Relationship of 2,3-Diphosphoglycerate and Other Blood
RELATIONSHIP OF 2,3-DIPHOSPHOGLYCERATE AND OTHER BLOOD
PARAMETERS TO TRAINING, SMOKING AND ACUTE EXERCISE
By
Leonard Roy Marchant
B.P.E., University of British Columbia, 1971
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF PHYSICAL EDUCATION
in the School
of
Physical Education
and
Recreation
We accept this thesis as conforming to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
September, 1973 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study.
I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of pfrYstc&JL. ^oac/^r>&W
The University of British Columbia Vancouver 8, Canada i
ABSTRACT
The purpose of this study was to examine differences in 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 in relation to the variables of physical activity 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 fit subjects have significantly higher
2,3-DPG concentrations and sedentary subjects have significantly lower 2,3-DPG concentrations than moderately fit subjects; smokers have significantly higher
2,3-DPG levels than nonsmokers; exercise produces significant increases in
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. ii
A priori orthogonal comparisons of pre exercise red cell 2,3-DPG levels indicated that differences between groups were not significant, i.e. highly fit groups did not demonstrate 2,3-DPG levels significantly higher, nor did sedentary groups demonstrate 2,3-DPG levels significantly lower than moderately fit 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 in 2,3-DPG levels should not be totally rejected. Demonstration of differences in the carrying capacity of the blood, as reflected by differences in 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 is 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 is 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 Iii 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 in 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 is initiated. 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, statistician unsurpassed, for his help in analysis of the data; Dr. Joseph Angel and
Dr. David Randall for their expertise in matters biochemical and physiological respectively.
My appreciation is also extended to Dr. Melvin Lee and Dr. I. Desai, of the School of Home Economics, for providing research facilities used to conduct biochemical determinations.
Finally, a special thanks is 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
Statistical Analysis 49
IV. RESULTS AND DISCUSSION 52
Results. 52
Descriptive Statistics 52
Homogenity of Variance 67
Statistical 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. Statistical Analysis 110
B. Individual Scores 114
C. Statistical Comparison of High Work Capacity VS Low Work Capacity (Post Hoc) 125 vii
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 Levels.... 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 Levels...... 71
XVII Analysis of Change of Hematocrit as a Result of Exercise...... 72
XVIII Orthogonal Comparisons of Pre Exercise MCHC Levels...... 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 P5Q as a Result of 2,3-DPG and MCHC. 86
XXIV Theoretical Changes in ?-n as a Result of Changes in 2,3-DPG and MCHC 1. 96 viii
LIST OF FIGURES
Figure Page
1 The Oxygen Dissociation Curve of Hemoglobin 16
2 Red Blood Cell 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 all 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 cell 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 is a major constituent of mammalian red blood cells, causes a concentration dependent diminution of the oxygen affinity of hemoglobin by its binding to special sites on the beta chains of the hemoglobin molecule (Benescji et al., 1968). Many kinds of hypoxia appear to induce an increase of 2,3-DPG levels in red blood cells (Baumann et al.,1967; Eaton and
Brewer, 1968; Lenfant et al., 1968; Oski et al., 1969; Torrance and Bartlett,
1970; Valeri and Fortier, 1969). In all 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 it is obvious that the adaptive changes of 2,3-DPG in red blood cells are of special physiological importance. Exercise is 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 al., 1970;
Mitchell et al., 1958; Rowell, 1969). It has also been reported (Rowell et al.,
1964) that highly trained individuals have a reduced saturation of hemoglobin in the lung alveoli. Investigations have been made to determine whether or not increases in 2,3-DPG were responsible for these phenomena (Dempsey et al.,
1971; Eaton et al., 1969; Faulkner et al., 1970; Shappell et al., 1971); how• ever, the results are conflicting.
Several factors could have produced the ambiguity observed in the four studies dealing with the 2,3-DPG response to exercise. First, one would presume that a stress placed on a physiological system governs the response elicited. To determine the response mechanism, in this case changes in the concentration of red blood cell 2,3-DPG, all subjects should have been stressed equally, based on a criteria relating to their individual capabilities.
Secondly, it is possible that the basal, unstressed level of 2,3-DPG in the red blood cell might influence the amount of additional 2,3-DPG which can be produced when the system is placed under stress; especially if unbound
2,3-DPG inhibits the enzyme (DPG mutase) responsible for further 2,3-DPG synthesis (Benesch et al., 1970). It appears that training may increase basal levels of 2,3-DPG (Shappell et al., 1971), which could account for a lack of response when stress, in the form of exercise, is placed on highly trained individuals (Dempsey et al., 1971). 3
Third, it is possible that smoking could affect basal levels of
2,3-DPG. Smokers tend to have a left shift in their oxygen dissociation curve caused by binding of carbon monoxide (from cigarette smoke) to hemoglobin
(Lenfant et al,, 1970). Therefore an increase of 2,3-DPG would tend to comp• ensate for the effect of smoking by moving the curve back to its normal position.
The small size of samples used in previous studies, ranging from three to ten subjects, could also be a factor leading to the incongruity of the results. Individual differences in numbers of red blood cells, 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 if larger sample sizes were utilized.
In conclusion, it is apparent that in order to ascertain whether or not the response of the 2,3-DPG mechanism to exercise-induced hypoxia is a
significant physiological phenomenon, a study using a larger number of subjects9 with subgroups of different fitness levels, stressed at an equal percentage of their maximum capacity, is necessary,
Statement of the Problem
The purpose of this study is to investigate the effects of acute exercise on the levels of red cell 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 if red blood cell 2,3-DPG increases as a result of training, thereby facilitating oxygen delivery for a given amount of hemoglobin,
2. To determine if red blood cell 2,3-DPG is increased in smokers, compensating for a left shift in the oxygen dissociation curve due to carboxy- hemoglobin.
3. To determine if an increase in 2,3-DPG occurs during a strenuous one hour bout of exercise, facilitating oxygen delivery to active muscles,
4. To determine relationships, if any, between red blood cell 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 in 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 eliciting 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 eliciting an average heart rate of approximately 150 beats per minute, produces an increase in red blood cell 2,3-diphosphoglycerate levels in all subjects.
2. The mean resting level of red blood cell 2,3-diphosphoglycerate is highest in the highly trained subjects and lowest in the sedentary subjects.
3. The mean resting level of red blood cell 2,3-diphosphoglycerate is higher in smoking subjects than in nonsmoking subjects of the same fitness level.
4. The increase in red blood cell 2,3-diphosphoglycerate, as a result of exercise, is larger in those subjects with lower resting 2,3-DPG levels. That is, there exists a negative correlation between resting 2,3-DPG levels and the increase in 2,3-DPG levels due to exercise.
5. A negative correlation exists between 2,3-DPG levels and blood hemoglobin levels. That is, subjects with high hemoglobin levels have low red blood cell 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 HCOPO3H2
I
CH2OPO3H2
2,3-diphosphoglycerate is bound preferentially to deoxyhemoglobin and in so doing, affects the affinity of hemoglobin for oxygen so that an increased amount of oxygen will be released at a constant partial pressure of oxygen.
The reaction may be summarized as follows:
HbDPG + 02 J Hb02 + 2,3-DPG.
Hematocrit. The volume occupied by packed red blood cells in a given quantity of blood after centrifugation in a special graduated tube.
The parameter is 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 is made up of about 10,000 atoms, including four atoms of Iron. Each atom of iron lies at the center of the group of atoms that form the pigment called heme-, which gives blood its red color and its ability 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 is 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 in the ability of the blood to dissolve and transport oxygen. Each of the four atoms of iron in the hemoglobin molecule can take up one molecule (two atoms) of oxygen. The reaction is reversible in the sense that oxygen is taken up where it is plentiful, as in the lungs, and released where it is scarce, as in the tissues.
Hb + 02 J Hb02
Mean Corpuscular Hemoglobin Concentration (MCHC). An expression, in absolute terms, of the average hemoglobin concentration per unit volume (per
100 ml) of packed red blood cells, calculated from the equation
MCHC _ hemoglobin (gm/100 ml) x 100 hematocrit and stated in grams per 100 ml of packed red blood cells.
Oxygen Dissociation Curve of Hemoglobin. A graphical presentation of the relationship between the amount of oxygen bound to hemoglobin vs. the
partial pressure of oxygen in the blood (P02). The blood leaving the lungs
usually has a P02 of about 100 Torr, and the amount of hemoglobin that is
bound with oxygen at this P02, called the hemoglobin saturation, is about 97 i
per cent. On the other hand, in normal venous blood the P02 is about 40 Torr and the per cent saturation is 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 is reduced to approximately 14.4 ml (PO^ of 40 Torr, 75% saturated) or a total loss of about five millilters of oxygen from 100 millilters of bloodi In exercise, two mechanisms operate upon the hemoglobin of the red blood cell to increase oxygen delivery to the tissues. First, the interstitial
fluid P02 may fall 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 affinity of hemoglobin,requir•
ing an increased P02 to maintain the bond between the two molecules. Because
the P02 at the tissues is low, much more oxygen will dissociate from the hemoglobin when the affinity is 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 it occurs multiplied by the percentage of the total volume that is occupies. Thus, if the total pressure of all the atmospheric gases is about 760 Torr, and if the oxygen content is
about 20 per cent of this mixture, then the partial pressure of oxygen (P02) in the atmosphere is equal to 760 x 0.20, or 152 Torr.
PJ.Q This parameter is utilized by physiologists to provide a frame of reference for the relative position of the oxygen dissociation curve of hemoglobin. It is the partial pressure of oxygen in the blood at which 50 i per cent of the hemoglobin is saturated with oxygen (ie one-half of the hemoglobin is bound to oxygen, one-half the hemoglobin is unbound).
Kilopond Meter (kpm). One kp is the force acting on the mass of one kg at normal acceleration of gravity. One kpm is the work done in moving one kp a distance of one meter. 9
Delimitations
1. The data under consideration in this study is based upon blood samples taken from venous blood of the upper limbs of the subjects. Interpret• ation of the data is 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 in humans - the amount of oxygen carried by the blood and the rate at which it is carried. This study is concerned only with the first mechanism, therefore any conclusions will be based only on a partial investigation of blood oxygen transport, with the relationship to the blood flow response remaining unanswer• ed.
Limitations
1. The investigation is 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 criteria of five per cent, then two additional determinations were made.
