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Relationship of 2,3-diphosphoglycerate and other blood parameters to training, smoking and acute exercise Marchant, Leonard Roy 1973

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RELATIONSHIP OF 2,3-DIPHOSPHOGLYCERATE AND OTHER BLOOD PARAMETERS TO TRAINING, SMOKING AND ACUTE EXERCISE By Leonard Roy Marchant B.P.E., U n i v e r s i t y of B r i t i s h Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF PHYSICAL EDUCATION i n the School of Physical Education and Recreation We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1973 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f pfrYstc&JL. ^oac/^r>&W The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT The purpose of this study was to examine differences i n 2,3-diphospho-glycerate concentrations among groups of smokers and nonsmokers, to determine relationships between 2,3-DPG concentrations and other blood parameters affecting oxygen transport, and to examine the effects of acute exercise on 2,3-DPG concentrations. Antecubital venous blood from each subject, before and after exercise, was analyzed for 2,3-DPG, hematocrit, hemoglobin and blood pH. Mean corpuscular hemoglobin concentration (MCHC) was calculated by dividing hemoglobin by hematocrit. Forty university-aged males constituted the sample population. Each subject was assigned to one of five groups, eight subjects per group, based on his status i n relation to the variables of physical ac t i v i t y and cigarette smoking. The task consisted of one hour of exercise on a bicycle ergometer at a work rate producing a heartrate of approximately 150 beats per minute (70 per cent of maximal aerobic capacity). Blood samples were taken immediately prior to and immediately following the bout of exercise. A 12 hour fast preceded the work phase of the experiment. The hypotheses were: highly f i t subjects have significantly higher 2,3-DPG concentrations and sedentary subjects have significantly lower 2,3-DPG concentrations than moderately f i t subjects; smokers have significantly higher 2,3-DPG levels than nonsmokers; exercise produces significant increases i n 2,3-DPG; negative relationships exist between 2,3-DPG levels and hemoglobin levels as well as between pre exercise 2,3-DPG levels and change of 2,3-DPG as a result of exercise. i i A p r i o r i orthogonal comparisons of pre exercise red c e l l 2,3-DPG levels indicated that differences between groups were not significant, i.e. highly f i t groups did not demonstrate 2,3-DPG levels significantly higher, nor did sedentary groups demonstrate 2,3-DPG levels significantly lower than moderately f i t groups. A definite trend towards higher 2,3-DPG levels was observed as training intensity increased, indicating that the hypothesis of physical training producing an increase i n 2,3-DPG levels should not be totally rejected. Demonstration of differences in the carrying capacity of the blood, as reflected by differences i n MCHC, hemoglobin and hematocrit, between groups appeared to be related to the trend observed in 2,3-DPG levels. Differences between smokers and nonsmokers in relation to 2,3-DPG concentrations were not significant,indicating that the hypoxia produced through cigarette smoking i s not an important stimulator of 2,3-DPG production. Multivariate analysis of results indicated that 2,3-DPG levels were not significantly increased as a result of one hour of exercise at 70 per cent of maximal aerobic capacity. This i s indicative of a slow-acting response mechanism affecting 2,3-DPG production, which requires more than one hour, or a more severe stress, to produce a physiological beneficial effect on oxygen transport by the blood. A significant negative correlation was observed between pre exercise levels of 2,3-DPG and hemoglobin levels. This was also reflected in the significant negative correlation between 2,3-DPG and hematocrit and 2,3-DPG and MCHC. A negative correlation was also observed between the change in 2,3-DPG and the change of MCHC that occurred as a result of exercise. The I i i results are interpreted as showing a compensatory effect of 2,3-DPG in producing increased unloading of oxygen when the carrying capacity of the blood is reduced through a reduction in hemoglobin levels. An intimate relationship between 2,3-DPG and MCHC, tending to produce homeostasis in the position of the oxygen dissociation curve of hemoglobin, has been postulated. Changes i n 2,3-DPG as a result of exercise were not related to the pre exercise concentration of 2,3-DPG indicating that change of 2,3-DPG is not significantly affected by the amount of 2,3-DPG present before physical activity i s ini t i a t e d . iv ACKNOWLEDGMENTS The author would like to express his sincere gratitude to the members of his thesis committee for their help and support throughout the preparation of this study; Dr. Kenneth Coutts, committee chairman, for his astute knowledge of exercise physiology; Dr. Robert Schutz, s t a t i s t i c i a n unsurpassed, for his help in analysis of the data; Dr. Joseph Angel and Dr. David Randall for their expertise i n matters biochemical and physiological respectively. My appreciation i s also extended to Dr. Melvin Lee and Dr. I. Desai, of the School of Home Economics, for providing research f a c i l i t i e s used to conduct biochemical determinations. Finally, a special thanks i s conveyed to my brother, Wayne Marchant, for the excellent preparation of the various figures found within the study;r and to a dear friend, Robin Kitagawa, for her many hours of assistance in the experimental phase of the study. The study would not have been possible with-out Robin's help. V TABLE OF CONTENTS Chapter Page I. STATEMENT OF THE PROBLEM 1 Introduction . ........ 1 Statement of the Problem 3 Subproblems . ° • ° 4 Hypotheses . 5 Definition of Terms <> 6 Delimitations . 9 Limitations . 9 Significance of the Study 10 II. REVIEW OF THE LITERATURE . 11 Introduction 11 The Chemical Reserve Mechanism in Oxygen Transport 11 The Temperature Effect 12 The Bohr Effect 13 The 2,3-Disphosphoglycerate Effect 14 The Binding of 2,3-Diphosphoglycerate to Hemoglobin... 15 The Blood pH Effect .. 18 The Carbon Dioxide Effect 19 The Hemoglobin Concentration Effect 20 The Postulated Control of the 2,3-DPG Response Mechanism..... 21 The Biochemical Control of 2,3-DPG Concentrations... 21 The Physiological Control of 2,3-DPG Concentrations. 24 2,3-Diphosphoglycerate Changes in Long Standing Hypoxias..... 29 Altitude Exposure. • 31 vi Chapter Page Exercise Exposure ...... . 32 Cigarette Smoking and the 2,3-Diphosphoglycerate Response ..... 35 III. MATERIALS AND METHODS 39 Subjects 39 Experimental Procedures . 41 Biochemical Determinations 45 Physiological Determinations . 48 Experimental Design 48 St a t i s t i c a l Analysis 49 IV. RESULTS AND DISCUSSION 52 Results. 52 Descriptive Statistics 52 Homogenity of Variance 67 St a t i s t i c a l Analysis of the Data - Test of Hypotheses 68 Discussion 76 Pre Exercise Parameters 76 The Effect of Exercise 89 V. SUMMARY AND CONCLUSIONS - 98 Summary 98 Conclusions 100 BIBLIOGRAPHY 101 APPENDICES 110 A. S t a t i s t i c a l Analysis 110 B. Individual Scores 114 C. St a t i s t i c a l Comparison of High Work Capacity VS Low Work Capacity (Post Hoc) 125 v i i LIST OF TABLES Table Page I Analysis Format of Dependent Variables . 51 II Means and Standard Deviations of 2,3-DPG Concentrations.. . 53 III Means and Standard Deviations of Hemoglobin Concentrations 55 IV Means and Standard Deviations of Hematocrit Levels 57 V Means and Standard Deviations of Mean Corpuscular Hemoglobin Concentrations (MCHC) 59 VI Means and Standard Deviations of Blood pH 61 VII Means and Standard Deviations of Body Weight and Average Work Per Heartbeat 63 T IX Correlation Coefficients Between Pre Exercise 2,3-DPG Levels and Other Dependent Variables . . . . 65 X Correlation Coefficients Between Post Exercise 2,3-DPG Levels and Other Dependent Variables . . . 65 XI Correlation Coefficients Between Change in 2,3-DPG, as a Result of Exercise, and Other Dependent Variables 66 XII Orthogonal Comparisons of Pre Exercise 2,3-DPG L e v e l s . . . . 68 XIII Analysis of Change of 2,3-DPG as a Result of Exercise 69 XIV Orthogonal Comparisons of Pre Exercise Hemoglobin Concentrations.. . 70 XV Analysis of Change of Hemoglobin as a Result of Exercise 70 XVI Orthogonal Comparisons of Pre Exercise Hematocrit L e v e l s . . . . . . . . . . . 71 XVII Analysis of Change of Hematocrit as a Result of E x e r c i s e . . . . . . . . . . . 72 XVIII Orthogonal Comparisons of Pre Exercise MCHC L e v e l s . . . . . . . . . . 72 XIX Analysis of Change of MCHC as a Result of Exercise. . 73 XX Orthogonal Comparisons of Pre Exercise Blood pH 74 XXI Analysis of Change of Blood pH as a Result of Exercise 74 XXII Orthogonal Comparisons of Average Work Per Heartbeat 76 XXIII Theoretical Differences in P 5 Q as a Result of 2,3-DPG and MCHC. 86 XXIV Theoretical Changes i n ?-n as a Result of Changes i n 2,3-DPG and MCHC 1. 96 v i i i LIST OF FIGURES Figure Page 1 The Oxygen Dissociation Curve of Hemoglobin 16 2 Red Blood C e l l Metabolic Pathways 23 3 Factors Altering 2,3-DPG Concentrations 30 4.1 The Experimental Design 51 4.2 Format of Orthogonal Comparisons 51 5 Graphical Presentation of 2,3-DPG Concentrations.... 54 6 Graphical Presentation of Hemoglobin Levels 56 7 Graphical Presentation of Hematocrit Levels 58 8 Graphical Presentation of MCHC Levels 60 9 Graphical Presentation of Blood pH Measurements 62 10 Graphical Presentation of Average Work Per Heartbeat..... 64 11 Relationship Between Change in 2,3-DPG and Change in MCHC as a Result of Exercise 94 CHAPTER I STATEMENT OF THE PROBLEM Introduction Acute and chronic hypoxia induce a number of adaptive mechanisms which are a l l directed against an impairment of the oxygen supply to the tissues. Among these processes an increase in cardiac output, a stimulation of respiration and, during chronic hypoxia, a rise in the circulating red c e l l mass have been well known for several years. Recently a further mechanism of adaptation has gained particular interest, namely, the shift to the right of the oxyhemoglobin dissociation curve occurring under various conditions of hypoxia. As was shown by Chanutin and Curnish (1967) and by Benesch and Benesch (1967) 2,3-diphospho-glycerate (2,3-DPG), which i s a major constituent of mammalian red blood c e l l s , causes a concentration dependent diminution of the oxygen a f f i n i t y of hemoglobin by i t s binding to special sites on the beta chains of the hemoglobin molecule (Benescji et a l . , 1968). Many kinds of hypoxia appear to induce an increase of 2,3-DPG levels i n red blood c e l l s (Baumann et al.,1967; Eaton and Brewer, 1968; Lenfant et a l . , 1968; Oski et a l . , 1969; Torrance and Bartlett, 1970; Valeri and Fortier, 1969). In a l l these cases the shift of the oxyhemo-globin dissociation curve was considered to be causally related to the concentration changes of 2,3-DPG. From these observations i t i s obvious that the adaptive changes of 2,3-DPG i n red blood cells are of special physiological importance. Exercise i s capable of inducing both acute (a single exercise bout) and chronic (training over prolonged periods) hypoxia. In addition, a right shift of the oxygen dissociation curve of hemoglobin beyond that attributable to a decrease in blood pH and increase in blood temperature has been observed, during strenuous exercise, by several investigators (Faulkner et a l . , 1970; Mitchell et a l . , 1958; Rowell, 1969). It has also been reported (Rowell et a l . , 1964) that highly trained individuals have a reduced saturation of hemoglobin in the lung a l v e o l i . Investigations have been made to determine whether or not increases in 2,3-DPG were responsible for these phenomena (Dempsey et a l . , 1971; Eaton et a l . , 1969; Faulkner et a l . , 1970; Shappell et a l . , 1971); how-ever, the results are conflicting. Several factors could have produced the ambiguity observed i n the four studies dealing with the 2,3-DPG response to exercise. F i r s t , one would presume that a stress placed on a physiological system governs the response e l i c i t e d . To determine the response mechanism, i n this case changes in the concentration of red blood c e l l 2,3-DPG, a l l subjects should have been stressed equally, based on a c r i t e r i a relating to their individual capabilities. Secondly, i t i s possible that the basal, unstressed level of 2,3-DPG in the red blood c e l l might influence the amount of additional 2,3-DPG which can be produced when the system i s placed under stress; especially i f unbound 2,3-DPG inhibits the enzyme (DPG mutase) responsible for further 2,3-DPG synthesis (Benesch et a l . , 1970). It appears that training may increase basal levels of 2,3-DPG (Shappell et a l . , 1971), which could account for a lack of response when stress, i n the form of exercise, i s placed on highly trained individuals (Dempsey et a l . , 1971). 3 Third, i t i s possible that smoking could affect basal levels of 2,3-DPG. Smokers tend to have a l e f t shift in their oxygen dissociation curve caused by binding of carbon monoxide (from cigarette smoke) to hemoglobin (Lenfant et a l , , 1970). Therefore an increase of 2,3-DPG would tend to comp-ensate for the effect of smoking by moving the curve back to i t s normal position. The small size of samples used i n previous studies, ranging from three to ten subjects, could also be a factor leading to the incongruity of the results. Individual differences i n numbers of red blood c e l l s , blood pH, hemoglobin levels and other factors thought to affect 2,3-DPG levels would contribute to heterogeneity of resting 2,3-DPG levels, thereby masking trends which might become apparent i f larger sample sizes were u t i l i z e d . In conclusion, i t i s apparent that i n order to ascertain whether or not the response of the 2,3-DPG mechanism to exercise-induced hypoxia i s a significant physiological phenomenon, a study using a larger number of subjects 9 with subgroups of different fitness levels, stressed at an equal percentage of their maximum capacity, i s necessary, Statement of the Problem The purpose of this study i s to investigate the effects of acute exercise on the levels of red c e l l 2,3-diphosphoglycerate of human subjects. Further, this study attempts to delineate differences in 2,3-DPG and other blood parameters between smoking and nonsmoking subjects of differing fitness levels. Subproblems The subproblems are: 1. To determine i f red blood c e l l 2,3-DPG increases as a result of training, thereby f a c i l i t a t i n g oxygen delivery for a given amount of hemoglobin, 2. To determine i f red blood c e l l 2,3-DPG i s increased in smokers, compensating for a l e f t shift i n the oxygen dissociation curve due to carboxy-hemoglobin. 3. To determine i f an increase in 2,3-DPG occurs during a strenuous one hour bout of exercise, f a c i l i t a t i n g oxygen delivery to active muscles, 4. To determine relationships, i f any, between red blood c e l l 2,3-DPG levels and the levels of hematocrit, hemoglobin, mean corpuscular hemoglobin concentration (MCHC) and blood pH„ 5. To determine the changes in hematocrit, hemoglobin, MCHC and blood pH that occur as a result of acute exercise. 6.' To determine differences i n hematocrit, hemoglobin, MCHC and blood pH among smoking and nonsmoking subjects of different fitness levels, 7. To determine the work capacity of smokers and nonsmokers of various states of fitness when they are equated on an equivalent physiological stress ( i.e. a work rate e l i c i t i n g a heart rate of 150 beats per minute for one hour duration), Hypotheses The hypotheses are: 1. A one hour bout of exercise, at a work load e l i c i t i n g an average heart rate of approximately 150 beats per minute, produces an increase in red blood c e l l 2,3-diphosphoglycerate levels in a l l subjects. 2. The mean resting level of red blood c e l l 2,3-diphosphoglycerate is highest i n the highly trained subjects and lowest in the sedentary subjects. 3. The mean resting level of red blood c e l l 2,3-diphosphoglycerate is higher i n smoking subjects than in nonsmoking subjects of the same fitness le v e l . 4. The increase i n red blood c e l l 2,3-diphosphoglycerate, as a result of exercise, i s larger in those subjects with lower resting 2,3-DPG levels. That i s , there exists a negative correlation between resting 2,3-DPG levels and the increase i n 2,3-DPG levels due to exercise. 5. A negative correlation exists between 2,3-DPG levels and blood hemoglobin levels. That i s , subjects with high hemoglobin levels have low red blood c e l l 2,3-DPG levels and vice versa. Definition of Terras 2,3-Diphosphoglycerate (2,3-DPG). A metabolic intermediate of the glycolytic (Emden-Meyeroff) pathway in the mammalian erythrocyte. It is produced by conversion of 1,3-diphosphoglycerate by the enzyme DPG mutase, via the Rapoport-Luebering shunt (Figure 2). The chemical structure of the 2,3-DPG molecule may be represented as follows: COOH I H C O P O 3 H 2 I C H 2 O P O 3 H 2 2,3-diphosphoglycerate is bound preferentially to deoxyhemoglobin and in so doing, affects the a f f i n i t y of hemoglobin for oxygen so that an increased amount of oxygen w i l l be released at a constant partial pressure of oxygen. The reaction may be summarized as follows: HbDPG + 0 2 J Hb02 + 2,3-DPG. Hematocrit. The volume occupied by packed red blood c e l l s i n a given quantity of blood after centrifugation i n a special graduated tube. The parameter i s expressed as a percentage. Hemoglobin. The main component of the mammalian erythrocyte which functions in the transport of oxygen from the lungs, through the arteries, to the tissues. A single erythrocyte contains about 200 million molecules of 1 ! hemoglobin. Each molecule i s made up of about 10,000 atoms, including four atoms of Iron. Each atom of iron l i e s at the center of the group of atoms that form the pigment called heme-, which gives blood i t s red color and i t s a b i l i t y to combine with oxygen. Each heme group is enfolded in one of the four chains of amino acid units that collectively constitute the protein part of the molecule, which i s called globin. The four chains of globin consist of two identical pairs. The members of one pair are known as alpha (a) chains and those of the other as beta (3) chains. Together the four chains contain a total of 574 amino acids (Perutz, 1968). Hemoglobin is responsible for a 70-fold increase i n the a b i l i t y of the blood to dissolve and transport oxygen. Each of the four atoms of iron i n the hemoglobin molecule can take up one molecule (two atoms) of oxygen. The reaction is reversible i n the sense that oxygen is taken up where i t i s p l e n t i f u l , as i n the lungs, and released where i t i s scarce, as in the tissues. Hb + 0 2 J Hb02 Mean Corpuscular Hemoglobin Concentration (MCHC). An expression, i n absolute terms, of the average hemoglobin concentration per unit volume (per 100 ml) of packed red blood c e l l s , calculated from the equation M C H C _ hemoglobin (gm/100 ml) x 100 hematocrit and stated in grams per 100 ml of packed red blood c e l l s . Oxygen Dissociation Curve of Hemoglobin. A graphical presentation of the relationship between the amount of oxygen bound to hemoglobin vs. the pa r t i a l pressure of oxygen in the blood (P0 2). The blood leaving the lungs usually has a P0 2 of about 100 Torr, and the amount of hemoglobin that i s bound with oxygen at this P0 2, called the hemoglobin saturation, i s about 97 i per cent. On the other hand, in normal venous blood the P0 2 i s about 40 Torr and the per cent saturation i s about 75 per cent. The blood of a normal person contains approximately 15 grams of hemoglobin in each 100 ml of blood, and each gram of hemoglobin can bind with a maximum of about 1.34 ml of oxygen. Therefore, on the average, the hemoglobin in 100 ml of blood can combine with a total of about 20 ml of oxygen. On passing through the tissue capillaries 8 this amount i s reduced to approximately 14.4 ml (PO^ of 40 Torr, 75% saturated) or a total loss of about five m i l l i l t e r s of oxygen from 100 m i l l i l t e r s of bloodi In exercise, two mechanisms operate upon the hemoglobin of the red blood c e l l to increase oxygen delivery to the tissues. F i r s t , the i n t e r s t i t i a l f l u i d P0 2 may f a l l as low as 15 Torr so that only 4.4 ml of oxygen remains bound with hemoglobin in each 100 ml of blood. Second, the oxygen dissociation curve may shift to the right due to conformational changes induced upon the hemoglobin molecules. This decreases the oxygen a f f i n i t y of hemoglobin,requir-ing an increased P0 2 to maintain the bond between the two molecules. Because the P0 2 at the tissues i s low, much more oxygen w i l l dissociate from the hemoglobin when the a f f i n i t y i s reduced. This rightward shift of the dissociation curve is known as the Bohr effect (Guyton, 1971). ):r Partial Pressure.