Red blood cell distribution width (RDW) is a quantitative

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1 Effect of Exercise Training on Red Blood Cell Distribution Width as a Marker of Impaired Exercise Tolerance in Patients With Coronary Artery Disease Summary Yasuhiro Nishiyama, 1 MD, Hiroshi Niiyama, 1 MD, Haruhito Harada, 1 MD, Atsushi Katou, 1 MD, Noriko Yoshida, 1 MD, and Hisao Ikeda, 1 MD Red blood cell distribution width (RDW) can predict mortality in cardiovascular disease. However, the underlying mechanisms of the beneficial prognostic marker remain unknown. The purpose of this study was to investigate whether the RDW is related to impaired exercise tolerance and exercise training (ET) effect on RDW in patients with coronary artery disease (CAD). Seventy-eight patients who underwent ET by supervised bicycle ergometer during 3 weeks served as the ET group whereas 30 patients who did not undergo ET were the control group. Exercise stress test with cardiopulmonary analysis was performed in the ET group. Peak oxygen uptake (from 14.1 ± 4.0 to 15.1 ± 3.8 ml/kg/minute, P < 0.05) significantly increased in the ET group. Although RDW and serum erythropoietin concentration (EP) before the observation period did not differ between the ET and control groups, RDW (from 44.4 ± 4.7 to 43.4 ± 3.8 fl, P < 0.01) and EP (from 27.9 ± 15.8 to 22.9 ± 8.2 miu/ml, P < 0.005) significantly decreased in the ET group, however, these parameters did not change in the control group. In the ET group, RDW was negatively correlated with peak oxygen uptake (r = -0.55, P < 0.01) and the changes in RDW before and after ET were positively correlated with the changes in EP (r = 0.39, P < 0.005). Thus, ET increases exercise tolerance and decreases RDW in association with increased oxygen uptake in patients with CAD. (Int Heart J 2016; 57: ) Key words: Erythropoietin, Exercise stress test, Oxygen uptake, Oxygen transport Red blood cell distribution width (RDW) is a quantitative measure of anisocytosis, the variability in size of the circulating erythrocytes. In general, higher RDW reflects increased red blood cell destruction such as hemolytic disorders and nutritional deficiency including iron, vitamin B 12, and folate deficiency. Recent studies showed a strong independent association between higher levels of RDW and the risk of adverse cardiovascular outcomes in patients with heart failure, coronary artery disease, and pulmonary hypertension. 1-5) However, the underlying mechanisms of the beneficial prognostic marker remain unknown. Red blood cells transport oxygen to tissues such as peripheral muscle. Increased RDW signifies increased red blood cells with incomplete oxygen binding with hemoglobin such as premature erythrocytes or iron deficiency anemia. Accordingly, we speculate that higher levels of RDW may affect oxygen transport capacity, which is associated with adverse clinical outcomes. 1-5) Exercise training increases aerobic capacity and exercise tolerance, 6) and improves prognosis in patients with coronary artery disease (CAD). A previous study has shown that higher levels of RDW were related to impaired exercise capacity and exercise training decreased RDW in patients with chronic heart failure, 7) but no study has reported them in patients with CAD. Therefore, we investigated whether RDW is related to impaired exercise tolerance and exercise training effect on RDW in patients with CAD. Methods Study patients: The study population consisted of 108 patients with stable CAD who were admitted to Kurume University Medical Center. CAD was defined as the presence of visible luminal narrowing (> 50%) in at least one of the 15 coronary segments according to the American Heart Association and having the symptoms of chest pain with ischemic electrocardiographic changes. All subjects received coronary angiography to diagnose CAD. Of these, 20 had a previous myocardial infarction, and 88 patients had a history of angina with no evidence of previous infarction. Forty-six patients had prior percutaneous intervention and 10 had coronary artery bypass graft surgery. All patients were Grade 1 of Canadian Car- From the 1 Department of Cardiology, Kurume University Medical Center, Fukuoka, Japan. Address for correspondence: Yasuhiro Nishiyama, MD, Department of Cardiology, Kurume University Medical Center, Kokubu-machi, Kurume, Fukuoka , Japan. ynishi@orange.ocn.ne.jp Received for publication January 8, Revised and accepted March 9, Released in advance online on J-STAGE September 1, All rights reserved by the International Heart Journal Association. 553