For statistical analysis, the mean value of two determinations, varying by less than five per cent, was utilized. The study is 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 is required to elicit changes in the concentration of red blood cell 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 is required to elicit 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 cell 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 is known that facilitation 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 is also known that 2,3-DPG facilitates oxygen delivery by causing a right shift in the oxygen dissociation curve of hemoglobin in 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 is responsible for the shift that occurs during exercise. This investigation is an attempt to clarify 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 life to continue. There are several mechanisms for increasing the capacity of the red blood cell to deliver oxygen to metabolically active tissue, namely, an increase in hemoglobin concentration or in the number of red blood cells (ERYTHROPOIETIC
RESERVE), an increase in the rate of blood flow (FLOW RESERVE), a slight increase in the oxygen concentration of arterial blood which is accomplished only by hyperventilation in normal, healthy individuals (RESPIRATORY
RESERVE), and lastly, an alteration in the hemoglobin affinity 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 is 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 initiated, an increased affinity for oxygen is 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 is determined experimentally with
respect to the partial pressure of oxygen, the curve so obtained (called
the oxygen dissociation curve of hemoglobin) is found to be sigmoidal.
Physiologically, this means that hemoglobin is an efficient transport system.
In the lungs a high oxygen tension forces oxygen onto the low affinity deoxy
structure and converts it to the high affinity oxy structure. In the tissues
a low oxygen tension causes the oxygen to be released and the structure to
revert to the low affinity 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 in 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 partial pressure
of oxygen (Po2). A rise in blood temperature from 37 to 41 degrees, at
constant pH (7.2) and venous Po2 (27 Torr), was seen to cause a decrease in venous blood oxygen saturation from 30 to 20 percent (Shappell et al., 1971).
Recently, Lenfant et al.,(1972) have shown that the temperature effect on binding of oxygen to hemoglobin is reduced with increasing concentrations
of 2,3-diphosphoglycerate (2,3-DPG). The corollary of this relationship is,
as pointed out by Benesch et al.(1969), the effect of 2,3-DPG in lowering the
oxygen affinity of hemoglobin decreases with increasing temperature. 13
The Bohr Effect. In vivo, both the partial pressure of carbon dioxide (PCO2) and pH affect the affinity of hemoglobin for oxygen. The
Bohr effect is defined as the difference in the total charge carried by oxy and deoxyhemoglobin at constant pH (Kilmartin, 1972). Three amino acid residues have been implicated in 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 (1972) is defined as the increase in the amount of oxygen delivered from 100 milliliters of blood when acidification by 0.1 or 0.2 pH units takes place at 50 percent hemoglobin saturation. The influence of the Bohr effect on the oxygen affinity, expressed as P^Q (i.e. the oxygen partial pressure at which 50 percent of hemoglobin is 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 is 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 al.(1972) pointed out that alterations in pH play the major role in 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 C02
and pH). They found a decrease in 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 in unloading of oxygen by 26 percent observed by Doll et al. (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 al. (1969) who found a decrease in the Bohr effect with increasing 2,3-DPG.
The 2,3-diphosphoglycerate Effect. A third mechanism of shifting the hemoglobin oxygen dissociation curve is mediated through the concentration of inorganic phosphates within the red blood cell. It was known for several years (Greenwald, 1925) that large quantities of 2,3-DPG are present in the red blood cells of pigs. Later Rapoport and Guest (1940) reported that high levels of 2,3-DPG are also present in human erythrocytes. However, it was not until 1967 that the physiological role for 2,3-DPG was clarified. Benesch and Benesch (1967) and Chanutin and Curnish (1967) reported independently that the oxygen affinity of human hemoglobin could be altered by the addition of 2,3-DPG, suggesting that Z.3-DPG performed a regulatory function in oxygen transport by the red blood cell.
The change in oxygen saturation brought about by 2,3-DPG at high oxygen tensions is comparatively small so that little effect is observed on
oxygen loading in the alveoli of the lungs. In contrast at a Po2 value of
30 Torr, which is 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 concentration of 2,3-DPG. At an extracellular pH of 7.40 a change of P^_ of one Torr resulted from a change of about
0.4 uMoles 2,3-DPG per gram of red blood cells (RBC). The same increase in P^Q at 2,3-DPG .concentrations above 8 uM/g RBC occurred only when the 2,3-DPG concentration was changed by about 1.5 uM/g RBC. It was concluded that 2,3-DPG exerts a dual effect on the oxygen affinity of human red blood cells.
The specific interaction of 2,3-DPG reached its maximal effect at 2,3-DPG concentrations of about 8 uM/g RBC. The second effect was an unspecified one, due to intracellular pH changes.
This indirect effect, which is common to all non-penetrating anions, was solely responsible for the further decrease of the oxygen affinity of blood at 2,3-DPG concentrations exceeding
8 uMoles per gram red blood cells.
The Binding of 2,3-diphosphoglycerate to Hemoglobin
Mole for mole 2,3-diphosphoglycerate binds preferent• ially with deoxyhemoglobin (deoxy HbA) in normal adults, not with fully oxygenated HbA. This binding is assumed to occur in the central cavity of the two beta chains of the tetrameric hemoglobin molecule, with the formation of salt bridges involving the two N-terminal amino groups, histidine 143 and lysine 82
(Perutz, 1970). It has been suggested that the entrance to the central cavity of the beta chains is too small to admit the DPG molecule, but as a result of the six angstrom increase in the size of the cavity entrance which occurs upon deoxygenation,
(Muirhead et al., 1967) the nine angstrom 2,3-DPG molecule could 16
i
FIGURE I CJ «S t— m THE OXYGEN DISSOCIATION CURVE uj olo OF HUMAN BLOOD AND SOME 8 S3SI VARIABLES INVOLVED IN OXYGEN SUPPLY TO PERIPHERAL CAPILLARIES CE UJ X _J — U-UJ LJ cr o OH oo
100 ARTERIAL pH 7.44
—I V 90 Ca02-Cv02 > =0.041 80 (21%)
70
CM 60 O I
-o 50
*40 30
20
10
0L 100
P02-TORR
THE FIGURE IS BASED ON DATA OF NORMAL, RESTING INDIVIDUALS.
THE ABSCISSA IS IN UNITS OF OXYGEN TENSION (TORR) WHEREAS THE
ORDINATE IS EXPRESSED BOTH IN PERCENT 02 SATURATION AND 02 CON•
CENTRATION. THE OXYGEN AFFINITY FOR BLOOD IS INDICATED BY l°Q= 27 TORR.
THE OXYGEN CAPACITY IS 19.6 VOL % (0.196 ML. 0^/ ML BLOOD). THE
ARTERIOVENOUS OXYGEN DIFFERENCE ( Ca02 - Cv02) IS 4 . 1 VOL %( 0.041 ML
02/ML BLOOD), EQUIVALENT TO 21 PERCENT EXTRACTION BY TISSUES. THE OX•
YGEN TENSION IN MIXED VENOUS BLOOD .(Pv02) IS 39 TORR. THE THREE
RESERVE MECHANISMS SHOWN ACT TO INCREASE OXYGEN TRANSPORT INDE•
PENDENT OF BLOOD FLOW AND WITHOUT A DROP IN P02 . ADAPTED FROM
METCALFE AND DHINDSA (1972).- 17 easily be accomodated by deoxyhemoglobin (Brewer and Eaton,1971).
This mechanism would explain the apparent partial competition between oxygen and 2,3-DPG.
The increase in P5Q, or the right shift in the oxygen dissociation curve, produced in the presence of 2,3-DPG in the red blood cell is thought to be caused by an increase in the rate
of dissociation of Hb02 to Hb and 02; with the overall process including a reaction of 2,3-DPG with the hemoglobin molecule, an interaction between the binding sites of 2,3-DPG and those of oxygen, and finally the subsequent net dissociation of oxygen from hemoglobin (Forster, 1972), Bauer (1972) concluded that the influence of 2,3-DPG (and hydrogen ions) on the oxygen affinity of hemoglobin was brought about by a change of both the assoc• iation and dissociation velocity constants of the hemoglobin- oxygen reaction, with the former decreasing and the latter increasing with increasing concentrations of the substrate.
The reaction of oxygen with a single hemoglobin molecule
(composed of tetrameric subunits) has been demonstrated to proceed as follows by Perutz (1970);
lc. k * lc«
a a (aia23ie2) X ( i°20i23i32) X (ai02a2o23i 32) X ( i°-a2023i0232) !4
•*- (ai02a2o23i023202)
where k^, k2, k^, k^ signify the first, second, third and
fourth association velocity constant respectively and aia2 and
3i32 signify the alpha and beta subunits of hemoglobin. 18
Tyuma (1972) found that 2,3-DPG reduces and k2 to about one-tenth and reduces k» much more, however k. was insen- 3 4 sitive to the phosphate and also to pH changes in the range of
7.0 to 7.8, irrespective of the presence or absence of 2,3-DPG.
This lack of response of k^ to 2,3-DPG was consistent with the results and ideas described In two recent papers (Gibson, 1970;
Perutz, 1970). In one, it was shown that inorganic phosphate increases the rate of dissociation of the second, third and fourth molecules leaving oxyhemoglobin A without affecting the rate of the first molecule (Gibson, 1970). In the other, Perutz
(1970) proposed a model for the sequence of co-operative binding of hemoglobin A in which 2,3-DPG combined with deoxy HbA is expelled after the second heme group has been oxygenated.
Several factors, other than the interaction with oxygen affect the binding of 2,3-DPG to hemoglobin. Blood pH, carbon dioxide and hemoglobin concentration would appear to be those of greatest physiological importance.
The Blood pH Effect. The difference in binding between
Hb02 and Hb was seen to decrease from a value of about 0.3 moles per mole Hb tetramer at pH 7 to less than 0.1 moles/mole Hb tetra- mer at pH 7.7 (Garby and deVerdier, 1972). The pH dependence of the data was accounted for on the basis of oxygen linked binding of 2,3-DPG to the residues B-terminal valine, histidine 143 and lysine 82. These residues were found to have pKa values of 7.6,
6.8 and 10.5 respectively; a situation which would make binding at pH 8 very small. In addition, the 2,3-DPG molecule was found 19
to have two negatively charged groups with an average pKa of about
7.1 which would account for the vanishing binding of 2,3-DPG to hemoglobin towards pH 6.