(P) The partial pressure of a gas is the total pressure of the mixture of gases in which i t occurs multiplied by the percentage of the total volume that i s occupies. Thus, i f the total pressure of a l l the atmospheric gases is about 760 Torr, and i f the oxygen content i s about 20 per cent of this mixture, then the par t i a l pressure of oxygen (P0 2) in the atmosphere i s equal to 760 x 0.20, or 152 Torr. PJ.Q This parameter i s u t i l i z e d by physiologists to provide a frame of reference for the relative position of the oxygen dissociation curve of hemoglobin. It i s the partial pressure of oxygen in the blood at which 50 i per cent of the hemoglobin i s saturated with oxygen (ie one-half of the hemoglobin i s bound to oxygen, one-half the hemoglobin i s unbound). Kilopond Meter (kpm). One kp i s the force acting on the mass of one kg at normal acceleration of gravity. One kpm i s the work done in moving one kp a distance of one meter. Delimitations 9 1. The data under consideration in this study i s based upon blood samples taken from venous blood of the upper limbs of the subjects. Interpret-ation of the data i s limited, by necessity, to the specific anatomical site of blood sampling (ie the upper limbs). 2. There are two main components to the oxygen delivery mechanism i n humans - the amount of oxygen carried by the blood and the rate at which i t i s carried. This study i s concerned only with the f i r s t mechanism, therefore any conclusions w i l l be based only on a parti a l investigation of blood oxygen transport, with the relationship to the blood flow response remaining unanswer-ed. Limitations 1. The investigation i s limited by the sample size of 40 subjects. In addition, subjects were assigned to subgroups according to a status variable which may have obscured cause and effect relationships of the physiological parameters under study. 2. In analyzing the blood samples for hematocrit, hemoglobin, MCHC, pH and 2,3-DPG two determinations were made on each variable for each blood j sample. If the difference obtained between the two measurements exceeded a preset c r i t e r i a of five per cent, then two additional determinations were made. For s t a t i s t i c a l analysis, the mean value of two determinations, varying by less than five per cent, was u t i l i z e d . The study i s limited by the accuracy of the analytical equipment and the method of reporting the results. 10 3. The investigation does not show what duration of time i s required to e l i c i t changes in the concentration of red blood c e l l 2,3-DPG, but only whether or not 2,3-DPG concentrations change after one hour of exercise. Similarly, the investigation does not show what percentage of stress i s required to e l i c i t changes in 2,3-DPG concentrations, but only whether exercise stress producing a heart rate of 150 beats per minute induces a change in the concentration of red blood c e l l 2,3-DPG. Significance of the Study If physical, educators and exercise physiologists are to be concerned with improving human fitness and physical performance they must have knowledge of the physiological phenomena that occur as a result of exercise and/or training. They must also strive to find factors which favorably influence those factors which enhance fitness and performance. It i s known that f a c i l i t a t i o n of the oxygen transport mechanism occurs during exercise by means of a right shift of the oxygen dissociation curve of hemoglobin beyond that which can be explained by known physiological responses. It i s also known that 2,3-DPG f a c i l i t a t e s oxygen delivery by causing a right shift in the oxygen dissociation curve of hemoglobin i n certain pathalogical and non-pathalogical conditions of hypoxia. However, studies to the present time have not provided conclusive evidence to show whether or not 2,3-DPG i s responsible for the sh i f t that occurs during exercise. This investigation i s an attempt to c l a r i f y the ambiguity of the role of 2,3-DPG in exercise. In addition the study considers whether 2,3-DPG acts as an adaptive mechanism under another condition of hypoxia, namely smoking. CHAPTER II REVIEW OF THE LITERATURE Introduction Tissue oxygen consumption represents a prime requirement which must be met for l i f e to continue. There are several mechanisms for increasing the capacity of the red blood c e l l to deliver oxygen to metabolically active tissue, namely, an increase i n hemoglobin concentration or in the number of red blood c e l l s (ERYTHROPOIETIC RESERVE), an increase in the rate of blood flow (FLOW RESERVE), a slight increase i n the oxygen concentration of a r t e r i a l blood which i s accomplished only by hyperventilation in normal, healthy individuals (RESPIRATORY RESERVE), and l a s t l y , an alteration in the hemoglobin a f f i n i t y for oxygen resulting in the displacement of the oxygen dissociation curve to the right which in turn permits a greater extraction of oxygen from the blood (CHEMICAL RESERVE) (Metcalfe and Dhindsa, 1972). The Chemical Reserve Mechanism in Oxygen Transport Oxygen uptake and transport by hemoglobin i s complicated by the presence of four heme groups in each hemoglobin (Hb) molecule and the j consequent fact that each reduced (ferrous) hemoglobin molecule may bind none, one, two, three or four molecules of oxygen (Harris and Kellermeyer, 1970). When the binding of oxygen to hemoglobin is in i t i a t e d , an increased a f f i n i t y for oxygen i s induced in the remaining heme groups, allowing them to bind much more readily to oxygen (Ten Eyck, 1972). This heme-heme 11 12 interaction can be recognized by a consideration of the oxygen association or dissociation curves for human hemoglobin (Figure 1). When the percentage of hemoglobin saturated with oxygen i s determined experimentally with respect to the pa r t i a l pressure of oxygen, the curve so obtained (called the oxygen dissociation curve of hemoglobin) i s found to be sigmoidal. Physiologically, this means that hemoglobin i s an efficient transport system. In the lungs a high oxygen tension forces oxygen onto the low a f f i n i t y deoxy structure and converts i t to the high a f f i n i t y oxy structure. In the tissues a low oxygen tension causes the oxygen to be released and the structure to revert to the low a f f i n i t y form. This effectively provides a one-way system from the lungs to the body tissues (Guyton, 1971). The alteration of both the position and shape of the curve, increasing the amount of oxygen delivered to active tissues, can occur through modifications in temperature, blood pH and levels of organic phosphates (Bartels, 1972). The Temperature Effect. In humans, blood temperature increases during exercise causing a right shift i n the oxygen dissociation curve (Astrand and Rodahl, 1970). Such a rise in temperature of the blood perfusing muscles weakens the binding of oxygen to hemoglobin so that more oxygen can be released into the working muscles without a change in the pa r t i a l pressure of oxygen (Po 2). A rise in blood temperature from 37 to 41 degrees, at constant pH (7.2) and venous Po 2 (27 Torr), was seen to cause a decrease in venous blood oxygen saturation from 30 to 20 percent (Shappell et a l . , 1971). Recently, Lenfant et al.,(1972) have shown that the temperature effect on binding of oxygen to hemoglobin i s reduced with increasing concentrations of 2,3-diphosphoglycerate (2,3-DPG). The corollary of this relationship i s , as pointed out by Benesch et al.(1969), the effect of 2,3-DPG i n lowering the oxygen a f f i n i t y of hemoglobin decreases with increasing temperature. 13 The Bohr Effect. In vivo, both the part i a l pressure of carbon dioxide (PCO2) and pH affect the a f f i n i t y of hemoglobin for oxygen. The Bohr effect i s defined as the difference in the total charge carried by oxy and deoxyhemoglobin at constant pH (Kilmartin, 1 9 7 2 ) . Three amino acid residues have been implicated i n the Bohr effect of human hemoglobin: histidine 146 (on the Beta subunit), valine 1 and histidine 122 ( on the Alpha subunit). The "effective" Bohr effect, according to Bartels ( 1 9 7 2 ) i s defined as the increase i n the amount of oxygen delivered from 100 m i l l i l i t e r s of blood when aci d i f i c a t i o n by 0.1 or 0.2 pH units takes place at 50 percent hemoglobin saturation. The influence of the Bohr effect on the oxygen a f f i n i t y , expressed as P^Q (i.e. the oxygen pa r t i a l pressure at which 5 0 percent of hemoglobin i s saturated with oxygen), was found to be more pronounced as the carrying capacity (hemoglobin concentration) increased. In addition, the optimal "effective" Bohr effect with varying oxygen capacities occurred at a P^Q of 33 Torr. (In normal resting man P^Q i s approximately 27 Torr). At 40 Torr venous oxygen pressure (similar to man at rest) the oxygen unloading capacity also increased with increasing hemoglobin concentration but the influence was not as great as found at 33 Torr. Lenfant et a l . ( 1 9 7 2 ) pointed out that alterations in pH play the major role i n the Bohr effect, with the contribution from being approximately 20 percent. (The Bohr coefficient generally used (change in log Po2/change in log pH),-0.480, includes both the contributions from C 0 2 and pH). They found a decrease i n pH from 7.40 to 7.20, at constant temperature (37 degrees) and venous Po„ (27 Torr) caused a decrease in venous oxygen 14 r saturation from 48 percent to 30 percent. This concurs with increase i n unloading of oxygen by 26 percent observed by Doll et a l . (1968) when blood pH decreased from 7.42 to 7.17. Recent evidence suggests that higher 2,3-DPG levels entail a higher Bohr effect (Bauer, 1969; Siggard-Andersen and Sailing, 1971). This contradicts the earlier work of Benesch et a l . (1969) who found a decrease i n the Bohr effect with increasing 2,3-DPG. The 2,3-diphosphoglycerate Effect. A third mechanism of shifting the hemoglobin oxygen dissociation curve i s mediated through the concentration of inorganic phosphates within the red blood c e l l . It was known for several years (Greenwald, 1925) that large quantities of 2,3-DPG are present i n the red blood c e l l s of pigs. Later Rapoport and Guest (1940) reported that high levels of 2,3-DPG are also present i n human erythrocytes. However, i t was not u n t i l 1967 that the physiological role for 2,3-DPG was c l a r i f i e d . Benesch and Benesch (1967) and Chanutin and Curnish (1967) reported independently that the oxygen a f f i n i t y of human hemoglobin could be altered by the addition of 2,3-DPG, suggesting that Z.3-DPG performed a regulatory function i n oxygen transport by the red blood c e l l . The change in oxygen saturation brought about by 2,3-DPG at high oxygen tensions i s comparatively small so that l i t t l e effect i s observed on oxygen loading i n the alveoli of the lungs. In contrast at a Po 2 value of 30 Torr, which i s within the physiological range of metabolically active tissue, oxygen saturation decreased from 83 percent to 28 percent when the concentration of 2,3-DPG was raised from 0.1 to 23 uMoles 2,3-DPG per gram of hemoglobin (Duhm, 1971). Duhm (1972) demonstrated a curvilinear relationship between P__ and 15 the c o n c e n t r a t i o n of 2,3-DPG. At an e x t r a c e l l u l a r pH of 7.40 a change of P^_ of one Torr r e s u l t e d from a change of about 0.4 uMoles 2,3-DPG per gram of red blood c e l l s (RBC). The same i n c r e a s e i n P^Q at 2,3-DPG .con c e n t r a t i o n s above 8 uM/g RBC occurred only when the 2,3-DPG c o n c e n t r a t i o n was changed by about 1.5 uM/g RBC. I t was concluded that 2,3-DPG exerts a dual e f f e c t on the oxygen a f f i n i t y of human red blood c e l l s . The s p e c i f i c i n t e r a c t i o n of 2,3-DPG reached i t s maximal e f f e c t at 2,3-DPG c o n c e n t r a t i o n s of about 8 uM/g RBC. The second e f f e c t was an u n s p e c i f i e d one, due to i n t r a c e l l u l a r pH changes. T h i s i n d i r e c t e f f e c t , which i s common to a l l n o n - p e n e t r a t i n g anions, was s o l e l y r e s p o n s i b l e f o r the f u r t h e r decrease of the oxygen a f f i n i t y of blood at 2,3-DPG c o n c e n t r a t i o n s exceeding 8 uMoles per gram red blood c e l l s . The B i n d i n g of 2,3-diphosphoglycerate to Hemoglobin Mole f o r mole 2,3-diphosphoglycerate binds p r e f e r e n t -i a l l y with deoxyhemoglobin (deoxy HbA) i n normal a d u l t s , not with f u l l y oxygenated HbA. T h i s b i n d i n g i s assumed to occur i n the c e n t r a l c a v i t y of the two beta chains of the t e t r a m e r i c hemoglobin molecule, with the form a t i o n of s a l t b r i d g e s i n v o l v i n g the two N-terminal amino groups, h i s t i d i n e 143 and l y s i n e 82 (Perutz, 1970). I t has been suggested that the entrance to the c e n t r a l c a v i t y of the beta chains i s too s m a l l to admit the DPG molecule, but as a r e s u l t of the s i x angstrom i n c r e a s e i n the s i z e of the c a v i t y entrance which occurs upon deoxygenation, (Muirhead et a l . , 1967) the nine angstrom 2,3-DPG molecule could 16 FIGURE I T H E O X Y G E N D I S S O C I A T I O N C U R V E O F H U M A N B L O O D A N D S O M E V A R I A B L E S I N V O L V E D I N O X Y G E N S U P P L Y T O P E R I P H E R A L C A P I L L A R I E S 100 90 80 70 CM 60 O I -o 50 * 4 0 30 20 10 0 L i CJ «S t— m uj o l o 8 S 3 S I CE UJ X _J — U-UJ L J cr o O H o o ARTERIAL pH 7.44 — I V C a 0 2 - C v 0 2 > =0.041 (21%) 100 P 0 2 - T O R R T H E FIGURE IS BASED ON DATA OF N O R M A L , RESTING INDIVIDUALS. THE ABSCISSA IS IN UNITS OF OXYGEN TENSION ( T O R R ) W H E R E A S T H E ORDINATE IS EXPRESSED BOTH IN P E R C E N T 0 2 SATURATION AND 0 2 C O N -CENTRATION. T H E OXYGEN AFFINITY FOR BLOOD IS INDICATED BY l ° Q = 27 TORR. T H E OXYGEN CAPACITY IS 19.6 VOL % ( 0 . 1 9 6 ML. 0^/ ML B L O O D ) . THE ARTERIOVENOUS OXYGEN D I F F E R E N C E ( C a 0 2 - C v 0 2 ) IS 4 . 1 V O L % ( 0.041 ML 0 2 / M L B L O O D ) , EQUIVALENT TO 21 PERCENT EXTRACTION BY TISSUES. THE O X -YGEN TENSION IN MIXED VENOUS BLOOD . (Pv0 2 ) IS 39 TORR. T H E T H R E E RESERVE MECHANISMS SHOWN ACT TO INCREASE OXYGEN TRANSPORT I N D E -PENDENT OF BLOOD FLOW AND WITHOUT A DROP IN P 0 2 . ADAPTED FROM M E T C A L F E AND DHINDSA (1972).-17 e a s i l y be accomodated by deoxyhemoglobin (Brewer and Eaton,1971). T h i s mechanism would e x p l a i n the apparent p a r t i a l c o m p e t i t i o n between oxygen and 2,3-DPG. The i n c r e a s e i n P 5 Q , or the r i g h t s h i f t i n the oxygen d i s s o c i a t i o n curve, produced i n the presence of 2,3-DPG i n the red blood c e l l i s thought to be caused by an i n c r e a s e i n the r a t e of d i s s o c i a t i o n of Hb0 2 to Hb and 0 2; with the o v e r a l l process i n c l u d i n g a r e a c t i o n of 2,3-DPG with the hemoglobin molecule, an i n t e r a c t i o n between the b i n d i n g s i t e s of 2,3-DPG and those of oxygen, and f i n a l l y the subsequent net d i s s o c i a t i o n of oxygen from hemoglobin ( F o r s t e r , 1972), Bauer (1972) concluded that the i n f l u e n c e of 2,3-DPG (and hydrogen i o n s ) on the oxygen a f f i n i t y of hemoglobin was brought about by a change of both the asso c -i a t i o n and d i s s o c i a t i o n v e l o c i t y c o n s t a n t s of the hemoglobin-oxygen r e a c t i o n , with the former d e c r e a s i n g and the l a t t e r i n c r e a s i n g with i n c r e a s i n g c o n c e n t r a t i o n s of the s u b s t r a t e . The r e a c t i o n of oxygen with a s i n g l e hemoglobin molecule (composed of t e t r a m e r i c s u b u n i t s ) has been demonstrated to proceed as f o l l o w s by Perutz (1970); lc. k * lc« ( a i a 2 3 i e 2 ) X ( a i ° 2 0 i 2 3 i 3 2 ) X ( a i 0 2 a 2 o 2 3 i 3 2) X ( a i ° - a 2 0 2 3 i 0 2 3 2 ) !4 •*- ( a i 0 2 a 2 o 2 3 i 0 2 3 2 0 2 ) where k^, k 2, k^, k^ s i g n i f y the f i r s t , second, t h i r d and f o u r t h a s s o c i a t i o n v e l o c i t y constant r e s p e c t i v e l y and a i a 2 and 3i32 s i g n i f y the alpha and beta s u b u n i t s of hemoglobin. 18 Tyuma (1972) found that 2,3-DPG reduces and k 2 to about one-tenth and reduces k» much more, however k. was i n s e n -3 4 s i t i v e to the phosphate and a l s o to pH changes i n the range of 7.0 to 7.8, i r r e s p e c t i v e of the presence or absence of 2,3-DPG. Th i s l a c k of response of k^ to 2,3-DPG was c o n s i s t e n t with the r e s u l t s and ideas d e s c r i b e d In two recent papers (Gibson, 1970; Per u t z , 1970). In one, i t was shown that i n o r g a n i c phosphate i n c r e a s e s the r a t e of d i s s o c i a t i o n of the second, t h i r d and f o u r t h molecules l e a v i n g oxyhemoglobin A without a f f e c t i n g the r a t e of the f i r s t molecule (Gibson, 1970). In the ot h e r , Perutz (1970) proposed a model f o r the sequence of c o - o p e r a t i v e b i n d i n g of hemoglobin A i n which 2,3-DPG combined w i t h deoxy HbA i s e x p e l l e d a f t e r the second heme group has been oxygenated. S e v e r a l f a c t o r s , other than the i n t e r a c t i o n with oxygen a f f e c t the b i n d i n g of 2,3-DPG to hemoglobin. Blood pH, carbon d i o x i d e and hemoglobin c o n c e n t r a t i o n would appear to be those of g r e a t e s t p h y s i o l o g i c a l importance. The Blood pH E f f e c t . The d i f f e r e n c e i n b i n d i n g between Hb0 2 and Hb was seen to decrease from a va l u e of about 0.3 moles per mole Hb tetramer at pH 7 to l e s s than 0.1 moles/mole Hb t e t r a -mer at pH 7.7 (Garby and d e V e r d i e r , 1972). The pH dependence of the data was accounted f o r on the b a s i s of oxygen l i n k e d b i n d i n g of 2,3-DPG to the r e s i d u e s B-terminal v a l i n e , h i s t i d i n e 143 and l y s i n e 82. These r e s i d u e s were found to have pKa v a l u e s of 7.6, 6.8 and 10.5 r e s p e c t i v e l y ; a s i t u a t i o n which would make b i n d i n g at pH 8 very s m a l l . In a d d i t i o n , the 2,3-DPG molecule was found 19 t o have two n e g a t i v e l y c h a r g e d g r o u p s w i t h an a v e r a g e pKa of ab o u t 7.1 w h i c h would a c c o u n t f o r t h e v a n i s h i n g b i n d i n g o f 2,3-DPG t o h e m o g l o b i n t o w a r d s pH 6. The C a r b o n D i o x i d e E f f e c t . R e c e n t d a t a of Bauer (1970) and T o m i t a and R i g g s (1971) showed t h a t 2,3-DPG i n t e r f e r e s w i t h t h e c a r b a m i n o b i n d i n g to h e m o g l o b i n and t h a t C0^ i n h i b i t s t h e e f f e c t o f 2,3-DPG on ox y g e n a f f i n i t y . The d i f f e r e n c e i n b i n d i n g of 2,3-DPG between t h e two s t a t e s o f o x y g e n a t i o n o f h e m o g l o b i n d e c r e a s e s i n t h e p r e s e n c e o f C O 2 . A l l C 0 2 bound to h e m o g l o b i n i s i n t h e n e g a t i v e l y c h a r g e d c a r b a m a t e f o r m ( i e RNHCOO ) and, as p a r t of t h i s i s b e l i e v e d t o o c c u r a t t h e two N - t e r m i n a l g r o u p s of t h e 3 c h a i n s , i t would be e x p e c t e d t h a t c o m p e t i t i o n would e x i s t b etween t h e b i n d i n g o f C 0 2 and 2,3-DPG. R o s s i - B e r n a r d i e t a l . (1972) f o u n d t h a t a d d i t i o n o f one mole 2,3-DPG/ mole Hb l o w e r s t h e amount o f o x y g e n - l i n k e d c a r b a m a t e by a b o u t o n e - h a l f . When t h e p r o p o r t i o n o f 2,3-DPG was r e d u c e d t o 0.5 mole/mole Hb an i n t e r -m e d i a t e v a l u e was o b s e r v e d . F u r t h e r m o r e u n d e r a p p r o x i m a t e l y p h y s i o l o g i c a l c o n d i t i o n s , r e d u c e d Hb a p p e a r e d t o have a g r e a t e r a f f i n i t y f o r 2,3-DPG t h a n f o r C 0 2 . T h i s l i k e l y a c c o u n t s f o r t h e d e s c r e p a n c y o f t h e p r e v i o u s d a t a o f R o s s i - B e r n a r d i and R o u g h t o n (1967) who, when u s i n g d i a l y z e d Hb, showed t h a t H b C 0 2 i s r e s p o n -s i b l e f o r 25-27 p e r c e n t o f t h e p h y s i o l o g i c a l c a r b o n d i o x i d e t r a n s p o r t by t h e b l o o d . T h e i r r e c e n t e v i d e n c e ' i n d i c a t e s t h a t t h i s v a l u e was a p p r o x i m a t e l y 40 p e r c e n t t o o h i g h when whole b l o o d , c o n t a i n i n g 2,3-DPG, was u s e d ( R o s s i - B e r n a r d i , 1 9 7 2 ) . 