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The impact of splenic release of red cells on hematocrit changes during exercise Wolski, Lynneth Ann 1999

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T H E IMPACT OF SPLENIC RELEASE OF RED CELLS ON HEMATOCRIT CHANGES DURING EXERCISE by LYNNETH ANN WOLSKI B.Sc, University of Victoria, 1989 M.Sc, University of Victoria, 1992 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR T H E DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF GRADUATE STUDIES (Department of Interdisciplinary Studies) We accept this thesis a conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1998 © Lynneth Ann Wolski, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of lnhrd)3a,plfr>dru Tfturlh*^ The University of British Columbia Vancouver, Canada Date ~fon,,ary iQQQ DE-6 (2/88) ABSTRACT The purpose of this study was to determine the volume of red cells released by the spleen during exercise and to establish the impact of splenic emptying on peripheral hematocrit during exercise. The influences of training status and hypoxic exposure on splenic emptying and exercise hemodynamics were also examined. 6 trained (T) and 6 untrained (N) and 4 splenectomized (S) subjects completed a set of two tests: a maximal aerobic power test and a 30 minute exercise test (ES). The T and N groups also completed the two tests i n hypoxic conditions (FI02 = 0.16). The results of the maximal test determined the power outputs (PO) at which the subjects exercised during the exercise session. The ES consisted of 10 minutes at a FO requiring 25% of maximal V O 2 , ten minutes at a PO requiring 50% of maximal V O 2 and 10 minutes at a PO requiring 75% of maximal VO2. Red cell volume (RCV), plasma volume (PV) were directly measured pre- and post-exercise using radioisotope labeling ( 5 1Cr, 1 2 5 | -RHISA, 1 3 1I-RHISA). Hematocrit (Hct) was measured and the spleen was imaged, using 9 9 m Tc, pre-exercise and after each 10 minute workload. Spleen volume (SV) was calculated using the average count of the anterior and posterior scan and the count of a known volume of blood. There was no difference in pre-exercise RCV between the S group (2033 mL) and the N group (2058 mL). The N group showed a significant increase in RCV in both normoxic and hypoxic conditions post-exercise (p<0.05). The S group PV of 4255 mL was significantly higher than the N group PV of 3518 mL (p<0.01). Post-exercise PV in S was significantly lower than pre-exercise PV and difference between N and S pre-exercise PV disappeared. The N and T groups also showed a significant (p<0.05) decrease in PV pre-to post-exercise, in both the normoxic and hypoxic conditions, however there was no difference in that decrease between the two test conditions or between the groups. SV also decreased pre- to post-exercise for N and T in both conditions (p<0.01). The pre-exercise SV of the N group (360 mL) was higher than that of the T group (279 mL), but when the change in SV was shown as a percentage of original SV, the decrease in SV after each exercise load was identical for both groups in both normoxic and hypoxic conditions. The volume of red cells released by the spleen ranged from 142-187 mL, representing 7-9% of total red cell volume. Both N and T showed significant increases in Hct in both exercise conditions but there was no difference between the groups or between conditions. The S group Hct readings at all test times were significantly lower than the corresponding N group Hct and did not significantly increase during exercise: Reductions in PV in the N and T groups were calculated to only cause 68-78% of the change in Hct during both normoxic-and hypoxic exercise with the increase in circulating RCV causing the remainder of the change. The results of this study demonstrate that splenic release of red cells has a significant impact on peripheral hematocrit. Aerobic fitness and hypoxic exposure does not influence the reduction in spleen volume with exercise or it's impact on hematocrit changes. The results of this study also provide evidence that indirect calculations of plasma volume changes could result in prediction errors of 22 to 33%. V TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES vi LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x DEDICATION x INTRODUCTION 1 Statement of the Problem 4 Objectives 5 METHODOLOGY 8 Experimental Design 8 Subject Selection 9 Testing Procedures 1 0 Maximal Aerobic Power Tests normoxic and hypoxic 10 30 minute exercise test session 1 1 Protocols for Determination of Blood Volumes 1 4 Red Cell Volume 14 Plasma Volume 1 5 Total Blood Volume 1 5 Spleen volume 1 6 Hematocrit 1 7 Statistical Analysis 1 7 RESULTS 1 9 Group characteristics 1 9 Reliability of the measurement techniques 1 9 Untrained normal subject (N) and splenectomized subject (S) response to normoxic exercise 2 3 Normal and Trained subjects comparisons normoxia 2 9 Normal and Trained subjects comparisons hypoxia 3 7 Normal and Trained subjects normoxic and hypoxic comparison 4 5 DISCUSSION 5 4 Blood Volumes 5 4 Spleen Volumes 5 6 Hematocrits 5 9 Methodology Considerations 6 1 Conclusions 6 3 APPENDIX A. Review Of The Literature 6 5 References 8 0 APPENDIX B. Nuclear Medicine Techniques 8 5 v i APPENDIX C. Informed Consent 8 9 APPENDIX D. Examples of Regions Of Interest 9 4 APPENDIX E. Calculations 9 7 v i i L I S T O F T A B L E S Table 1. Order of test sessions 8 Table 2. Group physical characterstics 2 0 Table 3. Comparisons of average V02 max and Peak Power in normoxia and hypoxia 2 1 Table 4.. Reliability scores of directly measured pre-exercise blood volumes and spleen volumes 2 2 Table 5. Pre-exercise plasma volume and red cell volume per kilogram of body weight for groups N and T in normoxic conditions 2 9 Table 6. Pre-exercise plasma volume and red cell volume per kilogram of body weight for groups N and T in hypoxic conditions 3 7, Table 7. Pre-exercise red cell, plasma and total blood volumes in normoxia and hypoxia for T and N groups 4 5 Table 8. Relative contribution of plasma volume and circulating red cell changes to changes in hematocrit with exercise in normoxic and hypoxic conditions for groups N and T 5 3 v i i i LIST OF FIGURES Figure 1. Timeline for Nuclear Medicine Protocol 1 3 Figure 2. Pre-exercise RCV, PV and TBV for groups N and S 2 5 Figure 3. Changes in RCV and PV with 30 minutes of exercise in groups N andS 26 Figure 4. Spleen volume of N subjects pre-exercise and during exercise 2 7 Figure 5. Hematocrit changes during 30 minutes of exercise in groups N andS 28 Figure 6. Comparison of normoxic, pre-exercise RCV, PV and TBV of N and T groups 31 Figure 7. RCV pre- and post-exercise for N and T groups in normoxic conditions 3 2 Figure 8. PV changes from 30 minutes of exercise for groups N and T 3 3 Figure 9. Changes in spleen volume during 30 minutes of exercise in normoxia fro groups T and N 3 4 Figure 10. Spleen volume shown as a percentage of the original volume for groups T and N in normoxia 3 5 Figure 11. Changes in hematocrit during 30 minutes of exercise in normoxia for groups T and N 3 6 Figure 12. Hypoxic pre-exercise RCV, PV and TBV for groups N and T 3 9 Figure 13. RCV pre- and post-exercise for groups N and T in hypoxia 4 0 Figure 14. PV pre- and post-exercise for groups N and T in hypoxia 4 1 Figure 15. Changes in spleen volume during 30 minutes of exercise in hypoxia for groups N and T 4 2 Figure 16. Spleen volume shown as a percentage of the original volume for groups N and T in hypoxia 4 3 Figure 17. Changes in hematocrit during 30 minutes of exercise in hypoxia for groups N and T 4 4 i x Figure 18. RCV pre- and post-exercise for groups N and T in normoxia and hypoxia 4 7 Figure 19. PV pre- and post-exercise for groups N and T in normoxia and hypoxia 4 8 Figure 20. Spleen volume changes during 30 minutes of exercise in normoxia and hypoxia for group T 4 9 Figure 2T. Spleen volume (Changes? during 30 minutes of exercise in normoxia and hypoxia for group n 5 0 Figure 22. Comparison of changes in hematocrit in normoxia and hypoxia for group T 51 Figure 23. Comparison of changes in hematocrit in normoxia and hypoxia for group N 5 2 Figure 24. Diagramatic illustration of the spleen 6 9 X ACKNOWLEDGEMENTS The completion of this thesis was made possible by the contributions of many others. First and foremost, I would like to thank Dr. Donald McKenzie for providing guidance, support and wisdom throughout my degree. His valuable insights and astounding depth of knowledge in the field of exercise physiology has only deepened my love and interest for the area. I also extend my gratitude to my thesis committee members, Dr. Ken Courts, Dr. Donald Lyster, Dr. Howard Wengenand Dr. Peter Hochachka. The time and energy that each of these men spent to help refine the research design and methodology is gratefully acknowledged. I would also like to thank the members of my examining committee, Dr. Tim Noakes, Dr. Ted Rhodes and Dr. Jack Taunton for the time spent reading my thesis and preparing for the final oral examination. I would also like to recognize the valuable contribution of the lab staff and other graduate students at the Allan McGavin Sports Medicine Centre, especially Diana Jespersen and Sherri Niesen. Diana provided helpful assistance and direction in helping organize testing, equipment moving and supplies. Sherri, my office-mate, provided endless support, companionship and laughter. Thank you both. I was also extremely fortunate to have a home away from home in Vancouver while completing my degree. I am forever grateful to my dear friend, Joan Kraayvanger, for opening her home and heart to me. Lastly, a very special thank you to my husband, Mark, and my daughter, Cailyn whose love, support and encouragement made the completion of this thesis possible and worthwhile. DEDICATION This thesis is dedicated to my husband, Mark and daughter, Cailyn. You continually bring joy to my life. I love you both. I also dedicate this thesis to my dear friend, Joan whose support and friendship I can never begin to repay. 1 INTRODUCTION Exercise causes both acute and chronic changes in blood plasma volume, hematocrit and total red cell volume. These changes, in turn, have an impact on blood viscosity and oxygen delivery to the body tissues. While the changes in plasma volume with training and acute exercise have been extensively studied, the role of changes in total red cell volume and circulating red cell volume have not. Furthermore, the question of whether changes in circulating red cell volume with exercise contribute significantly to changes in hematocrit has not been clarified. A balance between oxygen carrying capacity and hyperviscosity is necessary for sustained aerobic activity. Acute, intense exercise can cause a 1 0 - 2 0 % contraction in plasma volume resulting in hyperviscosity (61). With training, the body attenuates the severity of this reaction by expanding the plasma volume, in response to higher levels of renin, aldosterone, vasopressin and albumin (27), to a state of pseudoanemia. Rather than being a pathological condition, pseudoanemia compensates for the hemoconcentration that is encountered with exercise. Hemoconcentration results in hyperviscosity which is detrimental to oxygen delivery (20). Studies using animal models such as racehorses (45), Weddell seals (4 6) and dogs (29), have shown that the spleen contracts during exercise and under stressful (fight or flight) situations resulting in a release of a stagnant pool of red blood cells into the circulation. Splenic contraction and the subsequent release of red blood cells is also believed to be an integral 2 component of the response to exercise and stress and is associated with a large increase in hematocrit. The adaptation to repetitive physical activity on splenic contraction has not been determined. It is not known if the splenic response to exercise stress decreases with training, acting to avoid hemoconcentration or, if splenic response increases with training in a parallel adaptation to the'training-induced plasma volume expansion. In humans, the normal spleen serves many functions. The most important ones are filtration, phagocytosis and destruction of red blood cells, storage of viable blood cells, antigen uptake, antibody formation, potential hemopoiesis, lymphocyte production and possible hemopoietic regulation (38). Generally, it has been accepted that red cell content of the spleen alters depending on sympathetic nervous