The Carbon Dioxide Effect. Recent data of Bauer (1970) and Tomita and Riggs (1971) showed that 2,3-DPG interferes with
the carbamino binding to hemoglobin and that C0^ inhibits the
effect of 2,3-DPG on oxygen affinity. The difference in binding of 2,3-DPG between the two states of oxygenation of hemoglobin
decreases in the presence of CO2. All C02 bound to hemoglobin
is in the negatively charged carbamate form (ie RNHCOO ) and, as part of this is believed to occur at the two N-terminal groups of the 3 chains, it would be expected that competition would
exist between the binding of C02 and 2,3-DPG. Rossi-Bernardi et al. (1972) found that addition of one mole 2,3-DPG/ mole Hb lowers the amount of oxygen-linked carbamate by about one-half. When the proportion of 2,3-DPG was reduced to 0.5 mole/mole Hb an inter• mediate value was observed. Furthermore under approximately physiological conditions, reduced Hb appeared to have a greater
affinity for 2,3-DPG than for C02. This likely accounts for the descrepancy of the previous data of Rossi-Bernardi and Roughton
(1967) who, when using dialyzed Hb, showed that HbC02 is respon•
sible for 25-27 percent of the physiological carbon dioxide
transport by the blood. Their recent evidence'indicates that this value was approximately 40 percent too high when whole blood,
containing 2,3-DPG, was used(Rossi-Bernardi, 1972). 20
The Hemoglobin Concentration Effect. With increasing
hemoglobin concentration there is a decreased affinity of 2,3-DPG
for deoxygenated hemoglobin (Garby and deVerdier, 1970). Under
conditions prevailing in the intact cell about 15 percent and 35
percent of the total 2,3-DPG was bound to fully oxygenated and
deoxygenated cells respectively. Therefore with higher concen•
trations of hemoglobin there is a reduced amount of binding per mole hemoglobin so that the overall amount bound remains relatively
constant. The difference in binding between Hb02 and Hb was
calculated to be 0.2 to 0.3 moles per mole Hb tetramer under
physiological conditions, with a maximum value at very low Hb
concentrations of 0.3 to 0.4 moles per mole Hb tetramer.
It would appear that at a higher mean cell hemoglobin
concentration oxygen affinity of hemoglobin is reduced independent
of 2,3-DPG. Numerous, sets of data have established that either
changes in P^Q can occur in the absence of a 2,3-DPG change or
that changes in 2,3-DPG are not necessarily accompanied by a
change in P^Q* Because hemoglobin concentration has been shown
to influence the affinity of hemoglobin for oxygen (Benesch et al.
1969), Lenfant et al. (1972) examined the possibility of this
factor being responsible for the many paradoxical findings. It
was found that MCHC did affect P5Q independent of 2,3-DPG,
agreeing with the data of Shappell et al.(1971) and Bellingham
et al. (1971) that the relationship between P,.-. and hemoglobin
concentration was linear. When Lenfant et al.(1972) corrected
P^0 to a MCHC of 33 percent the linear relationship between 2,3-
DPG and P^n was identical for all subjects. Recent data of 21
Forster (1972) indicated that the relationship between P Q and
MCHC was influenced by 2,3-DPG concentrations such that the greater the 2,3-DPG concentration the greater the coefficient
AP5Q/AHb.
The Postulated Control of the 2,3-DPG Response Mechanism.
The level of 2,3-DPG in the red blood cell is regulated by biochemical mechanisms inherent within the cell and physiological influences arising from outside the cell. While these two factors are Inseparable within the intact cell they have been discussed separately to provide a clearer understanding.
The Biochemical Control of 2,3-DPG Concentrations.
Brewer et al. (1972) suggested an increase in 2,3-DPG in the erythrocyte comes about either by an increase in glucose consumption and glycolytic rate or by a shift of glycolytic flow through the Rapoport-Luebering DPG shunt at the expense of the ATP-generating phosphoglycerate kinase reaction (Figure 2).
Under physiological conditions about 80 per cent of 1,3-diphospho- glycerate (1,3-DPG) is directly decomposed to 3-phosphoglycerate
(3 PGA) in the phosphoglycerate kinase reaction. Only 15 to 20 per cent of the total glycolytic flux passes through the 2,3-DPG pool (Gerlach and Duhm, 1972). Rapoport et al.(1972) has observed that the maximal activities of both enzymes controlling concentrations on 2,3-DPG, DPG mutase and DPG phosphatase, permit reaction rates that are 1-5 percent of the glycolytic rate.
However, in the intact cell these rates reach values up to twice 22 the rate of glycolysis. Therefore, additional factors modifying the activity of DPG mutase have to be postulated. Because
2,3-DPG acts as a product inhibitor of DPG mutase (Rose, 1970) it was suggested that a relief of this product inhibition due to increased binding of 2,3-DPG to deoxyhemoglobin might be the key factor in the acceleration of 2,3-DPG synthesis in deoxygenated cells (Benesch and Benesch, 1967; Eaton and Brewer, 1968). Such a mechanism has been considered to be very effective under the condition of hypoxia, which induces a diminution of the concentration of free 2,3-DPG due to its greater binding to deoxyhemoglobin (Oski et al., 1970). However, more recent studies indicate that binding by hemoglobin is not sufficient to explain the high rates of synthesis. Gerlach and Duhm (1972) investigated this problem under special conditions in intact human red cells. With no difference in intracellular pH between deoxy and oxyhemoglobin the rates of synthesis were almost identical, Indicating that In intact red blood cells product inhibition of DPG mutase is not influenced by concentration changes of free 2,3-DPG resulting from the different binding of
2,3-DPG to oxy and deoxyhemoglobin.
The rate of 2,3-DPG synthesis depends first of all on the concentration of 1,3-DPG. The level of this compound is controlled by two factors: (a) the glyceraldehyde-3-phosphate dehydrogenase reaction, which depends on the concentrations of
glyceraldehyde-3-phosphate (GA-3-P), NAD and orthophosphate (Pi);
(b) the concentrations of ADP, ATP and 3 PGA which, as reactants of the phosphoglycerate kinase reaction, are in equilibrium with 23
FIGURE 2
THE MAJOR PATHWAY OF CARBOHYDRATE METABOLISM IN MATURE
MAMMALIAN RED BLOOD CELLS . ENZYMES AFFECTING 2,3 DIPHO-
SPHOGLYCERATE CONCENTRATIONS ARE PRESENTED, ALONG WITH
FACTORS KNOWN TO AFFECT THEIR ACTIVITY.
GLUCOSE-I- PHOSPHATE f NADP, NADPH NADP NADPH 1 HEXOKINASJ" GLUCOSE- GLUCOSE - 6 - PHOSPHATE 6 PHOSPHOGLUCONATE PENTOSE PHOSHATES ADP FRUCTOSE-6- PHOSPHATE ^ACTIVATED BY: ATP V AMP, AOP, FOP AND INCREASE IN pH IPHOSPHQFRUCTOKINASE I INHIBITED BY'. • ATP, CITRATE AND DECREASE IN pH - DIPHOSPHATE
ACTIVATED BY'. I DIHYDROXYACETONE GLYCERALDEHYDE - 3 1, 3 OPS , 3 PGA PHOSPHATE PHOSPHATE (GA- 3 - P) AND INCREASE X IN pH I— NAD • \ < Pi GLYCERALOEHYOE - 3 INHIBITED BY: Q_ PHOSPHATEDEHYDROGENASE 2,3 DPS AND NADH- ORTHOPHOSPHATE '1,3 DIPHOSPHOGLYCERATE' ( 1,3 DPG ) DPG MUTASE 2,3 DIPH0SPH0 O ADP GLYCERATE X 3 PHOSPHOGLYCERATE ATP KINASE (2,3 DPG) or 3 PHOSPHOGLYCERATE^ LU (3 PGA ) 7F 3 DIPHOSPHOGLYCERATE >- PHOSPHATASE UJ PHOSPHOGLYCERATE MUTASE i INHIBITED BY'. LU 2 PHOSPHOGLYCERATE 3 PGA Q ACTIVATED BY. CO DECREASE IN pH, ORTHOPHOSPHA TE UJ PHOSPHOENOLPYRUVATJ E THE RAPOPORT- Ul ADP X LEUBERING SHUNT PYRUVATE ATP KINASE PYRUVATE
NADH- LACTATE DEHYDROGENASE NAD-^- -ALACTAT E 24
1,3-DPG (Gerlach and Duhm, 1972). On the basis of these
considerations, an increase of the 1,3-DPG concentration and
consecutive elevation of the 2,3-DPG levels were obtained when
GA-3-P, inorganic phosphate and NAD were made available in large
amounts (Deuticke et al., 1971; Deuticke and Duhm, 1972).
Furthermore, a small reduction of ADP was found to induce an
increase of the 1,3-DPG concentration followed by a greater
formation of 2,3-DPG (Duhm et al., 1968)
Rapoport et al. (1972) suggested that a high concentra•
tion of inorganic phosphate acts primarily to release the
inhibition of phosphofructokinase, which is the major control
point of glycolysis of most mammalian tissue. Finally, Brewer
and Eaton (1971) hypothesized a system in which 2,3-DPG inhibits
the action of hexokinase, the enzyme which initiates glycolysis
and which may be important as a rate limiting enzyme in red blood cell glycolysis.
The Physiological Control of 2,3-DPG Concentration.
It appears that numerous physiological and pathalogical
conditions evoke an increase in 2,3-DPG, which in turn mediates
a displacement to the right of the oxygen dissociation curve.
These conditions are, in nearly all instances, characterized by
hypoxia and/or acid-base disturbances. Among the numerous
factors which can influence 2,3-DPG metabolism alterations in
blood pH and changes in the oxygenation state of hemoglobin are
thought to be mainly responsible for the adaptive concentration 25 r changes (Gerlach and Duhm, 1972). 2,3-DPG levels are known to increase after an elevation of blood pH (Asakura et al., 1966), whereas a decrease in pH causes a decrease in 2,3-DPG concentra• tion (Astrup, 1970; Guest and Rapoport, 1941; Rorth, 1970). The biochemical mechanism whereby pH affects 2,3-DPG levels in vivo has not been elucidated so far. The affect appears to be due to an increased glucose consumption mediated through a pH activation of phosphoglycerate kinase (PFK) and DPG mutase (Rapoport and
Leubering, 1952; Brewer, 1972b),
The intracellular mechanism of change responds to influences arising outside the cell. For example, the intra• cellular pH is ultimately determined by the extracellular pH.
Therefore all the extracellular factors which influence pH will have some effect on 2,3-DPG levels. For example, hyperventila• tion will lower CO^ levels, increase pH and increase 2,3-DPG levels. Metabolic acidosis and alkalosis will likewise have their respective effects (Brewer et al., 1972).
