AJ, Ducharme NG, et al. Dietary therapy of equine polysaccharide storage myopathy. Equine Pract 1997; 19:30–37. 17. Freestone JF, Kamerling SG, Church G, et al. Exercise induced changes in creatine kinase and aspartate aminotransferase activities in the horse: Effects of conditioning, exercise tests and acepromazine. J Equine Vet Sci 1989;9:275–280. 18. De La Corte FD, Valberg SJ, MacLeay JM, et al. The effect of feeding a fat supplement to horses with polysaccharide storage myopathy. World Equine Vet Rev 1999;4:12–19. 19. Anderson GM. The influence of exercise on serum enzyme levels in the horse. Equine Vet J 1975;7:160–165. 20. Frauenfelder HC, Rossdale PD, Ricketts SW. Changes in serum muscle enzyme levels associated with training schedules and stage of the oestrus cycle in Thoroughbred racehorses. Equine Vet J 1986;18: 371–374. 21. Firshman AM, Valberg SJ, Finno C, et al. Epidemiological aspects of polysaccharide storage myopathy (PSSM) in Quarter Horse. Am J Vet Res 2003. In press. 22. Valentine BA, Van Saun RJ, Thompson KN, et al. Role of dietary carbohydrate and fat in horses with equine polysaccharide storage myopathy. J Am Vet Med Assoc 2001;219:1537–1544. 23. McKenzie EM, Valberg SJ, Godden SM, et al. Plasma and urine electrolyte and mineral concentrations in Thoroughbred horses with recurrent exertional rhabdomyolysis after consumption of diets varying in cation-anion balance. Am J Vet Res 2001;62:1053–1060. 24. National Research Council. Nutrient Requirements of the Horse. Washington, DC: National Academy Press; 1989. 25. Lowry OH, Passonneau JV. Measurement of enzyme activities with pyridine nucleotides. In: A Flexible System for Enzyme Analysis. New York, NY: Academic Press; 1973:93–108. 26. Statistical Analysis Systems Institute. SAS User’s Guide: Statistics, Version 8.0 Developer’s Edition. Cary, NC: SAS Institute; 1999. 27. Morris D, Divers TJ, Whitlock RH. Renal clearance and fractional excretion of electrolytes over a 24-hour period in horses. Am J Vet Res 1984;45:2431–2435. 28. Kohn CW, Strasser SL. 24-hour renal clearance and excretion of endogenous substances in the mare. Am J Vet Res 1986;47:1332– 1337. 29. Gray J, Harris P, Snow DH. Preliminary investigations into the calcium and magnesium status of the horse. In: Blackmore DJ, ed. Animal Clinical Biochemistry—The Future. Cambridge, UK: Cambridge University Press; 1988:307–317. 30. Caple IW, Bourke JM, Ellis PG. An examination of the calcium and phosphorus nutrition of Thoroughbred racehorses. Aust Vet J 1982;58:132–135. 31. MacLeay JM, Valberg SJ, Sorum SA, et al. Heritability of recurrent exertional rhabdomyolysis in Thoroughbred racehorses. Am J Vet Res 1999;60:250–256. 32. Oldham SL, Potter GD, Evans JW, et al. Storage and mobilization of muscle glycogen in exercising horses fed a fat-supplemented diet. J Equine Vet Sci 1990;10:353–359. 33. Julen TR, Potter GD, Greene LW. Adaptation to a fat-supplemented diet by cutting horses. J Equine Vet Sci 1995;15:436–440. 34. MacLeay JM, Valberg SJ, Pagan JD, et al. Effect of diet on Thoroughbred horses with recurrent exertional rhabdomyolysis performing a standardised exercise test. Equine Vet J 1999;30(Suppl): 458–462. 35. Pagan JD, Essen-Gustavsson B, Lindholm A, et al. The effect of dietary energy source on exercise performance in Standardbred horses. In: Gillespie JR, Robinson NE, ed. Equine Exercise Physiology 2. Davis, CA: ICEEP Publications; 1987:696–700. 36. Pagan JD, Burger I, Jackson SG. The long term effects of feeding fat to 2-year-old Thoroughbreds in training. Equine Vet J 1995; 18(Suppl):343–348. 37. Crandell KG, Pagan JD, Harris P, et al. A comparison of grain, added to meet National Research Council requirements), Kentucky Equine Research, Versailles, KY b Equi-jewel High Fat Stabilized Rice Bran, Producers Rice Mill, Inc, Stuttgart, AR c Laboratory analysis, Dairy One, Ithaca, NY d Equistat heart rate monitor, EQB Inc, Unionville, PA e Proc Mixed using the Repeated statement, SAS version 8.0, Statistical Analysis Systems Institute, Cary, NC f Proc Npar1way in SAS, version 8.0, Statistical Analysis Systems Institute, Cary, NC Acknowledgments Funded by the Southern California Equine Foundation. The authors thank Joanne Khaleel and Ana Kloster for technical assistance. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Veterinary Internal Medicine Wiley

Effect of Dietary Starch, Fat, and Bicarbonate Content on Exercise Responses and Serum Creatine Kinase Activity in Equine Recurrent Exertional Rhabdomyolysis

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1939-1676
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10.1111/j.1939-1676.2003.tb02502.x
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Abstract

To determine the effect of dietary starch, bicarbonate, and fat content on metabolic responses and serum creatine kinase (CK) activity in exercising Thoroughbreds with recurrent exertional rhabdomyolysis (RER), 5 RER horses were fed 3 isocaloric diets (28.8 Mcal/d [120.5 MJ/d]) for 3 weeks in a crossover design and exercised for 30 minutes on a treadmill 5 days/wk. On the last day of each diet, an incremental standardized exercise test (SET) was performed. The starch diet contained 40% digestible energy (DE) as starch and 5% as fat; the bicarbonate-starch diet was identical but was supplemented with sodium bicarbonate (4.2% of the pellet); and the fat diet provided 7% DE as starch and 20% as fat. Serum CK activity before the SET was similar among the diets. Serum CK activity (log transformed) after submaximal exercise differed dramatically among the diets and was greatest on the bicarbonate-starch diet (6.51 1.5) and lowest on the fat diet (5.71 0.6). Appreciable differences were observed in the severity of RER among individual horses. Postexercise plasma pH, bicarbonate concentration, and lactate concentration did not differ among the diets. Resting heart rates before the SET were markedly lower on the fat diet than on the starch diet. Muscle lactate and glycogen concentrations before and after the SET did not differ markedly among the diets. A