discharge, vasoconstriction of the splenic arteries and/or increased intra-abdominal pressure due to increased ventilation. Staubli et al. (56) showed that in hyperventilation, the normal spleen does contract, postulating that it is a combination of increased intra-abdominal pressure and catecholamine release that contributes to splenic contraction. However Laub et al. (38) did not find a clear relationship between splenic red cell content and adrenergic activity. The mechanism(s) responsible for splenic contraction and the subsequent release of red cells during exercise stress have not been fully identified. Although animal studies have shown that the normal spleen contracts under stressful condition there has been considerable dispute regarding the red cell reservoir function of the human spleen during exercise and stress. Some studies have shown no evidence for mobilization of blood reserve by exercise, 3 epinephrine or hemorrhage (2,15) while others have shown significant splenic shrinkage with exercise (23,51), epinephrine injection (6) and hyperventilation (56). Methodology may account for the discrepancies in these findings. Generally, studies that have calculated blood volume changes using hematocrit and plasma volume or have used isolated, perfused human spleens have found no evidence for mobilization of blood reserves or splenic contraction, whereas studies that have used radioactive labeling to obtain in vivo images of the spleen, along with measures in blood parameters, have shown significant changes in spleen size and peripheral hematocrit during exercise stress. For example, Laub et al. (38) found that average hematocrit increased from 44.6% to 48.0% at maximum work level in subjects exercising for thirty minutes, while splenic scans using s^Tc pertechnetate showed that splenic red cell content decreased linearly throughout exercise to 34.2% of resting value. Similarly, other researchers using radiopharmaceuticals have also found decreases of 34.4% - 49% in splenic radioactivity with exercise (24,51,64). In the above studies, the changes in spleen volume have only been described qualitatively, as a percent change from original levels. Laub et al. (38) calculated that splenic contraction may account for about one-fourth of the rise in hematocrit during exhaustive exercise, with plasma volume shifts accounting for the remainder. However, they did not label plasma in order to account for the changes in plasma volume with exercise. The change i n hematocrit with exercise is most likely due to a combination of an increased release of red blood cells by the spleen and shifts in plasma volume, but the role of each in increasing hematocrit has not been defined. 4 Exercise can cause changes in hemodynamics (splenic release of red cells, shifts in plasma volume, changes in blood viscosity) which may have an indirect impact on conditions such as cardiovascular disease and stroke. As the spleen plays such an important role in many functions of the blood, it is logical to begin; studying the hemodynamics of the spleen during exercise. Understanding the impact of training state and hypoxia on splenic contraction, and exactly how the spleen functions hemodynamically during exercise wi l l provide the groundwork for future study on the influence of exercise and training in other systems in which the spleen has an important function. In addition, the ability to quantify the exact changes in peripheral hematocrit that result from increased red cell volume as opposed to plasma volume shifts will provide valuable information on the vascular physiology of normal subjects during exercise. Statement of the Problem The purpose of this study was to determine if the spleen has a significant role in increasing hematocrit during acute exercise. Furthermore, the decrease in both the volume of the spleen and plasma volume during 30 minutes of exercise and the corresponding increase in hematocrit in untrained and trained subjects in both normoxic and hypoxic environments was determined. 5 Object ives Thus, the objective of this study was to answer the following research questions. 1. What is the change in spleen volume during graded exercise in normal, untrained human, subjects?:-2. What is the effect of increases in circulating red cell volume and plasma volume shifts during exercise on peripheral hematocrit ? As mentioned previously, the actual volume change of the spleen during exercise has not been determined. Results from previous studies have shown that spleen volume decreases with exercise and hematocrit correspondingly increases, but the increase in hematocrit that is due to splenic release of red cells and the increase that is due to plasma volume shifts has remained unresolved. In order to gain more information on the role of the spleen during exercise and subsequent changes in peripheral hematocrit, normal, healthy male and female subjects were studied during thirty minutes of graded exercise. Individuals without spleens acted as control subjects to determine if there are other potential sites of contribution to the total circulating red blood cell pool during exercise. It was hypothesized that the normal, untrained subjects would show a significant decrease in splenic volume with a corresponding significant increase in hematocrit 6 that would be attributable to both an infusion of red blood cells from the spleen and a decrease in plasma volume. Furthermore, it was hypothesized that the splenectomized control group would also demonstrate plasma volume decreases but no significant changes in hematocrit with exercise. 3. Does aerobic fitness have an influence on splenic volume changes and peripheral hematocrit during exercise? Evidence suggests that fitter people have a decreased response to sympathetic hormones (33,66) and increased plasma volume (8) . Well-trained subjects were compared to untrained subjects in normoxic conditions to determine if fitness level and training-induced changes i n blood volume had an influence on plasma volume shifts as well as splenic volume changes and subsequent release of red blood cells. Red blood cell mobilization by the spleen may depend upon sympathetic nervous discharge (38) and/or mechanical effects on the spleen of diaphragmatic contractions during ventilation (56). It was hypothesized that untrained subjects would demonstrate a significantly greater degree of splenic shrinkage and larger changes in peripheral hematocrit than the we II-trained subjects. Is splenic contribution to circulating red cell volume greater during exercise in a hypoxic environment? Does aerobic fitness level influence the relationship? Once the contribution of the spleen pool was quantified, normal, untrained subjects ; performed t exercise in hypoxic conditions to determine if splenic contribution of red blood cells to peripheral circulation is greater in a hypoxic situation. Hypoxic exposure increases catecholamine release in both trained and untrained subjects (57) and if splenic volume changes are caused by catecholamine stimulation as suggested by Staubli et al. (56), then hypoxia should result in an increased reduction in splenic volume compared to normoxia. Well-trained cyclists were also compared to untrained subjects in hypoxic conditions to determine if fitness level influences the degree of splenic volume change during hypoxic exercise. It was hypothesized again that the untrained subjects would have significantly larger reductions in spleen volume and larger increases in hematocrit than the well-trained subjects. 8 METHODOLOGY Experimental Design All hypotheses were tested simultaneously. Subjects completed two sets of two tests:: a maximal aerobic power test and a 30 minute exercise test. The test set was completed either in normoxic or hypoxic conditions and the order of the test set was always the maximal aerobic power test followed by the 3 0 minute exercise test with at least 48 hours between the two tests. The aerobic power test results were used to set the workloads used during the exercise test session. The order of normoxic and hypoxic test sets was randomized (Table 1). The minimum time allowed between the normoxic and hypoxic test series (VO2 max and the 30 minute exercise test) was 2 weeks to allow for decay of the radioactive isotopes used for blood labeling and recovery from exercise. Table 1: Order of test sessions Test 1 Test 2 Test 3 Test 4 VO2 max (norm) 30 min (norm) VO2 max (hyp) 30 min (hyp) VO2 max (hyp) 30 min (hyp) VO2 max (norm) 30 min (norm) VO2 max: maximal aerobic power test 30 min: 30 minute exercise test norm: normoxic exercise conditions hyp: hypoxic (F1O2 = 0.16) exercise conditions 9 Subject Selection: A total of sixteen subjects, six normal (N), four splenectomized (S) and six trained (T) signed informed consent (Appendix C) and completed this study that was approved by the University of British Columbia Ethics Committee. Competitive road cyclists or. long distance runners were recruited as the T subjects. Healthy, active males and females made up the N group and splenectomized subjects were recruited to be control subjects (S). Three of the splenectomized subjects had had their spleen removed due to acute trauma to the organ and one subject had undergone splenectomy because of genetically-inherited problems. Inclusion/exclusion criteria for the groups were as follows: Inclusion criteria 1. Ages 20-40 years 2. Clearance for participation by a medical doctor 3. Males: aerobic power scores between 30 and 50 mL-kg"1-min"1 for groups N and S and above 60 mL-kg"1-min"1 for group T. 4. Females: aerobic power scores between 35 and 45 mL-kg"1-min"1 for the groups N and S and above 55 mL-kg"1 -min"1 for group T. Exclusion criteria 1. Outside the age range chosen 2. Aerobic power score below the acceptable range 3. A cardiovascular, respiratory or musculoskeletal condition that would compromise their ability to perform all of the required testing 4. Exclusion by the physician 5. Exposure to radioactive nucleotides in the past three months 1 0 Testing Procedures Maximal Aerobic Power Tests: normoxic and hypoxic The subjects' height in centimeters and mass in kilograms was measured and recorded upon arrival to the laboratory for either the normoxic or hypoxic maximal aerobic power test. Group T and N subjects completed both a normoxic and a hypoxic maximal aerobic power test while Group S subjects only performed the normoxic test. The maximal aerobic power tests were performed on an electronically-braked Mjinhardt cycle ergometer (KEM-3). One test was completed in a normoxic environment (F1O2 = 0.2096) and one while breathing hypoxic gas (F|02 = 0.16). Expired gases were analyzed using a Rayfield Exercise Testing System with a mixing chamber and averaged every 20 seconds to determine ventilation (VE), carbon dioxide production (VCO2) and oxygen consumption (VO2). Criteria for attainment of VO2 max were three of the following: an increase of less than 0.1-0.2 L-min"1 or <2 mL-kg" 1 -min" 1 in V O 2 with an increase in work load; an RER value >1.1; attainment of 90% of predicted maximal heart rate; and volitional fatigue. Power outputs were ramped at 25 W-min"1 for all groups. Heart rate was monitored using a telemetric device (Polar Vantage XL Heart Monitor). In order to induce hypoxia, subjects breathed humidified, hypoxic gas (F|O2=0.16) for 5 minutes prior to starting the test (for equilibration) and throughout the test. The protocol and criteria for the hypoxic maximal 11 aerobic power test was the same as the normoxic maximal aerobic power test described above. 30 minute exercise test session Subjects in groups T and N also completed two 30 minute exercise tests (normoxic and hypoxic) at the UBC Nuclear Medicine Department. Group S subjects completed only the normoxic test. Subjects were instructed to be well hydrated (ie. have had 8 glasses of water in the past 12 hours), rested (no exercise for 24 hours) and to be 6 hours post-absorptive when they arrived at Nuclear Medicine. After the subject had been seated for 2 0 minutes, a 20g Jelco intravenous catheter with a Heparin lock was inserted into both the right and left anticubital vein. 20 rmLs of blood was withdrawn from left arm for 5 1 Cr and 9 9 m T c labelling. An additional 10 ml_s of blood was withdrawn for residual calculations. Once the red cells had been labeled with 5 1 C R , 10 mLs of 5 1 C R red blood cells were re-injected into the subject for determination of red cell volume. 