In order to demonstrate that the hypoxia-induced rise of the blood pH is essential for the elevation of 2,3-DPG,
Gerlach and Duhm (1972) exposed rats to gas mixtures of low oxygen content containing five per cent C02« It was found that the hypoxia-induced rise of 2,3-DPG does not occur in the presence of five per cent CO2, which prevented the occurrence of respiratory alkalosis. On the other hand, exposure of the animals to a gas mixture of normal oxygen content with five per cent CO- resulted in a moderate decrease of 2,3-DPG levels due 26
to the induced respiratory acidosis. The importance of respiratory alkalosis was also shown by Lenfant et al.(1970) who observed that in men the documented rise of 2,3-DPG at high altitude (Lenfant et al., 1968; Rorth, 1972; Torrence et al.,
1970) does not occur after the application of diamox which prevents the hypoxia-induced alkalosis. At high altitude the increase of 2,3-DPG was found to be 4.7 uMoles/gram Hb for an increase of 0.1 plasma pH units. Similarly, at sea level, normal subjects made acidic showed a decrease in 2,3-DPG of 2.4 uMdles/ gram Hb for a 0.1 decrease in plasma pH. The difference in the rate between the two conditions was accounted for by the increase in deoxyhemoglobin present at altitude (Lenfant et al., 1972).
Although a pH change occurs almost instantaneously, the effect of pH on 2,3-DPG synthesis is much slower. Bellingham et al.(1971) showed that when pH was increased from 7.32 to 7.40 within one- half hour, the 2,3-DPG concentration did not increase until two to three hours after the onset of the pH change.
From these results it is apparent that blood pH plays an important role in inducing concentration changes of 2,3-DPG in hypoxic hypoxemia. However 2,3-DPG concentration also changes in those kinds of hypoxemia in which blood pH remains at normal values or increases only slightly. It has been shown that the rate of 2,3-DPG synthesis is higher in deoxygenated than in oxygenated cells (Duhm and Gerlach, 1971); and in anemia an increased arterial desaturation and presumably an increased venous desaturation exists (Brewer et al., 1972). Since deoxy• hemoglobin is more alkaline than oxyhemoglobin (the Haldane 27 effect), chronic desaturation will therefore mean a relative intracellular alkalosis. In addition, it has been found that the difference between intracellular and extracellular pH (ApH) is smaller in deoxyenated blood (Duhm and Gerlach, 1971). For example, at an extracellular pH of 7.40, deoxygenation caused the ApH to decrease from -0.2 to -0.13. This means that the intracellular pH rises from 7.20 to 7.27 upon deoxygenation.
Although the shift of the intracellular pH of 0.07 units to the alkaline side seems to be rather small, it nevertheless exerts a remarkable influence on 2,3-DPG metabolism. It has been estimated that an elevation of the intracellular pH of this magnitude causes the 2,3-DPG concentration to increase by about 25 per cent
(ie 1 uMole 2,3-DPG/ml red blood cells) (Astrup, 1970). This agrees with Rorth and Brahe (1972) who found an increase in pH of
0.04 units will increase the 2,3-DPG level by 15-20 per cent.
Furthermore, as the 2,3-DPG increases, the resulting lowered oxygen affinity will cause the relative desaturation at a given
P02 to be greater, causing a further relative intracellular alkalosis (Gerlach and Duhm, 19720.
In summary, red cell pH is the essential mechanism which has been demonstrated to contribute to the elevation of red cell 2,3-DPG in various kinds of hypoxia. In acute hypoxic hypoxia, the intraerythrocytic pH rises due to an increase in blood pH caused by hyperventilation. In chronic hypoxic hypoxia or anemia hypoxia, elevation of intracellular pH is due to an increase in deoxyhemoglobin. Limitation of 2,3-DPG concentra• tion is mainly brought about by a feedback mechanism, which again 28 depends on changes of red blood cell 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 cell 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 in rats made polycythemic by intraperitoneal injection of erythrocytes. From these and other experiments it 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 al. (1972) hypothesized that the level of 2,3-DPG may have a deterministic effect on the mean corpuscular hemoglobin concentration (MCHC).
The red blood cell membrane is not permeable to 2,3-DPG, which comprises a large component of the total non-diffusable molecules within the cell. 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 in an increase in cell volume because of an increase in intra• 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 is to postulate a causal effect of MCHC on 2,3-DPG. The oxygen affinity of hemoglobin is concentration dependent exclusive of 2,3-DPG effects 29
(Benesch et al., 1969), even in the physiological range of hemoglobin concen• tration (Bellingham et al., 1971). 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 al., 1972). In examining the data it was
found that MCHC did vary and did affect P5Q 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 al. (1971) and Shappell et al.
(1971). When P^Q was corrected to a standard MCHC value (33 per cent), the relationship between P^Q and 2,3-DPG was found to be identical for all subjects (Lenfant et al., 1972). It is believed that changes in hemoglobin concentration induce changes in the conformation of the individual hemoglobin molecules, thereby affecting the affinity each molecule has for oxygen. Brewer et al. (1972) suggested that a change in conformation manifested by a lower
MCHC is 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 in Long Standing Hypoxias
Elevations in 2,3-DPG, with a corresponding right shift in the oxygen dissociation curve of hemoglobin to improve oxygen delivery, have been observed in numerous hypoxic diseases. These include severe pulmonary disease
(Eaton et al., 1970), hemolytic and non-hemolytic anemia (Hjelm, 1970), iron deficiency, leukemia, uremia and emphysema (Eaton et al., 1970). 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 in the rate of oxygen delivered (Brewer and Eaton, 1971). 30
FIGURE 3
FACTORS CONTRIBUTING TO THE CONTROL OF RED CELL 2,3-DPG
FROM BUNN AND JANDL , 1970 31
2,3-diphosphoglycerate Changes in Non-pathalogical Conditions
Altitude Exposure. At high altitude the affinity of hemoglobin for oxygen is decreased (ie an increase in is observed), making hemoglobin- bound oxygen more available to body tissues (Aste-Salazar and Hurtado, 1944).
Lenfant et al. (1968) exposed sea level residents to altitudes of 15,000 feet and found an increase in 2,3-DPG which occurred concomitant to an increase in
PJ-Q. Both occurred within 24 hours of exposure. Upon returning to sea level, parallel decreases in both 2,3-DPG and P^Q were observed. It was suggested that the increase in 2,3-DPG represented an important rapid adaptive mechanism to anoxia.
Torrance et al.(1970/71) compared sojourners at altitude with residents at altitude, finding a marked hyperventilation and increased levels of hemo• globin and 2,3-DPG in the latter group. The affinity 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 is was the same in the two groups at higher altitude. The increases in 2,3-DPG paralleled the decrease in the oxygen affinity of hemoglobin. pH and bicarbonate changes at altitude reflected an increasing respiratory alkalosis which likely triggered the 2,3-DPG response. The sojourners, who had the lowest hemoglobin concen• tration, had the greatest hyperventilation and the largest increase in 2,3-DPG
i when moving from sea level to altitude. j
The evidence that plasma pH changes are responsible for the rise in
2,3-DPG upon exposure to altitude is supported by the study of Lenfant et al.
(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 P5Q 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 is necessary in the altitude response of 2,3-DPG. However, it 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 liter of blood (Rorth and Brahe, 1972). In addition "stress" hormones such as epinephrine (Brewer, 1972a) and thyroid hormones (Miller et al., 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 al. (1970a).
They calculated that if one goes to high altitude and does not increase his hemoglobin, nor shifts his dissociation curve, his working ability will be decreased by about 50 per cent. If, however, there is a shift of the hemo• globin dissociation curve, his working ability would be about 75 per cent of what he can do at sea level. The last 25 per cent would be gained if 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 al., 1971; Dempsey et al.,1971), which will be discussed in the chronological order cited above. i |
Eaton et al. (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 in
2,3-DPG was related to the severity of the exercise.
Faulkner et al. (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 cell 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 cells, 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 is much more rapid than the six hour half response time observed during adaptation to high altitude (Lenfant et al.,
1968). 34
Shappell et al. (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 arterial
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, all values of 2,3-DPG, whether at rest or during exercise, were significantly higher in both arterial and venous blood.
The fact that the increase in 2,3-DPG as a result of training was not accompanied by an increase in P^g was explained on the basis of a signif•
icant decrease in MCHC after training. A significant linear regression between the fall in MCHC and the increase in 2,3-DPG suggested that one effect
counterbalanced the other.
Shappell's data indicated that an increase in 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
rise in hemoglobin concentration in light of the previously mentioned fall in
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 al., 1949), an increase (Knehr et al., 1942)
and a decrease (Oscai et al., 1968). However, all studies report an increase
in total blood volume as a result of training. 35 Dempsey et al. (1971) had four highly trained subjects perform
treadmill exercises of 65-70 per cent of their maximal aerobic power until
exhaustion. A decline of 2,3-DPG, ranging from 2-17 per cent was observed
in all 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 in 2,3-DPG with
exercise, one study reports no change with exercise but an increase with
training and one study reports a decrease in 2,3-DPG with exercise which
rises back to resting levels if the exercise is of sufficient duration.
Cigarette Smoking and the 2,3-diphospoglycerate Response. The
relationship between smoking and 2,3-DPG has received very little attention.
Smoking is unique in that it induces a hypoxia which is not associated with
an increased desaturation of hemoglobin (Brewer at al., 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 affinity 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 left, 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), it 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 al., 1970). Two important facts emerged from a study of these individuals. First, there was no right shift of the oxygen dissociation curve, in spite of the marked elevation in 2,3-DPG. Second, all but four of the 24 individuals studied were habitual smokers. (The exceptions were found to have lung disease - silicosis, 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 left sided curve displacements also have polycythemia and that all patients with right sided displacements had low hemoglobin concentra- I tions. It was suggested that the erythropoietin system is sensitive to altered tissue oxygen tensions which are due to oxygen dissociation curve displacement. 37
When Brewer et al. (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 left shifted compared to non-smokers residing at altitude). Both groups of smokers were also found to have a significantly lower arterial 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 left of the oxygen dissociation curve and a reduction in arterial oxygen tension.
In order to determine whether or not 2,3-DPG levels were regulated primarily through hemoglobin desaturation, Eaton et al. (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 in 2,3-DPG averaging 12 per cent was observed after three hours, with no significant change from hour three to hour six. Thus it appeared that in hypoxia a mechanism exists beyond simple desaturation of hemoglobin which is capable of stimulating an increase in the levels of red blood cell
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 felt this observed decrease in 2,3-DPG explained the increased
! affinity 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 in the literature which consider the combined effects of smoking and physical activity on levels of 2,3-DPG and/or hemoglobin.