20 The Hemoglobin C o n c e n t r a t i o n E f f e c t . With i n c r e a s i n g hemoglobin c o n c e n t r a t i o n there i s a decreased a f f i n i t y of 2,3-DPG f o r deoxygenated hemoglobin (Garby and d e V e r d i e r , 1970). Under c o n d i t i o n s p r e v a i l i n g i n the i n t a c t c e l l about 15 percent and 35 percent of the t o t a l 2,3-DPG was bound to f u l l y oxygenated and deoxygenated c e l l s r e s p e c t i v e l y . T h e r e f o r e with higher concen-t r a t i o n s of hemoglobin there i s a reduced amount of b i n d i n g per mole hemoglobin so that the o v e r a l l amount bound remains r e l a t i v e l y c o n s t a n t . The d i f f e r e n c e i n b i n d i n g between Hb02 and Hb was c a l c u l a t e d to be 0.2 to 0.3 moles per mole Hb tetramer under p h y s i o l o g i c a l c o n d i t i o n s , with a maximum v a l u e at very low Hb c o n c e n t r a t i o n s of 0.3 to 0.4 moles per mole Hb tetramer. I t would appear that at a higher mean c e l l hemoglobin c o n c e n t r a t i o n oxygen a f f i n i t y of hemoglobin i s reduced independent of 2,3-DPG. Numerous, s e t s of data have e s t a b l i s h e d that e i t h e r changes i n P^Q can occur i n the absence of a 2,3-DPG change or that changes i n 2,3-DPG are not n e c e s s a r i l y accompanied by a change i n P^Q* Because hemoglobin c o n c e n t r a t i o n has been shown to i n f l u e n c e the a f f i n i t y of hemoglobin f o r oxygen (Benesch et a l . 1969), Lenfant et a l . (1972) examined the p o s s i b i l i t y of t h i s f a c t o r being r e s p o n s i b l e f o r the many p a r a d o x i c a l f i n d i n g s . I t was found that MCHC d i d a f f e c t P 5 Q independent of 2,3-DPG, agr e e i n g with the data of S h a p p e l l et al.(1971) and Bellingham et a l . (1971) that the r e l a t i o n s h i p between P,.-. and hemoglobin c o n c e n t r a t i o n was l i n e a r . When Lenfant et al.(1972) c o r r e c t e d P^ 0 to a MCHC of 33 percent the l i n e a r r e l a t i o n s h i p between 2,3-DPG and P^n was i d e n t i c a l f o r a l l s u b j e c t s . Recent data of 21 F o r s t e r (1972) i n d i c a t e d that the r e l a t i o n s h i p between P Q and MCHC was i n f l u e n c e d by 2,3-DPG c o n c e n t r a t i o n s such that the gr e a t e r the 2,3-DPG c o n c e n t r a t i o n the g r e a t e r the c o e f f i c i e n t AP 5 Q/AHb. The P o s t u l a t e d C o n t r o l of the 2,3-DPG Response Mechanism. The l e v e l of 2,3-DPG i n the red blood c e l l i s r e g u l a t e d by b i o c h e m i c a l mechanisms i n h e r e n t w i t h i n the c e l l and p h y s i o l o g i c a l i n f l u e n c e s a r i s i n g from o u t s i d e the c e l l . While these two f a c t o r s are Inse p a r a b l e w i t h i n the i n t a c t c e l l they have been d i s c u s s e d s e p a r a t e l y to p r o v i d e a c l e a r e r u n d e r s t a n d i n g . The B i o c h e m i c a l C o n t r o l of 2,3-DPG C o n c e n t r a t i o n s . Brewer et a l . (1972) suggested an i n c r e a s e i n 2,3-DPG i n the e r y t h r o c y t e comes about e i t h e r by an i n c r e a s e i n glucose consumption and g l y c o l y t i c r a t e or by a s h i f t of g l y c o l y t i c flow through the Rapoport-Luebering DPG shunt at the expense of the ATP-generating phosphoglycerate k i n a s e r e a c t i o n ( F i g u r e 2 ) . Under p h y s i o l o g i c a l c o n d i t i o n s about 80 per cent of 1,3-diphospho-g l y c e r a t e (1,3-DPG) i s d i r e c t l y decomposed to 3-phosphoglycerate (3 PGA) i n the phosphoglycerate k i n a s e r e a c t i o n . Only 15 to 20 per cent of the t o t a l g l y c o l y t i c f l u x passes through the 2,3-DPG poo l ( G e r l a c h and Duhm, 1972). Rapoport et al.(1972) has observed that the maximal a c t i v i t i e s of both enzymes c o n t r o l l i n g c o n c e n t r a t i o n s on 2,3-DPG, DPG mutase and DPG phosphatase, permit r e a c t i o n r a t e s that are 1-5 percent of the g l y c o l y t i c r a t e . However, i n the i n t a c t c e l l these r a t e s reach v a l u e s up to twice 22 the r a t e of g l y c o l y s i s . T h e r e f o r e , a d d i t i o n a l f a c t o r s modifying the a c t i v i t y of DPG mutase have to be p o s t u l a t e d . Because 2,3-DPG ac t s as a product i n h i b i t o r of DPG mutase (Rose, 1970) i t was suggested that a r e l i e f of t h i s product i n h i b i t i o n due to i n c r e a s e d b i n d i n g of 2,3-DPG to deoxyhemoglobin might be the key f a c t o r i n the a c c e l e r a t i o n of 2,3-DPG s y n t h e s i s i n deoxygenated c e l l s (Benesch and Benesch, 1967; Eaton and Brewer, 1968). Such a mechanism has been c o n s i d e r e d to be very e f f e c t i v e under the c o n d i t i o n of hypoxia, which induces a d i m i n u t i o n of the c o n c e n t r a t i o n of f r e e 2,3-DPG due to i t s g r e a t e r b i n d i n g to deoxyhemoglobin (Oski et a l . , 1970). However, more recent s t u d i e s i n d i c a t e that b i n d i n g by hemoglobin i s not s u f f i c i e n t to e x p l a i n the high r a t e s of s y n t h e s i s . G e r l a c h and Duhm (1972) i n v e s t i g a t e d t h i s problem under s p e c i a l c o n d i t i o n s i n i n t a c t human red c e l l s . With no d i f f e r e n c e i n i n t r a c e l l u l a r pH between deoxy and oxyhemoglobin the r a t e s of s y n t h e s i s were almost i d e n t i c a l , I n d i c a t i n g that In i n t a c t red blood c e l l s product i n h i b i t i o n of DPG mutase i s not i n f l u e n c e d by c o n c e n t r a t i o n changes of f r e e 2,3-DPG r e s u l t i n g from the d i f f e r e n t b i n d i n g of 2,3-DPG to oxy and deoxyhemoglobin. The r a t e of 2,3-DPG s y n t h e s i s depends f i r s t of a l l on the c o n c e n t r a t i o n of 1,3-DPG. The l e v e l of t h i s compound i s c o n t r o l l e d by two f a c t o r s : (a) the glyceraldehyde-3-phosphate dehydrogenase r e a c t i o n , which depends on the c o n c e n t r a t i o n s of glyceraldehyde-3-phosphate (GA-3-P), NAD and orthophosphate ( P i ) ; (b) the c o n c e n t r a t i o n s of ADP, ATP and 3 PGA which, as r e a c t a n t s of the phosphoglycerate kinase r e a c t i o n , are i n e q u i l i b r i u m with 2 3 F I G U R E 2 T H E MAJOR PATHWAY OF CARBOHYDRATE METABOLISM IN MATURE MAMMALIAN RED BLOOD C E L L S . ENZYMES AFFECTING 2 , 3 D I P H O -SPHOGLYCERATE CONCENTRATIONS A R E P R E S E N T E D , ALONG WITH FACTORS KNOWN TO AFFECT THEIR ACTIVITY. 1 HEXOKINASJ" GLUCOSE- I - PHOSPHATE f NADP, GLUCOSE-I X I— < Q_ O X o r LU >-UJ LU Q CO UJ Ul X GLUCOSE - 6 - PHOSPHATE ADP NADPH NADP 6 PHOSPHOGLUCONATE NADPH PENTOSE PHOSHATES FRUCTOSE-6- PHOSPHATE ^ACTIVATED BY: V AMP, AOP, FOP AND INCREASE IN pH ATP IPHOSPHQFRUCTOKINASE I DIHYDROXYACETONE PHOSPHATE INHIBITED BY'. • ATP, CITRATE AND DECREASE IN pH - DIPHOSPHATE GLYCERALDEHYDE - 3 PHOSPHATE (GA- 3 - P) NAD • NADH-Pi GLYCERALOEHYOE - 3 PHOSPHATEDEHYDROGENASE ACTIVATED BY'. 1, 3 OPS , 3 PGA AND INCREASE IN pH \ '1,3 DIPHOSPHOGLYCERATE' ( 1,3 DPG ) ADP ATP DPG MUTASE INHIBITED BY: 2,3 DPS AND ORTHOPHOSPHATE 3 PHOSPHOGLYCERATE KINASE 2,3 DIPH0SPH0 GLYCERATE (2,3 DPG) 3 PHOSPHOGLYCERATE^ (3 PGA ) PHOSPHOGLYCERATE MUTASE i 7 F 3 DIPHOSPHOGLYCERATE PHOSPHATASE 2 PHOSPHOGLYCERATE J INHIBITED BY'. 3 PGA ACTIVATED BY. DECREASE IN pH, ORTHOPHOSPHA TE PHOSPHOENOLPYRUVATE ADP ATP PYRUVATE KINASE THE RAPOPORT-LEUBERING SHUNT PYRUVATE NADH-NAD-^- -A LACTATE DEHYDROGENASE LACTATE 24 1,3-DPG (Gerlach and Duhm, 1972). On the b a s i s of these c o n s i d e r a t i o n s , an i n c r e a s e of the 1,3-DPG c o n c e n t r a t i o n and co n s e c u t i v e e l e v a t i o n of the 2,3-DPG l e v e l s were obtained when GA-3-P, i n o r g a n i c phosphate and NAD were made a v a i l a b l e i n l a r g e amounts (Deuticke et a l . , 1971; Deuticke and Duhm, 1972). Furthermore, a smal l r e d u c t i o n of ADP was found to induce an i n c r e a s e of the 1,3-DPG c o n c e n t r a t i o n f o l l o w e d by a g r e a t e r f o r m a t i o n of 2,3-DPG (Duhm et a l . , 1968) Rapoport et a l . (1972) suggested that a high c o n c e n t r a -t i o n of i n o r g a n i c phosphate a c t s p r i m a r i l y to r e l e a s e the i n h i b i t i o n of ph o s p h o f r u c t o k i n a s e , which i s the major c o n t r o l p o i n t of g l y c o l y s i s of most mammalian t i s s u e . F i n a l l y , Brewer and Eaton (1971) hyp o t h e s i z e d a system i n which 2,3-DPG i n h i b i t s the a c t i o n of hexokinase, the enzyme which i n i t i a t e s g l y c o l y s i s and which may be important as a r a t e l i m i t i n g enzyme i n red blood c e l l g l y c o l y s i s . The P h y s i o l o g i c a l C o n t r o l of 2,3-DPG C o n c e n t r a t i o n . I t appears that numerous p h y s i o l o g i c a l and p a t h a l o g i c a l c o n d i t i o n s evoke an i n c r e a s e i n 2,3-DPG, which i n tu r n mediates a displacement to the r i g h t of the oxygen d i s s o c i a t i o n curve. These c o n d i t i o n s are, i n n e a r l y a l l i n s t a n c e s , c h a r a c t e r i z e d by hypoxia and/or acid-base d i s t u r b a n c e s . Among the numerous f a c t o r s which can i n f l u e n c e 2,3-DPG metabolism a l t e r a t i o n s i n blood pH and changes i n the oxygenation s t a t e of hemoglobin are thought to be mainly r e s p o n s i b l e f o r the ad a p t i v e c o n c e n t r a t i o n 25 r changes (Gerlach and Duhm, 1972). 2,3-DPG l e v e l s are known to i n c r e a s e a f t e r an e l e v a t i o n of blood pH (Asakura et a l . , 1966), whereas a decrease i n pH causes a decrease i n 2,3-DPG co n c e n t r a -t i o n (Astrup, 1970; Guest and Rapoport, 1941; Rorth, 1970). The b i o c h e m i c a l mechanism whereby pH a f f e c t s 2,3-DPG l e v e l s i n v i v o has not been e l u c i d a t e d so f a r . The a f f e c t appears to be due to an i n c r e a s e d glucose consumption mediated through a pH a c t i v a t i o n of phosphoglycerate kinase (PFK) and DPG mutase (Rapoport and Leubering, 1952; Brewer, 1972b), The i n t r a c e l l u l a r mechanism of change responds to i n f l u e n c e s a r i s i n g o u t s i d e the c e l l . For example, the i n t r a -c e l l u l a r pH i s u l t i m a t e l y determined by the e x t r a c e l l u l a r pH. The r e f o r e a l l the e x t r a c e l l u l a r f a c t o r s which i n f l u e n c e pH w i l l have some e f f e c t on 2,3-DPG l e v e l s . For example, h y p e r v e n t i l a -t i o n w i l l lower CO^ l e v e l s , i n c r e a s e pH and i n c r e a s e 2,3-DPG l e v e l s . M e t a b o l i c a c i d o s i s and a l k a l o s i s w i l l l i k e w i s e have t h e i r r e s p e c t i v e e f f e c t s (Brewer et a l . , 1972). In order to demonstrate that the hypoxia-induced r i s e of the blood pH i s e s s e n t i a l f o r the e l e v a t i o n of 2,3-DPG, Ge r l a c h and Duhm (1972) exposed r a t s to gas mixtures of low oxygen content c o n t a i n i n g f i v e per cent C02« I t was found that the hypoxia-induced r i s e of 2,3-DPG does not occur i n the presence of f i v e per cent CO2, which prevented the occurrence of r e s p i r a t o r y a l k a l o s i s . On the other hand, exposure of the animals to a gas mixture of normal oxygen content with f i v e per cent CO- r e s u l t e d i n a moderate decrease of 2,3-DPG l e v e l s due 26 to the induced r e s p i r a t o r y a c i d o s i s . The importance of r e s p i r a t o r y a l k a l o s i s was a l s o shown by Lenfant et al.(1970) who observed that i n men the documented r i s e of 2,3-DPG at high a l t i t u d e (Lenfant et a l . , 1968; Rorth, 1972; Torrence et a l . , 1970) does not occur a f t e r the a p p l i c a t i o n of diamox which prevents the hypoxia-induced a l k a l o s i s . At high a l t i t u d e the i n c r e a s e of 2,3-DPG was found to be 4.7 uMoles/gram Hb f o r an i n c r e a s e of 0.1 plasma pH u n i t s . S i m i l a r l y , at sea l e v e l , normal s u b j e c t s made a c i d i c showed a decrease i n 2,3-DPG of 2.4 uMdles/ gram Hb f o r a 0.1 decrease i n plasma pH. The d i f f e r e n c e i n the r a t e between the two c o n d i t i o n s was accounted f o r by the i n c r e a s e i n deoxyhemoglobin present at a l t i t u d e (Lenfant et a l . , 1972). Although a pH change occurs almost i n s t a n t a n e o u s l y , the e f f e c t of pH on 2,3-DPG s y n t h e s i s i s much slower. Bellingham et al.(1971) showed that when pH was i n c r e a s e d from 7.32 to 7.40 w i t h i n one-h a l f hour, the 2,3-DPG c o n c e n t r a t i o n d i d not i n c r e a s e u n t i l two to three hours a f t e r the onset of the pH change. From these r e s u l t s i t i s apparent that blood pH p l a y s an important r o l e i n i n d u c i n g c o n c e n t r a t i o n changes of 2,3-DPG i n hypoxic hypoxemia. However 2,3-DPG c o n c e n t r a t i o n a l s o changes i n those kinds of hypoxemia i n which blood pH remains at normal val u e s or i n c r e a s e s o n l y s l i g h t l y . It has been shown that the r a t e of 2,3-DPG s y n t h e s i s i s higher i n deoxygenated than i n oxygenated c e l l s (Duhm and G e r l a c h , 1971); and i n anemia an i n c r e a s e d a r t e r i a l d e s a t u r a t i o n and presumably an i n c r e a s e d venous d e s a t u r a t i o n e x i s t s (Brewer et a l . , 1972). Since deoxy-hemoglobin i s more a l k a l i n e than oxyhemoglobin (the Haldane 27 e f f e c t ) , c h r o n i c d e s a t u r a t i o n w i l l t h e r e f o r e mean a r e l a t i v e i n t r a c e l l u l a r a l k a l o s i s . In a d d i t i o n , i t has been found that the d i f f e r e n c e between i n t r a c e l l u l a r and e x t r a c e l l u l a r pH (ApH) i s s m a l l e r i n deoxyenated blood (Duhm and G e r l a c h , 1971). For example, at an e x t r a c e l l u l a r pH of 7.40, deoxygenation caused the ApH to decrease from -0.2 to -0.13. T h i s means that the i n t r a c e l l u l a r pH r i s e s from 7.20 to 7.27 upon deoxygenation. Although the s h i f t of the i n t r a c e l l u l a r pH of 0.07 u n i t s to the a l k a l i n e s i d e seems to be r a t h e r s m a l l , i t n e v e r t h e l e s s e x e r t s a remarkable i n f l u e n c e on 2,3-DPG metabolism. I t has been estimated that an e l e v a t i o n of the i n t r a c e l l u l a r pH of t h i s magnitude causes the 2,3-DPG c o n c e n t r a t i o n to i n c r e a s e by about 25 per cent ( i e 1 uMole 2,3-DPG/ml red blood c e l l s ) (Astrup, 1970). T h i s agrees with Rorth and Brahe (1972) who found an i n c r e a s e i n pH of 0.04 u n i t s w i l l i n c r e a s e the 2,3-DPG l e v e l by 15-20 per cent. Furthermore, as the 2,3-DPG i n c r e a s e s , the r e s u l t i n g lowered oxygen a f f i n i t y w i l l cause the r e l a t i v e d e s a t u r a t i o n at a given P 0 2 to be g r e a t e r , causing a f u r t h e r r e l a t i v e i n t r a c e l l u l a r a l k a l o s i s (Gerlach and Duhm, 19720. In summary, red c e l l pH i s the e s s e n t i a l mechanism which has been demonstrated to c o n t r i b u t e to the e l e v a t i o n of red c e l l 2,3-DPG i n v a r i o u s kinds of hypoxia. In acute hypoxic hypoxia, the i n t r a e r y t h r o c y t i c pH r i s e s due to an i n c r e a s e i n blood pH caused by h y p e r v e n t i l a t i o n . In c h r o n i c hypoxic hypoxia or anemia hypoxia, e l e v a t i o n of i n t r a c e l l u l a r pH i s due to an i n c r e a s e i n deoxyhemoglobin. L i m i t a t i o n of 2,3-DPG co n c e n t r a -t i o n i s mainly brought about by a feedback mechanism, which again 28 depends on changes of red blood c e l l pH. With rising concentrations of 2,3-DPG, which has acidic properties, the intracellular pH decreases (Gerlach and Duhm, 1972). Since the reduction of the intracellular pH operates against the hypoxia-induced rise in red blood c e l l pH, the increase of 2,3-DPG becomes limited. An additional mechanism may also operate to produce an increase in intracellular pH under hypoxic conditions. Gerlach and Duhm (1972) demon-strated an increase in 2,3-DPG levels in rats made anemic by repeated bleeding and a decrease in 2,3-DPG levels i n rats made polycythemic by intraperitoneal injection of erythrocytes. From these and other experiments i t was conluded that a negative correlation exists between 2,3-DPG and whole blood hemoglobin, which was in agreement with observations made on humans (Eaton and Brewer, 1968; Edwards, 1972; Valeri and Fortier, 1969). Brewer et a l . (1972) hypothesized that the level of 2,3-DPG may have a deterministic effect on the mean corpuscular hemoglobin concentration (MCHC). The red blood c e l l membrane i s not permeable to 2,3-DPG, which comprises a large component of the total non-diffusable molecules within the c e l l . Also, as the content of 2,3-DPG increases, there may be a concomitant increase in cation content in order that charge neutrality may be maintained. This in turn results i n an increase in c e l l volume because of an increase in int r a -cellular water required to maintain iso-osmolarity; thereby an overall drop in MCHC would be produced. Another way to explain the negative correlation between 2,3-DPG and MCHC i s to postulate a causal effect of MCHC on 2,3-DPG. The oxygen a f f i n i t y of hemoglobin i s concentration dependent exclusive of 2,3-DPG effects 29 (Benesch et a l . , 1 9 6 9 ) , even in the physiological range of hemoglobin concen-tration (Bellingham et a l . , 1 9 7 1 ) . Numerous experiments have shown that changes in P^Q can occur in the absence of 2,3-DPG change or that changes in 2,3-DPG are not necessarily accompanied by a change in P^Q. It was postulated that concentration changes of intra-erythocytic hemoglobin might be responsible for these observations (Lenfant et a l . , 1 9 7 2 ) . In examining the data i t was found that MCHC did vary and did affect P 5 Q independent of 2,3-DPG. A linear relationship was found to exist between P^Q and erythrocyte hemoglobin concen-tration, agreeing with results of Bellingham et a l . ( 1 9 7 1 ) and Shappell et a l . ( 1 9 7 1 ) . When P^Q was corrected to a standard MCHC value ( 3 3 per cent), the relationship between P^Q and 2,3-DPG was found to be identical for a l l subjects (Lenfant et a l . , 1 9 7 2 ) . It i s believed that changes in hemoglobin concentration induce changes in the conformation of the individual hemoglobin molecules, thereby affecting the a f f i n i t y each molecule has for oxygen. Brewer et a l . ( 1 9 7 2 ) suggested that a change i n conformation manifested by a lower MCHC i s one which increases the pK of the hemoglobin molecules leading to a relative intracellular alkalosis and an increased 2,3-DPG production. 2,3-Diphosphoglycerate Changes i n Long Standing Hypoxias Elevations i n 2,3-DPG, with a corresponding right shift i n the oxygen dissociation curve of hemoglobin to improve oxygen delivery, have been observed in numerous hypoxic diseases. These include severe pulmonary disease (Eaton et a l . , 1 9 7 0 ) , hemolytic and non-hemolytic anemia (Hjelm, 1 9 7 0 ) , iron deficiency, leukemia, uremia and emphysema (Eaton et a l . , 1 9 7 0 ) . The increase observed in these various pathalogical conditions has been found to be as high as 150 per cent, causing as much as a two-fold increase i n the rate of oxygen delivered (Brewer and Eaton, 1 9 7 1 ) . 