1 0 pCi of 1 2 5I -RIHSA was injected immediately afterwards for determination of pre-exercise plasma volume. 10 mL blood samples were taken at 10, 20 and 30 minutes post injection. The 30 minute post injection sample was used to calculate pre-exercise red ceil volume as well as plasma volume. After pre-exercise plasma volume and red cell volume blood samples had been taken, the subject was injected with 1 mL of 9 9 m Tc- label led red cells. Pre-exercise anterior and posterior spleen images were taken using a Siemens ZLC 3700 Orbiter Camera. Each image was scanned for 2 minutes. 1 2 After the pre-exercise scans were completed, the subject began the exercise session. The results of the maximal test determined the power outputs (PO) at which the subjects exercised during the exercise session (ES). The exercise consisted of 10 minutes at a PO requiring 25% of maximal VO2, ten minutes at a PO requiring t 50%, of maximal VO2 and 10 minutes at a PO requiring 75% of maximal VO2 with a four minute rest period between exercise loads to allow for completion of splenic scans. 10 mL blood samples were drawn during the last minute of exercise in each of the three workloads. The 30 minute blood sample was used for determination of post-exercise red cell volume. At the end of the third workload, 10 uCi of 1 3 1 I -RIHSA was injected immediately after the 30 minute blood sample was drawn for determination of post-exercise red cell volume. 10 mL blood samples were again taken 1 0, 20 and 30 minutes post-injection. A total of 120 mLs of blood was taken from the subject during the hypoxic test session. Hypoxic test: In order to stimulate hypoxia, subjects breathed humidified, hypoxic gas (F1O2 = 0.16) for 5 minutes prior to starting the ES (for equilibration) and throughout the ES. Exercise workloads for the ES were set at power outputs equivalent to 25, 50 and 75% of VO2 max as determined in the hypoxic aerobic power test. 13 1 4 Protocols for Determination of Blood Volumes Red Cell Volume: 15 mL of blood was withdrawn from the subject and incubated with 150 uCi of sodium chromate in an acid-citrate-dextrose solution for 20 minutes. 100 mg of ascorbic acid was then added and the solution incubated for a further 5 minutes. 1 mL of tagged blood was diluted with 99 mL of H2O and counted in a Pace Gamma Counter (Picker) to determine labeled blood concentration. 10 mL of labeled blood was then re-injected into the subject. After 30 minutes of equilibration time a 10 mL blood sample was drawn from the opposite arm and counted in the Pace Gamma Counter to determine new activity concentration. The labeling yield of red blood cells using this technique is greater than 90% (50). The formula for determining red cell volume is as follows: RCV (mL) = injected 51Cr RBC cpm/removed 51Cr RBC cpm/mL (36) 5 1 Cr reaches an equilibrium concentration throughout the body within 15-20 minutes (36). Baker and Remington (3) showed that splenic contraction does not cause a significant change in total red cell volume indicating that tagged 5 1 C r red cells equilibrate in the spleen as well as throughout the peripheral circulation. 1 5 Plasma Volume (Pre-exercise): 10 uCi of 1 2 5I-RIHSA was injected into the subject at the same time as the 5 1 Cr was injected. Blood samples were drawn from the opposite arm 10, 20 and 30 minutes following the initial injection and the activity counted in a Pace Gamma Counter. The log of the sample activity was plotted against time and extrapolated back to zero time to determine plasma volume (36). (Albumin slowly leaks from the intravascular space into the extravascular space, thus when measuring plasma volume with 1 2 5 1 -RIHSA or 131I-RIHSA , a plot of the log of sample activity against time is extrapolated back to zero time to obtain the true activity concentration ). Plasma volume Post-exercise: 1 3 1I-RIHSA was used to determine post-exercise plasma volume following the same procedures as above. The formula for determining plasma volume is: PV (mL) = injected 125I-RIHSA cmp/removed 125I-RIHSA cmp/mL (36) Total Blood Volume: Blood volume was calculated pre and post-exercise using pre- and post-exercise total red cell volume and plasma volume measurements. Because a relationship between plasma volume and red cell volume can be derived from knowledge of a subject's hematocrit, a single 1 6 determination of either plasma volume or red cell volume is often used to determine whole blood volume. However, this method for quantifying whole blood volume is potentially erroneous due to variations between central and peripheral hematocrit values. The accurate quantification of total blood volume therefore requires a determination •  of both; the plasma volume and the red cell volume (58) . Bv'(pre-exercise) = RCV + PV (pre-exercise) BV(post-exercise) = RCV + PV(post-exercise) (3 6) Spleen volume: 5 ml_s of red cells were labeled using 1.5 mg of stannous pyrophosphate followed by 25 mCi of " m T c pertechnetate. Labeling yield of red blood cells using this method is greater than 95% (50) . Anterior and posterior views of the spleen were taken using a ZLC 3700 Orbitor (Siemens). Each scan took four minutes to complete; two minutes for the posterior view and two minutes for the anterior view. In order to determine spleen counts for the anterior and posterior views, regions of interest were drawn around the spleen (Appendix D). Spleen radioactivity for pre-exercise and each exercise load was calculated from the average of the anterior and posterior spleen counts. 1 7 1 mL samples of red cells taken from the each of the blood samples (pre-exercise, after 10 minutes, 20 minutes and 30 minutes of exercise) were also counted using the ZLC Orbitor. The count of activity in the known volume (1 mL sample) was used to determine the spleen volume at each of the measurement times based upon the average spleen/activity. The samples only contained red cells because the hematocrit of the spleen has been estimated to be close to 9 5 - 9 7 % (26). Spinning down blood using a centrifuge will result in a red cell column that is approximately 96-97% pure red cell (some plasma is trapped during this procedure). Therefore, a sample of red cells only will have a hematocrit reading which is much closer to spleen hematocrit. Hematocrit Blood samples for hematocrits were taken from the anticubital vein. Hematocrit was measured in triplicate with a microtiematocrit centrifuge and corrected for 4% plasma trapped with the packed red cells. Statistical Ana lys is This study was a 3 (control/untrained/trained) x 2 (normoxic/hypoxic) x 4 (scans/session) factorial design with repeated measures on the second and third factors. 1 8 Descriptive statistics were determined for all variables measured. Reliability of the red cell volume, plasma volume, total blood volume, spleen volume and hematocrit were done using correlational analysis. The reliability was determined using the pre-exercise measures of the normoxic and hypoxic test sessions for the T and N subjects. In the case of the hypoxic test sessions,1 samples1 were taken prior to starting the hypoxic exposure. Differences in red cell volume, plasma volume and hematocrit pre- and post-exercise between groups N and S were analyzed using a repeated measures ANOVA. A two way factorial MANOVA with repeated measures was used to determine if there were differences in the exercise response between groups T and N during normoxic and hypoxic exposure. If a significant main effect was found, Tukey's post-hoc analysis was carried out to identify where the changes occurred. 1 9 RESULTS Group characteristics The physical characteristics of each group are shown in Table 2. The average body mass and the average height of the trained (T) group were significantly less (p<0.03) than the average body mass and height of both the normal (N) and splenectomized (S) groups. There was no difference in height and body mass between the N and S groups. There were also significant differences between the T and the N group's average maximal aerobic power (VO2 max) scores in both normoxic and hypoxic conditions, with the T group being higher in both. Peak power attained on the cycle ergometer were also significantly higher in both the normoxic and hypoxic conditions for T than for N. The S control subjects produced lower peak power at V O 2 max than N subjects despite there being no difference in V O 2 max (Table 3). Reliability of the measurement techniques The reliability coefficients for red cell volume, plasma volume, total blood volume, splenic volume and hematocrit each of the variables are shown in Table 4. Reliability for all the variables was high except for pre-exercise plasma volume. CO •o Q) _N E o o CD C a> a. ico a E i _ o iz l-o a> c (0 CO CO 00 a> o CO o> CO 1 c i 1 CO <o CO in • cc CM T— co co CO CO 0> c a CC ^ 111 CO I X a> c CO cc ui co I X CO CM CO co CO I CO CM O c\i co O) CM CM CO CM CO CO o o m m o CO O) CO CO m CO i n o m co co CO i n o CO o O) IV CO i— CO o> CO CM i n m co •* CO i n CM CM CM m O) (0 © >«i a> ° m CO CO TT II II II c c c h- Z GO CL Q. CL 3 3 3 2 2 2 CD CD CD CO T3 C (0 V) Q. 3 O o> <0 o mm 0 +* o (0 a U (0 o (0 CM JQ a 0) N E o o . CJ c a. Ico X o E o c X o Q. > . a •= E >- co ° X O E o c a x o Q. >» l-o 0> c X o E c o c o o 0 (0 d> X 111 +1 o -I-CO +1 C\J CO CO m oo T — +1 CO 00 CM CO T- <0 +1 +1 O) CM 00 +1 o> CO CO 00 +1 CT) 00 CM CO i - CM +1 CM CO 3 a C 3 x CM * > <0 O ^J? 5 > •* °- C-- I c n — a w Q. 3 O k_ o> CO TJ c a n x o E »_ o c 3 o CL a a> Q. •o C Q X (0 E CM o •> Q> a> CO > a w c o w co a. E o U CO CO C o CD a. 3 O T3 C a x o a C a 22 Table 4: Reliability scores of directly measured p r e exercise blood volumes and spleen volumes Var iable Trial 1 Trial 2 R e l i a b i l i t y Red cell volume. 2040 2007 r=0.977 (mLs) Plasma volume 3466 3436 r=0.642 (mLs) Total blood volume 5505 5443 r=0.884 (mLs) Hematocrit 38.5 38.4 r=0.883 (%) Spleen volume 29 7 319 r=0.900 (mLs) . n = 12 * reliability coefficient is significant at p<0.05 23 Untrained normal subject (N) and splenectomized subject (S) response to normoxic exercise. There was no difference in pre-exercise red cell volume (RCV) between the splenectomized control subjects: (S) and the normal subjects (N) with their volumes being 2033 mL and 2058 mL respectively (Figure 2). However, plasma (PV) and total blood volumes (TBV), measured pre-exercise were significantly higher in the S group than the N subjects (PV = 4255 mL and TBV = 6288 mL for the S group compared to PV = 3518 mL and TBV = 5575 mL for the N group). The RCV of the N group signicantly increased during 30 minutes of exercise from 2058 mL to 2215 mL while the S group RCV remained unchanged. PV in both groups dropped significantly following 30 minutes of exercise with N decreasing from 3518 mL to 3059 mL and S decreasing from 4255 mL to 3414 mL (Figure 3). The significant difference between N and S p r e -exercise PV disappeared in the post-exercise PV measurements. The spleen volume (SV) of the N subjects decreased significantly during each exercise load (Figure 4). The pre-exercise SV of 338 mL was reduced to 283 mL after 10 minutes of exercise, 219 mL after 20 minutes of exercise and 143 mL after 30 minutes of exercise. This represents a 58% reduction in SV. 24 Figure 5 shows the changes in hematocrit (HCT) during 30 minutes of exercise for both N and S groups. The S group HCT readings at all test times were significantly lower than the corresponding N group HCT, due to the significantly higher PV of the splenectomized group. During 30 minutes of exercise the S group HCT did not significantly increase. The N subjects HCT reading of 40.2% after 10 minutes of exercise was significantly higher than the pre-exercise value of 38.8%. The final HCT reading of 42.1% after 3 0 minutes of exercise was also significantly higher than the pre-exercise value and the 10 minute HCT reading. The change in PV in the S subjects accounts for 100% of the increase in HCT. In the N subjects, PV changes account for 70% of the rise in HCT (0.84%) and red cell mobilization from the spleen accounts for 30% of the HCT increase (0.36%). 25 8000-, o v-' 6000 4000-1 2000-1 [?!| Normal HI Splenectomized * shows a significant difference between the groups Figure 2: Pre-exercise red cell volume (RCV), plasma volume (PV) and total blood volume (TBV) for groups N and S. 26 5000 N 4000-1 30004 > 2000-1 E J Pre H Post 1000-1 0-RCV N RCV S PV N a , PV s Variable and group * shows a significant difference between pre- and post-exercise values. a shows a significant difference between groups. Figure 3: Changes in red cell (RCV) and plasma volumes (PV) with 30 minutes of exercise in normal (N) and splenectomized subjects (S). 