high-fat, low-starch diet results in dramatically lower postexercise CK activity in severely affected RER horses than does a low-fat, high-starch diet without measurably altering muscle lactate and glycogen concentrations. Dietary bicarbonate supplementation at the concentration administered in this study did not prevent increased serum CK activity on a high-starch diet. Key words: Exertional myopathy; Horse; Nutrition; Tying up. xertional rhabdomyolysis is a common and frustrating condition that affects many breeds of horses. Previously, it was thought to be a singular entity arising from the production of lactic acid and protons in muscle after excessive glycogenolysis in horses that were exercised after rest on a high-carbohydrate diet.1 It is now known that several distinct conditions comprise this syndrome, and lactic acidosis is not a factor in the pathophysiology of these diseases.2,3 Polysaccharide storage myopathy (PSSM) was 1st identified as a myopathy of quarter horses and, later, of draft breeds in the 1990s and is characterized by an increased clearance of glucose from the bloodstream and the accumulation of glycogen and abnormal complex polysaccharide within muscle cells.4,5 Recurrent exertional rhabdomyolysis (RER) has been identified as a common cause of pain and muscle necrosis in Thoroughbred racehorses, affecting approximately 5% of this population, and appears to constitute a heritable stress-related defect in intracellular From the Department of Clinical and Population Sciences, College of Veterinary Medicine, University of Minnesota, St Paul, MN (McKenzie, Valberg, Godden, MacLeay); Kentucky Equine Research Inc, Versailles, KY (Pagan, Geor); and the Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA (Carlson). Dr Geor is presently affiliated with R & J Veterinary Consultants Inc, Guelph, Ontario, Canada. Dr MacLeay is presently affiliated with the Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Fort Collins, CO. Previously presented at the 19th Annual Forum of the American College of Veterinary Medicine, Denver, CO, May 23–26, 2001. Reprint requests: Erica C. McKenzie, BSc, BVMS, Comparative Exercise Physiology Laboratory, 264 McElroy Hall, Oklahoma State University, Stillwater, OK 74078; e-mail: mcke0174@tc.umn.edu. Submitted October 25, 2002; Revised January 8, 2003; Accepted February 5, 2003. Copyright 2003 by the American College of Veterinary Internal Medicine 0891-6640/03/1705-0012/$3.00/0 calcium (Ca2 ) regulation.6,7 In horses with PSSM or RER, muscular pain and stiffness usually are elicited by exercise, with concurrent increases in serum creatine kinase (CK) activity. Subclinical episodes of muscular necrosis also may occur.8 Despite major advances in the understanding of the etiology and pathophysiology of these conditions, identifying consistently successful treatment and management strategies has proved challenging. A diet high in soluble carbohydrates (starch) is believed to be a predisposing factor for episodes of rhabdomyolysis in horses with PSSM or RER, but the pathophysiology and signalment associated with the 2 conditions appear to be entirely distinct.3,9 A reduction in daily grain intake is a common management strategy for both diseases. Dietary sodium bicarbonate supplementation also is frequently used in practice to manage RER horses, likely because of the false notion that lactic acid accumulation can cause rhabdomyolysis.1 Dietary fat supplementation in horses has been associated with enhanced aerobic and anaerobic performance, decreased thermal stress, decreased gut fill and water requirement, and calmer demeanor.10–13 Recently, dietary fat supplementation has been advocated for exertional rhabdomyolysis to allow exercising horses to maintain a high caloric intake without additional dietary starch.14,15 However, few standardized dietary trials to investigate the effect of fat on equine rhabdomyolysis have been performed. Many previous trials have used a variety of breeds of horses; different exercise protocols with varying degrees of fitness, exertion, and confinement; and several rations that varied in palatability as well as in type and amount of dietary fat. In these trials, success was judged subjectively by the owners and, in some instances, by the intermittent determination of serum CK activity after highly variable degrees of exertion.15,16 Exercise intensity and frequency, stall confinement, diet, and degree of fitness have been shown to influ- McKenzie et al ence the occurrence of subclinical and clinical rhabdomyolysis.3,17–20 Thus, to date, controlled dietary trials investigating the impact of dietary fat on equine rhabdomyolysis are few, and further investigation is warranted. In PSSM horses, a low-starch diet reduces glycogen accumulation in muscle and, when combined with a daily exercise regime, has been fairly successful in ameliorating the signs of rhabdomyolysis in these horses.15,16,18,21,22 A previous dietary trial in RER horses found that a high-energy, high-starch diet (28.8 Mcal/d [120.5 MJ/d]) resulted in dramatic increases in serum CK activity after treadmill exercise. Lowering the caloric intake to 21.4 Mcal/d (89.5 MJ/d) prevented marked increases in postexercise serum CK regardless of whether the calories were provided as starch or fat.3 However, to our knowledge, the effect of a high-energy, low-starch diet providing a large percentage of daily calories from fat on exercising horses with RER has not been investigated. The purpose of the current study was to assess the effects of 3 isocaloric high-energy diets (28.8 Mcal/d [120.5 MJ/d]) varying in starch, fat, and bicarbonate content on postexercise serum CK activity in Thoroughbred horses with RER undergoing treadmill exercise. The objective was to determine whether the source of calories in a high-calorie diet would affect the occurrence of episodes of muscle necrosis in RER horses and to determine the palliative effects, if any, of dietary sodium bicarbonate on the occurrence of RER. Table 1. Digestible energy (DE) and percentage of electrolyte and mineral composition of the 3 