30 FIGURE 3 F A C T O R S C O N T R I B U T I N G T O T H E C O N T R O L O F R E D C E L L 2 , 3 - D P G F R O M B U N N A N D J A N D L , 1 9 7 0 31 2,3-diphosphoglycerate Changes in Non-pathalogical Conditions Altitude Exposure. At high altitude the a f f i n i t y of hemoglobin for oxygen i s decreased (ie an increase in i s observed), making hemoglobin-bound oxygen more available to body tissues (Aste-Salazar and Hurtado, 1 9 4 4 ) . Lenfant et a l . (1968) exposed sea level residents to altitudes of 1 5 , 0 0 0 feet and found an increase in 2 , 3 - D P G which occurred concomitant to an increase i n PJ-Q. Both occurred within 24 hours of exposure. Upon returning to sea level, parallel decreases in both 2 , 3 - D P G and P^Q were observed. It was suggested that the increase in 2 , 3 - D P G represented an important rapid adaptive mechanism to anoxia. Torrance et al. ( 1 9 7 0 / 7 1 ) compared sojourners at altitude with residents at altitude, finding a marked hyperventilation and increased levels of hemo-globin and 2 , 3 - D P G in the latter group. The a f f i n i t y of hemoglobin for oxygen was decreased In both groups, with the magnitude of the decrease being greater in the sojourners than in the natives at lower altitudes, but i s was the same in the two groups at higher altitude. The increases i n 2 , 3 - D P G paralleled the decrease in the oxygen a f f i n i t y of hemoglobin. pH and bicarbonate changes at altitude reflected an increasing respiratory alkalosis which l i k e l y triggered the 2 , 3 - D P G response. The sojourners, who had the lowest hemoglobin concen-tration, had the greatest hyperventilation and the largest increase in 2 , 3 - D P G i when moving from sea level to altitude. j The evidence that plasma pH changes are responsible for the rise in 2 , 3 - D P G upon exposure to altitude i s supported by the study of Lenfant et a l . (1971) who found that administration of acetazolamide prior to ascent, to prevent plasma pH from rising above sea level value, prevented both the rise 32 t in P 5 Q and 2,3-DPG. Rorth (1970) and Rorth and Brahe (1972) have provided support to the pH hypothesis by finding that a pH induced stimulation of glycolysis i s necessary in the altitude response of 2,3-DPG. However, i t was also suggested that various hormonal agents might also mediate 2,3-DPG alterations. For example, prostaglandins were found to increase 2,3-DPG levels by 20 per cent within 30 minutes of administering a dosage of 10 ^ moles per l i t e r of blood (Rorth and Brahe, 1972). In addition "stress" hormones such as epinephrine (Brewer, 1972a) and thyroid hormones (Miller et a l . , 1970; Snyder and Reddy, 1970) have also been related to increases in 2,3-DPG. The importance of the hemoglobin increase and 2,3-DPG increase upon i exposure to high altitude has been demonstrated by Lenfant et a l . (1970a). They calculated that i f one goes to high altitude and does not increase his hemoglobin, nor shifts his dissociation curve, his working a b i l i t y w i l l be decreased by about 50 per cent. If, however, there i s a shift of the hemo-globin dissociation curve, his working a b i l i t y would be about 75 per cent of what he can do at sea level. The last 25 per cent would be gained i f he also has a hemoglobin increase. Exercise Exposure. The literature contains four reports on the response of 2,3-DPG to exercise (Eaton et al.,1969; Faulkner et al.,1970; Shappell et a l . , 1971; Dempsey et al.,1971), which w i l l be discussed in the chronological order cited above. i | Eaton et a l . (1969) observed an 18 per cent increase in 2,3-DPG levels in ten adult males ages 21-45 after 60 minutes of moderate exercise (basketball). To obtain a more controlled physiological stress they then had the ten men pedal, at 50 RPM, for 50 minutes on a bicycle ergometer; the load 33 was adjusted to provide 1200 kg/minute of work for five trained subjects and 600 kg/minute work for five untrained subjects. The work loads ranged from 55 per cent to 90 per cent of the subjects' maximal oxygen uptake, with a mean of 70 per cent. 2,3-DPG concentrations after 30 minutes of exercise did not vary from resting values. However, after 50 minutes of exercise increases in 2,3-DPG had occurred in seven of ten subjects. A strong positive correla-tion existed between the change in blood lactate and the change in 2,3-DPG after 50 minutes of exercise, indicating to the authors that the increase i n 2,3-DPG was related to the severity of the exercise. Faulkner et a l . (1970) reported that the 2,3-DPG response did not appear to be stimulated or sustained by low intensity work of long duration. The red blood c e l l 2,3-DPG concentration of three subjects who traversed a 14 mile distance at their fastest possible speed (1.75, 2 and 4 hours respect-ively) were the same as resting values. To determine the effect of short exhaustive work on the 2,3-DPG concentration in red blood c e l l s , Faulkner et al.(1970) ran three trained and three untrained subjects to voluntary fatigue, during a time course of 8-12 minutes, on a motor driven treadmill. The mean increase in 2,3-DPG after the exhaustive run was 10 per cent, with an increase of from 0.5 uMoles to 3.8 uMoles per gram hemoglobin occurring in five of six subjects. The rapid increase in 2,3-DPG in this study indicated that the response time during l adaptation to heavy exercise i s much more rapid than the six hour half response time observed during adaptation to high altitude (Lenfant et a l . , 1968). 34 Shappell et a l . (1971) examined the 2,3-DPG response to exercise in three healthy, untrained, nonsmoking males before and after an eight week course of physical training, during which time there was a 10 per cent increase in maximum oxygen uptake. No significant change was observed in the a r t e r i a l and venous P,.Q either with acute exercise (successive ten minute runs at 50, 50 and 75 per cent of VO2 max.) or as a result of training. Similarly, there was no significant change in 2,3-DPG during the acute experiment. However, after training, a l l values of 2,3-DPG, whether at rest or during exercise, were significantly higher in both a r t e r i a l and venous blood. The fact that the increase in 2,3-DPG as a result of training was not accompanied by an increase i n P^g was explained on the basis of a signif-icant decrease in MCHC after training. A significant linear regression between the f a l l i n MCHC and the increase i n 2,3-DPG suggested that one effect counterbalanced the other. Shappell's data indicated that an increase i n whole blood hemoglobin concentration occurred after training (before and after means were 14.1 and 14.4 grams Hb/100 mis whole blood respectively). In addition, the hemoconcen-tration that occurred as a result of exercise was larger after training, increasing from AO.8 to A1.2 grams Hb/100 mis whole blood. Considering this r i s e i n hemoglobin concentration in light of the previously mentioned f a l l i n MCHC, one would conclude that hematocrit rose as a result of training ( how-ever no data was provided). The effects on training on hemoglobin concentra-tion and hematocrit appear somewhat ambiguous. Previous investigations have reported no change (Kjellberg et a l . , 1949), an increase (Knehr et a l . , 1942) and a decrease (Oscai et a l . , 1968). However, a l l studies report an increase in total blood volume as a result of training. 35 Dempsey et a l . (1971) had four highly trained subjects perform treadmill exercises of 65-70 per cent of their maximal aerobic power u n t i l exhaustion. A decline of 2,3-DPG, ranging from 2-17 per cent was observed in a l l four subjects at the midpoint of exercise. At exhaustion, the 2,3-DPG levels had returned to near pre-exercise levels. Only one subject showed an increase (of nine per cent) above his resting level. Neither the direction nor magnitude of the 2,3-DPG change was related to the physiologic response to work. The literature on the effect of exercise upon the 2,3-DPG response may be summarized as follows: two studies report an increase i n 2,3-DPG with exercise, one study reports no change with exercise but an increase with training and one study reports a decrease i n 2,3-DPG with exercise which rises back to resting levels i f the exercise i s of sufficient duration. Cigarette Smoking and the 2,3-diphospoglycerate Response. The relationship between smoking and 2,3-DPG has received very l i t t l e attention. Smoking i s unique i n that i t induces a hypoxia which i s not associated with an increased desaturation of hemoglobin (Brewer at a l . , 1970). Cigarette smoke contains significant amounts of carbon monoxide (CO). A study has shown that subjects who smoked 10 to 12 cigarettes a day had 4.9 per cent carboxyhemoglobin, those who smoked 15 to 25 cigarettes a day had 6.3 per cent and those who smoked 30 to 40 cigarettes per day had 9.3 per cent (Astrand and Rodahl, 1970). Carbon monoxide combines with hemoglobin at the same site as does oxygen (both ligands attaching to the iron atom of the porphyrin group of the Hb molecule), however the a f f i n i t y of hemoglobin for carbon monoxide is 200 to 300 times greater than to oxygen. A substantial amount of CO loading as a result of cigarette smoking produces hypoxia in two ways: by 36 i removal of a portion of the hemoglobin from oxygen transport and by causing a shift in the oxygen dissociation curve of hemoglobin to the l e f t , inter-fering with the unloading of oxygen to the tissues (Harris and Kellermeyer, 1970). Brewer et al- (1970) reported no significant difference in the 2,3-DPG levels of smokers as compared to non-smokers when tested at sea level. How-ever, at high altitude (Leadville, Colorado- elevation 10,200 feet), i t was observed that residents suffering from chronic mountain sickness, typified by polycythemia, had 2,3-DPG levels significantly elevated over other acclimatized individuals (Eaton et a l . , 1970). Two important facts emerged from a study of these individuals. F i r s t , there was no right shift of the oxygen dissociation curve, in spite of the marked elevation i n 2,3-DPG. Second, a l l but four of the 24 individuals studied were habitual smokers. (The exceptions were found to have lung disease - s i l i c o s i s , emphysema or both.) It was concluded that the failure of the oxygen dissociation curve of hemoglobin to shift to the right was the result of carboxyhemoglobin and that this was the factor which produced the polycythemic syndrome. This hypothesis was supported by Astrup (1972) who found that exposure of animals to carbon monoxide or hypoxia increases the activity of the erythropoietin.system, leading to a higher hemoglobin concentration. In the same discussion, Astrup (1972) reported that human patients with l e f t sided curve displacements also have polycythemia and that a l l patients with right sided displacements had low hemoglobin concentra-I tions. It was suggested that the erythropoietin system i s sensitive to altered tissue oxygen tensions which are due to oxygen dissociation curve displacement. 37 When Brewer et a l . (1970) compared the polycythemic smokers with normal smokers residing at high altitude, no significant difference was ob-served in the position of their oxygen dissociation curves (ie both had curves that were l e f t shifted compared to non-smokers residing at altitude). Both groups of smokers were also found to have a significantly lower a r t e r i a l oxygen tension at high altitude. It was concluded that smokers subjected to altitude hypoxia have three effects operating against adequate oxygen delivery to their tissues; the binding of a proportion of Hb with carbon monoxide, a shift to the l e f t of the oxygen dissociation curve and a reduction i n a r t e r i a l oxygen tension. In order to determine whether or not 2,3-DPG levels were regulated primarily through hemoglobin desaturation, Eaton et a l . (1970) induced hypoxia in 10 human subjects through carbon monoxide loading. Carboxyhemoglobin levels ranged from 10-25 per cent and were sustained for six hours. An increase i n 2,3-DPG averaging 12 per cent was observed after three hours, with no significant change from hour three to hour six. Thus i t appeared that in hypoxia a mechanism exists beyond simple desaturation of hemoglobin which i s capable of stimulating an increase i n the levels of red blood c e l l 2,3-DPG. However, these results are at variance with the findings of Astrup (1970b) who found a 5-10 per cent reduction in 2,3-DPG after exposure of humans to carbon monoxide for 24 hours (producing 15-20 per cent carboxy-hemoglobin). He f e l t this observed decrease in 2,3-DPG explained the increased ! a f f i n i t y of hemoglobin for oxygen in the presence of carbon monoxide. An increase that could not be explained wholly by the Haldane effect of CO on hemoglobin. 38 Although the effects of cigarette smoking (carbon monoxide) on the 2,3-DPG response mechanism appear unresolved, the effect of smoking on increas-ing whole blood hemoglobin and hematocrit appears to be accepted. Isager and Hagerup (1971) and Burch and DePasquale (1962) found significantly higher hematocrits in cigarette smokers,which they hypothesized was due to elevated levels of carbon monoxide. In addition, a relationship between cigarette smoking and a decrease in MCHC was observed, suggesting a higher incidence of iron-deficient erythropoiesis among cigarette smokers. No studies appear i n the literature which consider the combined effects of smoking and physical a c t i v i t y on levels of 2,3-DPG and/or hemoglobin. Summary Oxygen delivery to tissues can be fa c i l i t a t e d by several mechanisms, one of which occurs through a decrease of the a f f i n i t y of hemoglobin for oxygen. This response i s produced through changes in body temperature, carbon dioxide levels in the blood, blood pH, and 2,3-DPG. The effect of 2,3-DPG is produced through binding to specific sites on the hemoglobin molecule. The binding a f f i n i t y i s altered by such factors as blood pH, carbon dioxide and hemoglobin concentrations. Biochemical control of 2,3-DPG levels i s exerted primarily through regulation of glycolytic flux, with less important control provided by the enzyme 2,3-DPG mutase. Physiological control of 2,3-DPG levels i s exerted primarily through changes in pH, either plasma pH changes or intracellular pH changes as affected by the levels of deoxyhemoglobin. Various long standing pathalogical conditions producing hypoxia have been shown to produce an increase in 2,3-DPG, as has exposure to altitude. Studies on the hypoxia produced through either exercise or cigarette smoking producing alterations in 2,3-DPG have been conflicting and at present appear to be unresolved. CHAPTER III MATERIALS AND METHODS Subjects The sample population was comprised of 40 male volunteers, age ranging from 21 to 36 years (X = 25), height ranging from 167.6 to 193.2 centimeters (X = 181.0), and weight ranging from 60.6 to 112.9 kilograms (X = 78.9). Subjects were assigned to one of five groups (eight subjects per group) based on their status as defined by the following c r i t e r i a : Group I - Sedentary Nonsmokers. Comprised of subjects who had not indulged i n a regular program of physical activity during the six months prior to testing and who had refrained from smoking tobacco during the same period. Group II - Moderately F i t Nonsmokers. Comprised of subjects who had indulged i n a regular program of physical activity, primarily of an non-endurance nature, during the six months prior to testing and who had refrained from smoking tobacco during the same period. Group III - Highly F i t Nonsmokers. Comprised of members of the University of B.C. cross country team (several who have competed internationally) who had indulged in a regular program of intensive endurance training during the six months prior to testing and who had refrained from smoking tobacco during j the same period. Group IV - Sedentary Smokers. Comprised of subjects who had not indulged in a regular program of physical activity during the six months prior to testing and who had smoked approximately 20 cigarettes per day during the same time period. 39 AO Group V - Moderately F i t Smokers. Comprised of subjects who had indulged in a regular program of physical activity, primarily of a non-endurance nature, during the six months prior to testing and who had smoked approximately 20 cigarettes per day during the same time period. The three "fitness" groups were chosen on the basis of the effects of endurance training on cardiovascular changes. A general principle of training i s that adaptation takes place at a given stress; in order to achieve further improvement the training intensity has to be increased. Therefore groups were chosen to represent the mid-point and either end of the endurance training "continuum". An additional advantage of chosing competitive cross-country runners to represent the "highly f i t " group, other than their intensive training program, i s that a large genetic component may contribute to their great maximal aerobic power (Astrand, 1970). Therefore any genetic effects enhancing 2,3-DPG concentration, as suggested by Brewer et a l . , (1972), might be observed i n these subjects. It was hypothesized that the capacity to perform endurance work would vary greatly between the three "fitness" groups. Since oxygen transport i s an essential component of endurance capacity, any beneficial effects of 2,3-DPG on improving oxygen delivery as a result of training should be observed in comparison of these three groups. The advantage of using groups of widely differing fitness levels i s pointed out by Ekblom et a l . , (1968) who found that i differences between the trained and untrained state are much more significant when trained endurance athletes are compared with subjects performing normal physical activity than with changes before and after training are compared in the same individual. 41 The c r i t e r i a of 20 cigarettes per day as a minimum consumption level for the smokers was based on the common occurrence among smokers to limit their cigarette consumption to one package, or less,per day. Choosing smokers who smoked a minimum of one package (20 cigarettes) per day would ensure production of a sufficient amount of carboxyhemoglobin to reduce the oxygen transporting capacity of the blood (Astrand, 1970). Experimental Procedures A l l subjects were tested once, in a random order, during the interval 30 June 1972 - 1 April 1973, i n the Human Performance Laboratory, University of B.C. Physical Education Building. Ambient temperature i n the laboratory during the testing periods ranged from 19.5 to 23.5°C and barometric pressure ranged from 757.8 to 764.8 Torr. Extraneous individuals were, as far as possible, kept from the laboratory during a testing session. Each subject was instructed to fast (refrain from consuming calorie-containing foodstuffs) for the 12 hour period immediately preceeding the test phase. This was done to prevent wide differences in blood glucose levels among the subjects, which could possibly contribute to differences in red blood c e l l metabolic activity. After an overnight fast,blood glucose levels usually range from 80 to 100 mg glucose/100 ml of blood (Davidson and Passmore, 1969). Prior to testing, each subject, wearing gym shorts and training shoes only, was measured for height, weight and resting heartrate. Next, a qualified nurse, who was present in a l l testing sessions, removed three m i l l i l i t e r s of blood from the antecubital vein of the subject's arm. This was accomplished through the use of heparinized vacutainers (Becton, Dickinson Cat.No. 4862) and 42 s t e r i l e , disposable needles ( size 20 x lh). The subject was positioned comfortably on a pre-calibrated Monark bicycle ergometer where Beckman Biopotential skin electrodes from a Sanborn 500 (Model 1500A) electrocardiogram were attached in the prescribed manner (Spinco Division Technical Publications, 1965) to monitor heart rate. Accuracy of the electrocardiogram readings were checked by making periodic comparisons with simultaneously obtained heart rates using a stethoscope. The experimental phase consisted of each subject pedalling the bicycle ergometer for one hour, at 50 RPM (to the sound of a calibrated metronome) and at a resistance which e l i c i t e d a heart rate of approximately 150 beats per minute. A l l subjects commenced work at a resistance of two kiloponds (2 KP).At the end of the third minute of exercise, and each minute thereafter, the resistance was increased u n t i l a heart rate of 150 beats/min. was obtained. Resistance and heart rate were recorded for each minute of exercise, with the load adjusted as required to maintain the heart rate at 150 beats/min. Average heart rates during the experiment ranged from 141 to 152 beats/min, with the only exceptions being two of the highly f i t , nonsmoking subjects who had insufficient strength to pedal at a resistance producing an average heart rate beyond that which they obtained (130 and 138 beats/min respectively). During prolonged heavy physical work, an individual's performance capacity depends largely on his a b i l i t y to take up, transport, and deliver oxygen to the working muscle. Consequently, the maximal oxygen uptake is probably the best laboratory measure of a person's physical fitness, as defined by the capacity of the individual for prolonged heavy work (Astrand, 1970). 