Summary
Oxygen delivery to tissues can be facilitated by several mechanisms, one of which occurs through a decrease of the affinity of hemoglobin for oxygen.
This response is 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 affinity is altered by such factors as blood pH, carbon dioxide and hemoglobin concentrations. Biochemical control of 2,3-DPG levels is 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 is 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 criteria:
Group I - Sedentary Nonsmokers. Comprised of subjects who had not
indulged in 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 Fit Nonsmokers. Comprised of subjects who had
indulged in 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 Fit 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 Fit 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 is 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 fit" group, other than their
intensive training program, is 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 al., (1972), might be observed in these subjects.
It was hypothesized that the capacity to perform endurance work would vary greatly between the three "fitness" groups. Since oxygen transport
is 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 is pointed out by Ekblom et al., (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 criteria 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
All subjects were tested once, in a random order, during the interval
30 June 1972 - 1 April 1973, in the Human Performance Laboratory, University of B.C. Physical Education Building. Ambient temperature in 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 cell 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 all testing sessions, removed three milliliters 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 sterile, 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 elicited a heart rate of approximately
150 beats per minute. All 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 until 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 fit, 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 ability 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 it 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 Vo2 (Astrand et al., 1964; Rowell et al., 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 in 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 in these subjects is such that the relation between heart rate and oxygen uptake remains virtually unchanged. (Top athletes in endurance events often have a maximal oxygen uptake that is about twice as high as that of an average man).
During exercise in the sitting 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 al., 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 is increased up to
or close to the same maximal value, for. all subjects, at maximal Vo2 (Astrand et al., 1964; Rowell et al., 1966; Wang et al., 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^, all subjects in this study would have approximately
the same a-v oxygen difference, or the same amount of hemoglobin desaturation which is 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 is independent of body weight. A
fixed pedal frequency of 50 RPM (300 meters per minute) was utilized because
optimal mechanical efficiency is obtained at this rate (Astrand, 1970).
A time interval of one hour was chosen because the contribution to
energy output is almost solely derived from aerobic processes (Astrand, 1970),
therefore differences in 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 is 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 is noted at about 50 per cent of
maximal aerobic power (Hermansen and Saltin, 1967; Williams et al., 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 45 to observe any adaptive changes that might occur within the red blood cell to enhance oxygen delivery, and thereby man's ability 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 first 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 its effect on this protein's ability to bind oxygen (Brewer and Eaton, 1971). Hematocrit was measured because the concentration of hemoglobin varies directly with the number of red blood cells present, and because mean corpuscular hemoglobin concentration (^^^ocrit^ has been found to affect the binding of oxygen to hemoglobin independent of
2,3-DPG (Bellingham et al., 1971). Blood pH was measured because it 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 in 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 milliliter of blood was diluted in five milliliters 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 milliliter of blood with three milliliters 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 all protein. The supernatant was stored at 10°C until analysis of 2,3-DPG was undertaken, which was within eight hours of blood sampling for all subjects.
2,3-DPG was determined using the enzymatic method described in Sigma
Chemical Company technical bulletin 35 UV (1971). The method is based on the decrease in optical density at 340 nm when NADH is 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 milliter of whole blood, micromoles per ml of packed blood cells and micromoles per gram of hemoglobin. The latter expression was used in the statistical compar• isons because the hemoglobin content of red blood cells 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 is expressed in relation to volume of cells or whole blood, changes will be observed purely as a result of changes in red cell volume or red cell number when no change in red cell content has occurred (Bellingham et al., 1971).
Two determinations were made on both the pre and post exercise levels of 2,3-DPG, hematocrit, hemoglobin and blood pH. A criteria 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 is approx• imately a five per cent variation from the normal mean reported in 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 al.,
(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 all 40 subjects the differences obtained for duplicate hematocrit, pH and 2,3-DPG determinations never exceeded the above criteria. However, on 1 three occasions hemoglobin determinations did exceed the criteria. In these cases hemoglobin determinations were repeated immediately, and in all cases the second set of duplicate readings were within the acceptable range. The mean value of duplicate determinations was utilized 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 cell "highly fit 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 is presented in Figure 4.1. Because of the missing
cell, the five groups were treated as one factor (no interaction considered)
for purposes of statistical 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 all five groups, thus resulting in a
5x2 design. The analysis format is shown in Table I.
Statistical 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 utilized 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 X5 Xi X5
X2 X6 X2 X6 X3 X7 X3 X7 Xif Xs Xi* Xs
Moderately Fit X9 X!3 X9 X13 Nonsmokers Xu Xu X10 Xn X;i X15 Xu X15 X12 Xl6 X12 Xl6
Highly Fit Nonsmokers • Xn X21 X17 X21 Xi8 X22 Xia X22 Xi9 X23 Xi9 X23
X20 X2I, X20 X2if
Bendentary Smokers X25 X29 X25 X29
X26 X30 X26 X30 X27 X31 X27 X31 X28 X32 X28 X32
Moderately Fit X33 X37 X33 X37 Smokers Xsif X38 X3i» X38 X35 X39 X35 X39 X36 Xi»o X36 XifO 51
FIGURE 4-1
EXERIMENTAL DESIGN
SEDENTARY MODERATELY FIT HIGHLY FIT
SEDENTARY MODERATELY FIT SMOKERS SMOKERS n = 8 SMOKERS n = 8
MODERATELY SEDENTARY HIGHLY FIT FIT NONSMOKERS NONSMOKERS NONSMOKERS NONSMOKERS n = 8 n = 8 n = 8
FIGURE 4-2
• ORTHOGONAL COMPARISONS
MODERATELY HIGHLY SEDENTARY SEDENTARY MODERATELY FIT FIT NON FIT NON NON SMOKERS SMOKERS SMOKERS SMOKERS SMOKERS COMPARISON I COMPARISON 3
COMPARISON 2 f COMPARISON 4 ORTHOGONALITY INDICATED BY NO INTERSECTING LINES CHAPTER IV
RESULTS AND DISCUSSION
Results
Descriptive Statistics
Individual results for all subjects for all 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 in Table II herein.
Values for each of the five groups, for groups combined orthogonally and for pooled samples are listed separately. The same information is also presented graphically in 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 in 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 in Table VII. The average work per heartbeat, for each of the five groups, is also graphically illustrated in
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 in 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 Deviations of 2,3-DPG Levels
Expressed in Micromoles per Gram Hemoglobin for
Each Group Before and After Exercise
Groups Before After
Sedentary Nonsmokers 12. 02 + 1. 94 12. 11 + 1. 90
Moderately Fit Nonsmokers 12. 72 + 1. 26 12. 61 + 1. 46
Highly Fit Nonsmokers 13. 29 + 1. 86 13. 64 + 2. 39
Sedentary Smokers 12. 15 + 1. 30 12. 60 + 1. 31
Moderately Fit Smokers 13. 41 + 1. 30 13. 76 + 1. 36
Sedentary and Moderately Fit Nonsmokers Combined 12. 37 + 1. 62 12. 36 + 1. 66
All Nonsmokers Combined 12. 67 + 1. 72 12. 79 + 1. 98
All Smokers Combined 12 .7 8 + 1. 41 13. 18 + 1. 42
All Subjects Combined 12. 72 + 1. 56 12. 94 + 1. 73 54 FIGURE 5
MEANS AND STANDARD DEVIATIONS OF 2,3-DPG LEVELS EXPRESSED IN MICROMOLES PER GRAM HEMOGLOBIN FOR EACH GROUP BEFORE AND AFTER EXERCISE
I6.O-1
15.0- Q BEFORE EXERCISE AFTER EXERCISE
14.0-
2 < o 13.0- tr Uo_J —Z m I w9 UJ i or
x 2 x ^ 2 2 11.0- \ 10.0- i I I ! t^/////i SNS MFNS HFNLS ss- •MFS- Ly////A DPG 55
TABLE III
Means and Standard Deviations of Hemoglobin
Concentrations Expressed as Grams Per 100
Milliliters for All Groups Before and After
Exercise
Groups Before After
Sedentary Nonsmokers 15. 82 + 1. 20 16. 53 + 0. 97
Moderately Fit Nonsmokers 15. 51 + 1. 07 16. 28 + 0. 61
Highly Fit Nonsmokers 14. 30 + 1. 86 14. 93 + 1. 82
Sedentary Smokers 16. 37 ± 1. 07 16. 78 + 1. 00
Moderately Fit Smokers 16. 61 ± 0. 76 17. 45 + 0.76
Sedentary and Moderately Fit Nonsmokers Combined 15. 67 ± 1. 11 16. 40 + 0. 79
All Nonsmokers Combined 15. 21 ± 1. 51 15. 91 + 1. 38
All Smokers Combined 16. 49 + 0. 91 17. 11 + 0. 93
All Subjects Combined 15. 72 ± 1. 25 16. 39 + 1. 11 56 FIGURE 6
MEANS AND STANDARD DEVIATIONS OF HEMOGLOBIN CONCENTRATIONS
EXPRESSED AS GRAMS PER 100 MILLILITERS FOR ALL GROUPS BEFORE
AND AFTER EXERCISE
20.01
_] BEFORE EXERCISE 19.0- ^ AFTER EXERCISE 18.0- CC UJ 0_ V77777^ CO 17.0- / V < y7?<77) cr 777? ° I6.0H f £ 815.0-
DO