rations (hay plus pellet) on a dry matter basis. Ration Type Variable % sodium % potassium % chloride % magnesium % phosphorus % calcium % sulfur Total DE in Mcal/d (MJ/d) % DE from starch % DE from fat DE, digestible energy. Starch 0.84 1.39 1.56 0.23 0.47 0.69 0.14 28.8 (120.5) 40.0 5.0 BicarbonateStarch 1.3 1.35 1.55 0.22 0.64 0.71 0.15 28.8 (120.5) 40.0 5.0 Fat 0.80 1.54 1.47 0.27 0.42 0.97 0.19 28.8 (120.5) 7.0 20.0 incorporated into this pellet to form the bicarbonate-starch feed. Starch supplied 40% and fat 5% of the daily DE for these 2 diets. The highfat pellet was composed of 54% soy hulls, 25% rice bran,b 7% solvent extracted soybean meal, 6% allofat, 5% wheat middlings, and 2.5% pellet binder. On the fat diet, starch supplied 7% and fat 20% of the daily DE. After formulation, all diets were analyzed by a commercial laboratoryc for electrolyte and mineral concentrations and starch and fat content. Materials and Methods Horses A 3-year-old Thoroughbred stallion and 5 Thoroughbred mares (2, 6, 9, 11, and 13 years old) with RER were used. Horses were selected on the basis of criteria used in previous studies to define RER, including a history of clinical rhabdomyolysis, increases in serum CK after exercise, and an abnormally low threshold for intercostal muscle contracture in the presence of caffeine and halothane.6,23 Experimental Design A replicated 3 3 randomized design was used with 6 horses. The pelleted feeds were fed at 0.036 Mcal/kg (0.151 MJ/kg) body weight (1% of body weight), divided into 2 feedings per day. Horses were introduced to the pellet with a gradually increasing amount of the starch pellet over a 5-day period before commencement of the trial. Two horses began on the bicarbonate-starch diet, 2 horses began on the fat diet, and 2 horses began on the starch diet. After 5 days, a 6year-old mare on the starch diet was removed from the trial because of injury, and the remaining horses continued on the diets as planned. Each diet was fed for a total of 21 days. Gradually increasing amounts of the next diet to be consumed were mixed with the previous diet over a 1-week washout period to avoid sudden changes. All 3 pellets were highly palatable and were readily consumed by all horses. Training To establish a similar plane of fitness, horses were exercised 5 days/ wk for a total of 5 weeks on a high-speed treadmill before the trial. Horses performed alternating intervals of walk (1.9 m/s), trot (4.0 m/s), and canter (7.0 m/s). Daily exercise gradually was increased to a maximum of 30 minutes/d. On the Wednesday of the last 2 training weeks, the final canter interval was replaced by a 2-minute gallop (11 m/s) with the treadmill inclined to a 6% slope. Throughout the study, all horses were confined to a stall when they were not exercising on the treadmill and were rested on the Saturday and Sunday of every week. Daily Exercise Regime Daily exercise throughout the trial consisted of alternating 2-minute intervals of walk, trot, and canter for a total of 30 minutes, 5 days/ wk. On the Wednesday of the 1st 2 weeks on each diet, the last canter interval was replaced by a 2-minute gallop (11 m/s) on a 6% slope, designed to achieve a heart rate of 200 beats/min. During the week, when diet changeovers were occurring, the horses continued to undergo the same daily exercise routine, but postexercise CK activity was not measured. Horses did not gallop on the Wednesday during the 3rd week of each diet when the standardized exercise test (SET) was to be performed. Diets All horses were fed grass hay at 0.022 Mcal/kg (0.092 MJ/kg) body weight per day (1.2% of body weight). Three isocaloric pelleted feeds were designed that, in combination with hay, provided 28.8 Mcal (120.5 MJ) in digestible energy (DE) per day (Table 1). All 3 pellets were supplemented with additional dicalcium phosphate, sodium chloride, and a combined vitamin, electrolyte, and mineral supplementa to meet National Research Council recommendations.24 The pellet of the starch and bicarbonate-starch diets was composed of approximately 38% ground corn, 32% wheat middlings, 15% oats, 10% soy meal and hulls, and 4% molasses. Sodium bicarbonate at 4.2% dry matter was Daily Sample Collection Immediately postexercise throughout the trial, jugular venous blood was collected into lithium heparin blood gas syringes for the measurement of plasma pH, bicarbonate (HCO3 ), ionized calcium (Ca2 ), sodium (Na ), and potassium (K ) concentrations. On the last 5 exercise days of each diet, venous blood gas samples also were obtained Diet and Equine Exertional Rhabdomyolysis Table 2. Mean ( SD) preexercise plasma acid-base values and electrolyte concentrations in 5 Thoroughbreds with recurrent exertional rhabdomyolysis (RER) consuming 3 diets varying in starch, bicarbonate, and fat content. Variable Blood pH Bicarbonate (mmol/L) Total CO2 (mmol/L) Base excess Ionized calcium (mmol/L) Sodium (mmol/L) Potassium (mmol/L) N 7.40 30.4 31.9 5.6 1.53 129 4.2 Starch 5 (n 30) 0.02 1.95 2.03 1.79 0.10 3.35 0.67 Bicarbonate-Starch N 5 (n 30) 7.40 30.8 32.3 6.1 1.53 129 4.0 0.02 1.84 1.93 1.60 0.08 3.43 0.51 A A A A A A A Fat 5 (n 30) A B B B A A B AB AB AB A A A N, number of horses; n, total number of samples; RER, recurrent exertional rhabdomyolysis. a Differing letters indicate significant differences attributable to diet among horses. immediately before exercise. Four hours after daily exercise, blood was obtained for the measurement of serum CK activity. compound symmetry correlation structure, and repeated on subject horse. The main effects that were offered to the models used in the analysis included diet (starch, bicarbonate-starch, or fat), day (Monday–Friday), and a diet day interaction term. A preliminary review of descriptive statistics by the Proc Univariate showed that raw serum CK data were not normally distributed (skewness statistic value for all measures, 10.6; mean skewness value by diet, 5.0 [range, 3.4–7.7]; mean skewness by horse, 2.9 [range, 1.6–4.9]). All other blood parameters measured were normally distributed. Because normality is one