43 Because determination of maximum oxygen uptake necessitates repeated testing, elaborate equipment and several experimental personnel i t was decided to make use of the fact that a linear relationship exists between per cent of maximal oxygen uptake and heart rate, up to about 70 per cent of maximal Vo 2 (Astrand et a l . , 1964; Rowell et a l . , 1964). Using 86 subjects, Astrand (1952) found that a pulse rate of 154 represented 70 per cent of the maximal oxygen uptake. Therefore, a heart rate of 150 beats per minute, equivalent to 65-70 per cent of the subjects' maximal oxygen uptake, was employed i n this experiment. Even though habitual endurance training enables a person to achieve a certain cardiac output during rest, as well as at work, with a slower heart rate and a larger stroke volume (Astrand, 1970), the increased maximal oxygen uptake observed i n these subjects i s such that the relation between heart rate and oxygen uptake remains v i r t u a l l y unchanged. (Top athletes in endurance events often have a maximal oxygen uptake that i s about twice as high as that of an average man). During exercise in the si t t i n g position an "optimal" stroke volume is reached when oxygen uptake exceeds 40 per cent of maximal aerobic power, with a variation of only ± four percent occurring as oxygen uptake increases further (Astrand et a l . , 1964). Therefore, with heart rate also held constant during the one hour test, cardiac output remains approximately constant, (Cardiac Output (Q) = Stroke Volume (SV) x Heart Rate (HR)). Thus any changes that occur in 2,3-DPG concentrations could be considered independent of changes in cardiac output. The relationship between arteriovenous (a-v) oxygen difference and oxygen uptake in normal men reveals that oxygen extraction i s increased up to or close to the same maximal value, for. a l l subjects, at maximal Vo 2 (Astrand et a l . , 1964; Rowell et a l . , 1966; Wang et a l . , 1961). When a-v oxygen 44 difference was related to the relative oxygen uptake (per cent of maximal Vo^) the relationship was found to be linear (Rowell, 1969). Therefore, at 70 per cent of vc^, a l l subjects in this study would have approximately the same a-v oxygen difference, or the same amount of hemoglobin desaturation which i s one of the hypothesized regulatory mechanisms controlling 2,3-DPG concentrations. The bicycle ergometer was chosen because the work output can be predicted with greater accuracy than for any other type of exercise. Also, within limits, the mechanical efficiency i s independent of body weight. A fixed pedal frequency of 50 RPM (300 meters per minute) was u t i l i z e d because optimal mechanical efficiency i s obtained at this rate (Astrand, 1970). A time interval of one hour was chosen because the contribution to energy output i s almost solely derived from aerobic processes (Astrand, 1970), therefore differences i n anaerobic capacity would not confound the work rates subjects could perform at. Tolerance time during exercise appears to be more related to psychological factors (motivation) than physiological factors. Well trained, highly motivated subjects may maintain oxygen uptakes of approximately 90 per cent for one hour, whereas untrained subjects often cannot work beyond one hour at 50 per cent of their maximum fc^. Experimental data on tolerance time i s extremely scanty. It has been reported that a trained individual can work at a relatively high oxygen uptake in relation to his maximum (60 to 65 per cent) without any elevation in blood lactate concentration. When untrained, a rise i s noted at about 50 per cent of maximal aerobic power (Hermansen and Saltin, 1967; Williams et a l . , 1967). In summary, one hour of exercise was near the upper limit of tolerance time for many of the subjects, but hopefully of sufficient duration and intensity 4 5 to observe any adaptive changes that might occur within the red blood c e l l to enhance oxygen delivery, and thereby man's a b i l i t y to perform strenuous activity. Within one minute of cessation of exercise a second blood sample was obtained from the subject, from the anticubital vein of the alternate arm from which the f i r s t sample was taken. Biochemical Determinations The blood parameters measured through biochemical techniques, in addition to 2,3-DPG, included hematocrit, hemoglobin and blood pH. Hemoglobin was measured becuase 2,3-DPG exerts i t s effect on this protein's a b i l i t y to bind oxygen (Brewer and Eaton, 1971). Hematocrit was measured because the concentration of hemoglobin varies directly with the number of red blood c e l l s present, and because mean corpuscular hemoglobin concentration ( ^ ^ ^ o c r i t ^ has been found to affect the binding of oxygen to hemoglobin independent of 2,3-DPG (Bellingham et a l . , 1971). Blood pH was measured because i t has been reported to be the most important factor in controlling 2,3-DPG concen-trations (Astrup, 1970a). Blood aliquots were placed on an ice bath immediately after removal from the subject. Determinations for blood pH, hematocrit, hemoglobin, as well as isolation of the protein-free blood supernatant used for 2,3-DPG analysis were completed within 30 minutes from the time of sampling. Venous blood pH was measured by the Micro Astrup technique as described i n the Radiometer instruction manual (1963). 46 Hematocrit was read from a micro hematocrit tube (Becton, Dickinson Cat. No. 1025) after centrifugation at 11,500 x g for five minutes (method of Albert, 1965). Hemoglobin was determined by the spectrophotometric method described by Van Assendelft (1970). 0.02 m i l l i l i t e r of blood was diluted in five m i l l i l i t e r s of Drabkin's Reagent (Fisher Scientific Cat. No. D-120), then read at 540 nm using a Beckman DU-2 spectrophotometer. Mean corpuscular hemoglobin concentration (MCHC) was determined by dividing the hemoglobin concentration by the hematocrit. Isolation of the protein-free blood supernatant was accomplished by combining one m i l l i l i t e r of blood with three m i l l i l i t e r s of eight per cent trichloracetic acid. The resultant mixture was shaken vigorously, placed on ice for 10 minutes then centrifuged for 10 minutes at 3000 RPM to completely precipitate a l l protein. The supernatant was stored at 10°C u n t i l analysis of 2,3-DPG was undertaken, which was within eight hours of blood sampling for a l l subjects. 2,3-DPG was determined using the enzymatic method described in Sigma Chemical Company technical bulletin 35 UV (1971). The method i s based on the decrease in optical density at 340 nm when NADH i s oxidized to NAD in the reaction converting 1,3-DPG to glyceraldehyde-3-phosphate (see Figure 2). The Beckman DU-2 spectrophotometer was also used in the determination of 2,3-DPG. i i 2,3-DPG concentrations were expressed in micromoles (uM) per m i l l i t e r of whole blood, micromoles per ml of packed blood c e l l s and micromoles per gram of hemoglobin. The latter expression was used in the s t a t i s t i c a l compar-isons because the hemoglobin content of red blood c e l l s remains unchanged. 47 When MCHC is simultaneously reported the effect on P50 can be derived for changes in the concentration of 2,3-DPG. When 2,3-DPG i s expressed in relation to volume of cells or whole blood, changes w i l l be observed purely as a result of changes in red c e l l volume or red c e l l number when no change in red c e l l content has occurred (Bellingham et a l . , 1971). Two determinations were made on both the pre and post exercise levels of 2,3-DPG, hematocrit, hemoglobin and blood pH. A c r i t e r i a of five per cent variation in duplicate readings was chosen as an acceptable degree of accuracy. This was based "on results of eight measurements of 2,3-DPG from the same blood sample; a standard deviation of ±0.65 uM/g Hb was obtained, which i s approx-imately a five per cent variation from the normal mean reported i n the literature (13.00 uM/gm Hb). The accuracy of repeated measurement compared favorably with that reported by Bellingham et al.,(1971) and Shappell et a l . , (1971). The margin of difference allowed between duplicate measurements for each variable was as follows: Hematocrit ±2.15 per cent Blood pH ±0.02 pH units Hemoglobin ±0.020 (Optical Density of test - blank) 2,3-DPG ±0.015 (Optical Density of test - blank) For a l l 40 subjects the differences obtained for duplicate hematocrit, pH and 2,3-DPG determinations never exceeded the above c r i t e r i a . However, on 1 three occasions hemoglobin determinations did exceed the c r i t e r i a . In these cases hemoglobin determinations were repeated immediately, and in a l l cases the second set of duplicate readings were within the acceptable range. The mean value of duplicate determinations was ut i l i z e d as the recorded score (Tables I through V of Appendix B). 48 i Physiological Determinations In order to ascertain whether or not status variables used to assign subjects to groups actually separated the groups on the basis of capacity to perform strenuous endurance work, a measure of average work per heartbeat, over the one hour of exercise, was employed. Average work performed per minute was calculated by summing the resistance, in kiloponds, for each minute of exercise, multiplied by the distance travelled per minute (300 meters) and divided by the duration of work (60 minutes) Z(Kp x 300) . . 60 w Average heart rate per minute was calculated by the formula Average work per heartbeat was calculated by dividing equation (a) by equation (b). Individual scores for average work per minute, average heart rate per minute and average work per heartbeat are presented in Tables VI through X of Appendix B. i i Experimental Design The investigation employed a two by three (2 x 3) incomplete randomized group design, i . e . three levels of fitness and two levels of smoking, with the c e l l "highly f i t smokers" remaining empty because subjects of that status were unavailable for testing. Each of the other five cells 49 contained eight subjects, who were tested randomly during the experimental period. The group design i s presented in Figure 4.1. Because of the missing c e l l , the five groups were treated as one factor (no interaction considered) for purposes of s t a t i s t i c a l treatment. The dependent variables of 2,3-DPG, hemoglobin, hematocrit, MCHC and blood pH were tested under two treatment conditions: pre arid post exercise, for a l l five groups, thus resulting in a 5 x 2 design. The analysis format is shown in Table I. S t a t i s t i c a l Analysis Raw scores for each subject for each dependent variable, before and after exercise, were key-punched onto computer data cards for analysis by U.B.C.'s IBM 360/67 computer. The Fortran IV program - "Multivariate Univariate and Multivariate Analysis of Variance and Covariance" (Finn, 1968) was u t i l i z e d to analyze the data. The program computed means and standard deviations for each group and for groups combined orthogonally, for each dependent variable, before and after exercise. It also produced a correlation analysis to show relationships among dependent measures (testing Hypotheses 4 and 5). Third, the program performed univariate orthogonal comparisons on pre exercise levels of the dependent variables to test the effects of training and/ or smoking. It also generated orthonormalized transformations on the difference scores for the dependent variables (pre exercise levels minus post exercise levels) then performed univariate orthogonal comparisons on these orthonormal-i ized changes. (Thus the data was treated as a multivariate rather than a repeated measures univariate design). Comparisons 1, 2 and 3 (Figure 4.2) test Hypothesis 2, while comparison 4 tests Hypothesis 3. Finally, the program tested whether the generated orthonormalized changes as a result of exercise were significant at the .05 level (Hypothesis .1). - 50 TABLE I Analysis Format of Dependent Variables _ Treatment Conditions Group Pre Exercise Post Exercise Sedentary Nonsmokers Xi X 5 Xi X5 X2 X6 X 2 X6 X3 X7 X3 X7 Xif Xs Xi* Xs Moderately F i t X 9 X!3 X 9 X13 Nonsmokers X u X u X10 X n X;i X15 X u X15 X12 Xl6 X12 Xl6 Highly F i t Nonsmokers • X n X21 X17 X21 X i 8 X22 Xia X 2 2 Xi9 X23 Xi9 X23 X20 X2I, X20 X 2if Bendentary Smokers X 2 5 X29 X25 X29 X26 X30 X 26 X30 X 2 7 X31 X27 X31 X28 X 3 2 X28 X32 Moderately F i t Smokers X33 X37 X33 X37 Xsif X38 X3i» X 3 8 X 3 5 X39 X 3 5 X39 X36 Xi»o X 3 6 XifO 51 FIGURE 4-1 E X E R I M E N T A L D E S I G N SEDENTARY MODERATELY FIT HIGHLY FIT SEDENTARY SMOKERS n = 8 MODERATELY FIT SMOKERS n = 8 SEDENTARY NONSMOKERS n = 8 MODERATELY FIT NONSMOKERS n = 8 HIGHLY F IT NONSMOKERS n = 8 S M O K E R S NONSMOKERS FIGURE 4 -2 • O R T H O G O N A L C O M P A R I S O N S SEDENTARY NON SMOKERS MODERATELY FIT NON SMOKERS COMPARISON I HIGHLY FIT NON SMOKERS COMPARISON 2 SEDENTARY MODERATELY FIT SMOKERS SMOKERS COMPARISON 3 COMPARISON 4 f ORTHOGONALITY INDICATED BY NO INTERSECTING LINES CHAPTER IV RESULTS AND DISCUSSION Results Descriptive Statistics Individual results for a l l subjects for a l l dependent variables are presented in Tables I through X of Appendix B. From these results, means and standard deviations of 2,3-DPG concentrations, determined from pre and post exercise blood samples were calculated and are presented i n Table II herein. Values for each of the five groups, for groups combined orthogonally and for pooled samples are l i s t e d separately. The same information i s also presented graphically i n Figure 5. Similarly, means and standard deviations of pre and post exercise determinations of blood hemoglobin, hematocrit, mean corpuscular hemoglobin concentration and pH are presented i n Tables III,IV,V and VI; and are graphically displayed in Figures 6,7,8 and 9 respectively. The means and standard deviations of body weight and average work per heartbeat, for each of the five groups, for groups combined orthogonally and for the pooled sample population, are presented i n Table VII. The average work per heartbeat, for each of the five groups, is also graphically illustrated i n Figure 10. Correlations between pre exercise 2,3-DPG levels and pre exercise levels of other dependent variables are presented in Table IX. Post exercise correlations are presented i n Figure X, while correlations with change in 2,3-DPG, due to exercise, are shown in Table XI. 52 53 TABLE II Means and Standard D e v i a t i o n s of 2,3-DPG L e v e l s Expressed i n Micromoles per Gram Hemoglobin f o r Each Group Before and A f t e r E x e r c i s e Groups Before A f t e r Sedentary Nonsmokers 12. 02 + 1. 94 12. 11 + 1. 90 Moderately F i t Nonsmokers 12. 72 + 1. 26 12. 61 + 1. 46 H i g h l y F i t Nonsmokers 13. 29 + 1. 86 13. 64 + 2. 39 Sedentary Smokers 12. 15 + 1. 30 12. 60 + 1. 31 Moderately F i t Smokers 13. 41 + 1. 30 13. 76 + 1. 36 Sedentary and Moderately F i t Nonsmokers Combined 12. 37 + 1. 62 12. 36 + 1. 66 A l l Nonsmokers Combined 12. 67 + 1. 72 12. 79 + 1. 98 A l l Smokers Combined 12 . 78 + 1. 41 13. 18 + 1. 42 A l l S ubjects Combined 12. 72 + 1. 56 12. 94 + 1. 73 54 FIGURE 5 M E A N S A N D S T A N D A R D D E V I A T I O N S O F 2 , 3 - D P G L E V E L S E X P R E S S E D IN M I C R O M O L E S P E R G R A M H E M O G L O B I N F O R E A C H G R O U P B E F O R E A N D A F T E R E X E R C I S E I6.O-1 15.0-14.0-2 < o tr U J Z o_ — m w9 13.0-U J or o 11.0-10.0-Q BEFORE EXERCISE AFTER EXERCISE \ i I I 2 x ^ L y////A L ! 2 x 2 t^ /////i I i SNS MFNS HFNS ss- •MFS-D P G 55 TABLE I I I Means and Standard D e v i a t i o n s of Hemoglobin C o n c e n t r a t i o n s Expressed as Grams Per 100 M i l l i l i t e r s f o r A l l Groups Before and A f t e r E x e r c i s e Groups Before A f t e r Sedentary Nonsmokers 15. 82 + 1. 20 16. 53 + 0. 97 Moderately F i t Nonsmokers 15. 51 + 1. 07 16. 28 + 0. 61 H i g h l y F i t Nonsmokers 14. 30 + 1. 86 14. 93 + 1. 82 Sedentary Smokers 16. 37 ± 1. 07 16. 78 + 1. 00 Moderately F i t Smokers 16. 61 ± 0. 76 17. 45 + 0.76 Sedentary and Moderately F i t Nonsmokers Combined 15. 67 ± 1. 11 16. 40 + 0. 79 A l l Nonsmokers Combined 15. 21 ± 1. 51 15. 91 + 1. 38 A l l Smokers Combined 16. 49 + 0. 91 17. 11 + 0. 93 A l l Subjects Combined 15. 72 ± 1. 25 16. 39 + 1. 11 56 FIGURE 6 M E A N S A N D S T A N D A R D D E V I A T I O N S O F H E M O G L O B I N C O N C E N T R A T I O N S E X P R E S S E D A S G R A M S P E R 1 0 0 M I L L I L I T E R S F O R A L L G R O U P S B E F O R E A N D A F T E R E X E R C I S E 20.01 19.0- _ ] BEFORE EXERCISE ^ AFTER EXERCISE CC UJ 0_ 18.0-17.0-CO < cr ° I6.0H £ 815.0-DO S _l4.0 i t o 13.0-LU — a  E o o <J o X3 5 12.0-CQ o _j o o LU X 11.0-10.0-9.0H y7?<77) 777? I i I / / V77777^ / V f L V////A SNS MFNS HFNS- S S MFS H E M O G L O B I N 57 TABLE IV Means and Standard D e v i a t i o n s of Hematocrit L e v e l s Expressed as Percent Volume f o r A l l Groups Before and A f t e r E x e r c i s e Groups Before A f t e r Sedentary Nonsmokers 45. 81 + 2. 23 46. 61 + 2. 29 Moderately F i t Nonsmokers 44. 85 + 2. 94 45. 81 + 2. 45 H i g h l y F i t Nonsmokers 44. 25 + 2. 26 45. 48 + 2. 68 Sedentary Smokers 46. 94 + 2. 74 47. 40 + 2. 61 Moderately F i t Smokers 47. 68 + 1. 56 48. 18 + 1. 41 Sedentary and Moderately F i t Nonsmokers Combined 45. 33 + 2. 57 46. 21 + 2. 33 A l l Nonsmokers Combined 44. 97 + 2. 48 45. 93 + 2. 42 A l l Smokers Combined 47. 31 + 2. 19 47. 79 + 2. 07 A l l S ubjects Combined 45. 90 + 2. 39 46. 68 + 2. 33 58 FIGURE 7 M E A N S A N D S T A N D A R D D E V I A T I O N S O F H E M A T O C R I T L E V E L S E X -P R E S S E D A S P E R C E N T V O L U M E F O R A L L G R O U P S B E F O R E A N D A F T E R E X E R C I S E 55.0i 52.5- • BEFORE EXERCISE W\ AFTER EXERCISE 50.0-(f) _ UJ - J - • 2 4 7 . 5 t-cr_ O LU o o fe a 2 a, LU x 5 45.0-K M 42.5-4Q0-,1 '////// f l 2 v, ? x 2 / V////A y i / V / / / / / / / / y / y / y / y y y / y v / y y y y y y y y y y y y y y y '/ 1 ? 1 y y y i y yy y y y y y y v,,,,yA -SNS MFNS- HFNS- S S MFS-H E M A T O C R I T 59 TABLE V Means and Standard D e v i a t i o n s of Mean Co r p u s c u l a r Hemoglobin C o n c e n t r a t i o n (MCHC) Expressed i n Percent f o r a l l Groups Before and A f t e r E x e r c i s e Group Before A f t e r Sedentary Nonsmokers 34. 45 + 1. 44 35. 40 + 1. 22 Moderately F i t Nonsmokers 34. 53 + 0. 66 35. 57 + 1. 37 H i g h l y F i t Nonsmokers 32. 20 + 2. 67 32. 81 + 1. 86 Sedentary Smokers 34. 89 + 1. 19 35. 41 + 1. 08 Moderately F i t Smokers 34. 83 + 1. 27 36. 21 + 1. 23 Sedentary and Moderately F i t Nonsmokers Combined 34. 49 + 1. 08 35. 49 + 1. 26 A l l Nonsmokers Combined 33. 72 + 2. 04 34. 60 + 2. 05 A l l Smokers Combined 34. 86 + 1. 19 35. 81 + 1. 19 A l l S ubjects Combined 34. 18 + 1. 59 35. 08 + 1. 49 60 FIGURE 8 M E A N S A N D S T A N D A R D D E V I A T I O N S O F M E A N C O R P U S C U L A R H E M O G L O -B I N C O N C E N T R A T I O N ( M C H C ) E X P R E S S E D IN P E R C E N T F O R A L L G R O U P S B E F O R E A N D A F T E R E X E R C I S E 40.0-39.0-• BEFORE EXERCISE ffli AFTER EXERCISE 38.0 370 § u j 3 6 . 0 j 3S | 2 35.0-U J ~" o cc x34.0-go (_> — w 33.0-o_ cc o ° 320-31.0-300-1 W' Y////A V / / / / V / V v/////< 9 1 '/ / / / / !l '$ / / / / / / / / / / '////A V/////. Vi V v / V / V / V / V V V / V / V V, V. SNS- MFNS -HFNS-M C H C ss- -+ -MFS 61 TABLE VI Means and Standard D e v i a t i o n s of Blood pH f o r a l l Groups Before and A f t e r E x e r c i s e Groups Before A f t e r Sedentary Nonsmokers 7. 41 + 0. 06 7. 42 + 0. 01 Moderately F i t Nonsmokers 7. 38 + 0. 03 7. 43 + 0. 03 H i g h l y F i t Nonsmokers 7. 35 + 0. 03 7. 42 + 0. 05 Sedentary Smokers 7. 37 + 0. 04 7 . 44 + 0. 04 Moderately F i t Smokers 7. 36 + 0. 05 7 . 40 + 0, 07 Sedentary and Moderately F i t Nonsmokers Combined 7. 39 + 0. 05 7. 43 + 0. 02 A l l Nonsmokers Combined 7. 38 + 0. 05 7. 43 + 0. 03 A l l Smokers Combined 7. 37 + 0. 04 7. 42 + 0. 05 A l l S ubjects Combined 7. 37 + 0. 04 7 . 42 + 0. 04 62 FIGURE 9 M E A N S A N D S T A N D A R D D E V I A T I O N S O F B L O O D p H F O R A L L G R O U P S B E F O R E A N D A F T E R E X E R C I S E 7 50 7.45 o o o -i CQ CO Z3 O z LU > 7.40 7.35 7.30-• BEFORE EXERCISE __| AFTER EXERCISE K I ^  / r L •SNS- -+—MFNS- HFNS SS MFS-P H 6 3 i TABLE VII Means and Standard D e v i a t i o n s of Body Weight Expressed i n Kilograms and Average Work per Heartbeat Expressed i n Kilopond Meters f o r a l l Groups G r o u P s Body Weight (Kg) Average Work (KPM) Sedentary Nonsmokers 7 8 . 3 3 + 1 2 . 1 9 5 . 2 4 + 1 . 4 8 Moderately F i t Nonsmokers 8 4 . 0 1 + 6 . 7 3 6 . 7 4 + 0 . 4 9 H i g h l y F i t Nonsmokers 6 9 . 6 6 + 1 0 . 1 7 8 . 0 0 + 1 . 0 5 Sedentary Smokers 7 9 . 9 9 + 7 . 0 7 5 . 1 4 + 0 . 5 9 Moderately F i t Smokers 8 2 . 2 6 + 1 3 . 0 5 5 . 4 8 + 0 o 8 4 Sedentary and Moderately F i t Nonsmokers Combined 8 1 . 1 7 + 9 . 9 5 5 . 9 9 + 1 . 3 2 A l l Nonsmokers Combined 7 7 . 3 3 + 1 1 . 2 6 6 . 6 6 + 1 . 5 5 A l l Smokers Combined 8 1 . 1 2 + 1 0 . 2 0 5 . 3 1 + 0 . 7 3 A l l S u b j e c t s Combined 7 8 . 8 5 + 1 0 . 1 7 6 . 1 2 + 0 . 