27 3 5 0 n 10 0-" , 1 1 1 Pre exercise 10 min 20 min 30 min Sample Time * denotes a significant difference between the volumes (p<0.05). Figure 4:Spleen volume of N subjects pre-exercise and after 10, 20 and 30 minutes of exercise. 28 44 ^ Sample Time denotes a significant increase in hct with exercise in group N (p<0.05). Figure 5: Hematocrit changes during 30 minutes of exercise in groups N and S. 29 Normal and Trained subjects comparisons: normoxia The pre-exercise RCV, PV and TBV values for the normoxic tests of the T and N groups are shown in Figure 6. There were no differences in any of the pre-exercise blood measures between the T subjects and the N subjects. When plasma volume andredcell volume, were calculated per unit of body weight for the two groups, there was a significant difference in both volumes between the groups (Table 5). Table 5: Pre-exercise plasma volume and red cell vo lume calculated per kilogram of bodyveight for groups N and T in normoxic conditions. GROUP PV/KG Signi f icance RCV/KG S ign i f i cance (mLs/Kg) ( m L s / K g ) N 44.8 25.7 p<0.02 p<0.03 T 53.5 31.5 After 30 minutes of exercise the RCV of the N subjects significantly increased (p<0.02), however, the RCV of the T subjects did not (Figure 7). PV in both groups was significantly (p<0.0001) lower than pre-exercise values following 30 minutes of exercise (Figure 8), however there was still no difference in PV between the N and T groups after 30 minutes of exercise. 30 Within both groups, SV was significantly less than the previously measured volume at each measurement time (Figure 9). The SVof the T subjects was significantly lower than the SV of the N subjects pre-exercise and after each 10 minute exercise load (Figure 9). However, when SV was shown as a percentage of original SV, the decrease in SV after each exercise load was identical for. both groups (Figure 10). Both the N and the T groups had significant increases in HCT between all test time with 30 minutes of exercise (Figure 11). The pre-exercise HCT scores of 38.2% for T increased after each 10 minute exercise load to 3 9 . 1 % , 40.2% and 41.6%. The N group showed a similar trend with the p re -exercise HCT of 38.8% increasing to 39.1% after 10 minutes of exercise, 41.0% after 20 minutes of exercise and 42.2% after 30 minutes of exercise. There was no difference in HCT between the N and T groups at any test time. 31 Figure 6: Comparison of the normoxic pre-exercise red cell volume plasma volume and total blood volume of N and T groups. 32 Group denotes a significant increase in volume pre-to post-exercise (p<0.02). Figure 7: Red cell volumes pre- and post-exercise for N and T groups in normoxic conditions. 33 4000-, 3000-1 o w 2000-4 1000-1 Group P V p r e PV post shows a significant drop in plasma volume pre-to post-exercise within a group(p<0.0001). Figure 8: Plasma volume changes from 30 minutes of exercise for groups T and N. 34 Time * denote spleen volumes significantly different to each other between and within groups(p<0.05). Figure 9: Changes in spleen volume during 30 minutes of exercise in normoxia for groups T and N. 35 110-, Pre exercise 10 min 20 min 30 min Time * show a significant difference in spleen volume within a group. Note: data for the 2 groups overlap exactly Figure 10: Spleen volume shown as a percentage of the original volume for groups T and N normoxic exercise. 43 n Pre exercise 10 min 20 min 30 min Time * & ° symbols denote significant differences between hct readings within a group (p<0.05). Figure 11: Changes in hematocrit during 30 minutes of exercise in normoxia for groups T and N. 37 Normal and Trained subjects comparisons: hypoxia 3 In hypoxia, similar trends to the normoxic conditions in reduction of PV and increases in RCV with exercise occurred. Again, there was no difference i n pre-exercise RCV, PV or TBV between the two groups (Figure 12). However, if plasma volume and red cell volume are calculated per kilogram of body weight, significant differences between the groups appear with the trained group having significantly higher plasma and red cell volumes (Table 6). Table 6: Pre-exercise plasma volume and red cell vo lume calculated per kilogram of bodyveight for groups N and T in hypoxic conditions. GROUP PV/KG Signi f icance RCV/KG S ign i f i cance (mLs/Kg) ( m L s / K g ) N 43.1 25.4 p<0.01 p<0.02 T 54.9 31.5 After 30 minutes of exercise, the RCV of the N subjects had significantly (p<0.04) increased from 2029 mL to 2334 mL whereas the post-exercise RCV of 2145 mL for the T group was not different to the pre-exercise volume of 1987 mL (Figure 13). Both the N and T groups showed significant reductions in PV pre- to post-exercise in the hypoxic condition (Figure 14). 38 TBV of the T group also showed a significant reduction following exercise (p<0.008). The hypoxic pre-exercise SV of the N group was significantly higher than the pre-exercise SV of the T group (360 mL versus 279 mL). However, after 10 minutes of exercise the difference between the groups disappeared (Figure 15). Both theN and T groups showed significant decreases in SV from pre-exercise through each exercise load. When the reduction in SV following exercise is shown as a percentage of original SV, there are no differences between the groups at any test time (Figure 16). During hypoxic exercise, SV of both N and T subjects decreased 13-22% after 10 minutes of exercise, 39-44% after 20 minutes of exercise and 62-65% after 3 0 minutes of exercise. There were no differences between the N and T groups HCT readings p re -exercise or after any of the exercise loads (Figure 17). Within each group, HCT significantly increased during each exercise load. 39 0 N Group RCV pre PVpre BVpre Figure 12: Hypoxic pre-exercise red cell, plasma and total blood volumes for groups N and T. I s 3000 2500 4 2000 J 1500-J 1000-1 500-1 0 40 RCV pre RCV post 1 I l l T 1 N Group * denotes values that are significantly different from each other at p<0.05. Figure 13: Red cell volumes pre- and post-exercise for groups N and T in hypoxic conditions. 4 1 4000-, 3000 A i i 2000-I looo A Group * denote significant differences in PV within a group (p<0.05). Figure 14: Plasma volumes pre- and post-exercise for groups N and T in hypoxic conditions. 42 400-, * o Pre exercise 10 min 20 min 30 min Time * symbol denotes spleen volumes significantly different to each other between groups (p<0.05). 0 symbol denotes spleen volumes significantly different to each other within a group (p<0.05). Figure 15: Changes in spleen volume during 30 minutes of exercise in hypoxia for groups N and T 4 3 120 n Time * show significant difference in SV within a group (p<0.05). Figure 16: Spleen volume shown as a percentage of the original volume for groups N and T hypoxic exercise. 44 43 n * denote a significant difference in hct between readings within a group (p<0.05). Figure 17: Comparison of changes in hematocrit for groups N and T during 30 minutes of exercise in hypoxic conditions. 45 Normal and Trained subjects: normoxic and hypoxic comparison Pre-exercise RCV, PV and TBV were the same within each group in normoxia and hypoxia (Table 7). The N group showed a significant increase in RCV following exercise however the increase in both the normoxic and hypoxic conditions was the same (Figure 18). Both the N and T group showed significant decreases in PV pre-to post-exercise in both the normoxic and hypoxic conditions, however there was no difference in that decrease between the two test conditions (Figure 19). TBV change pre- to post-exercise was the same in the normoxic and hypoxic conditions within each group. Table 7: Pre-exercise red cell, plasma and total blood volumes in normoxia and hypoxia for T and N groups. RCV PV TBV MLs).. (mLs) (mLs) Group normoxia hypoxia normoxia hypoxia normox ia hypoxia T 2022±143 1987±149 34141171 34871121 54361299 54741261 N 2058±193 2029±216 35181114 33821182 5576+297 54111376 As previously shown, the T group SV decreased significantly from p re -exercise through all exercise loads. When comparing SV changes during normoxia and hypoxia, there were no differences either pre-exercise or after any of the 10 minute exercise loads (Figure 20). The N group also had 46 no difference in SV between normoxic and hypoxic exercise at any of the sample times (Figure 21). HCT changes for both the T group and the N group showed similar trends in both the normoxic and hypoxic conditions. Again, even though both groups showed significant increases'in? HCT with exercise, the values at each test time were the same in the normoxic and hypoxic conditions (Figures 22 and 23) . Table 8 shows the change in HCT that occurred for the T and N groups during normoxic and hypoxic exercise. Reductions in PV were found to have a relative contribution of 71% to changes in hematocrit for both groups in normoxic exercise. During hypoxic exercise, the contribution of PV changes to hematocrit changes increase to 78% in the T group and decreased to 6 7 % in the N group. Changes in circulating RCV caused the remainder of the changes in HCT. 47 3000 n ] RCV pre normoxic | RCV post normoxic RCV pre hypoxic RCV post hypoxic Group and ° denote values significantly different from each other @ p<0.05. Figure 18: Red cell volumes pre- and post-exercise for group N and T in normoxic and hypoxic conditions. 48 4000 n PV pre normoxic PV post normoxic PV pre hypoxic PV post hypoxic Group Figure 19: Plasma volume pre- and post-exercise for groups N and T in normoxic and hypoxic conditions. 49 300-, Pre exercise 10 min 20 min 30 min Time Figure 20: Spleen volumes during 30 minutes of exercise in normoxic and hypoxic conditions for group T. 50 Pre exercise 10 min 20 min 30 min Time Figure 21: Spleen volumes during 30 minutes of exercise in normoxic and hypoxic conditions for group N. 5 1 43 n Pre exercise 10 min 20 min 30 min Time Figure 22: Comparison of changes in hematocrit in normoxic and hypoxic conditions for group T. 52 43 n Time Figure 23: Comparison of changes in hematocrit in normoxic and hypoxic conditions for group N. 5 3 54 DISCUSSION Blood Volumes Pre-exercise red cell volume, plasma volume and total blood volume N and T groups were similar whereas the plasma volume and total blood volume of the S group was significantly higher (see Figures 2 and 6). The red cell volume, plasma volume and total blood volume of the T group were expected to be higher than the N or S groups as it has been shown that all three volumes increase with aerobic training (48,53). However, the N subjects were significantly taller and heavier than the T subjects (Table 2). When red cell volume and plasma volume were corrected for body mass in the N and T groups, T subjects demonstrated the expected higher red cell volume and plasma volume per unit of body mass (Table 5). As has been found in previous studies (5,10,19,40) the endurance trained individuals compared with untrained controls did not, in most cases, confirm the dominance of plasma volume changes, since hematocrits were not statistically different between the T and N groups (Figures 11 and 17). A possible explanation might be the assumption of increased erythropoiesis occurring when an elevated training level is maintained over a longer period of time. With the groups of subjects used in this study, RCV was significantly higher in the T subjects when volume was corrected for body mass. With the similar level trained state shown with the N and S groups (Table 3), it was expected that both the red cell and plasma volumes of the two groups 55 would be similar. However, the plasma volume of the S group was significantly higher than the plasma volume of the N group. There have been no other reported data on this phenomena in humans. However, one of the functions of the spleen is hemopoietic regulation (38), thus loss of the spleen may have impacted plasma volume in the S subjects After 30 minutes of exercise the plasma volume difference found between the N and S groups pre-exercise disappeared (Figure 3). This means that the decrease in plasma volume of the S group was proportionally greater than the decrease in plasma volume of the N group. Why plasma volume would show a greater decrease in S subjects compared to N subjects during exercise of the same relative intensity is not clear. It may be that the hemopoietic regulatory function