2 554 NISHIYAMA, ET AL Int Heart J September 2016 diovascular Society stable angina. 8) All patients underwent complete routine physical and laboratory examinations, and their complete anamnesis was obtained. Of the 108 patients, 78 were eligible for cardiac rehabilitation and the remaining 30 who were unable to perform the exercise stress test and cardiac rehabilitation because of orthopedic or neuromuscular disease were allocated to the control group. Patients with atrioventricular block and pacemaker implantation, severe renal failure, severe hepatic disease, malignant neoplasmas, or who received blood transfusion within 3 months were excluded from the study. All patients received conventional drug therapy. In these patients, medications included angiotensin converting enzyme inhibitors or angiotensin receptor blockers (n = 72), aspirin (n = 65), HMG-CoA inhibitors (n = 57), antithrombotic agents (n = 48), calcium channel antagonists (n = 47), vasodilators (n = 41), β blockers (n = 37), and diuretics (n = 27). Medication therapy was not changed during the study period. Informed consent was obtained from all patients, and the study was approved by the Human Study Committee of our institution. Blood samples and hormone measurements: Blood samples were collected in the morning following an overnight fast before and after exercise training in the training group, or at the same period in the control group in the hospital. After supine rest for at least 20 minutes, a sample was taken from the antecubital vein and after immediate centrifugation, aliquots were stored at 70 C until analysis. Complete blood count was determined using an XT-2000 automated cell counter (Sysmex, Kobe, Japan) with standard calibration. The RDW standard deviation was taken at the 20% relation height level, with the red cell histogram peak considered to be 100%. The red cell histogram crosses the 20% level twice. The distance between the two cross points was defined as RDW and is reported in femtoliters (fl). The erythropoietin levels were measured by radioimmunoassay. Exercise stress test and exercise training: Seventy-eight patients underwent a symptom-limited exercise stress test before and after exercise training. Exercise stress tests were performed with an electrically braked bicycle ergometer (BE250 Fukuda Denshi, Tokyo). A ramp protocol, starting at a workload of 20 watts and increased incrementally by 1 watt every 6 seconds (10W/minute) was used. The ECG and SBP were recorded every minute before, during, and after exercise (ML- 9000, Fukuda Denshi, Tokyo). Criteria for stopping the exercise test included life-threatening arrhythmias, ST-segment depression or elevation > 0.2 mv, a fall in SBP > 20 mmhg, or predicted target heart rate (HR) [(220 age) 0.85]. In 62 patients, expired gas was collected and analyzed continuously with an AE-310S gas analyzer (Minato Co, Osaka, Japan). Peak oxygen uptake (VO 2 ) was defined as the highest VO 2 value achieved at peak exercise. The anaerobic threshold (AT) was defined using the V-slope method. The slope of the relationship between ventilation (VE) and carbon dioxide production (VCO 2 ) (VE/VCO 2 slope) as a marker of ventilator inefficiency, was determined by linear regression. Exercise tolerance (metabolic equivalents, METs) was calculated by the following formula: Workload (watts) / Body Weight (kg) 35, or Peak VO 2 / 3.5. Twenty minutes of supervised bicycle training was performed every day for 3 weeks in the hospital. Exercise intensity was 70% of peak workload or AT level, determined by the entry exercise test. Statistical analysis: Values are presented as the mean ± standard deviation (SD). The comparisons between groups were made by the unpaired t-test and χ 2 test. Serial changes were compared by the paired t-test. Correlations of RDW with various parameters were examined by Pearson correlation analysis. We investigated the association with RDW and peak VO 2 before and after exercise training with analysis of covariance (ANCOVA). All statistical analyses were performed using an SPSS system. Skewed variables were log-transformed. A probability value of < 0.05 was considered significant. Results Baseline characteristics: Baseline characteristics of the enrolled patients are shown in