9 6 64 FIGURE 10 M E A N S A N D S T A N D A R D D E V I A T O N S O F A V E R A G E W O R K P E R H E A R T -B E A T E X P R E S S E D IN K I L O P O N D M E T E R S F O R A L L G R O U P S 10.0 9.0-8.0-2 7 0 ~ 6.0 m _ 5.0 < U J X _ 4.0 o 3.0H _5» 2.0-1.0-SNS — M F N S - j «—HFNS -f* S S +> MFS H W O R K 65 TABLE IX Correlation Coefficients Between Pre Exercise 2,3-DPG Concentrations and Other Pre Exercise Variables, Body Weight and Average Work Per Heartbeat Pre Exercise Variable Total Hemoglobin -0.672 <.01 Hematocrit -0.557 <.01 MCHC -0.524 <.01 Blood pH 0.063 NS Work Per Heartbeat -0.110 NS Body Weight -0.030 NS TABLE X Correlation Coefficients Between Post Exercise 2,3-DPG Concentrations and Other Post Exercise Variables, Body Weight and Average Work Per Heartbeat Post Exercise Variable Total Hemoglobin -0.652 <.01 Hematocrit -0.477 <.01 MCHC -0.506 <.01 Blood pH -0.165 NS Work Per Heartbeat -0.234 NS Body Weight 0.020 NS df = 38 (n-2) a .05 = 0.312 a .01 = 0.403 66 i TABLE XI Correlation Coefficients Between the Orthonormalized Change of 2,3-DPG and Other Blood Parameters, Body Weight and Average Work Per Heartbeat Variable r p Hemoglobin-before -0.164 NS Hemoglobin-after -0.020 NS Hemoglobin-change -0.312 NS Hematocrit-before 0.182 NS Hematocrit-after 0.239 NS Hematocrit-change 0.105 NS MCHC-before 0.074 NS MCHC-after -0.264 NS MCHC-change -0.489 <.01 Blood pH-before -0.173 NS Blood pH-after -0.186 NS Blood pH-change -0.009 NS 2,3-DPG-before -0.032 NS 2,3-DPG-after 0.438 <.01 Work Per Heartbeat -0.289 NS Body Weight 0.102 NS df=38 (n-2) a.05=0.312 a.01=0.403 67 Pre exercise 2,3-DPG concentrations were negatively correlated (p<0.01) with pre exercise levels of hemoglobin (r = -0.672), hematocrit, (r = -0.557) and mean corpuscular hemoglobin concentration (r = -0.524). Correlations between pre exercise 2,3-DPG levels and other dependent variables were nonsignificant at the 0.05 level. Post exercise 2,3-DPG concentrations also correlated negatively with post exercise levels of hemoglobin (r = -0.652), hematocrit (r = -0.472) and MCHC (r = -0.489), with nonsignificant correlations existing for the other dependent variables and post exercise 2,3-DPG concentrations. The change i n 2,3-DPG concentrations, as a result of exercise, was negatively correlated (p<0.01) with the change in mean corpuscular hemoglobin concentration (r = -0.489). A positive correlation (r = 0.438) was observed between the change in 2,3-DPG as the post exercise level of 2,3-DPG. A complete correlation matrix of a l l dependent variables may be found i n Table II, of Appendix A. Homogenity of Variance One of the assumptions underlying s t a t i s t i c a l comparisons of samples is homogenity of variance, therefore an ^ m a x ~ t e s t w a s performed on the six dependent variables: 2,3-DPG, hemoglobin, hematocrit, MCHC, blood pH and average work per heartbeat. Results are shown i n Table I, Appendix A. The observed variance ratios were not significant at p<0.05, thus i t was assumed that the variances of the ten samples for each of the six dependent variables were homogeneous for the purpose of s t a t i s t i c a l analysis. 68 S t a t i s t i c a l Analysis of the Data- Test of Hypotheses 2,3-Diphosphoglycerate Concentrations. The results of a p r i o r i orthogonal comparisons of the resting levels of 2,3-DPG are presented in Table XII. No s t a t i s t i c a l l y significant differences were obtained for any of the comparisons. TABLE XII Orthogonal Comparisons of Pre Exercise 2,3-DPG Levels Comparison df MS Sedentary Nonsmokers VS Moderately F i t Nonsmokers 1,14 1.932 <1 Sedentary and Moderately F i t Nonsmokers VS Highly F i t Nonsmokers 1,21 4.526 1.863 >.05 Sedentary Smokers VS Moderately F i t Smokers 1,14 6.439 2.650 >.05 Smokers VS Nonsmokers 1,35 0.105 <1 Results of univariate analysis of the orthonormalized changes of 2,3-DPG levels, as a result of exercise, for a l l subjects, as well as the a p r i o r i orthogonal comparisons between groups, are presented in Table XIII. No significant change in 2,3-DPG concentration occurred as a result of exercise; nor was the change in 2,3-DPG, as a result of exercise, significant-ly different between any of. the groups compared orthogonally. 69 TABLE XIII Univariate Analysis of Orthonormalized Change of 2,3-DPG as a Result of Exercise for A l l Subjects and Orthogonal Comparisons of Changes in 2,3-DPG Between Groups Comparison df MS F P A l l Subjects Pooled 1,35 1.017 3.103 >.05 Sedentary Nonsmokers VS Moderately F i t Nonsmokers 1,14 0.077 <1 Sedentary and Moderately F i t Nonsmokers VS Highly F i t Nonsmokers 1,21 0.330 1.007 >.05 Sedentary Smokers VS Moderately F i t Smokers 1,14 0.024 <1 Smokers VS Nonsmokers 1,35 0.389 1.186 >.05 Hemoglobin Concentrations. The results of a p r i o r i orthogonal comparisons of the resting levels of hemoglobin are presented in Table XIV, Hemoglobin levels of highly f i t nonsmokers were significantly lower than the hemoglobin of the other nonsmokers. Also, hemoglobin levels of nonsmokers were significantly lower than Hb levels of smokers. I Results of univariate analysis of the: orthonormalized changes i n ! hemoglobin levels are presented in Table XV. A significant increase in hemoglobin levels occurred as a result of exercise, but the increase was not significantly different between the groups compared orthogonally. 70 TABLE XIV Orthogonal Comparisons of Pre Exercise Hemoglobin Concentrations Comparison df MS Sedentary Nonsmokers VS Moderately F i t Nonsmokers Sedentary and Moderately F i t Nonsmokers VS Highly F i t Nonsmokers Sedentary Smokers VS Moderately F i t Smokers Smokers VS Nonsmokers 1,14 0.391 <1 1,21 10.010 6.450 <.05 1,14 0.216 <1 1,35 15.677 10.102 <.01 TABLE XV Univariate Analysis of Orthonormalized Changes of Hemoglobin as a Result of Exercise for A l l Subjects and Orthogonal Comparisons of Changes i n Hemoglobin Between Groups Comparison df MS A l l Subjects Pooled 1,35 Sedentary Nonsmokers VS Moderately F i t Nonsmokers 1»14 Sedentary and Moderately F i t Nonsmokers VS Highly F i t Nonsmokers 1,21 Sedentary Smokers VS Moderately F i t Smokers 1»14 Smokers VS Nonsmokers 1,35 8.965 53.212 <.01 0.007 <1 0.025 <1 0.368 2.182 >.05 0.027 <1 71 Hematocrit Levels. Smokers were found to have a significantly higher pre exercise hematocrit level than nonsmokers. Differences between other groups were non significant (Table XVI). TABLE XVI Orthogonal Comparisons of Pre Exercise Hematocrit Concentrations Comparison df MS Sedentary Nonsmokers VS Moderately F i t Nonsmokers 1,14 3.715 <1 Sedentary and Moderately F i t Nonsmokers VS Highly F i t Nonsmokers 1,21 6.214 1.084 >.05 Sedentary Smokers VS Moderately F i t Smokers 1,14 2.176 <1 Smokers VS Nonsmokers 1,35 52.415 9.147 <.01 A significant increase was observed i n hematocrit levels as a result of the one hour exercise stress. No difference in the change in hematocrit as a result of exercise was observed between any of the groups compared orthogonally (Table XVII). Mean Corpuscular Hemoglobin Concentration. Highly f i t nonsmokers were found to have a significantly lower pre exercise mean corpuscular hemoglobin concentration than other nonsmoking subjects. In addition, smokers were found to have a significantly higher pre exercise MCHC than non smokers (Table XVIII). 72 TABLE XVII Univariate Analysis of Orthonormalized Changes in Hematocrit as a Result of Exercise for A l l Subjects and Orthogonal Comparisons of Changes in Hematocrit Between Groups Comparison df MS A l l Subjects Pooled Sedentary Nonsmokers VS Moderately F i t Nonsmokers Sedentary and Moderately F i t Nonsmokers VS Highly F i t Nonsmokers Sedentary Smokers VS Moderately F i t Smokers Smokers VS Nonsmokers 1,35 11.943 18.023 <.01 1,14 0.055 <1 1,21 0.158 <1 1,14 0.005 <1 1,35 1.085 1.637 >.05 TABLE XVIII Orthogonal Comparisons of Pre Exercise Mean Corpuscular Hemoglobin Concentrations Comparison df MS Sedentary Nonsmokers VS Moderately F i t Nonsmokers Sedentary and Moderately F i t Nonsmokers VS Highly F i t Nonsmokers Sedentary Smokers VS Moderately F i t Smokers Smokers VS Nonsmokers 1,14 0.024 <1 1,21 27.999 11.049 <.01 1,14 0.013 <1 1,35 12.395 4.891 <.05 73 A significant increase in mean corpuscular hemoglobin concentration was observed to occur as a result of the one hour exercise stress, however the increase was not significantly different between groups compared orthog-onally. TABLE XIX Univariate Analysis of Orthonormalized Changes in Mean Corpuscular Hemoglobin Concentration as a Result of Exercise for A l l Subjects and Orthogonal Comparisons of Changes i n Mean Corpuscular Hemoglobin Concentration Between Groups Comparison df MS F P A l l subjects Pooled 1,35 16.299 29.809 <.01 Sedentary Nonsmokers VS Moderately F i t Nonsmokers 1,14 0.018 <1 Sedentary and Moderately F i t Nonsmokers VS Highly F i t Nonsmokers 1,21 0.389 <1 Sedentary Smokers VS Moderately F i t Smokers 1,14 1.462 2.674 >.05 Smokers VS Nonsmokers 1,35 0.028 <1 Blood pH. The only significant difference i n the resting, pre exercise values of blood pH were between the highly f i t nonsmokers and other nonsmoking subjects (Table XX). A significant increase in blood pH occurred as a result of one hour of exercise, however the increase was not significantly different between groups compared orthogonally (Table XXI). 74 TABLE XX Orthogonal Comparisons of Pre Exercise Blood pH Condition df MS Sedentary Nonsmokers VS Moderately F i t Nonsmokers 1,14 0.004 2.108 >.05 Sedentary and Moderately F i t Nonsmokers VS Highly F i t Nonsmokers 1,21 0.013 7.105 <.05 Sedentary Smokers VS Moderately F i t Smokers 1,14 0.003 <1 Smokers VS Nonsmokers 1,35 0.001 <1 TABLE XXI Univariate Analysis of Orthonormalized Changes in Blood pH as a Result of Exercise for A l l Subjects and Orthogonal Comparisons of Changes i n Blood pH Between Groups Condition df MS A l l Subjects Pooled 1,35 0.050 31.361 <.01 Sedentary Nonsmokers VS Moderately F i t Nonsmokers 1,14 0.002 1.502 >.05 Sedentary and Moderately F i t Nonsmokers VS Highly F i t Nonsmokers 1,21 0.003 1.826 >.05 Sedentary Smokers VS Moderately F i t Smokers 1,14 0.001 <1 Smokers VS Nonsmokers 1,35 0.000 <1 75 Average Work Per Heartbeat. A p r i o r i orthogonal comparisons of the average work per heartbeat for various groups are presented in Table XXII. Moderately f i t nonsmokers were able to work at a significantly higher level per heartbeat than sedentary nonsmokers. Highly f i t nonsmokers had a signif-icantly higher work per heartbeat level than the other nonsmoking subjects. The nonsmokers had a significantly higher work per heartbeat level than the smokers. No significant difference was observed between sedentary smokers and moderately f i t smokers. TABLE XXII Orthogonal Comparisons of Average Work Per Heartbeat Comparison df MS Sedentary Nonsmokers VS Moderately F i t Nonsmokers 1,14 9.075 9.909 <.01 Sedentary and Moderately F i t Nonsmokers VS Highly F i t Nonsmokers 1,21 21.494 23.468 <.01 Sedentary Smokers VS Moderately F i t Smokers 1,14 0.462 <1 Smokers VS Nonsmokers 1,35 17.458 19.061 <.01 76 Discussion Pre Exercise Parameters The mean resting level of 2,3-DPG for subjects participating in this study ( 1 2 . 7 2 ± 1.56 uM/g Hb, Page 5 3 ) was similar to that found by most investigators (Eaton and Brewer, 1968; Eaton et a l . , 1970; Faulkner et a l . , 1 9 7 0 ; Valeri and Collins, 1 9 7 1 ) . However, in other studies considerably higher mean resting 2,3-DPG concentrations were obtained. Bellingham et al„ ; ( 1 9 7 1 ) reported a mean 2,3-DPG level of 14.52 uM/g Hb; Shappell et a l , ( 1 9 7 1 ) , in a study on the effects of exercise, reported a mean 2,3-DPG level of 1 4 . 9 0 uM/g Hb, The small sample size of these latter studies, (three and seven subjects respectively), may have contributed to the selection, by chance, of subjects with above normal 2,3-DPG concentrations. In this study the range of pre exercise 2,3-DPG concentrations was 10. 1 5 to 15.1 7 uM/g Hb, with one exception, (Subject 'SD*, a highly f i t non-smoker had a 2,3-DPG level of 16.92 uM/g Hb.) Individual results are shown i n Table III, Appendix B. The range reported here i s within that reported by Eaton and Brewer ( 1 9 6 8 ) from 100 male Caucasians, of 8.5 to 1 5 . 8 6 uM/g Hb. Considering the overwelming evidence of a linear relationship between P^Q of the oxygen dissociation curve of hemoglobin and the concentration of 2,3-DPG (Messier and Schaefter, 1967; Torrence et a l . , 1 9 7 0 / 7 1 ; Valeri and Collins, 1 9 7 1 ) , as well as the findings of Duhm ( 1 9 7 2 ) , Oski ( 1 9 6 9 ) and Valeri and Collins ( 1 9 7 1 ) that a shift of one Torr i n P^Q i s accomplished through a change of 2,3-DPG concentration of approximately 1.40 uM/g Hb, i t would appear that a range of approximately four Torr exists in P^p.in the 40 subjects participating in this study, which could be directly related to the range i n resting 2,3-DPG concentrations, 77 i No significant difference ( p <.05) was observed between groups in relation to pre exercise 2,3-DPG concentrations (Table XII, Page 68)5 i.e. 2,3-DPG concentrations were not significantly higher in highly trained subjects nor lower in sedentary subjects (Hypothesis 2, Orthogonal comparisons 1, 2 and 3); nor were 2,3-DPG concentrations significantly higher in smoking subjects (Hypothesis 3, Orthogonal comparison 4), However, when the means for each group of subjects are examined (Table II, Figure 5, Pages 53-4) a definite trend i s observed. Among the nonsmokers an increase i n 2,3-DPG (as defined by the mean) i s apparent as one progresses from sedentary through highly f i t groups (12.02 ±1.94 uM/g Hb 13.29 ± 1.86 uM/g Hb). The same trend i s observed i n the smoking groups, 2,3-DPG means increase from 12.15-± 1.30 uM/g Hb to 13.41 ± 1.30 uM/g Hb. Also consistent with this trend i s the observation that 10 of 16 sedentary subjects had resting 2,3-DPG levels below 12.00 uM/g Hb whereas only 7 of 24 active subjects had 2,3-DPG levels below 12.00 uM/g Hb, Thus while one cannot accept the hypothesis that trained individuals have higher 2,3-DPG concentrations, as found by Shappell (1971), the hypothesis should not be totally rejected. In this study only four subjects demonstrated a resting 2,3-DPG level equal to, or exceeding, the pre-training level reported by Shappell and only one subject had a resting 2,3-DPG level of the magnitude of the post-training level, reported by Shappell,of 15.97 uM/g Hb. At least three p o s s i b i l i t i e s exist to explain the divergent results. F i r s t , Shappell's three subjects, perhaps fortuitously, demonstrated extremely high pre-training 2,3-DPG levels which may have reflected a genetically enhanced metabolic enzyme pathway more amenable to induction by physiological stimuli produced through physical training. 78 It Is possible that 2,3-DPG increments occur as a result of an increase in the intensity of physical ac t i v i t y . Once the acti v i t y level i s maintained for a sufficient duration to allow other, slower acting adaptive mechanisms to respond (such as an increase in stroke volume, increase i n respiratory enzymes, e t c ) 2,3-DPG levels tend to return to pre-training levels due to a reduction in hypoxia. In this study subjects tended to maintain a constant training level for six months prior to testing, whereas in Shappell's study subjects were tested before and after eight weeks of increased physical activity, A third explanation might be that training exerts a minor stimulatory effect on 2,3-DPG of red blood c e l l s . Because the highly trained subjects in this study had been involved i n a training program which was eminently superior to Shappell's in both intensity and duration, i t was expected that large differences i n 2,3-DPG levels would be observed, i f training produces an increase in 2,3-DPG, when these subjects were compared to sedentary subjects. However, the mean difference between highly f i t and sedentary nonsmokers was only 1.39 uM/g Hb, which was not s t a t i s t i c a l l y significant, Shappell's data shows an increase of 2,3-DPG, as a result of training, of only 1,07 uM/g Hb, which was s t a t i s t i c a l l y significant. Therefore, i t would appear that i f training does cause an increase i n 2,3-DPG concentrations, the increase i s rather small, perhaps i n the v i c i n i t y of 1,0 to 1.5 uM/g Hb, reflecting a change in P^Q of approximately one Torr, The correlation between 2,3-DPG and fitness, as measured by average work per heartbeat over one hour of exercise, was not s t a t i s t i c a l l y significant (r- -.110, Table II, Appendix A), indicating that 2,3-DPG i s not a valuable predictor of aerobic fitness even though i t may be slightly responsive to the hypoxia of endurance training. 79 Although the hypothesis that smokers have a higher 2,3-DPG level than nonsmokers also cannot be accepted, supporting the findings of Brewer et a l , (1970), the tendency of the data i s quite suggestive. Among the two sedentary groups l i t t l e difference i s observed i n mean pre exercise 2,3-DPG levels (SNS = 12.02 ± 1.94 uM/g Hb, SS » 12.15 ± 1.30 uM/g Hb), however a larger difference i s observed when comparing the moderately f i t groups, with the smokers having the higher mean 2,3-DPG level (MFNS = 12.72 ± 1,26 uM/g Hb, MFS = 13.41 ± 1.30 uM/g Hb). In fact the moderately f i t smokers exhibited the highest mean resting 2,3-DPG level of a l l five groups. These results indicate that smokers, when subjected to the hypoxia of repeated physical ac t i v i t y , respond with a larger increase in 2,3-DPG than nonsmokers subjected to the same conditions. This agrees with Eaton et a l . (1970) who found significantly higher levels of 2,3-DPG in polycythemic smokers at altitude than in normal altitude dwellers. Because smokers have part of their blood hemoglobin bound as carboxyhemoglobin and unavailable for oxygen transport, the hypoxia of exercise (and altitude) could produce a greater desaturation of the remaining oxyhemoglobin at the tissue level than would occur to nonsmokers with a l l their hemoglobin available for oxygen transport. Chronic desaturation leads to a relative intracellular alkalosis and consequently a higher rate of 2,3-DPG synthesis (Brewer and Eaton, 1971; Duhm and Gerlach, 1971; Rorth and Brahe, 1972), A highly significant negative correlation was observed in this study between resting concentrations of 2,3-DPG and blood hemoglobin levels (r = -.672, Table IX, Page 65) supporting hypothesis 5. This correlation was larger than found by Lenfant et a l . (1970) and Eaton and Brewer (1968)„ but similar to the negative correlation found.by Hjelm (1970) in healthy adult males, In effect this relationship indicates that as the oxygen carrying capacity of the blood 80 increases (higher hemoglobin concentrations), a decreased desaturation of hemoglobin, or oxygen unloading per gram of hemoglobin, results (as reflected by lower concentrations of 2,3-DPG per gram of hemoglobin). The inverse correlation between 2,3-DPG and hemoglobin also indicates that an increased concentration of deoxyhemoglobin and increased binding of 2,3-DPG at a lower hemoglobin concentration i s responsible for the increase in 2,3-DPG, This could occur by both the removal of inhibition of DPG mutase by unbound 2,3-DPG (Rose, 1970) and an increase in glycolytic flux produced through an increase in intracellular pH due to increased amounts of deoxyhemoglobin (Eaton and Brewer, 1968), These mechanisms are demonstrated i n Figure 3, Page 30, In comparing the hemoglobin concentrations of the three nonsmoking groups i n this study (Table III, Figure 6, Pages 55-6) the opposite trend to the 2,3-DPG trend i s observed as one would expect by the significant negative correlation between the two parameters. That i s , hemoglobin concentrations f a l l as fitness levels increase. The hemoglobin concentrations of the highly f i t nonsmokers was significantly lower than hemoglobin concentrations of the other nonsmoking subjects (p < .05, Table XIV, Page 70). This agrees with the finding of Oscai et a l . (1968) but i s contrary to the results of Knehr et a l , (1942) and Shappell et a l (1971), In contrast, active smokers tended to have slightly higher (but not significant) hemoglobin levels than sedentary smokers, i n spite of also having a-tendency towards higher 2,3-DPG concentrations. This i s reflected by the fact that although a significant negative correlation exists between hemoglobin and 2,3-DPG when a l l subjects are considered together, the correlation between the two parameters for the sedentary smokers i s only r = -.416 and for the 81 moderately f i t smokers the correlation between hemoglobin and 2,3-DPG drops to r = -.278. In addition, although no significant difference was observed bet-ween smokers and nonsmokers in terms of 2,3-DPG, smokers were found to have a significantly higher (p < .01, Table IV, Page 70) blood hemoglobin level. This latter finding i s contrary to the findings of Albert (1965) and Isager and Hagerup (1971) and may be pa r t i a l l y attributed to the very low hemoglobin levels of the highly f i t nonsmokers, although in both sedentary and moderately f i t groups the smokers tended to have sli g h t l y higher hemoglobin levels than the nonsmokers. Because blood hemoglobin levels are a function of both the number of red blood c e l l s (hematocrit) and the hemoglobin concentration within each c e l l (MCHC) i t i s pertinent to examine differences in these parameters to c l a r i f y the overall hemoglobin response and how i t relates to 2,3-DPG, The significant negative correlation between 2,3j-DPG and hematocrit (r = -.557) and 2,3-DPG and MCHC (r = -.524) may be related to the