S _l4.0i t o LU — E a 13.0- o o CQ o _j 11.0- o o LU 10.0- i X 9.0H I / / L SNS MFNIS HFNS- SS MFV////AS HEMOGLOBIN 57 TABLE IV Means and Standard Deviations of Hematocrit Levels Expressed as Percent Volume for All Groups Before and After Exercise Groups Before After Sedentary Nonsmokers 45. 81 + 2. 23 46. 61 + 2. 29 Moderately Fit Nonsmokers 44. 85 + 2. 94 45. 81 + 2. 45 Highly Fit Nonsmokers 44. 25 + 2. 26 45. 48 + 2. 68 Sedentary Smokers 46. 94 + 2. 74 47. 40 + 2. 61 Moderately Fit Smokers 47. 68 + 1. 56 48. 18 + 1. 41 Sedentary and Moderately Fit Nonsmokers Combined 45. 33 + 2. 57 46. 21 + 2. 33 All Nonsmokers Combined 44. 97 + 2. 48 45. 93 + 2. 42 All Smokers Combined 47. 31 + 2. 19 47. 79 + 2. 07 All Subjects Combined 45. 90 + 2. 39 46. 68 + 2. 33 58 FIGURE 7 MEANS AND STANDARD DEVIATIONS OF HEMATOCRIT LEVELS EX - PRESSED AS PERCENT VOLUME FOR ALL GROUPS BEFORE AND AFTER EXERCISE 55.0i 52.5- • BEFORE EXERCISE W\ AFTER EXERCISE 50.0- (f) _ UJ -J -•247.5 t- cr_ / O LU o o y i a / V fe / / / / a, / / 2 / / LU x x 5 45.0- f ? 2 y / KM y / y / y y y / y v / y y y 42.5- y y y y y y y y y y y y l '/ 1 y? y1 4Q0- y i y yy y y y y y y 2 v, v,,,,yA ,1 '////// V////A -SNS MFNS- HFNS- SS MFS- HEMATOCRIT 59 TABLE V Means and Standard Deviations of Mean Corpuscular Hemoglobin Concentration (MCHC) Expressed in Percent for all Groups Before and After Exercise Group Before After Sedentary Nonsmokers 34. 45 + 1. 44 35. 40 + 1. 22 Moderately Fit Nonsmokers 34. 53 + 0. 66 35. 57 + 1. 37 Highly Fit Nonsmokers 32. 20 + 2. 67 32. 81 + 1. 86 Sedentary Smokers 34. 89 + 1. 19 35. 41 + 1. 08 Moderately Fit Smokers 34. 83 + 1. 27 36. 21 + 1. 23 Sedentary and Moderately Fit Nonsmokers Combined 34. 49 + 1. 08 35. 49 + 1. 26 All Nonsmokers Combined 33. 72 + 2. 04 34. 60 + 2. 05 All Smokers Combined 34. 86 + 1. 19 35. 81 + 1. 19 All Subjects Combined 34. 18 + 1. 59 35. 08 + 1. 49 60 FIGURE 8 MEANS AND STANDARD DEVIATIONS OF MEAN CORPUSCULAR HEMOGLO• BIN CONCENTRATION (MCHC) EXPRESSED IN PERCENT FOR ALL GROUPS BEFORE AND AFTER EXERCISE 40.0- • BEFORE EXERCISE ffli AFTER EXERCISE 39.0- 38.0 370 §uj36.0j 3S W' | 235.0- UJ ~" 1 Vi o 9 '/ cc x34.0- go / / V v / / (_> — w 33.0- o_ / V cc / V o / V ° 320- !l 31.0- / V '$ V V 300- / / V / / / / / / / / V / / / V / / / V / V V, V. 1 Y////A v/////< '////A V/////. SNS- MFNS -HFNS- ss- -+ -MFS MCHC 61 TABLE VI Means and Standard Deviations of Blood pH for all Groups Before and After Exercise Groups Before After Sedentary Nonsmokers 7. 41 + 0. 06 7. 42 + 0. 01 Moderately Fit Nonsmokers 7. 38 + 0. 03 7. 43 + 0. 03 Highly Fit Nonsmokers 7. 35 + 0. 03 7. 42 + 0. 05 Sedentary Smokers 7. 37 + 0. 04 7 .4 4 + 0. 04 Moderately Fit Smokers 7. 36 + 0. 05 7 .4 0 + 0, 07 Sedentary and Moderately Fit Nonsmokers Combined 7. 39 + 0. 05 7. 43 + 0. 02 All Nonsmokers Combined 7. 38 + 0. 05 7. 43 + 0. 03 All Smokers Combined 7. 37 + 0. 04 7. 42 + 0. 05 All Subjects Combined 7. 37 + 0. 04 7 .4 2 + 0. 04 62 FIGURE 9 MEANS AND STANDARD DEVIATIONS OF BLOOD pH FOR ALL GROUPS BEFORE AND AFTER EXERCISE 7 50 • BEFORE EXERCISE __| AFTER EXERCISE 7.45 o o o -i K I ^ CQ 7.40 CO / Z3 zO LU > r 7.35 7.30- L •SNS- -+—MFNS- HFNS SS MFS- PH 63 i TABLE VII Means and Standard Deviations of Body Weight Expressed in Kilograms and Average Work per Heartbeat Expressed in Kilopond Meters for all Groups GrouPs Body Weight (Kg) Average Work (KPM) Sedentary Nonsmokers 78. 33 + 12. 19 5. 24 + 1. 48 Moderately Fit Nonsmokers 84. 01 + 6. 73 6. 74 + 0. 49 Highly Fit Nonsmokers 69. 66 + 10. 17 8. 00 + 1. 05 Sedentary Smokers 79. 99 + 7. 07 5. 14 + 0. 59 Moderately Fit Smokers 82. 26 + 13. 05 5. 48 + 0o 84 Sedentary and Moderately Fit Nonsmokers Combined 81. 17 + 9. 95 5. 99 + 1. 32 All Nonsmokers Combined 77 . 33 + 11. 26 6. 66 + 1. 55 All Smokers Combined 81. 12 + 10. 20 5. 31 + 0. 73 All Subjects Combined 78. 85 + 10. 17 6. 12 + 0. 96 64 FIGURE 10 MEANS AND STANDARD DEVIATONS OF AVERAGE WORK PER HEART• BEAT EXPRESSED IN KILOPOND METERS FOR ALL GROUPS 10.0 9.0- 8.0- 27 0 ~ 6.0 m _ 5.0 < UJ X _ 4.0 o 3.0H _5» 2.0- 1.0- SNS —MFNS -j«—HFNS -f* SS +> MFS H WORK 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 in 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 all dependent variables may be found in Table II, of Appendix A. Homogenity of Variance One of the assumptions underlying statistical comparisons of samples test was is homogenity of variance, therefore an ^max~ performed on the six dependent variables: 2,3-DPG, hemoglobin, hematocrit, MCHC, blood pH and average work per heartbeat. Results are shown in Table I, Appendix A. The observed variance ratios were not significant at p<0.05, thus it was assumed that the variances of the ten samples for each of the six dependent variables were homogeneous for the purpose of statistical analysis. 68 Statistical Analysis of the Data- Test of Hypotheses 2,3-Diphosphoglycerate Concentrations. The results of a priori orthogonal comparisons of the resting levels of 2,3-DPG are presented in Table XII. No statistically 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 Fit Nonsmokers 1,14 1.932 <1 Sedentary and Moderately Fit Nonsmokers VS Highly Fit Nonsmokers 1,21 4.526 1.863 >.05 Sedentary Smokers VS Moderately Fit 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 all subjects, as well as the a priori 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 All Subjects and Orthogonal Comparisons of Changes in 2,3-DPG Between Groups Comparison df MS F P All Subjects Pooled 1,35 1.017 3.103 >.05 Sedentary Nonsmokers VS Moderately Fit Nonsmokers 1,14 0.077 <1 Sedentary and Moderately Fit Nonsmokers VS Highly Fit Nonsmokers 1,21 0.330 1.007 >.05 Sedentary Smokers VS Moderately Fit Smokers 1,14 0.024 <1 Smokers VS Nonsmokers 1,35 0.389 1.186 >.05 Hemoglobin Concentrations. The results of a priori orthogonal comparisons of the resting levels of hemoglobin are presented in Table XIV, Hemoglobin levels of highly fit 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 in ! 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 Fit Nonsmokers 1,14 0.391 <1 Sedentary and Moderately Fit Nonsmokers VS Highly Fit Nonsmokers 1,21 10.010 6.450 <.05 Sedentary Smokers VS Moderately Fit Smokers 1,14 0.216 <1 Smokers VS Nonsmokers 1,35 15.677 10.102 <.01 TABLE XV Univariate Analysis of Orthonormalized Changes of Hemoglobin as a Result of Exercise for All Subjects and Orthogonal Comparisons of Changes in Hemoglobin Between Groups Comparison df MS All Subjects Pooled 1,35 8.965 53.212 <.01 Sedentary Nonsmokers VS Moderately Fit Nonsmokers 1»14 0.007 <1 Sedentary and Moderately Fit Nonsmokers VS Highly Fit Nonsmokers 1,21 0.025 <1 Sedentary Smokers VS 0.368 2.182 >.05 Moderately Fit Smokers 1»14 0.027 <1 Smokers VS Nonsmokers 1,35 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 Fit Nonsmokers 1,14 3.715 <1 Sedentary and Moderately Fit Nonsmokers VS Highly Fit Nonsmokers 1,21 6.214 1.084 >.05 Sedentary Smokers VS Moderately Fit Smokers 1,14 2.176 <1 Smokers VS Nonsmokers 1,35 52.415 9.147 <.01 A significant increase was observed in 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 fit 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 All Subjects and Orthogonal Comparisons of Changes in Hematocrit Between Groups Comparison df MS All Subjects Pooled 1,35 11.943 18.023 <.01 Sedentary Nonsmokers VS Moderately Fit Nonsmokers 1,14 0.055 <1 Sedentary and Moderately Fit Nonsmokers VS Highly Fit Nonsmokers 1,21 0.158 <1 Sedentary Smokers VS Moderately Fit Smokers 1,14 0.005 <1 Smokers VS Nonsmokers 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 Fit Nonsmokers 1,14 0.024 <1 Sedentary and Moderately Fit Nonsmokers VS Highly Fit Nonsmokers 1,21 27.999 11.049 <.01 Sedentary Smokers VS Moderately Fit Smokers 1,14 0.013 <1 Smokers VS Nonsmokers 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 All Subjects and Orthogonal Comparisons of Changes in Mean Corpuscular Hemoglobin Concentration Between Groups Comparison df MS F P All subjects Pooled 1,35 16.299 29.809 <.01 Sedentary Nonsmokers VS Moderately Fit Nonsmokers 1,14 0.018 <1 Sedentary and Moderately Fit Nonsmokers VS Highly Fit Nonsmokers 1,21 0.389 <1 Sedentary Smokers VS Moderately Fit Smokers 1,14 1.462 2.674 >.05 Smokers VS Nonsmokers 1,35 0.028 <1 Blood pH. The only significant difference in the resting, pre exercise values of blood pH were between the highly fit 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 Fit Nonsmokers 1,14 0.004 2.108 >.05 Sedentary and Moderately Fit Nonsmokers VS Highly Fit Nonsmokers 1,21 0.013 7.105 <.05 Sedentary Smokers VS Moderately Fit 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 All Subjects and Orthogonal Comparisons of Changes in Blood pH Between Groups Condition df MS All Subjects Pooled 1,35 0.050 31.361 <.01 Sedentary Nonsmokers VS Moderately Fit Nonsmokers 1,14 0.002 1.502 >.05 Sedentary and Moderately Fit Nonsmokers VS Highly Fit Nonsmokers 1,21 0.003 1.826 >.05 Sedentary Smokers VS Moderately Fit Smokers 1,14 0.001 <1 Smokers VS Nonsmokers 1,35 0.000 <1 75 Average Work Per Heartbeat. A priori orthogonal comparisons of the average work per heartbeat for various groups are presented in Table XXII. Moderately fit nonsmokers were able to work at a significantly higher level per heartbeat than sedentary nonsmokers. Highly fit 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 fit smokers. TABLE XXII Orthogonal Comparisons of Average Work Per Heartbeat Comparison df MS Sedentary Nonsmokers VS Moderately Fit Nonsmokers 1,14 9.075 9.909 <.01 Sedentary and Moderately Fit Nonsmokers VS Highly Fit Nonsmokers 1,21 21.494 23.468 <.01 Sedentary Smokers VS Moderately Fit 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 (12.72 ± 1.56 uM/g Hb, Page 53) was similar to that found by most investigators (Eaton and Brewer, 1968; Eaton et al., 1970; Faulkner et al., 1970; Valeri and Collins, 1971). However, in other studies considerably higher mean resting 2,3-DPG concentrations were obtained. Bellingham et al„; (1971) reported a mean 2,3-DPG level of 14.52 uM/g Hb; Shappell et al, (1971), in a study on the effects of exercise, reported a mean 2,3-DPG level of 14.90 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 10.15 to 15.17 uM/g Hb, with one exception, (Subject 'SD*, a highly fit non- smoker had a 2,3-DPG level of 16.92 uM/g Hb.) Individual results are shown in Table III, Appendix B. The range reported here is within that reported by Eaton and Brewer (1968) from 100 male Caucasians, of 8.5 to 15.86 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 al., 1970/71; Valeri and Collins, 1971), as well as the findings of Duhm (1972), Oski (1969) and Valeri and Collins (1971) that a shift of one Torr in P^Q is accomplished through a change of 2,3-DPG concentration of approximately 1.40 uM/g Hb, it 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 in 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 is observed. Among the nonsmokers an increase in 2,3-DPG (as defined by the mean) is apparent as one progresses from sedentary through highly fit groups (12.02 ±1.94 uM/g Hb 13.29 ± 1.86 uM/g Hb). The same trend is observed in 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 is 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 possibilities exist to explain the divergent results. First, 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 activity. Once the activity level is maintained for a sufficient duration to allow other, slower acting adaptive mechanisms to respond (such as an increase in stroke volume, increase in respiratory enzymes, etc) 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 cells. Because the highly trained subjects in this study had been involved in a training program which was eminently superior to Shappell's in both intensity and duration, it was expected that large differences in 2,3-DPG levels would be observed, if training produces an increase in 2,3-DPG, when these subjects were compared to sedentary subjects. However, the mean difference between highly fit and sedentary nonsmokers was only 1.39 uM/g Hb, which was not statistically 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 statistically significant. Therefore, it would appear that if training does cause an increase in 2,3-DPG concentrations, the increase is rather small, perhaps in the vicinity 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 statistically significant (r- -.110, Table