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Reliability of acid-base variables of arterial blood using the astrup micro-equipment Stevenson, Christopher Leonard 1969

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THE RELIABILITY OF ACID-BASE VARIABLES OF ARTERIAL BLOOD USING THE ASTRUP MICRO-EQUIPMENT by CHRISTOPHER LEONARD STEVENSON B. Sc. University of London, 1965 D.L.C., Cert. Ed. Loughborough College of Education, A Thesis submitted In p a r t i a l f u l f i l l m e n t of the requirements f o r the Degree of Master of Physical Education i n the School of Physical Education and Recreation We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA May 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r pub 1 i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Phys^al Education and Recreation The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada D a t e ABSTRACT The test - retest r e l i a b i l i t y c o e f f i c i e n t s of the values of measurements made on the pH, the Pco2, the standard bicarbonate, the base excess, and the buffer base, of a r t e r i a l whole blood were estimated for a group of 30 male subjects with the use of the Astrup Micro-equipment, the Siggaard-Andersen revised Nomogram, and a t e s t i n g program on three successive mornings. R e l i a b i l i t y c o e f f i c i e n t s were estimated f o r these parameters i n both the re s t i n g and the post-exercise conditions, and the i n t r a - i n d i v l d u a l and the i n t e r - i n d i v i d u a l variances were estimated f o r each r e l i a b i l i t y c o e f f i c i e n t . It was found that the standard bicarbonate and the base excess had the more r e l i a b l e values, and that the pH and P C 0 2 had less r e l i a b l e values. It was shown that the values of measurements of pH, standard bicarbonate and base excess were s i g n i f i c a n t l y d i f f e r e n t on Day 1 from those values on Days 2 and J. This e f f e c t was attributed to apprehension towards the te s t i n g experience and to the strangeness of the testing environment. The measurement errors of the Astrup Micro-equipment and the Siggaard-Andersen revised Nomogram were estimated f o r the i n d i v i d u a l parameters. It was found that the buffer base was the only parameter l n which the pH meter measurement error variance was large enough to have a decided e f f e c t upon the value of the r e l i a b i l i t y c o e f f i c i e n t . The Nomogram measurement error variances were so small that they could be considered n e g l i g i b l e . The temporal measurement error of the c o l l e c t i o n of blood - the error inherent i n the time difference between the c o l l e c t i o n of successive tubes of blood - was investigated f o r the pH parameter. This temporal measurement error was shown to be p r a c t i c a l l y n e g l i g i b l e although s t a t i s t i c a l l y s i g n i f i c a n t i n the r e s t i n g condition, but was shown to have both p r a c t i c a l importance and s t a t i s t i c a l s i g nificance i n the post-exercise condition. ACKNOWLEDGMENT The Author would l i k e to express his apprecia-t i o n of the assistance given to him "by the members of his Committee, e s p e c i a l l y Dr. S. E. Brown, Chairman, and Dr. B. G. Marteniuk, f o r t h e i r invaluable assistance. Special thanks must go to Miss R. Stadfeld f o r her generous and f r i e n d l y help i n the Laboratory, and to Miss P. N u t t a l l f o r her patience and understanding. TABLE OF CONTENTS CHAPTER PAGE I. Statement of the Problem 1 I I . Review of the Literature 8 I I I . Methods and Procedures 26 IV. Results and Discussion 36 V. Summary and Conclusions 59 BIBLIOGRAPHY 63 APPENDICES A. S t a t i s t i c a l Treatments . . . 