of the spleen is also important during exercise. Further investigations into the role of the spleen in the control of plasma volume and plasma volume shifts during exercise are warranted. Red cell volume of the N group increased significantly pre-to post-exercise in both normoxic and hypoxic exercise. The S group also showed an increasing trend in red cell volume with exercise (Figure 3), however, there were not enough subjects to show significance. Allsop et al. (1) also found a consistent increase in erythrocyte indices after 10 minutes of maximal exercise. The increase in red cell volume suggests that another source of red cells, that was not available for equilibration with 9 9 m Tc- labeled red cells, might exist. Two possibilities are the bone marrow and the skin. Rushmer (49) has suggested that the subpopullary plexus of the skin has a potential role as a blood depot, but also that this function is intimately related to 56 dissipation of heat. In other words, this blood is rarely released into the general circulation at the expense of temperature regulation. However, i n the case of unfamiliar and stressful exercise, these extravascular pools of red cells may somehow be mobilized. The bone marrow may also release immature red cells in response to stressful, unfamiliar exercise to in order to increase circulating red cell volume and hence improve O2 delivery. It has also been suggested that red cell swelling with exercise may occur, resulting in an increased reading for red cell volume. However, Wade (63) states that it is unlikely that significant osmotic adjustments occur in circulating red blood cells in response to the plasma volume changes because vasopressin secretion (which normally rises in proportion to the increase in plasma osmolality) does not increase significantly. Hespel et al. (34) also supported this opinion when they showed that intraerythrocyte K + concentrations did not changes unless the workload exceeded 80% VO2 max. Further research needs to be completed to determine why there is an increase in red cell volume with exercise and to establish if there are extraneous pools of red cells available for mobilization during exercise stress. Spleen Volumes Although many studies have reported changes in splenic radioactivity with exercise (24,51,64), no study has reported the actual change in spleen volume and the corresponding volume of red cells released into the circulation as it is difficult to do so. Oh et al. (44) and Hurford etal. (35) found the range of normal spleen volumes in Korean adults to be 160+62 c m 3 in females and 173 ±59 cm 3 in males. Wade et al. (64) determined that the 57 circulating splanchnic volume of their subjects had a range of 550 mLs to 1550 mLs. They also estimated that, at rest, 12-25% of the total blood volume was in the splanchnic bed. In this study, pre-exercise spleen volume in the N group was calculated to be 338 mLs. Exercise caused a progressive decline in splenic volume down to 143 mL (Figure 4). Therefore 195 mLs of blood were released from the spleen over the course of 30 minutes of exercise. If an average splenic hematocrit of 96% is used, then approximately 187 mLs of red cells were released into the circulation. Total red cell volume was 2058 mLs, thus the released 187 mLs of red cells represents 9% of the total red cell volume. Similarly, the spleen volume of the T group decreased from 255 mLs to 1 07 mLs after 30 minutes of exercise. Total red cell volume of the T group was 2022 mLs, therefore the spleen released 142 mLs of red cells, comprising 7% of the total red cell volume, into the circulation. These values correspond to the suggested storage capacity of the human spleen (26). The spleen volume of the T subjects was significantly less than the spleen volume of the N subjects pre-exercise and after all exercise loads in normoxic exercise (Figure 9). This means that the N subjects had a greater proportion of their blood volume, particularly their red cell volume, in the spleen. It is possible that since T subjects have less blood stored in the spleen, that some of the increase seen in red cell volume with aerobic training (when measured indirectly from hemoglobin and hematocrit) actually come from the reduced storage capacity in the spleen rather than from training-induced increases per se. 58 The mechanism responsible for the reduction in spleen volume during exercise is unclear. Previous researchers (38,51) have suggested that spleen volume may be reduced as a consequence of sympathetic stimulation. Since trained individuals have a depressed sympathetic response to exercise stress compared to untrained individuals, it was hypothesized that the reduction in spleen volume and the subsequent volume of red cells released by the organ would be less in the T subjects than in the N subjects. N subjects did show higher volumes of red cells being released by the spleen, however, when the reduction in spleen volume was shown as a percentage of original spleen volume, there were no group differences (Figure 10). These results show that the T subjects had reduced red cell storage in the spleen and subsequently, a proportionally higher circulating red cell volume than the N subjects. However, the volume of red cells released by the spleen during exercise was proportionally the same for T subjects as for N, thus training status does not appear to impact the splenic response during exercise. Hypoxia also causes an increased sympathetic response and stresses the O2 delivery system. Thus, building upon the suggestions of previous researchers (38,51), it was hypothesized that hypoxic exercise would cause a larger reduction in splenic volume than normoxic exercise in both the N and T groups. In hypoxia the volume of red cells released from the spleen during exercise increased for both groups. The volume of red cells released from the spleen was 225 mLs in the N group, representing 11% of the total red cell volume. The T group again released a smaller volume of red cells than the N group, 164 mLs, representing 8% of total red cell volume. Thus, hypoxic 59 exposure appeared to result in a increased release of red cells from the spleen in both groups, however, the differences in the splenic volume before, during and after exercise (normoxic versus hypoxic) were not significant for either group (Figures 20 and 21). Pre-exercise spleen volumes were significantly different in the hypoxic condition between the groups, but the differences disappeared during exercise (Figure 15). This suggests that the additive stress of hypoxia and exercise induced greater splenic release of red cells in the N subjects than in the T subjects. However, when the reduction in spleen volume was calculated as a percent change from the original volume, there was, once again, no difference in the reduction in spleen size between the groups in hypoxic exercise (Figure 16). Hematocrits Both the N and S group showed a trend toward an increase in peripheral hematocrit with exercise, however only the changes for the N group were significant (Figure 5). For the S group, the change in hematocrit would be due to plasma volume decreases and perhaps minor F-cell shifts caused by compensatory mobilization of blood from the micro- to the macrocirculation (41). A clear-cut F-cell shift has been demonstrated in response to bleeding in animals (37) suggesting that similar changes might occur in other states of hypovolemia. The change in hematocrit for the N group would be due to a combination of plasma volume shifts, and major F-cell shifts caused red cell release by the spleen. These results suggest that for the exercise mode and 60 intensities used, significant changes in hematocrit only occur when there are both plasma volume shifts and release of red cells by the spleen. The reduction in plasma volume in the S subjects accounted to 100% of the increase in hematocrit over 30 minutes of exercise as, obviously, red cells could not be mobilized from the spleen. In the N subjects, plasma volume decreases accounted for about 71% of the rise in hematocrit (2.4% of the total 3.4% increase in hematocrit) and red cell mobilization from the spleen accounted for 29% of the hematocrit increase (1.0% of the total 3 .4% increase in hematocrit) in normoxic conditions. During hypoxic exercise hematocrit also increased a total of 3.4% over 30 minutes of exercise; 6 7 % of the increase in hematocrit was due to plasma volume decreases and 3 3 % was due to an increase in circulating red cell volume. The T group showed identical results for normoxic exercise. In hypoxic exercise, the decrease i n plasma volume was responsible for 78% of the change in hematocrit and the increase in circulating red cell volume was responsible for 22% of the increase. [See Appendix E for calculations]. Laub et al (38) had suggested that the reduction in spleen radioactivity could potentially account for up to 25% of the rise in hematocrit. The results of this study provide strong evidence that Laub et al. (38) were correct in their predictions and that equations that use hematocrit changes to predict plasma volume changes (60) or equations using hemoglobin and hematocrit (13) contain significant error. Clearly, these equations need to be modified in order to account for the increase in hematocrit that is attributable to red cell infusion from the spleen. 61 Hematocrit increased significantly in both the N and T groups during 3 0 minutes of exercise in the normoxic and hypoxic conditions, however, there was no difference in hematocrit values between the groups in either condition (Figures 11 and 17). Furthermore, hypoxic exercise did not cause a greater increase in hematocrit than did normoxic exercise for either group (Figures 22 and 23). It was expected that the hematocrit of the T group would be significantly less than that of the N group as previous research has shown that the plasma volume expansion that occurs with aerobic training exceeds the increase in red cell volume (22,27,48,53). However, no differences in the proportions of PV or RCV in TBV were found between the N and T groups. The fact that plasma volume was measured directly may be the reason for the discrepancy between these results and previous studies results. As has been shown by the results of this study, plasma volume predicted using hematocrit or hematocrit and hemoglobin changes is subject to large error and may have over-predicted plasma volume. Methodology Considerations Nuclear medicine techniques using multiple radiopharmaceuticals for the simultaneous labeling of various blood components presented a unique challenge. The counts of the radioisotopes used had to be different enough so that they did not interfere with the reading. After careful research and a practice trial, it was determined that the four radioisotopes used in this study could be used in conjunction with each other. Prior to collecting data it had to be determined: 1) if the labeling of each component would be reliable from 62 test to test and 2) if the first test labeling of the blood components would interfere with the second testing session. The results of the test/retest reliability for red cell volume, spleen volume and total blood volume was acceptable with r values ranging from 0.883 to 0.977. Plasma volume reliability was somewhat lower, but the volatility of plasma volume within a subject from day to day and test to test was expected. Subjects were instructed to be fully hydrated upon arrival for testing, however several other factors can impact plasma volume, including the intensity of the last training session, illness, amount of activity (ie. walking versus driving in the car) just prior to coming into the lab, and food consumption. The impact of the residual radioactivity from the first nuclear medicine test session was also a concern. In the case of some of the isotopes ( 1 2 5 I -RHISA and 5 1 Cr) the decay half-life exceeded the planned time-between-tests. Additionally, because 1 2 5I-RHISA is attached to albumin protein which moves in and out of the vascular space, it had to be determined if, by the second testing session, the previous 1 2 5 I -RHISA had equilibrated in the intra- and extra-vascular fluid and thus would not cause errors in reading plasma volume in the second test. To determine this two subjects did a 30 minute exercise session one week after their first nuclear medicine test session, during which they had been injected with radioisotopes. Blood samples were drawn pre-and