of the 3 assumptions required for the results of repeated-measures analysis of variance to be valid, serum CK data were transformed to the natural logarithm (ln) before statistical analysis. Significance was set at P .05. Further analysis of variance (Proc Mixed in SAS26) included horse as a fixed effect in the model statement as well as a horse diet interaction term. As a final alternative approach to transforming the CK data, a nonparametric approach to analysis of the raw CK data also was completed26,f by the Wilcoxon statistic, after categorizing the CK data into one of 6 categories: (1) 404 U/L, (2) 404– 999 U/L, (3) 1,000–4,999 U/L, (4) 5,000–99,999 U/L, (5) 10,000– 999,999 U/L, and (6) 100,000 U/L. Standardized Exercise Test On the final Thursday (horses 1–3) or Friday (horses 4–6) of each diet, a near-maximal SET was performed by each horse. Before the SET, concurrent urine and serum samples were obtained from all horses to calculate the urinary fractional excretion of Ca2 , phosphorus (P), magnesium (Mg2 ), Na , K , and chloride (Cl ). The SET consisted of a 12-minute step test. Initially, horses walked at 1.9 m/s for 2 minutes. The treadmill then was inclined to a 6% slope, with 2 additional minutes of walking. Horses subsequently performed 2-minute increments of trot (4.5 m/s) and canter (7 m/s) and two 2-minute gallop intervals at 10 and then 11 m/s, gauged to achieve a peak heart rate of approximately 200 beats/min. Heart rate was recorded as the horses stood on the treadmill before commencement of the SET, during the last 15 seconds of each speed increment during the SET, and 5 minutes after the SET with the Equistat heart rate monitor (model HR8AE).d Serial blood samples for measurement of blood lactate concentrations were drawn through a preplaced jugular catheter at the same intervals as for heart rate measurement. Before and 4 hours after the SET, a serum sample was obtained for analysis of serum CK activity. Immediately before and after the SET, samples of the middle gluteal muscle were obtained 15 cm along a straight line from the top of the tuber coxa to the point of the tail through a single incision at a depth of 8 cm with a modified Bergstrom needle. Muscle samples for measurement of muscle lactate and glycogen concentrations were immediately frozen in liquid nitrogen and stored at 80 C for later analysis. Results Five of the 6 horses completed the trial successfully. One mare required 2 days of rest while consuming the starch diet because of postexercise stiffness and muscle cramping on the second Monday of the starch diet (CK, 109,650 U/L). Body weight did not differ markedly among the diets throughout the study (starch, 496.8 75.9 kg; bicarbonatestarch, 493.6 82.5 kg; and fat, 494.1 74.7 kg). Analysis of Samples Blood gas samples were stored on ice and analyzed within 30 minutes with a blood gas analyzer. Serum concentrations of Ca2 , P, Mg2 , Na , K , and Cl as well as serum CK activity were measured on an automated chemistry analyzer. Urine concentrations of Na , K , Ca2 , P, and Mg2 were determined by emission spectrometry, and urine Cl and creatinine concentrations were measured on an automated chemistry analyzer after preparation by a method described in a previously published study.23 Plasma lactate concentrations were determined with an automated lactate analyzer. Muscle for glycogen and lactate analysis was freeze-dried and dissected free of blood, fat, and connective tissue. Samples for glycogen analysis were boiled for 2 hours in 1 M HCl to produce glucose residues. Glucose residues and lactate concentrations were determined by fluorometric analysis according to the methods of Lowry and Passonneau (1973).25 Preexercise Plasma Acid-Base Status and Electrolyte Concentrations At rest, consumption of the bicarbonate-starch diet resulted in significantly mild increases in plasma HCO3 and total carbon dioxide concentrations and plasma base excess compared to the fat diet (Table 2). The plasma K concentration was markedly greater in horses consuming the fat diet than in horses consuming the other 2 diets. Postexercise Plasma Acid-Base Status, Electrolytes, and Minerals Exercise resulted in an increase in the plasma K concentration and decreases in plasma base excess, ionized Ca2 , total carbon dioxide, and HCO3 concentrations on all diets compared to preexercise values (Tables 2, 3). No differences were observed among any diets in post- Statistical Analysis Serum results were analyzed by repeated-measures analysis of variance26,e by means of a maximum likelihood estimation, specifying a McKenzie et al Table 3. Mean ( SD) and postexercise plasma acid-base values and electrolyte concentrations in 5 Thoroughbreds with RER consuming 3 diets varying in starch, bicarbonate, and fat content performing submaximal exercise or submaximal exercise including a 2-minute gallop on a 6% incline. Submaximal Exercise Variable Blood pH Bicarbonate (mmol/L) Total CO2 (mmol/L) Base excess Ionized calcium (mmol/L) Sodium (mmol/L) Potassium (mmol/L) Raw serum CK (U/L) ln(CK) N Starch 5 (n 55) 0.03 A 3.2 A 3.2 A 3.2 A 0.07 A 3.5 A 0.7 A 14,954 A 1.28 A Submaximal Exercise plus Gallop Interval N Fat 5 (n 7.41 29.2 30.6 5.0 1.46 129 5.3 387 5.71 54) 0.04 A 3.1 A 3.2 A 3.2 A 0.09 B 3.1 B 1.0 A 403 A 0.63 C N Starch 5 (n 10) 7.35 24.4 25.6 0.01 1.40 129 5.9 877 6.07 0.05 A 5.5 A 5.7 A 5.4 A 0.05 A 3.2 A 1.0 A 1,535 AB 1.06 B N Bicarbonate 5 (n 10) 7.35 23.9 25.1 0.35 1.38 130 5.5 2,560 6.64 0.06 A 5.4 A 5.6 A 5.4 A 0.05 A 3.5 A 1.1 A 4,063 A 1.69 A N Fat 5 (n 7.34 23.1 24.3 1.51 1.40 129 5.6 574 5.93 10) 0.05 A 4.4 A 4.5 A 4.5 A 0.07 A 2.5 A 1.3 A 680 B 0.87 B Bicarbonate 5 (n 54) 7.40 30.0 30.3 4.7 1.41 129 5.3 0.04 A 3.3 A 3.4 A 3.3 A 0.08 A 3.3 B 0.9 A 6,186 A 1.50 B 7.40 28.8 30.1 4.6 1.41 127 5.5 3,064 6.24 2,618 6.51 N, number of horses; n, total number of samples; RER, recurrent exertional rhabdomyolysis; CK, creatine kinase; ln, natural logarithm. a Differing letters indicate significant differences in parameters between diets in horses undergoing submaximal exercise or submaximal exercise plus