69 B. Sample Data Sheet 72 C. Raw Scores 74 LIST OF TABLES TABLE PAGE I. The Normal Values and Ranges of the Acid-Base Parameters of A r t e r i a l Blood 9 I I . General Characteristics of the Group and of the Environment During the Testing Period 36 I I I . Means and Standard Deviations of the Acid-Base Parameters of A r t e r i a l Blood, At Rest (37°C) . . 39 IV. Means and Standard Deviations of the Acid-Base Parameters of A r t e r i a l Blood, A f t e r Exercise (37°C) 40 V. Differences Between the Resting and Post-Exercise Mean Values of the Parameters of Blood 41 VI. Differences-Between-Days Over the Three Day Testing Period l n the Variables of the Blood . . 42 VII. Results of t Tests on the Variables Having S i g n i f i c a n t F Ratio Values 42 VIII. Between-Days R e l i a b i l i t i e s of the Values of the Parameters of A r t e r i a l Blood 45 IX. Results of t and z Tests on the Differences Between Correlation Series 47 X. The TOVAR Components of the Between-Days R e l i a b i l i t i e s of the Parameters of Blood . . . . 48 XI. The COVAR Component of the Between-Days R e l i a b i l i t i e s of the Parameters of Blood . . . . 48 V TABLE PAGE XII. The Values of the WIVAR Plus Measurement Error Component of the Between-Days R e l i a b i l i t i e s of the Parameters of Blood 49 XIII. The Changes i n the COVAR and WIVAR Values i n the Comparison of the Resting and Post-Exercise Conditions, as Percentages of Their Ori g i n a l Values 51 XIV. The Changes i n the COVAR and WIVAR Values, i n the Comparison of the Day 1 - Day 2 Series with the Day 2 - Day 3 Correlation Series, as Percentages of Their Ori g i n a l Values 51 XV. The pH Meter Measurement Errors f o r the Acid-Base Parameters 53 XVI. The Adjusted Values of the Between-Days Correlation by the Removal of Measurement Error Variance from the Denominator i n the COVAR/TOVAR Ratio 54 XVII. The Differences Between the Origi n a l Values of the Between-Days Correlations and the Adjusted Values f o r the Acid-Base Parameters . 55 XVIII. The Measurement Errors of the Siggaard-Andersen Revised Nomogram . . . . 56 XIX. Results of t Test on the Difference Between the pH of Two Tubes of Blood Due to the Temporal Error of C o l l e c t i o n 56 CHAPTER I STATEMENT OF THE PROBLEM The Problem The pH, the carbon dioxide tension ( P C 0 2 ) , the standard bicarbonate concentration, the base excess concentration, and the buffer base concentration of blood are physiological para-meters that are used extensively i n medicine. C l i n i c a l l y the values of the parameters of the blood often provide the basis f o r diagnoses of the condition of hos p i t a l patients, and f o r t h e i r subsequent treatment. The functional a p p l i c a t i o n of mea-surements of the parameters, both i n appropriate spheres of medical research and i n the related f i e l d of exercise physiology, depends upon the recognition of, and the estimation of, devia-tions of the values of the parameters from t h e i r accepted normal values. In order to use the measurements of the parameters pro-perly i n diagnosis and research, i t i s necessary to know within what l i m i t s of accuracy the measures, that are made on samples of blood taken from an in d i v i d u a l under standardized conditions, can be accepted. The s p e c i f i c problems of t h i s study were the estimation of the r e l i a b i l i t y of sample data that can be obtained using the Astrup micro equipment, and the estimation of the values of ce r t a i n errors that are present i n the method, including the 2. equipment associated with i t . The attack on these s p e c i f i c pro-blems can be outlined i n terms of the following objectives: 1) The determination of the error inherent in the temporal aspect of blood c o l l e c t i o n . 2) The determination of the measurement error of the pH meter (Radiometer PHM 22). '3) The determination of the error involved i n the use of the Siggaard-Andersen revised nomogram. 