post-exercise and counts for all radioisotopes were completed. There was no change in any of the values pre- to post-exercise indicating that all isotopes had reached equilibrium within the one week 63 period. Thus, in the second testing session, pre-test radioactivity levels for each isotope (the residual decay from the first test) simply had to be measured and subtracted from the counts taken at the second test session. The four minute rest period should not have caused significant changes i n spleen volume. Sandler et al. (51) showed that splenic radioactivity gradually increased during a ten minute post-exercise period however, splenic radioactivity shown to be 57% of pre-exercise value immediately after exercise was still down at 58% of pre-exercise value after two minutes of recovery. Both males and females were used as subjects in this study. Using a mixed gender subject pool was not expected to have an impact on the results. No study has found the hematocrit/plasma volume response of males and females to be different (22), however no study has directly compared the plasma volume changes of men and women under the same experimental conditions. In this study there were not enough male and female subjects of the same training level to compare gender. Conclus ions The results of this study demonstrate that spleen volume does decrease significantly during 30 minutes of upright cycling in both N and T subjects, releasing between 7 and 9% of the total red cell volume into the circulation. The splenic release of red cells had a significant impact on peripheral hematocrit as it was shown that splenectomized subjects did not demonstrate 64 the significant increase in hematocrit with 30 minutes of exercise that the normal subjects did. Aerobic fitness did not influence the reduction in spleen volume with exercise or it's impact on hematocrit changes. However, the spleen volumes of the T subjects were significantly less than those of the N subjects suggesting that the T subjects had more of the total red cell volume in circulation. Splenic contribution to circulating red cell volume during exercise was not affected by hypoxic exposure in either N or T subjects. The results of this study also provide evidence that indirect calculations of plasma volume changes could result in prediction errors of 22 to 33%. APPENDIX A. Review Of The Literature 66 1. Training-Induced changes In hematological profiles Exercise causes both acute and chronic changes in blood plasma volume, hematocrit and total red cell volume. These changes, in turn, have an impact on blood viscosity and oxygen delivery to the body tissues. In order to have optimal oxygen: delivery to body tissues during exercise, a balance between a high hematocrit (increased O2 carrying capacity per given volume of blood) and blood viscosity is necessary. While the changes in plasma volume with both training and acute exercise have been extensively studied, the impact of exercise on changes in circulating red cell volume has not been clarified. Acute intense exercise causes a 10 to 20% reduction in plasma volume (43,50). The decrease in plasma volume acts to increase peripheral hematocrit and subsequently, blood viscosity. The mechanisms that are responsible for this reduction are an increasing capillary hydrostatic pressure which forces an ultrafiltrate of plasma extravascularly, accumulating muscle metabolites which draws plasma into the muscle and thermoregulatory fluid losses (54,65). With training, the body attenuates the severity of hyperviscosity by expanding the plasma volume (hypervolemia). When compared with sedentary controls, endurance-trained athletes have an enhanced blood volume, although the total erythrocyte volume is not increased as much as plasma volume (48,53). Hypervolemia is a supercompensatory alteration to exercise as it causes an acute reduction in plasma volume and increase i n 6 7 osmolality (22). The specific mechanisms of training-induced hemodilution are not completely understood, however, it is believed to be the result of higher levels of renin, aldosterone, vasopressin and intravascular retention of albumin and other proteins and electrolytes (22,27). Controversy exists as to whether plasma volume expansion has the potential to increase maximal aerobic power (VC>2 max). Coyle et al. (12) showed that there is an optimal plasma volume for increasing VO2 max in untrained men which is approximately 200-300ml_ above their normal levels. Higher levels of plasma expansion results in excessive hemodilution and a subsequent decline in VO2 max. The potential for plasma volume expansion to increase VO2 max depends upon the balance between the extent to which stroke volume is increased compared to the reduction in hemoglobin concentration and thus, arterial O2 content. In exhaustive exercise with involvement of a large volume of skeletal muscle, blood flow increases by approximately 1200% (43). This is predominantly because of a reduction of vascular hindrance and increased driving pressure. However, as blood flow is linearly related to blood fluidity, the reciprocal value of blood viscosity, hemorrheological changes may be a limiting factor for tissue perfusion in maximal exercise. It is not clear to what extent an increase in blood viscosity under conditions of long term exercise can be compensated for by cardiovascular mechanisms without a reduction of work capacity. Furthermore, studies have not consistently found compromised blood flow properties during long term exercise and the magnitude of the changes is small. Hence from the hemorrheological view, a significant 68 impairment of tissue perfusion and aerobic work capacity cannot be concluded with certainty. Plasma volumes historically have been measured directly using either 1 3 1 1 -RHISA or Evan's blue-dye techniques or indirectly using hematocrit changes. One of the most often used methods, Dill and Costill's (13) prediction of plasma volume changes using hematocrits, assumes a constant number of red blood cells and a constant red cell volume. Research completed at the time using direct measures of red cell volume and plasma volume to calculate hematocrit showed no change in red cell volume with exercise (28). Because of limitations of the techniques available at the time, differentiation between total red cell volume and circulating red cell volume could not be made. However, other researchers (3,31) have shown that 99nriT c and 5 1 Cr, radioisotopes commonly used to determine total red cell volume, equilibrate in the red cells stored in the spleen. In equations in which circulating red cell volume is assumed to be consistent with total red cell volume, the potential dynamic storage function of the spleen has not been considered. Recent studies on both animals and humans have shown that the spleen stores red cells that can be released during times of stress (exercise, hemorrhage). Thus, there is a potential difference between total red cell volume and circulating red cell volume. When using hematocrit changes to determine the extent of plasma volume shifts during exercise, the influx of red cells from the spleen into the peripheral circulation should be considered. 69 Figure 24: Diagramatic views of the spleen 7 0 2. Anatomy And Function Of The Spleen The spleen is not a organ that normally comes under scrut iny by exerc i se scientists and thus, usually not much is known about it's anatomy and function and how it responds to exercise stress. It is situated obl iquely under the eighth through eleventh ribs on the left side of the body, lying between the fundus of the stomach and the diaphram. It is the largest lymphoid organ i n the body and measures five to six inches in length , three to four inches i n width and one and a half inches thick (26). The weight of the sp l een , proport ional to body weight, in adults is approximately 1 to 3 2 0 - 4 0 0 . However, the s ize and weight of the spleen are susceptible to ext reme variations at different periods of life, in different individuals and in the same individual under different condit ions. The spleen has a collagenous framework with a reticular network suspended within. It is surrounded by a splenic capsule which contains e last ic connective tissue and some smooth muscle. At one point on the surface of the spleen there is a deep indentation, the h i lum, where blood vesse ls enter and leave. Internally, trabeculae incompletely partition the spleen into lobules . A lobule is about 1 mm in diameter and is bounded by several t rabeculae. Each lobule is supplied by a central artery and is drained by ve ins which r u n in trabeculae to leave the lobule. The lobules are not distinct s ince they are not outlined completely by trabeculae. The parenchyma (splenic pulp) is of two distinct types, red pulp and whi te pulp. Red pulp is the major component of the spleen and consists of large thin-walled s inuses filled with blood separated by cords of lymphoid t i ssue . 71 It occupies all space not utilized by trabeculae and white pulp. The white pulp is typical lymphatic tissue and consists of clusters of lymphocytes anchored by reticular cells and fibers around a small central artery. Denser accumulations of B-lymphocytes are contained in splenic nodules along the strands of white pulp. The splenic artery enters the spleen at the hilum and divides into branches which pass along the trabeculae as trabecular arteries. As the trabeculae branch, so do the arteries until they are reduced to a diameter of approximately 0.2 mm. At this point, they leave the trabeculae and enter the splenic parenchyma. As they do so, the tunica adventitia of the arteries loosens, takes on the character of recticular tissue and becomes infiltrated with lymphocytes. After numerous divisions, the arterioles become reduced in size, lose their investment of white pulp and enter the red pulp. It has still not been established if the arterial capillaries open directly into the pulp reticulum and the blood gradually filters into the venous sinuses (open circulation theory) or if the arterial capillaries empty directly into the venous sinuses (the closed circulation theory) (39). In humans, the normal spleen serves many functions. It is an important hemopoietic organ, producing both lymphocytes and monocytes. The formation of antibodies also thought to be another important function of the spleen (38). B-lymphocytes develop into antibody-secreting plasma cells in response to antigen (47). The spleen also plays an essential role in filtration, phagocytosis and destruction of red blood cells and storage of iron and viable blood cells. As blood enters the spleen and passes through the red 72 pulp, aged red cells, foreign particles and bacteria are destroyed by resident macrophages. The structure of the sinusoids in the vascular bed of the spleen allows red blood cells to penetrate the vascular wall and to be packed and stored in the parenchyma of the spleen (38). It has long been recognized that the spleen acts as a depot from which blood may be expressed in times of stress. Red cell content of the spleen alters depending on sympathetic nervous discharge o r pressure in the splenic veins and the intra-abdominal pressure (56) . Emptying of the stagnant red cells in the spleen to the general circulation would increase the circulating red cell volume of the blood and thereby the circulating oxygen transport capacity. Studies examining the potential role of the spleen in the storage and release of red cells have been undertaken i n both animal and human models. 