a 2-minute gallop interval. exercise acid-base parameters in horses undergoing daily submaximal exercise (Table 3). The postexercise plasmaionized Ca2 concentration was significantly increased on the fat diet compared to the other 2 diets. A slightly but significantly lower postexercise plasma Na concentration was observed with the starch diet than with the other diets after submaximal exercise. The inclusion of a 2-minute gallop interval to the exercise routine resulted in a decrease in plasma pH and base excess as well as in plasma concentrations of HCO3 and total carbon dioxide compared to nongallop days (Table 3). Higher plasma concentrations of Na and K were observed after exercise on gallop days than on other days of the week. No significant difference was observed among the diets with regard to acid-base parameters or plasma electrolyte concentrations on gallop days (Table 3). Four-Hour Postexercise Serum CK The mare that was consuming the high-starch diet and dropped from the trial after 5 days of exercise had a mean CK of 383 U/L for the 5-day period (range, 285–948 U/L). For the 5 horses that completed the study, the raw mean postexercise serum CK activity after daily submaximal exercise was within the normal range (57–404 U/L) when they consumed the high-fat diet but was 7.9 times higher when they consumed the starch and bicarbonate-starch diets (Table 3). Repeated-measures analysis of variance identified a significant effect of both diet and day on postsubmaximal exercise values for the ln(CK). No significant interaction was observed between the terms describing diet and day. Subsequent contrast analysis showed that a significant difference in the ln(CK) existed among all 3 diets, with the postexercise ln(CK) being highest on the bicarbonate-starch diet and lowest on the fat diet (Fig 1). The statistical power to detect a treatment difference with the respective means, standard deviations, and sample sizes for the ln(CK) (1tailed test, .05) was .36 between the starch and bicarbonate-starch diets, .98 between the bicarbonate-starch and fat diets, and .82 between the starch and fat diets. Nonparametric testing of the untransformed CK data provided identical inferences (data not shown). Postgallop serum CK activity (ln) was significantly greater in horses consuming the bicarbonate-starch diet than in horses consuming the other diets (Table 3). Within each diet, no significant difference in serum CK activity (raw means or ln) was ob- Fig 1. Scatterplot of 4-hour postexercise serum creatine kinase (CK) activity in 5 horses with recurrent exertional rhabdomyolysis (RER) consuming 3 diets that varied in starch, fat, and bicarbonate content. Horizontal bars indicate median serum CK activity for each diet. Differing superscript letters indicate significant differences attributable to diet in mean natural logarithm (ln) CK activity 4 hours postexercise. N, number of plasma samples; N 65 (starch diet); N 64 (bicarbonate-starch and fat diets). Diet and Equine Exertional Rhabdomyolysis Day-of-Week Effect Across diets, a significant effect of day of the week on serum CK activity was observed on the analysis of logtransformed postexercise serum CK data. Serum CK activity (ln) on Monday was significantly higher than on Tuesday (P .06) as well as on the remaining days of the week (P .05). Individual statistical examination of the diets revealed that on the bicarbonate-starch diet, the ln(CK) on Monday was significantly higher than on Wednesday, Thursday, and Friday (Table 4). In horses consuming the fat diet, the ln(CK) on Monday was different from that on all other days except for Wednesday, when horses galloped. Fig 2. Scatterplot of 4-hour postexercise serum creatine kinase (CK) activity for 5 individual horses (TO, MG, MT, NA, and ST) with recurrent exertional rhabdomyolysis (RER) consuming 3 diets that varied in starch (triangles), fat (circles), and bicarbonate content (squares). Horizontal bars indicate median serum CK activity for each horse. Standardized Exercise Test served between 30 minutes of submaximal exercise compared to 28 minutes of submaximal exercise plus a 2-minute gallop interval. Subsequent analysis of variance indicated that a significant horse diet interaction existed; therefore, analysis was repeated after stratifying the data by horse. For stratified analysis, a significant effect of diet on ln(CK) values for horses 1 (NA), 3 (TO), and 4 (MA) was observed, with values being the lowest for the fat diet. There was a tendency for an effect of diet on the ln(CK) for horse 5 (MT, P .1) and no effect of diet on the ln(CK) for horse 2 (ST, P .3). Significant individual variation was observed in postexercise serum CK activity (Fig 2). For example, horse TO had the highest postexercise serum CK activity of all the horses when consuming the starch diet (mean CK, 12,014 U/L) and bicarbonate-starch diet (mean CK, 9,803 U/L) but had lower postexercise serum CK activity when consuming the high-fat diet (mean CK, 958 U/L). In comparison, 2 horses (NA and ST) had serum CK activity 1,000 U/L on all diets; therefore, diet had little effect regarding abnormal increases in postexercise serum CK activity for these 2 horses. Pre-SET fractional excretion values of Cl , K , Ca2 , Mg2 , and P did not differ significantly among the diets and were within previously published normal ranges for horses.27–30 The pre-SET fractional excretion of Na was significantly increased in horses consuming the bicarbonate-starch diet (0.55 0.49%) compared to that of horses consuming the starch diet (0.05 0.03%) and the fat diet (0.07 0.07%). Diet had no significant impact on postSET plasma acid-base parameters or plasma electrolyte concentrations (data not shown). The resting heart rate (beats per minute) measured in horses standing on the treadmill before each SET was significantly lower in horses consuming the fat diet (36 5.4 beats/min) than in horses consuming the starch diet (43 2.7 beats/min). The pre-SET heart rate in horses consuming the bicarbonate-starch diet was not different from that in horses consuming the