4) The determination of the presence of differences-between-days i n the values of the acid-base parameters of a r t e r i a l blood over the successive periods of t e s t i n g . •5) The determination of the r e l i a b i l i t i e s of consecutive day-to-day measurements of the acid-base parameters of a r t e r i a l blood, at rest and a f t e r sub-maximal exerc i s e . 6) The determination of the i n t r a - i n d i v i d u a l differences of the acid-base parameters of a r t e r i a l blood, at rest and a f t e r sub-maximal exercise. .7) The determination of the i n t e r - i n d i v i d u a l differences of the acid-base parameters of a r t e r i a l blood, at rest and a f t e r sub-maximal exercise. The problems were investigated by using a volunteer group of 30 male subjects. The f i r s t objective was investigated by the comparison of two a r t e r i a l blood samples taken immediately one a f t e r the other from the same subject under the same conditions. The second and t h i r d objectives were investigated 3. by the comparison of two readings from the same tube of a r t e r i a l blood under constant conditions, and the comparison of two sets of readings from the same tubes of blood under constant condi-t i o n s . Objectives 4-7 were investigated by the use of appropriate s t a t i s t i c a l analyses upon the r e s u l t s of the analyses of a r t e r i a l blood samples taken from the group of subjects each of whom was tested on three consecutive mornings. Def i n i t i o n s 1) R e l i a b i l i t y i s a measure of the r a t i o of i n t e r -i n d i v i d u a l variance to the t o t a l variance i n test scores. (1) 2) Total variance may be defined as the sum of a l l the v a r i a t i o n s l n the test scores. It includes the variations due to i n t e r - i n d i v i d u a l differences, i n t r a - i n d i v l d u a l differences and measurement error. (1) 3) I n t e r - i n d i v i d u a l difference variance may be defined as the v a r i a t i o n between individuals that would be observed i f an i n f i n i t e number of scores f o r each in d i v i d u a l were pooled so that the v a r i a t i o n s within the subject, as well as variable error of measurement, would be averaged out and disappear. (1) 4) I n t r a - i n d i v i d u a l difference variance may be defined as variance due to changes within the i n d i v i d u a l , as well as variance i n h i s response to the test s i t u a t i o n . (1) \5) Measurement error variance i s the sum of the error of observation and the variable error of the 4. instrument. (1) 6) Differences-between-days refers to differences i n the mean values of the parameters f o r the group, of such magnitude that the s t a t i s t i c a l p r o b a b i l i t i e s would indicate that the days could be considered to have had sub s t a n t i a l l y d i f f e r e n t effects upon the values of the parameters. '7) pH i s a measure of the a c i d i t y of a system. The conventional d e f i n i t i o n of pH has pH equal to the negative logarithm to the base ten of the hydrogen ion a c t i v i t y (2), however i n practice an "operational" scale of pH has been developed by assigning c e r t a i n pH values to c e r t a i n buffer solutions. The electrometrlc pH i s operationally defined by the equation: pH-r s pH s + f (gx ~ Es) * X S In 10 . RT where pHs i s the pH assigned to a standard buffer solution, and Ex and E s the electromotive force of a c e l l when the electrodes are immersed i n the test so l u t i o n (E x) and the buffer standard ( E s ) , respectively. F i s Faraday's number, R the gas constant and T the temperature (Kelvin). (3) 8) The carbon dioxide tension, or Pc©2, may be defined as being equal to the p a r t i a l pressure of carbon dioxide i n a gas phase with which the sample i s i n equilibrium. (4) Pco2 i s an i n d i c a t i o n of the concentration of carbon dioxide i n the blood, i t i s measured i n millimeters of mercury. 