3. Animal Studies Barcroft et al. (4) were the first to suggest that the spleen may have a red cell storage function in dogs. They found that red blood cells were held in the spleen "in reserve" and released during times of stress. However, others (30) showed 'rapid-mixing" of the spleen and suggested that red blood cells were not sequestered. As more advanced techniques became available, Baker & Remington (3) used radioactive isotopes to determine plasma volume and red cell volume in dogs. They found that when the plasma volume and the cell volume techniques were applied to the same animal, only rarely were the two blood volumes the same. This disparity was taken to indicate the presence of body reservoirs for both red cells and plasma, available for the dilution of 73 tagged cells or dye, but having a different cell/blood ratio from that found i n the large vessels. Furthermore, splenic contraction produced a rise in arterial hematocrit and in activity, but no significant change in the calculated total red cell volume (3). This indicated that the tagged cells reached an equilibrium concentration in the spleen fairly rapidly. Their findings supported Barcroft's earlier work (4) suggesting that in intact dogs have significant splenic reservoir capacity for red blood cells. Furthermore, Guntheroth and Mullings (29) found that splenic volume in dogs decreased i n response to epinephrine, fright hemorrhage and exercise. The degree of splenic contraction during exercise decreased both with time and training (29) . Also building on the early findings of Barcroft et al., Turner & Hodgetts (59) studied the relationship of the spleen to hematocrit changes in sheep. Using 5 1 Cr red cell labeling, they were able to show that red cells accumulated in large numbers in the spleen, that individual cells were retained there for periods ranging up to 30 minutes or longer, and that temporarily retained cells were subsequently displaced and replaced by cells from the afferent blood. They concluded that a state of dynamic red cell storage existed i n sheep, similar to that in dogs as found by Barcroft et al. (4). Turner and Hodgets (59) were also able to demonstrate that hematocrit increased and 5 1 Cr counts in the splenic region decreased after the sheep were exposed to stressful situations (being held or chased). From this, they concluded that red cells were mobilized from the spleen and were, in part, responsible for the increase in hematocrit. These observations were 74 supported by the findings of Vatner et al. (62) who studied the impact of severe, spontaneous exercise before and after splenectomy in six unrestrained, healthy dogs. Before splenectomy, exercise increased hematocrit from 40 to 49% while after splenectomy, hematocrit failed to increase. From their findings, they concluded that the contribution of red cells from the spleen during exercise made an important and significant contribution to increased hematocrit. Subsequent research has shown that the spleen contracts during exercise and under stressful (fight or flight) situations resulting in a release of a sequestered pool of red blood cells into the circulation in other animals such as racehorses (45) and Weddell seals (46) as well as in dogs and sheep. In all of the animals studied, the spleen has a relatively high spleen to body weight ratio and can store a substantial amount of the total red cell volume. The Weddell seal has the highest spleen weight as a percentage of body weight of any reported mammal, comprising 0.89% of body weight (46). It is estimated that 20 litres of injectable red blood cells are stored in the spleen of a 350 kg Weddell seal. Ovist et al (46) found that hemoglobin concentrations increased by nearly 60% during the first 10-12 minutes of both long and short dives and suggested that the source of the influx of oxygenated red blood cells was the spleen. In seals the diving reflex is characterized by profound sympathetic vasoconstriction of the peripheral vasculature. Ovist et al. (46) suggest that during diving the Weddell seal capitalizes on this sympathetic response to induce constriction of its very large spleen, thus injecting large quantities of oxygentated red blood cells 75 into the central circulation. Splenic smooth muscle may also contract at low PaC»2 levels or because of increased circulating catecholamine levels. In contrast, to the Weddell seal, the other animals that have demonstrated splenic storage of red cells have relatively smaller spleens. A horse spleen comprises 0.3% of total body weight and has a red blood cell storage capacity of 54% of total red cell volume (45). A sheep's spleen is approximately 0.20% of body weight and has a storage capacity of 26% of the total red cell volume (59) and a dog's spleen weight is about 0.22% of body weight and has a storage of 20% of total red blood cell count (4). The relative size of the sheep and dog spleen is close to that of the human which has led to the suggestion that the human spleen may also have a red blood cell storage function. 4 Human Studies The human spleen makes up approximately 0.25% to 0.29% of total body weight, but is believed to have a red blood cell storage capacity of less than 10% of total red cell volume (26), which is significantly less than the 2 0 % to 26% storage capacity of sheep and dog spleen (4,59). Generally, the storage function is not considered to be well developed in the human spleen since, on average, it is believed to contain only 200-250 mL of blood (4 9) . Furthermore, it has been suggested that the human spleen is incapable of contracting as it has fewer trabecular fibers than the animal models studied. As previously mentioned, hematocrit does increase in humans during exercise, but the increase has always been attributed to decreases in plasma volume. 76 Past research has produced equivocal results concerning the potential storage function of the human spleen. Ayers et al. (2) stated that old studies show that the normal human spleen does not take up normal red cells from the circulation as an acute store for emergencies. They found that stimulation of the postganglionic sympathetic nerves to the human spleen at frequencies of 3-1 OHz evoked graded vasoconstriction but very small changes in spleen volume. The injection of adrenaline and noradrenaline in doses of 0 . 2 5 - 2 5 pg to the spleen produced graded increases in splenic vascular resistance with a small decrease in spleen volume. In addition, administration of the a -adrenoreceptor blocking drug, phenoxybenzamine, completely abolished Or reduced the vascular responses of the human spleen to sympathetic nerve stimulation or noradrenaline. They concluded that these results provided direct evidence that the normal human spleen does not have a reservoir function. Other researchers also have found no evidence for mobilization of blood reserve by exercise, epinephrine or hemorrhage (15). However, others have shown significant splenic shrinkage with exercise (23,51), epinephrine injection (6) and hyperventilation (56). Schaffner et al. (52) refuted the suggestion that because the human spleen has relatively few trabecular muscle fibers, it is incapable of contracting (and thus having a reservoir function) like the spleen of a dog or race horse. They found that the relative paucity of trabecular muscle fibers in the human spleen does not appear to be crucial, since the extent of splenic contraction i n their subjects was significant (estimated reduction of 50% of spleen volume). 77 Several studies have used radioisotopes to image the spleen and to determine the reduction in spleen radioactivity with diving (35) and with exercise (24,38). Hurford and associates (35) studied the diving response of the Korean ama to determine if the diving response (ie. splenic contraction) was similar to that found in the Weddell seal (46). They found that during a 3 hour shift of repetitive diving, splenic volume, measured using ultrasound, decreased 19.5% and hematocrit increased 9.5%. In Japanese male divers, splenic size and hematocrit were unaffected by repetitive breath-hold diving. They concluded that splenic contraction may be an integral part of the diving response in trained Korean ama. Other studies have looked at splenic contraction in response to exercise alone. Sandler et al. (51), using technetium-99m ( 9 9 m T c ) , were the first to demonstrate splenic shrinkage after exercise. They found that splenic radioactivity decreased by 49% following 5-6 minutes of maximal, symptom-limited, supine cycling. They suggested that the data could be interpreted one of two ways. First, the spleen may actively contract. Second, the change in splenic activity may be passive with progressive egress of blood flow after splanchnic flow is reduced. They also suggested that the significant increase in catecholamine concentration found during exercise may induce both splenic and splanchnic vasoconstriction. Their conclusion was that, regardless of the mechanism, it was apparent that the spleen has a reservoir function during supine exercise. 78 Other studies have also had success in demonstrating a decrease in splenic radioactivity with different types of exercise. Foelich et al. (24) also used " m Tc4abe l led red blood cell's to determine changes in blood volume of liver, spleen, kidneys and cardiopulmonary with graded continuous exercise on a cycle ergometer. The counts for each of the organs were expressed as a percent of activity at zero workload. They found a 39% decrease i n splanchnic blood volume and no significant decreases in liver and kidney blood volume. Similarly, Laub et al. (38) studied 5 subjects exercising on a bike for three 10 minute periods of different power outputs (75, 150 and 200 watts) to determine the percent reduction in splenic activity. They found that the average hematocrit increased from 44.6% to 48.0% at the highest exercise load. Epinephrine and norephinephrine concentrations also rose gradually and significantly during exercise. During exercise the splenic erythrocyte content linearly decreased to 34.2% of resting value. They concluded that there did appear to be a relationship between hematocrit increase and the release of red cells from the spleen. However, they did not find a clear relationship between splenic red cell content and adrenergic activity and suggested that regulation of the splenic size could depend on other mechanisms, but whether these are neural or humoral in origin needs to be studied with better time resolution. 79 5. Summary The more recent literature clearly demonstrates that the human spleen, like that of the dog and sheep, does release red cells in response to exercise stress. However, the changes in spleen volume with exercise have only been described qualitatively, as a percent change from the original, rest levels and have not been quantitatively determined. Measurement of actual splenic volumes during exercise has not been done. Additionally, these studies have not used direct measurements of plasma volume to determine what plasma volume shifts occur during exercise. Thus the relative contributions of splenic infusion of red cells and decreases in plasma volume to increases i n hematocrit during exercise cannot be accurately calculated. Laub et al. (38) estimated that splenic contraction may account for about one-forth of the rise in hematocrit during exhaustive exercise. Clearly, if red cell mobilization from the spleen does provide a significant contribution to increases in hematocrit during exercise, there are important implications for the use of equations that use hematocrit alone to predict plasma volume changes. 80 REFERENCES 1. Allsop P, Peters AM, Arnot RN, Stuttle AW, Deenmamode M, Gwilliam ME, Myers MJ & Hall GM. Intrasplenic blood cell kinetics in man before and after brief maximal exercise. Clin Sci 83(1): 47-54, 1992. 2. 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Greenleaf JE, Convertino VA, Mangseth GR. Plasma volume during stress in man: osmolality and red cell volume. J Appl Physiol 47: 10 3 1 -1038, 1979. 29. Guntheroth WG, McGough GA & Mullins GL. Continuous recording of splenic diameter, vein flow, and hematocrit in intact dogs. Am. J. Physiol. 231(3): 690-694, 1967. 30. Hahn PF, Bale WF,, Bonner JF. Removal of red cells from the active circulation by sodium pentobarbital. Am J Physiol 138: 4 1 5 - 4 2 0 , 1943. 3 1 . Hall GM. Intrasplenic blood cell kinetics in man before and after brief maximal exercise. Clin Sci 83(1): 47-54, 1992. 32. Harrison MH, Graveney MJ & Cochrane LA. Some soureces of error in the calculation of relative change in plasma volume. Eur J Appl Physiol 50: 13-21, 1982. 3 3 . Hartley LJ, Mason JW, Hogan RP, Jones LG, Kotchen TA, Mougey EH, Wherry RE, Pernington LL & Ricketts PT. Multiple hormonal responses to prolonged exercise in relation to physical training. J Appl Physiol 33: 607-610, 1972. 34. Hespel P, Lijnen P, Fiocchi R e t a l . Cationic concentrations and transmembrane fluxes in erythrocytes of humans during exercise. J Appl Physiol 61: 37-43, 1986. 35. Hurford WE, Hong SK, Park YS, Ahn DW, Shiraki K, Mohri M & Zapol W. Splenic contraction during breath-hold diving in the Korean ama. J Appl Physiol 69(3): 932-936, 1990. 36. Kowalsky RJ & Perry JR. Radiopharmaceuticals in Nuclear Medicine Practice. East Norwalk, Conneticut: Appleton and Lange, p. 41 1 - 4 2 2 , 1987. 