fact that both these parameters are related to blood hemoglobin levels (i.e. the correlation between hemoglobin and hematocrit (r = .842) and hemoglobin and MCHC (r = .760) are highly si g n i f -icant, Table II, Appendix A), However the relationships are complicated by the fact that MCHC affects the oxygen a f f i n i t y for hemoglobin independent of 2,3-DPG, as well as affecting the carrying capacity of hemoglobin (Bellingham et a l . , 1971; Lenfant et a l . , 1972; Shappell et a l . , 1971), In this study smokers were found to have significantly higher hematocrit levels than nonsmokers (p < .01, Table XVI, Page 71), differences between fitness groups were not significantly different although the trend of hematocrits closely parallel trends of hemoglobin, decreasing through increasing fitness levels of nonsmokers an increasing with increasing fitness levels of 82 smokers. The higher hematocrits of smokers, which agrees with the literature (Isager and Hagerup, 1971; Burch and DePasquale, 1962) has been identified as a factor producing a higher incidence of coronary heart disease i n the smoking population (Brewer, 1972). The average hematocrit for a l l subjects pooled (45.90 ± 2.39) agrees with results of other investigators (Garby, 1970; Kilpatrick, 1961; Larsen, 1966; Lenfant et a l . , 1969; McDonough et a l . , 1965; Natvig, 1966; Natvig and Vellar, 1967). The viscosity, or resistance to flow, of blood is mainly dependent on the plasma proteins and the c e l l content. The higher the hematocrit the higher the viscosity. In dogs, by measurement of oxygen consumption, a r t e r i a l and venous oxygen concentrations, a r t e r i a l pH, cardiac output, peripheral resistance and maximal oxygen-transporting capacity at different hematocrit values, i t was demonstrated that oxygen consumption increased as the hematocrit increased u n t i l the latter reached 42 per cent. At progressively higher hematocrit values oxygen consumption decreased so that oxygen transport was maximal at 42 per cent. Increasing the number of red blood c e l l s increased the oxygen carrying capacity but decreased the rate of flow by increasing the blood viscosity (Harris and Kellermeyer, 1970:294). Assuming this study i s applicable to human subjects, i t indicates that the highly f i t nonsmokers have hematocrit levels that are closest to the "optimal" level, even though this level i s below the normal population mean. Smokers,on the other hand, have hematocrit levels which would tend to reduce oxygen delivery because of increased viscosity, i i i Guyton (1960:86) reported that the degree of physical a c t i v i t y of a person determines to a great extent the rate at which red blood c e l l s are produced. The fact that exercise increased the rate of blood c e l l production was indicative that i t i s anoxia of tissues that causes red blood c e l l produc-83 tion, because the tissues become depleted of oxygen in exercise. However, opposing the increase in red c e l l production with exercise, i s the finding that physical a c t i v i t y causes a decrease in the integrity of the red blood c e l l membrane which has been attributed to an increased circulatory rate, temperature, acidity and compression of c e l l s (Faulkner et a l . , 1970). It i s hypothesized that as training becomes more intense hematocrit i s reduced and maintained closer to the optimal level to allow red c e l l production to keep up with red c e l l destruction. Thus although training appears to cause a decrease in oxygen transport , the decreased blood viscosity and the tendency towards a decreased a f f i n i t y of hemoglobin for oxygen ( increased 2,3-DPG) may actually contribute to enhancement of oxygen delivery to the tissues. Also, the beneficial effect of training i n decreasing the risk to coronary heart disease may be exerted through this reduction in hematocrit. The higher hematocrits of smokers can also be related to a stimulation of erythropoiesis. Numerous studies on secondary polycythemia have shown that the severity of the polycythemic changes can be correlated to the degree of hypoxemia (Pugh, 1964). Smokers, both moderately f i t and sedentary, demonstrate increased red c e l l production due to hypoxia produced through binding of carbon monoxide to hemoglobin thereby removing i t from oxygen transport. The ac t i v i t y level of these individuals would l i k e l y not be of sufficient intensity to cause increased red c e l l destruction, thus hematocrits remain significantly higher. I Active smokers, who are not involved in a program of ac t i v i t y which is intensive ! i enough to cause significant increases in red c e l l f r a g i l i t y , but which produces additional hypoxia above that which i s due to smoking, would tend to have a further stimulation of red c e l l production and a higher hematocrit. Oxygen transport to tissues would be further impaired by the increase i n blood viscosity. 84 r The tendency towards a higher 2,3-DPG concentration in the moderately f i t smokers could p a r t i a l l y compensate for the effect of increased viscosity. Mean Corpuscular Hemoglobin Concentrations (MCHC) also exhibit a trend similar to hemoglobin levels (Table V, Figure 8, Pages 59-60) with highly f i t nonsmokers having a significantly lower (p < .01) MCHC than other non-smokers and smokers having a significantly higher MCHC than nonsmokers (p < ,05), (Table XVIII, Page 72), The higher MCHC levels of the smokers in this study are contrary to the results of Albert (1971) and Isager and Hagerup (1971) who found that smokers had significantly higher hematocrits, lower MCHC and unchanged hemo-globin levels. The average MCHC reported i n these studies of 33.18 ± 1,78 were considerably lower than the results of this investigation (34,86 ± 1.19), whereas hematocrit levels were quite similar. No explanation is apparent to account for the discrepancy of the data. The average MCHC obtained in this study for the nonsmoking subjects (33.72 ± 2.04) was similar to that obtained in other normal population studies (Garby, 1970; Natvig and Vellar, 1967), however the literature on this parameter is somewhat ambiguous. For example Guyton (1971:111) reported that concentrations of 34 grams per 108 mis of red blood c e l l s are near the metabolic limit of the hemoglobin forming mechanism of the cell,with values rarely exceeding this level. On the other hand, Larson (1966) reported an average MCHC of 34.4 ± 3.2 grams per 100 mis of packed c e l l s , which is in agreement with the results reported here. (In this study 26 of 40 subjects had a MCHC in excess of 34.0.) The lower MCHC of the highly f i t subjects i s i n agreement with the results of Shappell (1971), who found a decrease in MCHC from 33.85 ± 0,74 to 85 3 3 . 0 9 ± 0 .48 grams per 100 mis of blood c e l l s in 22 subjects after an eight week training period. The highly f i t subjects in this study demonstrated a MCHC of 3 2 . 2 0 ± 2 . 6 7 , which i s consistent with their more intensive training program. The hypothesis that erythropoietic a c t i v i t y is barely able to keep up with red c e l l destruction when training becomes very intensive i s further supported by the very low MCHC levels of the highly f i t subjects, In fact, two of the highly f i t subjects had MCHC levels below 3 0 . 0 grams per 100 mis, the value generally accepted as symptomatic of hyperchromia. The assumption i s that,because of the extremely high turnover rate of red c e l l s i n these individuals,the hemoglobin producing mechanism lags behind c e l l production, leading to red blood c e l l s with reduced hemoglobin levels. The lower MCHC of the highly f i t nonsmokers produces an adverse effect on oxygenation of tissues in that i t leads to an increased a f f i n i t y of hemoglobin for oxygen, i.e. i t causes a l e f t s h i f t i n the oxygen dissociation curve (Benesch et a l , 8 1969 ; Bellingham et a l . , 1 9 7 1 ) . The tendency towards higher 2 , 3 - D P G levels, although not s t a t i s t i c a l l y significant i n this study, could p a r t i a l l y offset the l e f t s h i f t of the oxygen dissociation curve. In order to c l a r i f y the overall effect on the oxygen delivery of blood as affected by 2 , 3 - D P G , MCHC and hematocrit a summary i s presented in Table XXIII. Use was made of the calculations of Bellingham et a l , , (1971) that change in P^Q due to 2 , 3 - D P G could be determined using the equation: [ P 5 Q = 0 . 6 9 4 DPG + 1 7 . 6 3 ] and change in P 5 Q due to MCHC could be determined by use of the equation [&P5Q = 0 .471 AMCHC - 0 , 2 1 6 ] , The results show that the moderately f i t smokers have the largest right shift in P^Q of any group, but they also have the largest increment in blood viscosity above the "optimal" level of 42 per cent. Moderately f i t nonsmokers and highly f i t nonsmokers have approximately the same shift in P , - N but the shift occurs by different 86 mechanisms. The highly f i t group obtains the beneficial effect solely through 2,3-DPG, whereas the moderately f i t group's P,.- is primarily affected by MCHC. In addition, the highly f i t nonsmokers possess the most advantageous hemato-c r i t . Sedentary smokers had slighly higher P,.. values than sedentary nonsmokers, which may have compensated for the same trend in hematocrits. It would appear that the highly f i t nonsmokers would have the parameter levels producing the most ef f i c i e n t oxygen delivery system. Table XXIII Theoretical Determinations of Increases i n P_-Due to Differences i n 2,3-DPG and MCHC and Hematocrit Levels Above the Theoretical Optimal Level Increases i n P,.^  Increases i n P ^ Hematocrit Percent Group due to 2,3-DPG due to MCHC above above optimal of above 12.02 uM/g Hb 32.20 g/100 mis 42% Sedentary Nonsmokers Moderately F i t Nonsmokers Highly F i t Nonsmokers Sedentary Smokers Moderately F i t Smokers 0.00U + 0.486 + 0.881 + 0.090 + 0.965 + 0.844 + 0,881 0.000 + 1.051 + 1.023 3.81 2,85 2.25 4.94 5.68 87 Highly f i t subject 'SD', who possessed an above normal pre exercise 2,3-disphosphoglycerate concentration of 16.92 uM/g Hb. was one of two subjects with a hypochromic MCHC (29.20 ), although his hematocrit level was very close to the theoretical optimum of 42 per cent (42.9%). The hypothesis that an increased desaturation of hemoglobin, due to a decreased MCHC, stimulates an increase i n 2,3-DPG is supported by the data of this subject. The f i r s t limitation of this study (Page 9) pointed out that subjects were assigned to subgroups according to a status variable which may have obscured relationships under study. However, when groups were compared orthog-onally u t i l i z i n g the physiological measure of aerobic fitness of this study, (average work per heartbeat), a significant difference existed between the three non smoking groups (Table XXII, Page 75). Differences in work per heart-beat between smoking groups were not significant. These results confirm that among the nonsmokers, assignment to fitness group through the status variable of training intensity separated the subjects physiologically i n relation to average work per heartbeat. Unfortunately the two smoking groups did not exhibit physiological differences i n fitness. (Group data is presented in Table VII, Figure 10, Pages 62-3.) In order to determine whether or not 2,3-DPG and other blood parameter were significantly different between high f i t and low f i t subjects, as measured by average work per heartbeat, a post hoc analysis was carried out. The 15 subjects with the highest average work per heartbeat (seven highly f i t non-smokers, five moderately f i t nonsmokers, one moderately f i t smoker and two sedentary nonsmokers) were compared with the 15 subjects with the lowest averag work per heartbeat (five moderately f i t smokers, five sedentary smokers and five sedentary nonsmokers). Multivariate analysis, using computer program 88 Multivariance (Finn, 1968) was again performed. Results are presented i n Tables I and II of Appendix C. No significant differences were observed between the two groups in 2,3-DPG concentrations and MCHC levels, but hemato-c r i t and hemoglobin levels were both significantly higher in the low f i t group. These results indicate that MCHC is more related to the training intensity one is engaged in than to a measure of one's aerobic capacity. Because natural endowment i s the most important factor determining an individual's aerobic capacity, with regular training being only capable of increasing aerobic capacity 10 to 20 per cent -(Astrand, 1970), some individuals could be expected tp have a relatively high aerobic capacity without being involved i n a training program causing alterations i n red blood c e l l turnover. Similarly, the indications presented in this study and supported by other studies, that 2,3-DPG enhancement occurs through chronic hypoxia support the hypothesis that 2,3-DPG concentrations would also be more related to training intensity than to aerobic capacity. Because blood pH has been suggested as the major regulator of 2,3-DPG concentrations (Asakura et a l . , 1966; Astrup, 1970; Rorth, 1970) a positive relationship was expected between pre exercise levels of blood pH and levels of 2,3-DPG (as found by Valeri and Collins, 1972). However, in this study no such relationship exists (r = .063, Table II, Appendix A). It i s observed that although the highly f i t nonsmokers have mean 2,3-DPG levels higher than other nonsmoking groups, they have significantly lower pre exercise blood pH levels (Table XX, Page 74)'. Several reports i n the literature indicate that 2,3-DPG changes that occur as a result of pH changes do so only after a considerable delay in time. For example, Messier and Schaefer (1971) subjected guinea pigs to chronic 89 hypercapnia (15 per cent CC^) leading to a precipitous f a l l in pH in the f i r s t hour with only a small drop in 2,3-DPG. The largest 2,3-DPG reduction occurred after six hours of exposure when the drop in pH was rather small or when pH had already began to r i s e . This suggested a metabolic reaction initiated by acidosis, such as rates of glucose u t i l i z a t i o n , that followed pH changes with a time lag. Thus, the lack of relationship between 2,3-DPG and pH in this study might be attributed to differences in rate of change between the two parameters. The mean pre exercise pH for a l l subjects i n this study (7.370) and the range of pre exercise venous blood pH levels (7.282 to 7.510) were similar to that reported in the literature (Federation of American Societies for Experimental Biology, 1961). No explanation i s apparent to explain the sign-i f i c a n t l y lower pre exercise venous blood pH of highly f i t nonsmokers, however the effect of acidosis on reducing 2,3-DPG concentrations may have masked higher 2,3-DPG levels in this group at a pH similar to other groups. The Effect of Exercise The time course of the 2,3-DPG response to hypoxia i s a major point of contention. Dempsey et a l . , (1971) observed no changes in 2,3-DPG after two hours of exercise, however Faulkner et a l . (1970) observed an 18 per cent increase within 60 minutes. In non-exercise studies, Hamasaki and Minakama ! (1971) observed a 20 per cent change in 2,3-DPG concentrations in the time blood circulates from artery to vein (seconds); Valeri and Fortier (1970) calculated a rate of increase of 0.80 uM/g Hb. for the f i r s t three hours after a blood transfusion of 2,3-DPG depleted c e l l s ; Gerlach et a l . (1970) exposed rats to hypoxia and found a significant increase in 2,3-DPG only after five 90 hours of elapsed time; Bellingham et a l . (1971) found no change in 2,3-DPG after four hours of induced alkalosis and acidosis; Lenfant et a l . (1970) found 2,3-DPG changes reach one-half their maximum after approximately six hours and reach their maximum only after 24 hours of altitude exposure. In this study, 2,3-diphosphoglycerate concentrations were not increased significantly (p = .087) as a result of one hour of exercise at approximately 65-70 per cent of maximal aerobic capacity (results i n Table XIII, Page 69), therefore Hypothesis 1 cannot be accepted. (Mean 2,3-DPG levels increased from 12.72 ± 1.56 to'12.94 ± 1.73 uM/g Hb., Table II, Figure 5, Pages 53-4.) Individual comparisons indicate that 26 subjects had an increase in 2,3-DPG ranging from 0.05 to 1.60 uM/g Hb., two subjects exhibit no change and 12 subjects had a decrease i n 2,3-DPG ranging from 0.06 to 1.40 uM/g Hb. (Table I through V, Appendix B). No significant difference was observed between groups on the effect of exercise on 2,3-DPG concentrations. The lack of a significant exercise-induced increase in 2,3-DPG in this study i s in agreement with Dempsey et a l . (1971) and Shappell et a l . (1971) but contrary to the findings of Eaton et a l . (1969) and Faulkner et a l . (1970). The fact that a majority of subjects (26) exhibited an increase in 2,3-DPG leads one to speculate that the mechanism producing increases in 2,3-DPG has been stimulated but that insufficient time has elapsed for a marked change to occur, which i s consistent with a time response of several hours. i Definitely, one hour of exercise does not produce a physiologically beneficial rise in 2,3-DPG. Two mechanisms i n this study could have triggered the 2,3-DPG response mechanism. As mentioned earlier, exercise theoretically produces a relative 92 If- ^ intracellular alkalosis through an increase in deoxyhemoglobin, which in turn favours 2,3-DPG elevation. Second,.a significant increase in venous blood pH (Table XXI, Page 74), again favouring 2,3-DPG elevation, occurred as a result of exercise. The fact that the 2,3-DPG response follows the pH response with a time lag could explain the lack of correlation between change in pH and change in 2,3-DPG (r = -.009, Table XI, Page 66). The increase in pH as a result of exercise was an unexpected finding. Exercise evokes an increase in CO2 production (which competitively binds with 2,3-DPG on specific sites on the hemoglobin molecule, Bauer (1970); Tomita and Riggs (1971))- and, in many cases when work rates exceed 50 per cent of maximal aerobic capacity, exercise leads to an increase in blood lactate. Both these substrates tend to lower blood pH and inhibit 2,3-DPG production. For example Gerlach et a l . (1970) demonstrated that the increase in 2,3-DPG produced by 24 hour exposure of rats to 11 per cent oxygen was abolished when five per cent CO2 was added. Two po s s i b i l i t i e s are offered to explain the rise in venous blood pH. F i r s t , exercise-evoked stimulation of respiration, leading to hyper-ventilation (Astrand, 1970:216), may have more than compensated for the metabolic acidosis produced through exercise. Another poss i b i l i t y i s that vasoconstric-tion of the vasculature of the nonworking arms causes blood flow to be redistributed away from this anatomical site during leg exercise (Simonson, 1971:145) thereby producing a localized environment i n the veins of the arm which would not reflect acid - base status at the working muscles and veins I carrying blood from them. For these reasons interpretation of the results i s limited to localized venous circulation of the arm. A most interesting finding of this study was that a significant increase in MCHC occurred as a result of exercise (Table XIX, Page 73). No 93 significant differences were observed between groups in the change in MCHC. Because the hemoglobin content within the c e l l is fixed, alterations in MCHC can occur only as a result of the red blood c e l l shrinking or swelling. The shrinking of c e l l s , leading to an increase in MCHC (from mean of 34.18 ± 1.59 to mean of 35.08 ± 1.49, Table V, Figure 8, Pages 59-60), produces an increase In P^Q, independent of 2,3-DPG, which in turn f a c i l i t a t e s oxygen delivery to active tissues. In addition to the significant increase in MCHC, a significant negative correlation exists between the change in MCHC and the change in 2,3-DPG as a result of exercise (r = -.489, Table XXI, Page 66). Only six subjects demonstrate a decrease in MCHC and a l l had an increase i n 2,3-DPG ranging from 0.32 to 1.31 uM/g Hb. Conversely, of the 12 subjects who have a decrease i n 2,3-DPG, a l l have an increase i n MCHC. This relationship between the change in 2,3-DPG and change in MCHC is demonstrated in Figure 11, Page 94. The increase in MCHC in this study i s contrary to the findings of Albert et a l . (1965) who reported that following exercise the diameter of red blood c e l l s increased by about one-half a micron due to the change of permea-b i l i t y of the c e l l membrane. However, Faulkner et a l . (1970) reported a compression of ce l l s as a result of exercise. A plausible explanation of the discrepancy of these results, which also appears applicable to this study, i s the work of Bellingham et a l . (1971) on the effects of induced alkalosis and acidosis. In the case of induced alkalosis, infusion of sodium bicarbonate produced a rapid increase in pH from 7.322 to 7.423 in the f i r s t hour, which | was followed by a slower rise to 7.450 during the next 24 hours. During the f i r s t hour of the acute change in plasma pH there was no alteration i n red c e l l 2,3-DPG however there was a change in oxygen a f f i n i t y and this corresponded to a significant increase of MCHC. (Similarly in induced acidosis there was a 94 F IGURE II R E L A T I O N S H I P B E T W E E N C H A N G E O F 2 , 3 D P G A N D C H A N G E IN M C H C A S A R E S U L T O F A O N E H O U R S T A N D A R D I Z E D B O U T O F E X E R C I S E + I50n r =-0.489 -100 +50 + 100 + 150 P50 F R O M 2 , 3 D P G ( T O R R ) 95 rapid f a l l i n MCHC). When acidosis of alkalosis was maintained, MCHC returned to normal levels but 2 , 3 - D P G levels were altered and correlated with changes in oxygen a f f i n i t y . Both MCHC and 2 , 3 - D P G changes produced a shift in the oxygen dissociation curve of hemoglobin which was opposite to the shift pro-duced by alterations i n pH so that, providing the pH change was not too rapid cir too large, P^ Q remained unchanged. A hypothesis, based on the results of this study and supported by the findings of Bellingham et a l . ( 1 9 7 1 ) , i s that MCHC changes occur rapidly in alkalosis so that P^Q remains unchanged while the 2 , 3 - D P G mechanism becomes activated. Of course there is obviously a limit to the change i n c e l l volume that can occur and large pH changes would be beyond the capabilities of this compensatory mechanism so that P^ Q would be altered u n t i l the 2 , 3 - D P G mechanism could readjust. When the 2 , 3 - D P G response is initiated i t i s hypothesized that MCHC drops, due to red blood c e l l swelling, i n proportion to the increase i n 2 , 3 - D P G . Because 2 , 3 - D P G i s an impermeable polyanionic molecule there may be a concomitant increase of cations within the c e l l when 2 , 3 - D P G levels increase, so that charge neutrality i s maintained (Brewer et a l . 