9) Standard bicarbonate may be defined as equal to the plasma bicarbonate concentration a f t e r the e q u i l i -brium of whole blood to a P C 0 2 of 40 mm. Hg. at 3?°C. (5) 10) Base excess may be defined as the base concentration as measured by t i t r a t i o n with strong a c i d to pH 7.40, at a P c o 2 of 40 mm. Hg. at 37°C. Base excess i s an i n d i c a t i o n of the excess or d e f i c i t of base i n the blood; i t i s measured i n m i l l l e q u i v a l e n t s per l i t r e . (4) 11) Buffer base i s defined as the sum of buffer ions i n the blood or plasma (6); the units are i n m i l l l -equivalents per l i t r e . 12) The temporal aspect of the c o l l e c t i o n of blood i s the time difference between the c o l l e c t i o n of one tube of blood and the c o l l e c t i o n of the next tube of blood. Limitations For a r e l i a b i l i t y study of t h i s type a more i d e a l arrangement would have been to have had 60 subjects. (7) Unfor-tunately t h i s would have required a very large amount of time and was not p r a c t i c a b l e . Delimitations The study was concerned s o l e l y with a r t e r i a l whole blood, 6. no investigations were made of either venous blood or blood plasma. The study was conducted e n t i r e l y using male subjects, consequently the corresponding r e s u l t s f o r females cannot be accurately assessed from the r e s u l t s . The study would have been more i d e a l i f the experiment had been repeated a f t e r an i n t e r v a l of a few months. (8) This, unfortunately, would have also required a large amount of time and was not pr a c t i c a b l e . 7. REFERENCES 1. Henry, F.M., " R e l i a b i l i t y , Measurement Error and Intra-Individual Differences," Research Quarterly. 30 , March 1959, P. 2 1 . 2. Sorensen, S.P.L., "Enzymstudien, I I , " Blochemlsche Z e l t s c h r l f t , 21 , 1909, p. 131 . 3. Kildeberg, P., C l i n i c a l Acid-Base Physiology, Copenhagen, Munksgaard, 1968. 4 . Astrup, P., Engel, K., Jorgensen, K., Siggaard-Andersen, 0., "Definitions and Terminology i n Blood Acid-Base Chemistry," Annals of the New York Academy of Science, 133. AA. 1, 1966, p. 59 . 5. Jorgensen, K., Astrup, P., "Standard Bicarbonate, i t s C l i n i c a l Significance and a New Method f o r i t s Determi-nation," Scandinavian Journal of C l i n i c a l and Laboratory  Investigation, 9, 1957. P« 122. 6 . Singer, R.B., Hastings, A.B., "An Improved C l i n i c a l Method fo r the Estimation of Disturbances of Acid-Base Balance of Human Blood," Medicine, 27 , 1948, p. 223. 7. Marteniuk, R.G., personal communication, September 1968. 8. Sanders, H.D., personal communication, September 1968. CHAPTER II REVIEW OF THE LITERATURE This review of the related l i t e r a t u r e has been divided into three areas: 1) Literature dealing with the effects of cer t a i n environmental agencies upon the acid-base parameters of blood; 2) Literature concerned with the instrumentational and the methodological errors; and 3) Reports of investigations which have been concerned with the d a i l y v a r i a t i o n s , and the i n t r a - i n d i v i d u a l and i n t e r - i n d i v i d u a l v a r i a t i o n s , i n the a c i d -base parameters of blood. 1) The Eff e c t s of Certain Environmental Agencies These are dealt with under the relevant subheadings. Normal Values of the Parameters The l i t e r a t u r e abounds with "normal values" and "normal ranges" f o r the various parameters of blood. The small variations that may be found among the reported values may be attributed to errors of sampling and to differences i n techniques and methodologies. The values and ranges i l l u s t r a t e d i n Table I are those reported by the Ad Hoc Committee on Methodology, S.R. Gamblno, Chairman. (1) 9. TABLE I THE NORMAL VALUES AND RANGES OF THE ACID-BASE PARAMETERS OF BLOOD (1) Parameter 38°C Value Range Value 37 °C Range PH 7-390 7.360 - 7.420 7.405 7 . 