Table I. The training and control groups did not differ in terms of age, sex, body mass index, myocardial infarction, PCI/CABG, EF, complications, and medication. Renal function, such as serum creatinine and estimated glomerular filtration (egfr), was significantly decreased in the control group compared to the training group. Clinical and hematological parameters: Clinical and hematological parameters of the enrolled patients are shown in Table II. Exercise tolerance (METs) significantly increased after exercise training. RDW, hemoglobin, and erythropoietin concentration significantly decreased after exercise training. These parameters did not change in the control group during the observation period. Mean corpuscular volume (MCV) did not change in either group after training or after the observation period. RDW, MCV, and erythropoietin concentration after the observation period in the control group were significantly larger than those after exercise training in the training group. Hemoglobin in the control group was significantly lower than those before and after exercise training in the training group. Cardiopulmonary parameters before and after exercise training: Exercise parameters in the cardiopulmonary exercise test are shown in Table III. Systolic blood pressure at rest and peak exercise significantly decreased after exercise training. Oxygen uptake at anaerobic threshold and peak exercise significantly increased after exercise training. However, heart rate at rest and peak exercise, peak workload, peak VO 2 /HR (oxygen pulse), ΔVO 2 /Δwork load, and VE/VCO 2 slope did not change before and after exercise training. Correlation between RDW and peak oxygen uptake: Figure 1 shows the correlation between RDW and peak VO 2. Significant inverse correlations were observed between peak VO 2 and RDW before (r = -0.36, P < 0.01) and after exercise training (r = -0.55, P < 0.01), respectively. These correlations were significantly stronger after exercise training than before exercise training (P < ). Correlation between the changes in RDW and erythropoietin before and after exercise training: Figure 2 shows the correlation between changes in RDW and changes in erythropoietin concentration after exercise training. A significant correlation was observed between the changes in RDW and changes in erythropoietin concentration (r = 0.39, P < 0.01). Discussion In the present study, we demonstrated that RDW was sig-

3 Vol 57 No 5 EFFECT OF EXERCISE TRAINING ON RDW 555 Table I. Baseline Characteristics of the Enrolled Patients Training (n = 78) Control (n = 30) P Age 69 ± ± 10 NS Sex (Male/Female) 48/30 19/11 NS Body mass index (kg/m 2 ) 23.7 ± ± 3.7 NS MI 16 4 NS PCI/CABG 36/7 10/3 NS EF 61 ± ± 14 NS Creatinine 0.8 ± ± 0.6 P < 0.05 egfr 66.7 ± ± 21.9 P < 0.05 Complication Hypertension 57 (73) 22 (73) NS Hyperlipidemia 39 (50) 15 (50) NS Diabetes mellitus 30 (38) 7 (23) NS Medication ACI/ARB 47 (60) 25 (83) NS Ca 30 (38) 17 (57) NS Statin 40 (51) 9 (30) NS β-blocker 29 (37) 8 (27) NS Vaso 25 (32) 16 (53) NS Diu 18 (23) 9 (30) NS A-P 49 (63) 16 (53) NS A-T 31 (40) 17 (57) NS Values are mean ± SD or number (%) as indicated. PCI indicates percutaneous coronary intervention; CABG, coronary artery bypass grafting; EF, ejection fraction; egfr, estimated glomerular filtration rate; ACI, angiotensin-converting enzyme inhibitor; ARB, angiotensin II receptor blocker; Ca, calcium-channel antagonist; Statin, HMG-CoA reductase inhibitor; Vaso, vasodilator; Diu, diuretics; A-P, antiplatelet agent; A-T, antithrombotic agent; and NS, not significant. Table II. Clinical and Hematological Parameters of the Enrolled Patients Training Control Before After Before After RDW (fl) 44.4 ± ± 3.8 ** 46.4 ± ± 6.1 ## Hemoglobin (g/dl) 13.0 ± ± 2.0 * 12.0 ± 1.5 # 11.7 ± 1.7 # MCV (fl) 93.2 ± ± ± ± 4.2 # Erythropoietin (miu/ml) 27.9 ± ± 8.2 * 28.6 ± ± 8.3 # METs 5.0 ± ± 2.0 ** Values are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, before versus after. # P < 0.05, ## P < 0.01, Training versus Control. RDW indicates red cell distribution width; MCV, mean corpuscular volume; and METs, metabolic equivalents. Table III. Cardiopulmonary Parameters Before and After Exercise Training Before After P Heart Rate (rest) 72 ± ± 11 NS. Heart Rate (peak exercise) 118 ± ± 19 NS. SBP (rest, mmhg) 123 ± ± 18 P < 0.01 SBP (peak exercise) 175 ± ± 32 P < 0.05 Peak Work Load (watts) 76 ± ± 28 NS AT VO 2 (ml/kg/minute) 9.5 ± ± 2.3 P < 0.05 Peak VO 2 (ml/kg/minute) 14.1 ± ± 3.8 P < 0.05 Peak VO 2 /HR (ml/beat) 7.7 ± ± 2.7 NS ΔVO 2 /Δ Load (ml/watt) 8.15 ± ± 2.95 NS VE/VCO 2 slope 32.4 ± ± 6.2 NS Values are mean ± SD. SBP indicates systolic blood pressure; AT, anaerobic threshold; VO 2, oxygen uptake; HR, heart rate; VE, ventilation; VCO 2, carbon dioxide production; and NS, not significant. nificantly related to peak VO 2 in patients with CAD. Furthermore, exercise training significantly increased exercise tolerance, and decreased RDW and erythropoietin concentrations. Thus, our findings, to the best of our knowledge, provide the first demonstration that exercise training increases exercise tolerance and decreases RDW in association with decreased erythropoietin concentration in patients with CAD. Increased RDW is a strong independent predictor of morbidity and mortality in patients with cardiovascular disease including heart failure, coronary artery disease, and pulmonary

4 556 NISHIYAMA, ET AL Int Heart J September 2016 Figure 1. Relationship of red blood cell distribution width (RDW) and peak VO 2. Note that a significant correlation was observed between RDW and peak VO 2. Figure 2. Relationship of changes in red blood cell distribution width (RDW) and the changes in erythropoietin concentration (EPO) before and after exercise training. Note that a significant correlation was observed between the changes in RDW and the changes in EPO before and after exercise training. hypertension. 1-5) Furthermore, Oh, et al showed a novel relation between higher RDW and elevated E/E (early mitral inflow velocity to early diastolic mitral annual velocity) in patients with acute heart failure, suggesting that RDW is associated with elevated left ventricular filling pressure. 9) Fukuta, et al showed that higher RDW was associated with elevated BNP in patients with CAD, suggesting that neurohumoral activation is a mechanistic link between increased RDW and adverse clinical outcomes in patients with CAD. 10) However, the possible mechanisms of higher RDW levels and adverse cardiovascular outcomes in patients with CAD remained unclear. Higher levels of RDW suggest immature red blood cell production in bone marrow and increased red blood cells with incomplete oxygen binding with hemoglobin. Therefore, increased RDW may affect oxygen transport and exercise tolerance. In our study, RDW negatively related to peak VO 2 in CAD patients, and was especially stronger after exercise training. Peak VO 2 is considered as a standard of exercise tolerance and reflects both cardiac performance and the capacity of peripheral working muscles to extract transported oxygen. 11) There was no significant relationship between the change in RDW and change in peak VO 2 before and after exercise training in our study. The reason is unclear. However, we thought that a decrease in RDW after exercise training would lead to increased oxygen transport and extraction of oxygen in contracting muscle and improved exercise capacity (increased peak VO 2 ). A decrease in RDW might be proportional to the increase in oxygen delivery, but not be proportional to extracted oxygen in contracting muscle. Our findings indicate that RDW reflects oxygen transport capacity and a reduction in oxygen transport capacity may be one of the mechanisms responsible for the relationship between increased RDW and poor prognosis in cardiovascular disease. Exercise training increased peak VO 2 and improved prognosis in CAD patients. Further investigations are needed to provide a more convincing explanation. Exercise training significantly decreased RDW and erythropoietin levels in this study. Although a previous study showed that exercise training decreased RDW in chronic heart failure patients, 7) no study has been conducted in patients with CAD. Additionally, our study showed a significant correlation between changes in RDW and changes in erythropoietin concentration. This suggests that the variability of RDW was induced by erythrocyte proliferation in the bone marrow. Thus, our study is the first report of an exercise training effect on RDW in CAD patients. A previous study demonstrated that exogenous erythropoietin treatment resulted in an increase of hemoglobin concentration leading to increased oxygen delivery and improved exercise capacity ) These results seem at first sight to be opposite to our results. However, we assessed that exercise training