II, Appendix A), indicating that 2,3-DPG is not a valuable predictor of aerobic fitness even though it 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 al, (1970), the tendency of the data is quite suggestive. Among the two sedentary groups little difference is observed in 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 is observed when comparing the moderately fit 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 fit smokers exhibited the highest mean resting 2,3-DPG level of all five groups. These results indicate that smokers, when subjected to the hypoxia of repeated physical activity, respond with a larger increase in 2,3-DPG than nonsmokers subjected to the same conditions. This agrees with Eaton et al. (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 all 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 al. (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 is 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 in Figure 3, Page 30, In comparing the hemoglobin concentrations of the three nonsmoking groups in this study (Table III, Figure 6, Pages 55-6) the opposite trend to the 2,3-DPG trend is observed as one would expect by the significant negative correlation between the two parameters. That is, hemoglobin concentrations fall as fitness levels increase. The hemoglobin concentrations of the highly fit 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 al. (1968) but is contrary to the results of Knehr et al, (1942) and Shappell et al (1971), In contrast, active smokers tended to have slightly higher (but not significant) hemoglobin levels than sedentary smokers, in spite of also having a-tendency towards higher 2,3-DPG concentrations. This is reflected by the fact that although a significant negative correlation exists between hemoglobin and 2,3-DPG when all subjects are considered together, the correlation between the two parameters for the sedentary smokers is only r = -.416 and for the 81 moderately fit 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 is contrary to the findings of Albert (1965) and Isager and Hagerup (1971) and may be partially attributed to the very low hemoglobin levels of the highly fit nonsmokers, although in both sedentary and moderately fit groups the smokers tended to have slightly higher hemoglobin levels than the nonsmokers. Because blood hemoglobin levels are a function of both the number of red blood cells (hematocrit) and the hemoglobin concentration within each cell (MCHC) it is pertinent to examine differences in these parameters to clarify the overall hemoglobin response and how it 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 signif• icant, Table II, Appendix A), However the relationships are complicated by the fact that MCHC affects the oxygen affinity for hemoglobin independent of 2,3-DPG, as well as affecting the carrying capacity of hemoglobin (Bellingham et al., 1971; Lenfant et al., 1972; Shappell et al., 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 in the smoking population (Brewer, 1972). The average hematocrit for all subjects pooled (45.90 ± 2.39) agrees with results of other investigators (Garby, 1970; Kilpatrick, 1961; Larsen, 1966; Lenfant et al., 1969; McDonough et al., 1965; Natvig, 1966; Natvig and Vellar, 1967). The viscosity, or resistance to flow, of blood is mainly dependent on the plasma proteins and the cell content. The higher the hematocrit the higher the viscosity. In dogs, by measurement of oxygen consumption, arterial and venous oxygen concentrations, arterial pH, cardiac output, peripheral resistance and maximal oxygen-transporting capacity at different hematocrit values, it was demonstrated that oxygen consumption increased as the hematocrit increased until 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 cells increased the oxygen carrying capacity but decreased the rate of flow by increasing the blood viscosity (Harris and Kellermeyer, 1970:294). Assuming this study is applicable to human subjects, it indicates that the highly fit nonsmokers have hematocrit levels that are closest to the "optimal" level, even though this level is 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 Guyton (1960:86) reported that the degree of physical activity of a person determines to a great extent the rate at which red blood cells are produced. The fact that exercise increased the rate of blood cell production was indicative that it is anoxia of tissues that causes red blood cell produc- 83 tion, because the tissues become depleted of oxygen in exercise. However, opposing the increase in red cell production with exercise, is the finding that physical activity causes a decrease in the integrity of the red blood cell membrane which has been attributed to an increased circulatory rate, temperature, acidity and compression of cells (Faulkner et al., 1970). It is hypothesized that as training becomes more intense hematocrit is reduced and maintained closer to the optimal level to allow red cell production to keep up with red cell destruction. Thus although training appears to cause a decrease in oxygen transport , the decreased blood viscosity and the tendency towards a decreased affinity 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 in 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 fit and sedentary, demonstrate increased red cell production due to hypoxia produced through binding of carbon monoxide to hemoglobin thereby removing it from oxygen transport. The activity level of these individuals would likely not be of sufficient intensity to cause increased red cell destruction, thus hematocrits remain significantly higher. I Active smokers, who are not involved in a program of activity which is intensive ! i enough to cause significant increases in red cell fragility, but which produces additional hypoxia above that which is due to smoking, would tend to have a further stimulation of red cell production and a higher hematocrit. Oxygen transport to tissues would be further impaired by the increase in blood viscosity. 84 r The tendency towards a higher 2,3-DPG concentration in the moderately fit smokers could partially 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 fit 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 in 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 cells 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 cells, 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 fit subjects is in agreement with the results of Shappell (1971), who found a decrease in MCHC from 33.85 ± 0,74 to 85 33.09 ± 0.48 grams per 100 mis of blood cells in 22 subjects after an eight week training period. The highly fit subjects in this study demonstrated a MCHC of 32.20 ± 2.67, which is consistent with their more intensive training program. The hypothesis that erythropoietic activity is barely able to keep up with red cell destruction when training becomes very intensive is further supported by the very low MCHC levels of the highly fit subjects, In fact, two of the highly fit subjects had MCHC levels below 30.0 grams per 100 mis, the value generally accepted as symptomatic of hyperchromia. The assumption is that,because of the extremely high turnover rate of red cells in these individuals,the hemoglobin producing mechanism lags behind cell production, leading to red blood cells with reduced hemoglobin levels. The lower MCHC of the highly fit nonsmokers produces an adverse effect on oxygenation of tissues in that it leads to an increased affinity of hemoglobin for oxygen, i.e. it causes a left shift in the oxygen dissociation curve (Benesch et al,8 1969; Bellingham et al., 1971). The tendency towards higher 2,3-DPG levels, although not statistically significant in this study, could partially offset the left shift of the oxygen dissociation curve. In order to clarify the overall effect on the oxygen delivery of blood as affected by 2,3-DPG, MCHC and hematocrit a summary is presented in Table XXIII. Use was made of the calculations of Bellingham et al,, (1971) that change in P^Q due to 2,3-DPG could be determined using the equation: [P5Q = 0.694 DPG + 17.63] and change in P5Q due to MCHC could be determined by use of the equation [&P5Q = 0.471 AMCHC - 0,216], The results show that the moderately fit 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 fit nonsmokers and highly fit nonsmokers have approximately the same shift in P,-N but the shift occurs by different 86 mechanisms. The highly fit group obtains the beneficial effect solely through 2,3-DPG, whereas the moderately fit group's P,.- is primarily affected by MCHC. In addition, the highly fit nonsmokers possess the most advantageous hemato• crit. 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 fit nonsmokers would have the parameter levels producing the most efficient oxygen delivery system. Table XXIII Theoretical Determinations of Increases in P_- Due to Differences in 2,3-DPG and MCHC and Hematocrit Levels Above the Theoretical Optimal Level Increases in P,.^ Increases in 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 0.00U + 0.844 3.81 Moderately Fit Nonsmokers + 0.486 + 0,881 2,85 Highly Fit Nonsmokers + 0.881 0.000 2.25 Sedentary Smokers + 0.090 + 1.051 4.94 Moderately Fit Smokers + 0.965 + 1.023 5.68 87 Highly fit 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 in 2,3-DPG is supported by the data of this subject. The first 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 utilizing 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 in relation to average work per heartbeat. Unfortunately the two smoking groups did not exhibit physiological differences in 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 fit and low fit 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 fit non- smokers, five moderately fit nonsmokers, one moderately fit smoker and two sedentary nonsmokers) were compared with the 15 subjects with the lowest averag work per heartbeat (five moderately fit smokers, five sedentary smokers and five sedentary nonsmokers). Multivariate analysis, using computer program 88 Multivariance (Finn, 1968) was again performed. Results are presented in 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• crit and hemoglobin levels were both significantly higher in the low fit 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 is 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 in a training program causing alterations in red blood cell 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 al., 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 is observed that although the highly fit 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 in 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 fall in pH in the first 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 rise. This suggested a metabolic reaction initiated by acidosis, such as rates of glucose utilization, 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 all subjects in 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 is apparent to explain the sign• ificantly lower pre exercise venous blood pH of highly fit 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 is a major point of contention. Dempsey et al., (1971) observed no changes in 2,3-DPG after two hours of exercise, however Faulkner et al. (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 first three hours after a blood transfusion of 2,3-DPG depleted