3 7 5 - 7 . 4 3 5 Pee>2 mm.Hg. 41.2 3 6 - 4 7 39.3 Standard Bicarbonate m.equ./L. 22.9 21.3 - 24.8 as at 38°C Base Excess m.equ./L. -0.1 -2.4 - + 2 . 3 as at 38°C B u f f e r Base m.equ./L. 46 - 52 as at 38 °C D i e t a r y Influences The work done by Hasselbalch (2 ) , and by Hasselbalch and Gammeltoft (3 ) . and reported by Siggaard-Andersen (4) and M o l l e r ( 5 ) , i n d i c a t e d t h a t a vegetable-based d i e t r e s u l t e d i n a P C 0 2 of blood t h a t was 2 - 3 nm. Hg. higher than a Pc© 2 on a meat-based d i e t . The pH of the blood, however, was found, by Hasselbalch (2 ) , t o be not s i g n i f i c a n t l y d i f f e r e n t on a vegetable-based d i e t or a meat-based d i e t . In a d d i t i o n , Lundbaek (6 ) , i n v e s t i g a t i n g carbohydrate 10. die t s , found that a high carbohydrate d i e t tended to rai s e the P C 0 2 of blood by 3 Hg. compared with a low carbohydrate d i e t . The Influence of the Digestion of Food The picture regarding the influence of meals upon the acid-base parameters of blood i s not a very c l e a r one. Jansen and Karbaum (7) produced evidence of a r i s e i n the pH of blood during the d i r s t few hours a f t e r a meal. However, Cullen and Earle (8), and Shock and Hastings (9). l a t e r concluded that there was no constant change i n pH of blood a f t e r meals. Hlgglns (10) reported evidence of a r i s e i n the Pco2 of blood a f t e r the ingestion of a meal. This finding was v e r i f i e d by Jansen and Karbaum (7). but was denied i n turn by Cullen and Earle (8), and Shock and Hastings (9). who found no consistent a l t e r a t i o n l n the Pco2« Hlgglns (10) also noted a f a l l i n the Pco2 of blood f o r approximately one hour a f t e r drinking black coffee. Siggaard-Andersen (4) has claimed that, although no change can be observed i n the base excess concentration of blood a f t e r a l i g h t meal, a f t e r a heavy meal the base excess concentration r i s e s by 3 - 4 m i l l i e q u i v a l e n t s per l i t r e (m.equ./L.). Moller (5) has claimed that a f t e r a very high protein meal the standard bicarbonate concentration of blood tends to r i s e by 2 - 3 m.equ./L. 11. Influence of Age and Sex Evidence that the pH of blood changes with increasing age has been produced by Eldahl (11), and by Shock and Ylengst (12). Eldahl found a r i s e i n the mean pH of his group of 0.12 pH units between the ages of 15 and 25 years, and a r i s e i n the mean pH of his group of 0.06 pH units between the ages of 25 - 60 years. Shock and Yiengst found a difference of 0.03 PH units between the mean pH of a group of 20 - 25 year olds and that of a group of 80 - 89 year olds. Moller (5). however, did not f i n d any s i g n i f i c a n t pH changes with age and, so, was not able to confirm these e a r l i e r f i n d i ngs. Shock (13) has produced evidence of a trend of increasing Pco2 of blood with age, showing i n his subjects a r i s e i n the mean Pco2 of 2.5 mm. Hg. between the ages of 11 and 2k years. Shock and Yiengst (12), however, found no s i g n i f i c a n t change. The pH of the blood of women has been reported to be 0.00 to 0.02 pH units higher than that of men (4). Moller (5), however, i n a l a t e r study did not f i n d any differences between the sexes l n pH. Shock and Hastings (9) found a 3.8 mm. Hg. difference, i n the Pco 2 of blood, between the sexes; Shock (13) showed that t h i s sex difference tended to set i n at about the age of 13 years, and that i t was maintained at approximately 2 mm. Hg. This reported difference was v e r i f i e d l a t e r by Moller (5), whose group of subjects also exhibited a 2 mm. Hg. difference i n the Pco 2 of blood. It has been claimed by Siggaard-Andersen (k) that women have a base excess concentration approximately 1 m.equ./L. lower 12. than men, having a value of -1.0 m.equ./L. compared with that of -0.1 m.equ./L. f o r men. The E f f e c t of Il l n e s s The deviations i n the values of the acid-base parameters of blood that are due to various i l l n e s s e s have been widely published and discussed i n s c i e n t i f i c journals. Singer (14) has summarized the basic categories of i l l n e s s which a f f e c t the parameters of blood, and has indicated what those effects are. Metabolic a c i d o s i s , a condition of non-volatile a c i d excess, r e s u l t s i n a lowering of the pH, the Pco2, and the Standard bicarbonate, the Base excess and the Buffer base, concentrations of the blood. Respiratory a c i d o s i s , a condition of carbonic a c i d excess, r e s u l t s i n a lowering of the pH, and a r a i s i n g of the Pc©2 and the Standard bicarbonate and Buffer base concentrations of the blood. Metabolic a l k a l o s i s , a condition of non-volatile a c i d d e f i c i t , r e s u l t s i n a raised pH, Pco2 and Standard bicarbonate. Base excess, and Buffer base, concentrations i n the blood. Respiratory a l k a l o s i s , a condition of carbonic a c i d d e f i c i t , r e s u l t s i n a raised pH but a lowered Pco2, Standard bicarbonate and Buffer base concentrations i n the blood. The e f f e c t of c l i n i c a l shock on the acid-base status of the blood has been studied by Cournand, et a l . (15) They found that i n early shock, ir r e s p e c t i v e of Its severity, the a c i d -base parameters of the blood were s t i l l normal. Later, an 13-uncompensated acidosis developed due to a high blood concentra-t i o n of L a c t i c Acid that resulted i n a lowering of the values of a l l the parameters. As recovery took place i t was found that the acidosis usually disappeared. The E f f e c t of Posture Rahn (16) has been quoted i n many a r t i c l e s and reviews as the source of the information that the Pco2 of the blood i n the l y i n g p o s i t i o n i s 3 - 4 mm. Hg. higher than the Pco2 i n the standing or s i t t i n g p ositions. However, e a r l i e r Higgins (10) had found evidence that the Pco2 of the blood i s higher i n the l y i n g p o s i t i o n than the s i t t i n g p osition, and higher i n the s i t -t i n g p o s i t i o n than i n the standing p o s i t i o n . The E f f e c t of Exercise There has been general agreement that the pH of blood remains more or less the same during mild exercise, that i t f a l l s s l i g h t l y during moderate exercise, and that during severe exercise i t f a l l s steeply. (17,18,19,20,21) There has, however, been disagreement over the effects of exercise on the Pco2 of blood. Some authors (22,23) have found that upon exercise the Pco2 rose, other authors (21,20,24) have found that the Pco2 f a l l s , and yet others have discovered no change i n the Pco2 upon exercise. (25) In addition, Terasllnna and McLeod (18) found that the Pco2 f e l l during the early stages of exercise, and then rose as the exercise continued. Holmgren and Svanborg (2 3) attributed these differences i n 14. findings to differences i n the types of exercises used, and differences i n methodologies and measuring techniques used, and to sampling er r o r s . There i s a consensus that the Standard bicarbonate concentration, the Base excess concentration, and the Buffer base concentration of blood f a l l s l i g h t l y with mild and moderate exercise, and f a l l more steeply with severe exercise ( 2 2 , 2 1 , 2 0 ) . The E f f e c t of A l t i t u d e Numerous authors have produced evidence that upon ascent to high a l t i t u d e a sea level-based i n d i v i d u a l suffers a form of compensated a l k a l o s i s ( 2 6 , 2 7 , 2 8 , 2 0 ) . The a l k a l o s i s has been shown to endure f o r the entire duration of the period at high a l t i t u d e ( 20 ,27 ) , even f o r periods up to eight weeks. (27) The E f f e c t of the External Temperature The work by Senay and Chrlstensen (29) on acute exposure to heat showed that the pH of blood rose during the exposure, irr e s p e c t i v e of whether the subject was working or r e s t i n g . Following an i n i t i a l rapid Increase, the pH rose l i n e a r l y over time f o r the duration of the exposure period. 