37. LaForteAJ, Lee LP, Rich GF, Skalak TC & Lee JS. Fluid restitution and shift of blood volume in anesthetized rabbits subject to cyclic hemorrhage. Am J Physiol 31: H190-H199, 1992. 38. Laub M, Hvid-Jacobsen K, Hovind P, Kanstrup I, Christensen NJ & Nielsen SL. Spleen emptying and venous hematocrit in humans during exercise. J Appl Physiol 74(3); 1024-1026, 1993. 39. Leeson TS & Leeson CR. Histology (2nd ed). Toronto Ont: W.B. Saunders pp 245-253, 1970. 40. Letcher RL, Pickering TG & Chien S. Effects of exercise on plasma viscosity in athletes and sedentary normal subjects. Clin Cardiol 4: 172-179, 1981. 83 4 1 . Lundvall J & Lindgren P. F-cell shift and protein loss strongly affect validity of PV reductions indicated by Hb/Hct and plasma proteins. J Appl Physiol 84(3): 822-829, 1998. 42 . McKeever KH, Hinchcliff KW, Fteed SM & Robertson JT. Role of decreased plasma volume in hematocrit alterations during incremental treadmill exercise in horses. Am J Physiol 265(2 Pt2): R 4 0 4 - 4 0 8 , 1993. 43 . Neuhaus D & Gehtgens P. Haemorrheology and long term exercise. Sports Med 18(1): 10-21, 1994. 44. Oh YH, Woo SK & Zeon SK. Measurement of spleen by computed tomography in normal Korean adults. J Korean Radiol Soc 25: 4 5 8 -464, 1989. 45. Persson SG, Ekman L, Lydin G, & Tufuesson G. Circulatory effects of splenectomy in the horse. I. Effect on red-cell distribution and variability of hematocrit in the peripheral blood. Zentrabl. Veterinaermed. 20: 441-455, 1973. 46. Ovist J , Hill RD, Schneider RC, Falke KJ, Liggins GC, Guppy M, Elliot RL, Hochachka PW & Zapol PM. Hemoglobin concentrations and blood gas tensions of fee-diving Weddell seals. J. Appl. Physiol. 61(4): 1 5 6 0 -1569, 1986. 47. Rhoades R & Pflanzer R. Human Physiology, 3rd ed. Orlando Florida: Saunders College Publishing. 831-32, 1996. 48. Rocker L, Kirsch KA, Heyduck B & Altenkirch HU. Influence of prolonged physical exercise on plasma volme, plasma proteins, electrolytes, and fluid regulating hormones. Intl J Sports Med 10: 270-274, 1989. 49. Rushmer R. Structure And Function Of The Cardiovascular System. Toronto Ont: W.B. Saunders Co, p 24-25, 1972. 50. Saba GB. Fundamentals of Nuclear Pharmacy. New York, N.Y.: Springer-Verlag, p. 95-112, 1979. 5 1 . Sandler MP, Kronenberg MW, Forman MB, Wolfe OH, Clanton JA & Partain CL. Dynamic fluctuations in blood and spleen radioactivity: Splenic contraction and relation to clinical radionuclide volume calculations. J. Am. Coll. Cardiol. 3: 1205-1211, 1984. 52. Schaffner A, Augustiny N, Otto RC & Fehr J. The hypersplenic spleen: a contractile reservoir of granulocytes and platelets. Arch Intern Med 145: 651-654, 1985. 84 53. Schmidt W, Maassen N, Tegtbur U & Brauman KM. Changes in plasma volume and red cell formation after a marathon competition. Eur J Appl Physiol 58: 453-458, 1989. 54. Selby GB & Eichner ER. Hematocrit and performance: the effect of endurance training on blood volume. Sem Hematol 31(2): 12 2 - 1 2 7 , 1994. 55. Spencer RP, Lange RC, Schwartz AD & Pearson HA. Radioisotopic studies of changes in splenic size in response to epinephrine and other stimuli. J Nucl Med 13(3): 211-214, 1971. 56. Staubli M, Bigger K, Kammer P, Rohner F, & Straub PW. Mechanisms of haematological changes induced by hyperventilation. Eur. J. Appl. Physiol. 58: 233-238, 1988. 57. Sutton JR. Effects of acute hypoxia on the hormonal response to exercise. J Appl Physiol. 42: 587-592, 1977. 58. Swanson DD, Chilton HM & Thrall JH. Pharmaceuticals in Medical Imaging. Toronto, Ont: MacMillan Publishing Co. p. 616-621, 1990. 59. Turner AW & Hodgetts VE. The dynamic red cell storate function of the spleen in sheep I. relationship to fluctuations of jugular hematocrit. Aust J Exp Biol 37, 399-420, 1959. 60. van Beaumont W. Evaluation of hemoconcentration from hematocrit measurements. J Appl Physiol 32(5): 721-713, 1972. 61 . Van RensburgJP, Kielbock AJ & van der Linde A. Physiologic and biochemical changes during a triathlon competition. Int J Sports Med 7: 30-35, 1986. 62. Vatner SF, Higgins CB, Millard RW & Franklin D. Role of the spleen in the peripheral vascular response to severe exercise in untethered dogs. Cardio Research 8: 276-282, 1974. 63. Wade CE. Response, regulation and actions of vasopressin during exercise: a review. Med Sci Sports Exer 16: 506-511, 1984. 64. Wade OL, Combes B, Childs AW, Wheeler HO, Cournand A& Bradley SE The effect of exercise on the splanchnic blood flow and splanchnic blood volume in normal man. Clin Sci 25: 457-463, 1956. 65. Wilkerson JE, Gurin B & Horvath SM. Exercise-induced changes in blood, red cell, and plasma volumes in man. Med Sci Sports 9: 15 5 -158, 1977. 66. Winder WW, Hagberg JM, Hickson RC, Ehsani AA & McLane JA. Time course of sympathoadrenal adaptation to endurance exercise training i n man. J Appl Physiol 45: 370-374, 1978. APPENDIX B. Nuclear Medicine Techniques 86 Radionuclide dilution techniques are routinely used to accurately determine total blood volume, plasma volume and red cell volume. These measurements require that the radioisotope mix with the components within the vascular compartment and that it distribute uniformly throughout the compartment. The tracer isotope should also have no impact on the volume of the compartment being measured. Additionally, the isotope must permit accurate detection and quantification at very low concentrations (58). Since blood consists of both plasma and cellular elements, determination of total blood volume should employ the combined use of two different isotopes: one that will mix with, and uniformly distribute in the plasma volume and one that will mix with, and uniformly distribute in the red cell volume (58). Radioiodinated Human Serum Albumin (RIHSA) are commonly used to provide accurate quantifications of plasma volume. A problem associated with using RHISA is that it is not solely confined to the vascular space and will diffuse into the extravascular space at a fractional rate of approximately 6 - 1 0 % per hour (58). Thus determination of plasma volume requires that at least two (preferably three) post-injection blood samples be drawn and counted. A semi-log plot of this data, backextrapolated to time zero (time of injection) will provide an accurate determination of plasma volume. 1 2 5 I -RIHSA and 1 3 1 I -RIHSA can both be used for determination of plasma volume, although 1 2 5 I -RIHSA has several advantages over 1 3 1 I -RIHSA. The low-energy photon emissions of 1 2 5I -RIHSA are substantially different from the 320 keV emission of 5 1 C R , thus permitting the simultaneous 87 quantification of both plasma and red cell volumes. 1 2 5 I -RIHSA decays by electron capture with a 60-day half-life emitting a 35 keV gamma and 2 7 keV x-rays (36). 1 3 1 I -RIHSA decays by beta-particle emission with a half-life of approximately 8.1 days also emitting a 364 keV gamma radiation (58) . In an exercise study, where pre- and post-exercise determination of plasma volume are required, both 1 2 5I-RIHSA and 1 3 1I -RIHSA must be utilized: one to measure pre-exercise plasma volume and one to measure post-exercise plasma volume. Because RIHSA leaks from the vascular space and the rate of leakage will not be linear with exercise, a blood sample drawn immediately upon cessation of exercise will not yield an accurate reading of post-exercise plasma volume. A new radionuclide must be injected at the end of exercise to create a new 'time zero' for measurement of post-exercise plasma volume. 5 1 C R is routinely used to label red cells for determination of total red cell volume. Uniform mixing of the 5 1CR-labeled cells is complete within 10 to 20 minutes post injection (58). 5 1 C R decays by electron capture with a 27.7 day half-life emitting a 320 keV gamma ray (36). The low photon abundance means that 5 1 C R is unsuitable as an imaging radionuclide, thus for imaging of the spleen, 9 9 m TC-labeled red cells must be utilized. Uptake of radioactive colloid by the spleen, conventionally used to assess the gross morphology of an organ, can also be employed to gauage the size of the spleen and its alteration following medications and other stimuli. 99mTc is 88 commonly used for imaging of the spleen as it binds to the hemoglobin of red cells (50). Hall (31) and Baker and Remington (3) found that erythrocytes labelled with 99myc equilibrate rapidly between the spleen and circulating blood after injection, thus accurate splenic images and counts can be done within 5 to 10 minutes after injection. Spencer et al (55) found no evidence that the spleen rotates or changes position, thus determination of total spleen radioactivity can be made using the average counts from the anterior and posterior images of the spleen. In order to calculate spleen volume from the splenic counts, the activity count of a known volume of red cells must be determined. This count can then be used to calculate spleen volume based upon the average spleen activity. unknown volume = # ofGQuntein unknown YQlumex known volume (mLs) # of counts in known volume APPENDIX C. Informed Consent 9 1 On two other occasions, you will be asked to spend about three hours, per session at the Vancouver Hospital, UBC site, Nuclear Medicine Department. Upon arrival you will sit down for about thirty minutes. An indwelling catheter will be put in each arm and approximately 20 mL of blood will be withdrawn, labeled and then reinjected. Your red blood cells and blood plasma will be labeled with radioactive substances to allow us to determine your red blood cell volume, your plasma volume and the volume of your spleen. Three blood samples will be withdrawn from the catheter at fifteen minute intervals after the blood sample has been injected. A gamma camera will be used to take images of your spleen. You will then start a 30 minute exercise session on a stationary bike at loads that will have been determined from your previous bike test at the Allan McGavin Sports Medicine Centre. Every ten minutes a 5 mL blood sample will be withdrawn from the catheter in your arm so we can determine your hematocrit. The gamma camera will also be used to take a back view of your spleen. At the end of the exercise test, a very small amount of another radioactive substance will be injected into your arm to remeasure your plasma volume. Blood samples will be withdrawn from the catheter 10, 20 and 30 minutes after the label is injected. At the second visit to the Nuclear Medicine Dept. you will follow the exact same procedures as the first session except that for five minutes prior to and during the thirty minute exercise test, you will be breathing the 16% (altitude) oxygen mixture. Risks: The exercise testing is considered to be very safe and we anticipate that you will experience only the normal temporary discomfort of minor muscle soreness associated with exercise up to maximal effort. There is also an extremely small risk that you may experience abnormal heart beats during maximal exercise. The equipment and procedures used to monitor your responses to maximal exercise will impose no added risk to your health and well-being. The doses of radiation that will be used in this study are equivalent to the amount of radiation you would be exposed to flying from Vancouver to Toronto and back twice. There is negligible risk associated with the radiation dose being used in this study. CONSENT In signing this form you are consenting to participate in this research project and acknowledge a receipt of a copy of this form. Subject name:_ Date: Subject signature: Witness signature: Investigator's signature: 94 APPENDIX D. Examples Of Regions Of Interest Drawn Around The Spleen Before Exercise And After Each Exercise Load. 95 Examples of posterior and anterior splenic scans taken pre-exercie (a), after 10 minutes at 25% \'(), max (b), after 10 minutes at 50% V O , max (c) and after 10 minutes at 75% VO, max (d). Posterior scans are frames 1,3,5 and 7. Anterior scans are frames 2,4,6 and 8. BLOOD-POOL-UNKNOWN-US SPORTS MEDICINE UBC5, OQQ005lSTATIC_SPLEENi95/1 2/07 97 A P P E N D I X E Calculations for the determination of the relative contributions of plasma volume changes and red cell volume changes to changes in peripheral hematocrit with exercise 98 A RCVcirculating = (RCV - SV p r e(0.96)) - (RCV - SV p Ost(0.96)) A PV = PVpre - PVpost Total A = A PV + A RCVcirculating A Hct = Hctpost - Hctpre PV%age of Total A= AP_V_ x 100% A Total A Hct due to PV A = PV%age X AHct RCV %age of Total A= ARCV x 100% A Total A Hct due to RCV A = RCV%age X AHct 

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