other 2 diets (40 2.9 beats/min). No significant dietary effect on maximal heart rate was observed during the SET or on the mean 5-minute post-SET heart rate (Table 5). Diet had no effect on resting, peak, or 5-minute post-SET plasma lactate concentrations and no effect on pre-SET or post-SET serum CK activity or the magnitude of change in CK activity with the SET. Table 4. Mean 4-hour postexercise serum creatine kinase activity (CK U/L) in 5 Thoroughbreds with RER consuming 3 diets that vary in starch, bicarbonate, and fat content undergoing daily exercise.a Starch Day Monday Tuesday Wednesday Thursday Friday CK (U/L) N 5, n 65 8,301 28,096 (173–109,650) 1,611 3,784 (173–14,907) 787 1,309 (173–5,173) 724 1,109 (211–3,816) 717 1,018 (184–3,356) ln(CK) N 5, n 65 6.59 6.23 6.02 6.05 6.05 1.73 1.29 0.99 0.91 0.96 Ab A A A A Bicarbonate-Starch CK (U/L) N 5, n 64 4,255 9,478 (152–36,060) 2,891 6,390 (160–24,740) 1,935 3,404 (142–12,840) 1,857 3,167 (164–10,200) 1,475 2,710 (165–9,024) ln(CK) N 5, n 64 6.78 6.54 6.48 6.37 6.36 1.74 1.61 1.47 1.55 1.31 A A B B B Fat CK (U/L) N 5, n 64 546 648 (186–2,688) 368 359 (147–1,483) 502 567 (160–2,285) 298 210 (158–775) 296 166 (151–632) N ln(CK) 5, n 64 0.74 0.64 0.77 0.55 0.50 A B A B B N, number of horses; n, total number of samples; CK, creatine kinase; RER, recurrent exertional rhabdomyolysis; ln, natural logarithm. a CK values are presented as raw and log-transformed (ln) mean ( SD). The range of CK values is presented in brackets. b Differing letters indicate significant differences in ln(CK) between Monday and other days of the week within each diet. McKenzie et al Table 5. Mean ( SD) parameters before and immediately after the standardized exercise test (SET) in Thoroughbred horses with recurrent exertional rhabdomyolysis consuming 3 diets varying in starch, bicarbonate, and fat content. Variablea Peak SET HR Post-SET HR Preplasma lactate Peak plasma lactate Postplasma lactate Pre-SET CK (U/L) Post-SET CK Premuscle glycogen Postmuscle glycogen muscle glycogen Premuscle lactate Postmuscle lactate muscle lactate N Starch 5, n 15 17.2 A 8.3 A 0.18 A 2.53 A 3.29 A 324 A 275 A 57.1 A 67.3 A 32.9 A 33.5 A 65.0 A 33.0 A Bicarbonate-Starch N 5, n 15 205 80 0.74 8.22 7.12 296 409 676.7 597.1 79.5 114.8 149.0 34.2 12.8 A 3.4 A 0.22 A 3.39 A 3.75 A 176 A 285 A 72.0 A 100.6 A 48.6 A 40.6 A 69.7 A 32.6 A N 199 77 0.78 6.56 5.23 222 293 692.5 570.6 121.9 112.6 161.6 49.0 Fat 5, n 11.7 A 10.9 A 0.15 A 3.40 A 3.37 A 35 A 90 A 147.0 A 146.7 A 126.1 A 12.42A 18.1 A 27.7 A SET, standardized exercise test; HR, heart rate in beats per minute; CK, serum creatine kinase activity; N, number of horses; n, total number of samples. a difference between preexercise and postexercise muscle glycogen and lactate values. Plasma lactate is measured as mmol/L and serum CK as U/L. Muscle glycogen and lactate concentrations are measured as mmol/kg dry weight. b Letters indicate statistical comparison in parameters between diets. Muscle Glycogen and Lactate Muscle glycogen concentration did not differ significantly among the diets before or after the SET (Table 5). No significant difference in pre-SET and post-SET muscle lactate concentrations was found among the diets. Diet did not significantly affect the magnitude of change in muscle glycogen and lactate concentrations with exercise (Table 5). Discussion In the present study, we showed that when RER-susceptible horses are fed a diet providing 40% of the DE in the form of starch, marked subclinical and clinical rhabdomyolysis may occur during exercise. The horses used in the present study have a well-defined form of RER that appears to involve an inherited defect in IM Ca2 regulation.6,31 An individual horse effect was noted, and for 1 stallion, no increases of CK were recorded regardless of diet. Equine diet trials such as these often are limited in the number of horses tested because of the expense and availability of suitable horses, and this limitation may affect the ability to achieve statistical significance for observed biologic differences. The power calculations performed with data from the ln(CK) in this study indicated that sufficient power was present to detect differences between the starch and fat diets and the bicarbonate-starch and fat diets. Rhabdomyolysis in RER horses may be affected by a combination of the total number of calories fed as well as by the starch composition of the diet. For example, in a previous crossover trial with some of the same RER horses, the postexercise serum CK activity was not increased (383 237 U/L) when a 21.4-Mcal/d (89.5 MJ/d) sweet feed diet was fed compared to a 28.8-Mcal/d (120.5 MJ/d) sweet feed diet (CK, 839 243 U/L).3 In the present study, the pelleted 28.8-Mcal/d (120.5 MJ/d) starch diet resulted in a mean postexercise serum CK 3,000 U/L. High-calorie starch diets containing corn, oats, and wheat middlings may be a critical factor in triggering muscular necrosis with exercise in susceptible Thoroughbreds. These findings become particularly important given that approximately 5% of Thoroughbred racehorses suffer from exertional rhabdomyolysis and that large amounts of sweet feed are commonly fed to racing Thoroughbreds.7 It is of great importance, therefore, that by replacing starch with a specifically designed fat ration, a high-calorie intake can be provided to racehorses without producing rhabdomyolysis. The high-fat diet used in the present study had to be specially formulated because it was not possible to provide a palatable 28.8-Mcal/d (120.5 MJ/d) low-starch, high-fat ration by feeding rice bran alone or by adding corn oil, which would require supplementation with approximately 800 mL of oil per day. This amount of supplementation is likely to be unpalatable to many horses.15 By providing 7% of the DE in starch and 20% of the DE from the fat found in rice bran and tallow, rhabdomyolysis was ameliorated in the horses of our study that developed high serum CK activities on the starch diet (Fig 2). It is unclear, however, whether the source of fat had any effect on the results of the