1 9 7 2 ) . Increases of these charged molecules within the c e l l would obviously produce a swelling of the c e l l thereby lowering MCHC. There i s biochemical evidence that the free 2 , 3 - D P G concentration of the red blood c e l l plays a role i n membrane ion transport (Benesch and Benesch, 1967) and could thus influence the composition of i t s own ionic environment (Benesch, Benesch and Yu, 1 9 6 9 ) . A relationship has also been reported between 2 , 3 - DPG changes and red c e l l cation permeabil-i t y i n a study on chronic hypercapnia (Messier and Schaefer, 1 9 7 1 ) . i I To place.the changes in MCHC and 2 , 3 - D P G , as a result of exercise, in a physiologic perspective, changes in P_ n f a c i l i t a t i n g oxygen delivery have 96 i been calculated, using the equations of Bellingham et a l . (1971), and are presented in Table XXIV. TABLE XXIV of Theoretical Changes i n 7 ^  of the Oxygen Dissociation Curve Hemoglobin as a Result of Changes in 2,3-DPG and MCHC Produced Through One Hour of Exercise Change in P,.- Change i n P,-0 Total Change i n P 5 Q Group due to change due to change due to changes in in 2.3-DPG in MCHC 2.3-DPG and MCHC Sedentary Nonsmokers 0.13 Moderately F i t Nonsmokers -0.07 Highly F i t Nonsmokers 0.24 Sedentary Smokers 0.'31 Moderately F i t Smokers 0.24 A l l Subjects Pooled 0.17 0.23 0.26 0.08 0.03 0.43 0.21 0.36 0.19 0.32 0.35 0.67 0.38 Because changes in P^Q were a l l less than one Torr the physiological i effect i n improving oxygen delivery would appear to be of very low magnitude. Hypothesis 4, that a negative correlation exists between resting 2,3-DPG levels and the increase i n 2,3-DPG levels as a result of exercise, must be rejected on the grounds that Hypothesis i was rejected, i.e. there was no significant increase of 2,3-DPG as a result of exercise. However, 97 i direct evidence of a non significant correlation between pre exercise 2,3-DPG concentrations and change in 2,3-DPG as a result of exercise (r = -0.032) also corroborates the rejection of Hypothesis 4. Pre exercise 2,3-DPG levels apparently do not influence the change of 2,3-DPG that occurs as a result of exercise. Increased inhibition of 2,3-DPG mutase by higher levels of unbound 2,3-DPG either does not occur, or i f i t does occur, i t does not constitute a major control mechanism over 2,3-DPG production. Finally, when high work capacity subjects were compared with low work capacity subjects through post hoc analysis, no new observations were forthcoming. Significant increases in blood pH and MCHC occurred, with no significant change observed in 2,3-DPG. The changes that occurred i n these blood parameters were again not significantly different between groups, which indicates the responses were general in nature. (Post hoc analysis of change is presented in Table III and Table IV of Appendix C.) 1 CHAPTER V SUMMARY AND CONCLUSIONS Summary The purpose of this study was to examine the effects of acute exercise on the level of red blood c e l l 2,3-Diphosphoglycerate. Further, the study examined differences in 2,3-DPG concentrations among groups of smokers and nonsmokers of different fitness levels and examined relationships between 2,3-DPG and other blood parameters affecting oxygen transport: hemoglobin, hematocrit, MCHC and blood pH. A total of 40 university-aged males were involved in the experiment as subjects. Each subject was assigned to one of five groups, (eight subjects per group), based on the status variables of physical a c t i v i t y and cigarette smoking. Resting blood samples were drawn from each subject after a 12 hour fast. Each subject then performed the task of pedalling a bicycle ergometer for one hour at a heart rate of approximately 150 beats per minute (equal to 70 per cent of maximal aerobic capacity). Immediately upon cessation of exercise blood samples were again obtained from the antecubital vein of each subject. Within 30 minutes of blood sampling, biochemical determinations were j made on levels of 2,3-DPG, blood pH, hemoglobin, and hematocrit. MCHC was calculated by dividing hemoglobin by hematocrit. Multivariate analysis of results indicated that 2,3-DPG levels were not significantly increased (p = 0.087) as a result of one hour of exercise, in spite of a rise in blood pH. The fact that; 2,3-DPG changes were not related 98 99 to pH changes was interpreted as evidence of a difference in the rate of change between the two parameters, with 2,3-DPG changes following pH changes after a considerable time lag. A significant increase in MCHC, causing a right shift in the oxygen dissociation curve of hemoglobin independent of 2,3-DPG, was observed as a result of exercise. This change in MCHC was negatively correlated with the change in 2,3-DPG, indicating a specific inter-action between these two parameters. No differences were observed between groups compared orthogonally in changes of 2,3-DPG, blood pH or MCHC as a result of exercise. Changes in 2,3-DPG were not related to pre exercise levels of 2,3-DPG indicating that change of 2,3-DPG, produced through various physiological stimuli, i s not significantly affected by the amount of 2,3-DPG present before the stimuli i s presented. No significant differences were observed between groups compared orthogonally in pre exercise 2,3-DPG concentrations; however, a definite trend towards higher 2,3-DPG levels was observed as training intensity increased. Levels of 2,3-DPG were not related to fitness as measured by average work per heartbeat during one hour of exercise. As training intensity increased there was evidence of alterations i n red c e l l turnover, affecting the levels of MCHC, hematocrit and hemoglobin. A negative correlation between these three para-meters and 2,3-DPG indicated that as blood transport of oxygen i s reduced, either through reduction in MCHC or an increase in blood viscosity, 2,3-DPG levels r i s e to compensate,by producing a decreased a f f i n i t y of hemoglobin for oxygen. Differences between smokers and nonsmokers in relation to 2,3-DPG were not significant indicating that the hypoxia produced through smoking i s not an important stimulator of 2,3-DPG production. 100 Conclusions 1. A one hour bout of exercise, at approximately 70 per cent of maximal aerobic capacity does not produce a physiologically advantageous increase i n 2,3-Diphosphoglyerate levels of red blood c e l l s . 2. 2,3-Diphosphoglycerate levels were not significantly higher in highly trained subjects, nor lower in sedentary subjects, although a definite trend towards higher 2,3-DPG levels with increasing a c t i v i t y level was observed. 3. Cigarette smokers do not have higher red c e l l 2,3-DPG levels than nonsmokers. 4. No relationship exists between pre exercise 2,3-DPG levels and changes in 2,3-DPG produced through a one hour bout of exercise. 5. A negative relationship exists between blood 2,3-DPG levels and blood hemoglobin levels. BIBLIOGRAPHY ALBERT, S,, JAIN, S,, SHIBUYA, J . , and ALBERT, C . , The-Hematocrit i n C l i n i c a l  Practice. Springfield, I l l i n o i s : Charles C. Thomas Co. , 1965. 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WANG, Y . , SHEPHERD, J . , MARSHALL, R., ROWELL, L. and TAYLOR, H . , Cardiac Response to Exercise i n Unconditioned Young Men and In Athletes. Circulat ion, 24: 1064, 1961. WILLIAMS, C G . , WYNDHAM, C . H . , KOK, R. and VON RAKDEN, M . J . , Effect of Training on Maximum Oxygen Uptake and on Anaerobic Metabolism i n Man. Int. Z.  Angew. Arbeitsphysiol . , 24: 18, 1967. APPENDIX A S t a t i s t i c a l Analysis 110 I l l TABLE I F -Test* max Homogeneity of Dependent Variables Variable Variance Ratio F max 2,3-DPG 5.71/1.59 3.59 >.05 Hemoglobin 3.46/0.37 9.35 >.05 Hematocrit 8.64/1.99 4.34 >.05 MCHC 7.13/0.44 16.20 >.01 Blood pH 0.0036/0.0004 9.00 >.05 Work 2.19/0.24 9.13 >.05 C r i t i c a l F 7 X= 14.3 C r i t i c a l F n w m 7 r 2 4 - ° max ,05(10,7) max .01(10,7) * The F -Test re l ie s on the tabled cumulative probabil i ty max * ' d i s t r ibut ion of a s t a t i s t i c which i s the variance of the largest to the smallest of the several sample variances. The maximum variance ra t io , S 2 / S 2 max min was compared to the cumulative probability dis tr ibut ion of F m a x ( a , n - 1 ) , where a = 10 (five status variables-before and after exercise) and n = 8 (eight sub-I jects per group). The distr ibution is found i n Table T of a text of s t a t i s t i c a l i tables (Rohlf and Sokal, 1969). TABLE II Correlation Matrix of Dependent Variables Hmct Hmct Hb Hb MCHC MCHC 2,3-DPG 2,3-DPG pH (B) (A) (B) (A) (B) (A) (B) (A) (B) 1. Hmct (before) 1.000 2. Hmct (after) 0.882* 1.000 3. Hb (before) 0.842* 0.837* 1.000 • 4. Hb (after) 0.667* 0.806* 0.885* 1.000 5\ MCHC (before) 0.295 0.435* 0.760* 0.780* 1.000 -6. MCHC (after) 0.032 0.123 0.449* 0.684* 0.771* 1.000 7. 2,3-DPG (before) -0.557* -0.654* -0.672* -0.735* -0.524* -0.425* 1.000 8. 2,3-DPG (after) -0.415* -0.477* -0.528* -0.652* -0.436* -0.506* 0.884* 1.000 9. pH (before) -0.059 0.036 -0.074 -Q.065 -0.087 -0.171 0.063 -0.024 1.000 10. pH (after) -0.034 -0.110 0.111 0.103 0.210 0.262 -0.086 -0.165 0.087 11. Work (KPM/beat) -0.075 0.102 -0.036 0.190 0.048 0.202 -0.110 -0.234 -0.355** 12. Body Weight -0.108 0.064 -0.042 0.045 0.056 -0.012 -0.030 0.020 -0.252 13. A Hmct -0.294 0.192 -0.057 0.245 0.269 0.182 -0.168 -0.102 0.199 14. A Hb -0.529* -0.250 -0.450* 0.018 -0.137 0.347** 0.034 -0.116 0.034 15. A MCHC -0.402** -0.487* -0.518* -0.213 -0.425* 0.248 0.192 -0.056 -0.114 16. A 2,3-DPG 0.182 0.239 0.164 0.021 0.074 -0.264 -0.032 0.438* -0.173 17. A pH 0.174 0.110 -0.137 -0.124 -0.218 -0.321** 0.110 0.104 0.678* TABLE II (continued) pH Work Weight A Hmct A Hb A MCHC A 2,3-DPG A pH (A) i o . pH (after) 1.000 11. Work (KPM/beat) -0.090 1.000 12. Body Weight -0.034 0.490* 1.000 13. A Hmct -0.150 0.362** 0.356** 1.000 14. A Hb -0.041 0.441* 0.177 0.593* 1.000 15." A MCHC 0.054 0.216 -0.102 -0.151 0.701* 1.000 16. A 2,3-DPG -0.186 -0.289 0.102 0.105 -0.312** -0.489* 1.000 17. A pH 0.670* 0.197 0.162 -0.259 -0.055 0.124 -0.009 df = 38 (n-2) a .05 = 0.312 a .01 = 0.403 * * Correlation s ignif icant at the .05 l e v e l . * Correlation significant at the .01 l e v e l . 1 APPENDIX B I n d i v i d u a l Scores 114 TABLE I Blood Parameter Data of Sedentary Nonsmokers Before and After Exercise Subject DG ER JW MC BT DM BF RS anatocrit (%) Hemoglobin (gms/100 ml) MCHC 2,3-DPG 2,3-DPG (uM/ml blood)(uM/ml cells) 2,3-DPG (uM/gm Hb) pH 46.2 16.68 36.10 1.73 3.74 10.37 7.362 B 48.8 17.34 35.53 1.91 3.91 11.01 7.447 A 45.7 16.45 36.00 • 1.71 3.74 10.40 7.412 B 48.0 17.98 37.46 1.96 4.08 10.90 7.430 A 45.6 14.50 31.79 2.20 4.82 15.17 7.351 B 44.1 15.03 34.08 2.28 5.17 15.17 7.417 A 43.2 14.44 33.43 2.04 4.72 14.13 7.347 B 44.1 15.91 36.08 2.23 5.06 14.02 7.440 A 47.1 16.07 34.11 1.86 3.95 11.57 7.457 B 47.2 16.14 34.20 1.90 4.03 11.77 7.433 A 50.9 17.75 34.87 1.86 3.65 10.48 7.449 B 50.8 17.29 34.03 1.82 3.58 10.53 7.432 A 43.0 14.61 33.98 1.96 4.56 13.42 7.510 B 44.4 15.84 35.67 2.16 4.86 13.64 7.426 A 45.6 16.10 35.31 1.71 3.75 10.62 7.389 B 46.2 16.68 36.10 1.78 3.85 10.67 7.409 A TABLE II Blood Parameter Data of Moderately F i t Nonsmokers Before and After Exercise Su,bj ect JG JM RB LC BH DJ BK RM natocrit (%) Hemoglobin (gms/100 ml) MCHC 2,3-DPG 2,3-DPG (uM/ml blood) (uM/ml ce l l s ) 2,3-DPG (uM/gm Hb) pH 45.9 15.57 33.92 2.05 4.47 13.17 7.409 B 47.9 15.83 33.05 2.25 4.70 14.21 7.465 A 41.3 14.44 34.96 • 2.17 5.25 15.03 7.400 B 44.1 15.93 36.13 2.43 5.51 15.25 7.455 A 46.2 16.41 35.52 2.06 4.46 12.55 7.368 B 47.0 17.17 36.54 2.06 4.38 12.00 7.395 A 45.3 15.87 35.03 1.84 4.06 11.59 7.387 B 44.9 16.49 36.72 1.87 4.17 11.34 7.458 A 50.8 17.40 34.26 1.97 3.88 11.32 7.341 B 49.4 17.06 34.53 2.06 4.17 12.08 7.419 A 45.6 15.26 33.47 1.81 3.97 11.86 7.322 B 46.0 16.26 35.34 2.09 4.54 12.85 7.417 A 43.4 15.07 34.72 1.85 4.26 12.28 7.394 B 45.8 16.05 35.04 1.75 3.82 10.90 7.409 A 41.0 14.08 34.33 1.96 4.78 13.92 7.412 B 41.4 15.41 37.23 1.89 4.57 12.26 7.449 A TABLE I I I Blood Parameter Data of Highly F i t Nonsmokers Before and After Exercise Subject GL SD NV RH KF DK ED GB uatocrit (%) Hemoglobin (gms/100 ml) MCHC 2,3-DPG uM/ml blood) 2,3-DPG (uM/ml cel l s ) 2,3-DPG (uM/gm Hb) pH 42.1 13.08 31.07 1.87 4.44 14.30 7.388 B 42.7 13.08 30.63 . 2.08 4.87 15.90 7.413 A 42.9 12.53 29.20 2.12 4.94 16.92 7.330 B-42.7 12.45 29.16 2.27 5.32 18.23 7.431 A 43.3 13.14 30.34 1.69 3.90 12.86 7.368 B 44.9 14.31 31.86 1.64 3.65 11.46 7.447 A 41.1 11.76 28.62 1.67 4.06 14.20 7.334 B 41.9 13.43 32.04 1.80 4.30 13.40 7.385 A 46.8 16.14 34.49 2.14 4.57 13.26 7.327 B 48.5 16.60 34.23 2.35 4.84 14.16 7.393 A 46.3 15.72 33.95 1.88 4.06 11.96 7.327 B 47.7 16.29 34.16 2.04 4.28 12.52 7.351 A 44.5 15.53 34.90 1.80 4.04 11.59 7.315 V 46.6 16.29 34.97 1.91 4.10 11.72 7.388 A 47.0 16.49 35.00 1.85 3.92 11.22 7.378 B 48.0 17.02 35.46 1.99 4.14 11.69 7.519 A TABLE IV Blood Parameter Data of Sedentary Smokers Before and After Exercise Subject WM EK LH HR DJ KD DW TG aatocrit '(%) Hemoglobin (gms/100 ml) MCHC 2,3-DPG (uM/ml blood) 2,3-DPG (uM/ml ce l l s ) 2,3-DPG (uM/gm Hb) PH 48.6 17.25 35.50 2.00 4.12 11.59 7.388 B 48.5 17.60 36.28 1.99 4.10 11.31 7.426 A 45.5 16.46 36.18 2.31 5.07 14.03 7.282 B 44.9 16.33 36.37 2.35 5.23 14.39 7.450 A 47.2 16.64 35.25 2.25 4.77 13.52 7.326 B 46.5 16.18 34.80 2.24 4.82 13.84 7.421 A 45.5 15.76 34.64 1.86 4.09 11.80 7.374 B 47.5 16.91 35.59 2.14 4.51 12.66 7.456 A 45.0 15.22 33.83 1.79 3.98 11.76 7.414 B 46.3 16.41 35.44 1.91 4.12 11.64 7.512 A 48.3 17.67 36.59 1.98 4.10 11.21 7.413 B 48.8 17.98 36.83 2.26 4.63 12.57 7.441 A 52.1 17.33 33.26 1.81 3.47 10.15 7.366 B 52.5 17.84 33.98 1.91 3.63 10.71 7.394 A 43.3 14.66 33.86 1.92 4.43 13.10 7.386 B 44.2 15.02 33.98 2.05 4.64 13.65 7.415 A TABLE V Blood Parameter Data of Moderately F i t Smokers Before and After Exercise Subject Hematocrit Hemoglobin (gms/100 ml) MCHC 2,3-DPG 2,3-DPG 2,3-DPG (uM/ml blood) (uM/ml cel l s ) (uM/gm Hb) pH BM TT RU GH WL GT KC NT 46.0 16.33 35.50 2.23 4.85 13.66 7.343 B 49.0 18.09 36.92 2.70 5.51 14.93 7.366 A 48.0 16.49 34.35 • 1.84 3.83 11.16 7.283 B 47.3 17.33 36.64 2.14 4.52 12.35 7.416 A 46.0 14.92 32.43 2.26 4.91 15.15 7.403 B 47.2 15.75 33.39 2.52 5.34 16.00 7.267 A 50.3 17.21 34.21 2.46 4.89 14.29 7.407 B 51.1 18.28 35.78 2.61 5.11 14.28 7.392 A 49.0 16.91 34.50 2.24 4.57 13.25 7.406 B 48.8 17.60 36.06 2.29 4.69 13.01 7.428 A 48.5 17.06 35.17 2.46 5.07 14.42 7.382 B 47.1 17.44 37.03 2.29 4.86 13.13 7.487 A 46.3 16.64 35.94 2.01 4.35 12.08 7.318 B 47.0 17.47 37.17 2.10 4.47 12.02 7.426 A 47.3 17.29 36.55 2.30 4.87 13.30 7.370 B 48.0 17.60 36.67 2.52 5.25 14.32 7.450 A TABLE VI Physical Data of Sedentary Nonsmokers Subject Age Height Weight Average Work Ave.Heartbeat Average Work (Cm) (Kg) Per Min. (KPM) Per Min. Per Heartbeat (KPM) DG 25 177.2 90.9 1035.0 144.9 7.14 ER 25 180.3 89.4 1016.3 141.2 7.19 JW 24 182.2 88.5 786.3 148.0 5.31 MC 22 182.9 74.8 827.5 148.3 5.58 BT 25 177.8 71.1 577.5 141.9 4.07 DM 24 174.6 64.2 578.8 147.2 3.93 BF 26 182.2 60.6 472.5 152.5 3.10 RS 23 184.5 87.1 813.8 146.1 5.57 TABLE VII Physical Data of Moderately F i t Nonsmokers Subject Age Height Weight Average Work Ave.Heartbeat Average Work • (Cm) (KgJ Per Min. (KPM) Per Min. Per Heartbeat(KPM) JG 21 174.6 90.7 895.0 144.9 6.18 JM 34 182.9 87.3 1037.1 146.0 7.10 RB 31 180.3 82.4 1092.5 145.5 7.51 LC 21 180.3 84.4 923.8 148.1 6.25 BH 30 184.3 89.1 980.0 149.1 6.57 DJ 29 188.0 79.2 1051.3 147.3 7.14 BK 23 188.0 88.7 1021.3 148.9 6.86 RM 22 175.3 70.3 936.3 147.7 6.34 TABLE VIII Physical Data of Highly F i t Nonsmokers Subject Age Height Weight Average Work Ave.Heartbeat Average Work (Cm) (Kg) Per Min. (KPM) Per Min. Per Heartbeat (KPM) GL 22 179.7 67.5 965.0 154.1 6.26 SD 29 182.9 69.9 1146.1 156.0 7.35 NV 36 193.2 93.0 1236.3 129.7 9.53 ' RH 26 176.5 65.2 1222.4 147.0 8.32 KF 26 182.2 66.9 1285.0 142.7 9.00 DK 20 174.0 58.5 1078.8 145.7 7.40 ED 23 175.2 71.2 1230.0 144.5 8.51 GB 22 181.0 65.1 1052.5 138.3 7.61 TABLE IX Physical Data of Sedentary Smokers Subject Age Height Weight Average Work Ave.Heartbeat Average Work ' (Cm) (Kg) Per Min. (KPM) Per Min. Per Heartbeat (KPM) WM 28 190.2 83.7 752.5 149.9 5.02 EK 29 167.6 84.4- 839.8 148.8 5.64 LH 23 185.4 68.9 671.3 151.0 4.45 • HR 25 180.2 76.7 827.5 149.2 5.54 DJ 22 186.9 73.9 860.0 148.2 5.80 KD 22 186.5 88.9 . 622.5 148.1 4.20 DW 24 177.8 76.2 722.5 147.9 4.89 TG 33 188.6 87.2 801.2 143.5 5.58 TABLE X Physical Data of Moderately F i t Smokers Subject Age Height Weight Average Work Ave.Heartbeat Average Work (Cm) (Kg) Per Min (KPM) Per Min. Per Heartbeat (KPM) BM 27 188.0 112.9 885.0 146.9 6.02 TT 21 177.8 74.3 776.0 154.0 5.04 RU 22 174.6 79.4 923.7 152.0 6.08 GH 22 168.9 72.8 827.5 153.0 5.41 WL 24 176.8 79.3 981.3 145.1 6.75 GT 25 186.7 76.2 807.5 148.4 5.44 KC 32 181.3 76.8 760.0 147.0 5.17 NT 30 181.6 86.4 593.8 150.9 3.93 • APPENDIX C St a t i s t i c a l Comparison of High Work Capacity VS Low Work Capacity 125 126 TABLE I Means and Standard Deviations of High Work Capacity and Low Work Capacity Subjects for each Dependent Variable Variable High Work Capacity Subjects Low Work Capacity Subjects Hematocrit Pre Exercise Post Exercise 4 5 . 2 9 ± 2 . 5 7 4 6 . 5 5 ± 2 . 3 0 4 7 . 5 7 ± 2 . 2 7 47.7512.25 Hemoglobin Pre Exercise Post Exercise 15.33+1.68 1 6 . 1 2 ± 1 . 5 6 16.5611.00 17.04+0.90 MCHC Pre Exercise Post Exercise 33.7412.38 34.5712.12 34.7711.25 35.66+1.17 2,3-DPG Pre Exercise Post Exercise .12.6011.76 12.6912.02 12.3211.58 12.6011.45 Blood pH Pre Exercise Post Exercise 7.3610.03 7.42+0.04 7.3910.06 7.4310.02 Average Work Per Heartbeat During Exercise 7.6010.86 4.7410.75 Body Weight " " 78.41111.37 76.8318.73 127 TABLE II Orthogonal Comparisons of High Work Capacity Subjects VS Low Work Capacity Subjects on Pre Exercise Levels of Dependent Variables Variable df MS F P MCHC 1, 28 7.998 2.216 >.05 2,3-DPG 1, 28 0.614 0.219 >.05 Hematocrit 1, 28 38.783 6.608 <.05 Hemoglobin 1, 28 11.334 5.928 <.01 Blood pH 1, 28 0.006 2.984 <.05 Work per Heartbeat 1, 28 61.361 94.916 <.001 TABLE III Orthogonal Comparisons of Orthonormalized Change of Dependent Variables Due to Exercise Variable df MS F p MCHC 1,28 11.162 22.741 <.01 2,3-DPG 1,28 0.502 1.750 >.05 Blood pH 1,28 0.039 32.773 <.01 Hematocrit 1,28 7.783 14.544 <.01 Hemoglobin 1,28 6.074 42.642 <.01 128 TABLE IV Orthogonal Comparisons of High Work. Capacity Subjects VS Low Work Capacity Subjects on the Orthonormalized Change of Dependent Variables Due to Exercise Variable df MS F p MCHC 1, ,28 0.014 0.029 >.05 2,3-DPG 1, ,28 0.139 0.485 >.05 Blood pH 1, ,28 0.002 1.447 >.05 Hematocrit 1. ,28 4.283 8.002 <.01 Hemoglobin 1. ,28 0.354 2.487 >.05 

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