cells; Gerlach et al. (1970) exposed rats to hypoxia and found a significant increase in 2,3-DPG only after five 90 hours of elapsed time; Bellingham et al. (1971) found no change in 2,3-DPG after four hours of induced alkalosis and acidosis; Lenfant et al. (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 in 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 in 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 is in agreement with Dempsey et al. (1971) and Shappell et al. (1971) but contrary to the findings of Eaton et al. (1969) and Faulkner et al. (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 is 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 in 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 al. (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 possibilities are offered to explain the rise in venous blood pH. First, 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 possibility is 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 in 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 is 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 cell is fixed, alterations in MCHC can occur only as a result of the red blood cell shrinking or swelling. The shrinking of cells, 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 facilitates 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 all had an increase in 2,3-DPG ranging from 0.32 to 1.31 uM/g Hb. Conversely, of the 12 subjects who have a decrease in 2,3-DPG, all have an increase in 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 is contrary to the findings of Albert et al. (1965) who reported that following exercise the diameter of red blood cells increased by about one-half a micron due to the change of permea• bility of the cell membrane. However, Faulkner et al. (1970) reported a compression of cells as a result of exercise. A plausible explanation of the discrepancy of these results, which also appears applicable to this study, is the work of Bellingham et al. (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 first hour, which | was followed by a slower rise to 7.450 during the next 24 hours. During the first hour of the acute change in plasma pH there was no alteration in red cell 2,3-DPG however there was a change in oxygen affinity and this corresponded to a significant increase of MCHC. (Similarly in induced acidosis there was a 94 FIGURE II RELATIONSHIP BETWEEN CHANGE OF 2 ,3 DPG AND CHANGE IN MCHC AS A RESULT OF A ONE HOUR STANDARDIZED BOUT OF EXERCISE + I50n r =-0.489 -100 +50 + 100 + 150 P50 FROM 2,3 DPG (TORR) 95 rapid fall in MCHC). When acidosis of alkalosis was maintained, MCHC returned to normal levels but 2,3-DPG levels were altered and correlated with changes in oxygen affinity. Both MCHC and 2,3-DPG changes produced a shift in the oxygen dissociation curve of hemoglobin which was opposite to the shift pro• duced by alterations in 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 al. (1971), is that MCHC changes occur rapidly in alkalosis so that P^Q remains unchanged while the 2,3-DPG mechanism becomes activated. Of course there is obviously a limit to the change in cell volume that can occur and large pH changes would be beyond the capabilities of this compensatory mechanism so that P^Q would be altered until the 2,3-DPG mechanism could readjust. When the 2,3-DPG response is initiated it is hypothesized that MCHC drops, due to red blood cell swelling, in proportion to the increase in 2,3-DPG. Because 2,3-DPG is an impermeable polyanionic molecule there may be a concomitant increase of cations within the cell when 2,3-DPG levels increase, so that charge neutrality is maintained (Brewer et al. 1972). Increases of these charged molecules within the cell would obviously produce a swelling of the cell thereby lowering MCHC. There is biochemical evidence that the free 2,3-DPG concentration of the red blood cell plays a role in membrane ion transport (Benesch and Benesch, 1967) and could thus influence the composition of its own ionic environment (Benesch, Benesch and Yu, 1969). A relationship has also been reported between 2,3- DPG changes and red cell cation permeabil• ity in a study on chronic hypercapnia (Messier and Schaefer, 1971). iI To place.the changes in MCHC and 2,3-DPG, as a result of exercise, in a physiologic perspective, changes in P_n facilitating oxygen delivery have 96 i been calculated, using the equations of Bellingham et al. (1971), and are presented in Table XXIV. TABLE XXIV Theoretical Changes in 7 ^ of the Oxygen Dissociation Curve of Hemoglobin as a Result of Changes in 2,3-DPG and MCHC Produced Through One Hour of Exercise Change in P,.- Change in P,-0 Total Change in P5Q 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 0.23 0.36 Moderately Fit Nonsmokers -0.07 0.26 0.19 Highly Fit 0.08 0.32 Nonsmokers 0.24 Sedentary 0.03 0.35 Smokers 0.'31 Moderately Fit Smokers 0.24 0.43 0.67 All Subjects Pooled 0.17 0.21 0.38 Because changes in P^Q were all less than one Torr the physiological i effect in 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 in 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 if it does occur, it 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 in 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 cell 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 activity 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, is not significantly affected by the amount of 2,3-DPG present before the stimuli is 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 in red cell 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 is reduced, either through reduction in MCHC or an increase in blood viscosity, 2,3-DPG levels rise to compensate,by producing a decreased affinity 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 is not an important stimulator of 2,3-DPG production. 100 Conclusions 1. 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The Nether- Lands: Charles C. Thomas, 1970. WANG, Y., SHEPHERD, J., MARSHALL, R., ROWELL, L. and TAYLOR, H., Cardiac Response to Exercise in Unconditioned Young Men and In Athletes. Circulation, 24: 1064, 1961. WILLIAMS, CG., WYNDHAM, C.H., KOK, R. and VON RAKDEN, M.J., Effect of Training on Maximum Oxygen Uptake and on Anaerobic Metabolism in Man. Int. Z. Angew. Arbeitsphysiol., 24: 18, 1967. APPENDIX A Statistical Analysis 110 Ill 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 24 Critical F 7X= 14.3 Critical F nwm 7r -° max ,05(10,7) max .01(10,7) * The F -Test relies on the tabled cumulative probability max * ' distribution of a statistic which is the variance of the largest to the smallest of the several sample variances. The maximum variance ratio, S2 /S2 max min was compared to the cumulative probability distribution of Fmax(a,n-1), where a = 10 (five status variables-before and after exercise) and n = 8 (eight sub- I jects per group). The distribution is found in Table T of a text of statistical 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) io. 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 ** Correlation significant at the .05 level. a .01 = 0.403 * Correlation significant at the .01 level. 1 APPENDIX B Individual Scores 114 TABLE I Blood Parameter Data of Sedentary Nonsmokers Before and After Exercise Subject anatocrit Hemoglobin MCHC 2,3-DPG 2,3-DPG 2,3-DPG pH (%) (gms/100 ml) (uM/ml blood)(uM/ml cells) (uM/gm Hb) 46.2 16.68 36.10 1.73 3.74 10.37 7.362 B DG 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 ER 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 JW 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 MC 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 BT 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 DM 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 BF 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 RS 46.2 16.68 36.10 1.78 3.85 10.67 7.409 A TABLE II Blood Parameter Data of Moderately Fit Nonsmokers Before and After Exercise Su,bj ect natocrit Hemoglobin MCHC 2,3-DPG 2,3-DPG 2,3-DPG pH (%) (gms/100 ml) (uM/ml blood) (uM/ml cells) (uM/gm Hb) 45.9 15.57 33.92 2.05 4.47 13.17 7.409 B JG 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 JM 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 RB 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 LC 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 BH 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 DJ 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 BK 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 RM 41.4 15.41 37.23 1.89 4.57 12.26 7.449 A TABLE III Blood Parameter Data of Highly Fit Nonsmokers Before and After Exercise Subject uatocrit Hemoglobin MCHC 2,3-DPG 2,3-DPG 2,3-DPG pH (%) (gms/100 ml) uM/ml blood) (uM/ml cells) (uM/gm Hb) 42.1 13.08 31.07 1.87 4.44 14.30 7.388 B GL 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- SD 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 NV 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 RH 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 KF 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 DK 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 ED 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 GB 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 aatocrit Hemoglobin MCHC 2,3-DPG 2,3-DPG 2,3-DPG PH '(%) (gms/100 ml) (uM/ml blood) (uM/ml cells) (uM/gm Hb) 48.6 17.25 35.50 2.00 4.12 11.59 7.388 B WM 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 EK 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 LH 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 HR 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 DJ 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 KD 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 DW 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 TG 44.2 15.02 33.98 2.05 4.64 13.65 7.415 A TABLE V Blood Parameter Data of Moderately Fit Smokers Before and After Exercise Subject Hematocrit Hemoglobin MCHC 2,3-DPG 2,3-DPG 2,3-DPG pH (gms/100 ml) (uM/ml blood) (uM/ml cells) (uM/gm Hb) 46.0 16.33 35.50 2.23 4.85 13.66 7.343 B BM 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 TT 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 RU 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 GH 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 WL 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 GT 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 KC 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 NT 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 Fit 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 Fit 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 Fit 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 Statistical 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 High Work Variable Low Work Capacity Subjects Capacity Subjects Pre Exercise 45.29±2.57 47.57±2.27 Hematocrit Post Exercise 46.55±2.30 47.7512.25 Pre Exercise 15.33+1.68 16.5611.00 Hemoglobin Post Exercise 16.12±1.56 17.04+0.90 Pre Exercise 33.7412.38 34.7711.25 MCHC Post Exercise 34.5712.12 35.66+1.17 .12.6011.76 12.3211.58 2,3-DPG Pre Exercise Post Exercise 12.6912.02 12.6011.45 Blood pH Pre Exercise 7.3610.03 7.3910.06 Post Exercise 7.42+0.04 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,,2 8 0.014 0.029 >.05 2,3-DPG 1,,2 8 0.139 0.485 >.05 Blood pH 1,,2 8 0.002 1.447 >.05 Hematocrit 1.,2 8 4.283 8.002 <.01 Hemoglobin 1.,2 8 0.354 2.487 >.05