present study. In addition, whether a high-fat diet provides a passive beneficial effect by the exclusion of dietary starch or offers unidentified specific protective effects directly from high amounts of dietary fat, as has been suggested, is not known.15,22 Previously, a minimum of 3 months of consuming a high-fat diet has been suggested to be required for metabolic adaptation and clinical improvement in signs of exertional rhabdomyolysis.15,22 However, in the current study, a rapid impact was observed, with dramatic reduction in or normalization of postexercise serum CK activity in all RER horses within the 1st week of consuming a high-fat diet, even in the most severely affected horse of the study. It seems unlikely that important changes in muscle metabolic processes are responsible for the palliative effects of a highfat, low-starch diet on RER because, in normal horses, a Diet and Equine Exertional Rhabdomyolysis minimum of 3–4 weeks are required to show metabolic adaptations to a high-fat diet, including alterations in muscle glycogen concentrations.32,33 In the present study, the high-fat or high-starch diets did not affect resting muscle lactate or muscle glycogen concentrations and did not create a significant difference in glycogen usage rates, in agreement with a previous dietary trial in RER horses.34 In contrast, 3 weeks of feeding rice bran as a dietary fat supplement to horses with PSSM resulted in a marked decrease in their abnormally high resting muscle glycogen concentrations.18 Thus, it appears unlikely that the addition of a fat supplement to RER horses provides a specific beneficial effect via alterations in muscle lactate or glycogen metabolism during exercise. Dietary fat supplementation has been reported to have a calming effect, whereas consumption of a high-starch diet is associated with excitable behavior.13,35 Recent epidemiologic studies have identified stress and anxiety as important predisposing factors in Thoroughbreds with exertional rhabdomyolysis.7 In the current study, horses consuming the fat diet subjectively were much calmer and easier to work with and had lower resting heart rates than the increased reactiveness to unexpected stimuli, misbehavior on the treadmill, and higher resting heart rates observed when RER horses consumed a high-starch diet. This finding is consistent with the lower resting heart rates and PCVs documented in RER horses consuming a fat-supplemented diet compared to a high-starch diet in a previous study.34 Thus, for high-calorie diets, one of the beneficial effects of replacing starch with fat for RER horses may be a modification of nervousness and excitability, which may make them less predisposed to rhabdomyolysis with exercise. Whether this beneficial effect is a reflection of decreased psychologic stress and anxiety in the calmer fat-fed horse or a result of beneficial influences on unidentified physiologic and neurohormonal mechanisms is currently not known. Dietary trials in normal horses have found high-fat diets to reduce preexercise and postexercise serum cortisol concentrations.36,37 Unfortunately, plasma cortisol concentrations were not measured in the current study. Another potential mechanism by which a high-fat diet may ameliorate rhabdomyolysis in RER horses is the modification of muscle membrane fatty acid composition with resultant alterations in inflammatory mediator production.38,39 Rice bran constituted a substantial portion of the high-fat diet in the current study and has a high oil content (18–22%), which includes 38.4% oleic acid (n-9 series), 34.4% linoleic acid (n-6 series), and 2.2% -linolenic acid (n-3 series).40 The ingestion of polyunsaturated fatty acids (PUFAs) of the (n-3) series has been associated with a modification of the inflammatory response in several species, including horses, after several weeks of intake.38,41 However, the high (n-6) : (n-3) PUFA ratio of rice bran and the rapid amelioration of rhabdomyolysis that was observed in this study shortly after horses commenced consuming the high-fat diet suggest that alteration of muscle membrane lipid composition is not an important reason for the beneficial effect of this diet. Other speculative modes by which a high-fat diet may affect exertional rhabdomyolysis include alteration of substrate usage during exercise and enhancement of muscular oxidative capacity.42,43 Measurements of fatty acid oxidation were not recorded in this study. Oleic acid has been shown in vitro to alter the threshold for halothane-induced Ca2 release from isolated human and porcine sarcoplasmic reticulum.44 RER horses also have altered muscle contracture thresholds in response to halothane, and another potential mechanism for the beneficial effect of fat diets in RER horses is modulation of IM Ca2 regulation.6 Alterations in electrolyte balance, such as those characterized by low fractional excretions of Na and K and high fractional excretions of P, have previously been implicated in the development of exertional rhabdomyolysis.45 The fractional excretion of electrolytes in the present study was within the normal range across the diets for all RER horses and did not appear to contribute to the differences in postexercise serum CK activity among the diets. In fact, horses consuming the bicarbonate-starch diet had a high fractional excretion of Na , with serum CK activity much higher than the normal reference range. Dietary supplementation with sodium bicarbonate at the concentration provided in this study appeared to have no beneficial effect on RER. These findings do not support the antiquated belief that lactic acidosis is the cause of clinical signs of rhabdomyolysis.46 One of the effects of the fat diet was to increase the plasmaionized Ca2 concentration after submaximal exercise, but it did not alter the magnitude of change in ionized Ca2 concentration with exercise. Although RER in Thoroughbreds appears to be due to an inherent defect in intracellular Ca2 regulation in the myocyte, it is not known whether plasma-ionized Ca2 concentrations are capable of influencing intracellular