decreased RDW leading to increase oxygen binding with hemoglobin and oxygen delivery, and improved exercise capacity. In the body, an increase of oxygen delivery causes negative feedback on endogenous erythropoietin concentration and a decrease in erythropoietin concentration after exercise training. Possible mechanisms underlying the beneficial effects of exercise training on RDW may be considered. In general, exercise training in CAD patients decreases proinflammatory cytokine production and induces inducible nitric oxide synthase expression and antioxidative effects, 16,17) leading to enhanced erythroid proliferation in red bone marrow. Zhao, et al showed that exercise training altered erythrocyte deformability and decreased the abnormality ratio of erythrocyte shape, resulting in enhanced oxygen delivery. 18) Schmidt, et al reported that exercise training increased the 2,3-diphosphoglycerate concentration of red blood cells and oxygen transport capacity. 19) Thus, several mechanisms for the effects of exercise training on RDW may be considered although further study is needed. In this study, hemoglobin concentration decreased after exercise training. A previous study demonstrated that the decrease in hemoglobin concentration was transient and occurred in the early phase of training and then returned to pretraining levels. 19) Additionally, another study reported that hypervolemia induced by exercise training led to a decrease in hemoglobin concentration ) If anemia (decrease in total hemoglobin volume) is true in this study, mean corpuscular volume should have decreased and erythropoietin concentration should have increased, but they did not in fact. Therefore, it is thought that the decrease in hemoglobin concentration after exercise training in our study did not affect RDW. Several limitations in this study are worth mentioning.

5 Vol 57 No 5 EFFECT OF EXERCISE TRAINING ON RDW 557 First, we did not directly measure oxygen delivery. However, since cardiopulmonary analysis exactly reflects oxygen delivery during exercise, this did not influence our conclusion. Second, most of the RDW values varied within the normal range. In the CARE study which showed a graded independent relation between higher levels of RDW and the risk of death and cardiovascular events in patients with CAD, 2) RDW levels were within the normal range in most of the patients. Therefore, our study findings have relevant clinical significance. Third, patients in the control group had a greater degree of renal dysfunction and lower hemoglobin than those in the training group. Although erythropoietin concentration, hemoglobin, and complications (diabetes mellitus and hyperlipidemia) at study entry were determining factors of RDW in stepwise multiple linear regression analysis (data not shown), erythropoietin concentration and rate of complication (diabetes mellitus and hyperlipidemia) did not differ between the groups. Additionally, RDW in the control group might be variable for lower hemoglobin, but did not change before and after the observation periods. Although medication such as statin or antiplatelet agents may have an effect on RDW, these medications were not determining factors in the stepwise multiple linear regression analysis and the rate of medication did not differ in either the control or exercise training group. Based on these results, RDW might decrease after exercise training. Finally, the number of patients was small and they were middle aged. Further investigations are needed to provide a more convincing conclusion in a larger number of patients with a wide range of age. In conclusion, the present study, to the best of our knowledge, provides the first demonstration that RDW is associated with impaired oxygen uptake and erythropoietin concentration in patients with CAD. This finding may be one of the underlying mechanisms of increased RDW and a poor prognosis in cardiovascular disease. Acknowledgment The authors wish to thank Ms. Emiko Shiotani for her excellent secretary assistance. References 1. Felker GM, Allen LA, Pocock SJ, et al; CHARM Investigators. Red cell distribution width as a novel prognostic marker in heart failure: data from the CHARM Program and the Duke Databank. J Am Coll Cardiol 2007; 50: Tonelli M, Sacks F, Arnold M, Moye L, Davis B, Pfeffer M; for the Cholesterol and Recurrent Events (CARE) Trial Investigators. 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