Ca2 concentrations.6 Serum CK activity in the current study was consistently higher on Mondays than on other days of the week. Anecdotally, subclinical and clinical rhabdomyolysis with return to exercise after short periods of rest has long been known to occur and also has been documented.1,20 That this observation occurred even in horses consuming the fat diet confirms that exercise after a period of rest is a predisposing factor for muscle damage. Whether the detrimental effects of exercise after rest are exerted independently from the detrimental effects of a high-starch diet is not known. Serum CK activity decreases over several weeks in PSSM horses, provided constant turnout, and remains abnormally high if the horses are confined to a stall.47 The successful management of RER horses also may require feeding a high-fat, low-starch diet accompanied by an appropriate exercise regimen that does not allow excessive periods of rest or stall confinement. Previous epidemiologic studies have suggested that holding back fit RER horses at a slower pace during exercise also predisposes them to rhabdomyolysis.7 Results of the current study indicate that in RER Thoroughbreds requiring a high caloric intake, a specially formulated low-starch, high-fat diet is a convenient and practical means to rapidly and dramatically reduce exercise-induced rhabdomyolysis as assessed by increases in serum CK activity. Additional research is necessary to determine the mechanism by which diet affects rhabdomyolysis. Footnotes Combined vitamin, electrolyte, and mineral supplement (KER 5X premix is a blend of essential vitamins and trace elements that was McKenzie et al 16. Valentine BA, Reynolds AJ, Ducharme NG, et al. Dietary therapy of equine polysaccharide storage myopathy. Equine Pract 1997; 19:30–37. 17. Freestone JF, Kamerling SG, Church G, et al. Exercise induced changes in creatine kinase and aspartate aminotransferase activities in the horse: Effects of conditioning, exercise tests and acepromazine. J Equine Vet Sci 1989;9:275–280. 18. De La Corte FD, Valberg SJ, MacLeay JM, et al. The effect of feeding a fat supplement to horses with polysaccharide storage myopathy. World Equine Vet Rev 1999;4:12–19. 19. Anderson GM. The influence of exercise on serum enzyme levels in the horse. Equine Vet J 1975;7:160–165. 20. Frauenfelder HC, Rossdale PD, Ricketts SW. Changes in serum muscle enzyme levels associated with training schedules and stage of the oestrus cycle in Thoroughbred racehorses. Equine Vet J 1986;18: 371–374. 21. Firshman AM, Valberg SJ, Finno C, et al. Epidemiological aspects of polysaccharide storage myopathy (PSSM) in Quarter Horse. Am J Vet Res 2003. In press. 22. Valentine BA, Van Saun RJ, Thompson KN, et al. Role of dietary carbohydrate and fat in horses with equine polysaccharide storage myopathy. J Am Vet Med Assoc 2001;219:1537–1544. 23. McKenzie EM, Valberg SJ, Godden SM, et al. Plasma and urine electrolyte and mineral concentrations in Thoroughbred horses with recurrent exertional rhabdomyolysis after consumption of diets varying in cation-anion balance. Am J Vet Res 2001;62:1053–1060. 24. National Research Council. Nutrient Requirements of the Horse. Washington, DC: National Academy Press; 1989. 25. Lowry OH, Passonneau JV. Measurement of enzyme activities with pyridine nucleotides. In: A Flexible System for Enzyme Analysis. New York, NY: Academic Press; 1973:93–108. 26. Statistical Analysis Systems Institute. SAS User’s Guide: Statistics, Version 8.0 Developer’s Edition. Cary, NC: SAS Institute; 1999. 27. Morris D, Divers TJ, Whitlock RH. Renal clearance and fractional excretion of electrolytes over a 24-hour period in horses. Am J Vet Res 1984;45:2431–2435. 28. Kohn CW, Strasser SL. 24-hour renal clearance and excretion of endogenous substances in the mare. Am J Vet Res 1986;47:1332– 1337. 29. Gray J, Harris P, Snow DH. Preliminary investigations into the calcium and magnesium status of the horse. In: Blackmore DJ, ed. Animal Clinical Biochemistry—The Future. Cambridge, UK: Cambridge University Press; 1988:307–317. 30. Caple IW, Bourke JM, Ellis PG. An examination of the calcium and phosphorus nutrition of Thoroughbred racehorses. Aust Vet J 1982;58:132–135. 31. MacLeay JM, Valberg SJ, Sorum SA, et al. Heritability of recurrent exertional rhabdomyolysis in Thoroughbred racehorses. Am J Vet Res 1999;60:250–256. 32. Oldham SL, Potter GD, Evans JW, et al. Storage and mobilization of muscle glycogen in exercising horses fed a fat-supplemented diet. J Equine Vet Sci 1990;10:353–359. 33. Julen TR, Potter GD, Greene LW. Adaptation to a fat-supplemented diet by cutting horses. J Equine Vet Sci 1995;15:436–440. 34. MacLeay JM, Valberg SJ, Pagan JD, et al. Effect of diet on Thoroughbred horses with recurrent exertional rhabdomyolysis performing a standardised exercise test. Equine Vet J 1999;30(Suppl): 458–462. 35. Pagan JD, Essen-Gustavsson B, Lindholm A, et al. The effect of dietary energy source on exercise performance in Standardbred horses. In: Gillespie JR, Robinson NE, ed. Equine Exercise Physiology 2. Davis, CA: ICEEP Publications; 1987:696–700. 36. Pagan JD, Burger I, Jackson SG. The long term effects of feeding fat to 2-year-old Thoroughbreds in training. Equine Vet J 1995; 18(Suppl):343–348. 37. Crandell KG, Pagan JD, Harris P, et al. A comparison of grain, added to meet National Research Council requirements), Kentucky Equine Research, Versailles, KY b Equi-jewel High Fat Stabilized Rice Bran, Producers Rice Mill, Inc, Stuttgart, AR c Laboratory analysis, Dairy One, Ithaca, NY d Equistat heart rate monitor, EQB Inc, Unionville, PA e Proc Mixed using the Repeated statement, SAS version 8.0, Statistical Analysis Systems Institute, Cary, NC f Proc Npar1way in SAS, version 8.0, Statistical Analysis Systems Institute, Cary, NC Acknowledgments Funded by the Southern California Equine Foundation. The authors thank Joanne Khaleel and Ana Kloster for technical assistance.

Journal

Journal of Veterinary Internal Medicine – Wiley

Published: Sep 1, 2003

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