2) Methodological Errors There are errors i n the method of analysis of blood, using the Astrup micro equipment and the Siggaard-Andersen revised nomogram, that lend themselves to quantitative a n a l y s i s . In t h i s section of the chapter there i s reviewed the errors involved i n the sampling of the blood, the e f f e c t of 15-temperature, the eff e c t of storage, and the use of the pH meter and the microtonometer. Sampling of the Blood The loss of carbon dioxide from the blood to the atmos-phere, while the sampling procedure was i n progress, was found to be n e g l i g i b l e by Siggaard-Andersen. (30) The same author has also claimed that a heparin preparation concentration of 1 gram/Litre would lead to a f a l l i n pH of 0.003 pH units, a r i s e i n Pc©2 of 0.1 mm. Hg., and a f a l l i n Base excess concentration of 0.2 m.equ./L. (30) The use of c a p i l l a r y blood from the finger or earlobe i n place of a r t e r i a l blood has been well supported In research. The absence of any s i g n i f i c a n t difference between c a p i l l a r y and a r t e r i a l blood has been shown by a number of authors (31,32,33. 3^,35). The mean differences that were found ranged between -0.001 ± 0.009 ( 3 D and -0.009 - 0.009 (33) PH units f o r the pH of c a p i l l a r y blood with respect to a r t e r i a l blood, and ranged between -O.32 ± 2.8 (31) and + 1.7 - 1-7 (33) mm. Hg. f o r the Pco2 of c a p i l l a r y blood with respect to a r t e r i a l blood. Koch (33) has claimed the standard bicarbonate concentration of c a p i l l a r y a r t e r i a l blood i s 0.25 - 0.75 m.equ./L. lower than that of a r t e r i a l blood. Ef f e c t of Temperature The pH of blood has been shown to f a l l 0.015 pH units f o r a 1°C r i s e i n temperature (36); however, t h i s a l t e r a t i o n i n pH 16 . has also been shown to vary i n magnitude with respect to the protein concentration l n the plasma, the haemoglobin concentra-t i o n and saturation, and the pH, of the blood. ( 1 ,36 ,37 ,38 ) It has been advocated (1) that the measurements of the acid-base parameters should be corrected to the body temperature of the subject only i f that body temperature deviates by more than two degrees Centigrade from the operating temperature of 37°C. The temperature correction f a c t o r f o r the Pc©2 of blood was found to be a r i s e of approximately 4.k% f o r a r i s e l n temperature of 1°C. This fig u r e has been accepted f o r current use i n acid-base work ( 1 , 4 ) . Bergman (39) has recently conclu-ded that: . . . systems concurrently i n use f o r correcting blood gas tensions f o r changes l n temperature are v a l i d f o r use i n blood samples having any Base excess concentra-t i o n , haemoglobin concentration and saturation l i k e l y to be encountered i n the physiological range. Astrup and Siggaard-Andersen (1) have reported that f o r 1°C changes i n temperature l n the area of the operating tempera ture, 37°C, the values of the Standard bicarbonate concentra-tions, the Base excess concentrations, and the Buffer base concentrations of blood were unaffected. They further claimed that the Siggaard-Andersen revised nomogram could be used at 37°C without a l t e r a t i o n . E f f e c t of Storage It has generally been accepted that f o r optimum accuracy the pH and the P C 0 2 of the blood should be measured within f i v e minutes of sampling. (1) 17. The changes i n the values of the acid-base parameters of blood over time, at various temperatures, have been studied by a number of investigators. (30,32,40) Siggaard-Andersen (30) has claimed that under conditions of anaerobic storage the change i n the values of the parameters at 22°C to 24°C was a f a l l of 0.024 pH units f o r pH, a f a l l of 0.5 m.equ./L. f o r the Base excess concentration, and a r i s e of 2.5 mm. Hg. f o r the Pco2; however, at 0°C to 4°C, he found a f a l l of only 0.006 pH units f o r pH, a f a l l of 0.1 m.equ./L. f o r the Base excess concentration, and a r i s e of only 0.6 mm. Hg. of the Pco2 of blood. Siggaard-Andersen concluded that the e f f e c t of anaerobic storage at 40°C was not s i g n i f i c a n t f o r a time period of up to three hours. Berglund, Malmberg and Stenhagen (40) have produced values of a pH f a l l during storage at 37°C of 0.0008 pH u n i t s / min. f o r the f i r s t 20 minutes, and a f a l l of 0.0009 pH units/aln. f o r the time thereafter. Maas and Van Heijst (32) have shown a reduction i n the pH value at a rate of 0.003 pH units/min. at 20°C, and a reduction of 0.001 pH units/min. at 5°C. The P H Meter Siggaard-Andersen (41) has claimed that the accuracy of the pH meter s o l e l y i s to an error of a i 0.003 pH un i t . Upon the i n c l u s i o n of sampling and methodological error, he claimed an accuracy f o r the pH meter to an error of t 0.005 of a pH un i t . 18 . The Mlcrotonometer The e f f e c t of both haemolysis and g l y c o l y s i s has been claimed (30) to be a n e g l i g i b l e factor; using the same gas mixtures and the same standard buffers, Siggaard-Andersen (4) has reproduced the carbon dioxide-equilibrated pH value to an error of t 0.002 of a pH u n i t . The Total Errors By taking dual readings on the same subjects at the same time, Siggaard-Andersen (4) has produced estimates of the errors to be expected from t h i s method of acid-base analysis of blood. He has claimed an error f o r pH of ± 0.005 of a pH unit, an error f o r P c o 2 of approximately two percent, an error f o r the Standard bicarbonate concentration of ± 0 .3 m.equ./L., and an error f o r the Base excess concentration of - 0.2 m.equ./L. Maas and Van Heijs t (42), and Naeraa (4-3) have also confirmed an error f o r the Pco£ by t h i s method of ± 2%; Berglund, Malmberg and Stenhagen (40) have confirmed the error over a P C 0 2 range of 31 to 56 mm. Hg. 3) Dally Variations There have been s u r p r i s i n g l y few studies that have focussed upon the estimation of the t e s t - r e t e s t r e l i a b i l i t i e s of the acid-base parameters of blood. Studies have been reported that have been concerned with twenty-four hour v a r i a t i o n s , and day-to-day v a r i a t i o n s , i n the acid-base chemistry of blood, but there have not been any studies that have dealt with the 1 9 . s t a t i s t i c a l concepts of i n t r a - i n d i v i d u a l variance and i n t e r -i n d i v i d u a l variance as components of the r e l i a b i l i t y . R e l i a b i l i t i e s Shock and Yiengst (12) computed the tes t - r e t e s t r e l i a -b i l i t i e s of the pH and the P e o 2 of blood from samples taken at two to three week i n t e r v a l s . Over t h i s period of time the r e l i a b i l i t i e s were O.36 f o r pH, and 0.59 f o r the P C 0 2 of blood. Shock (13) had e a r l i e r used the s t a t i s t i c of the Probable Error, which includes the te s t - r e t e s t c o r r e l a t i o n c o e f f i c i e n t , to i n d i -cate that i n his group, f o r the P C 0 2 of blood, tests repeated at intervals of one-half hour had errors of the same magnitude as tests repeated at interv a l s of one day. Day-to-Day Variations Shock and Hastings (9) measured the pH of a group of subjects, s i x days per week, over a 30 to 34 day period. The authors compared the standard deviations of the individuals* values with the standard deviation of the group. On t h i s basis they concluded that the day-to-day va r i a t i o n s , f o r some i n d i v i -duals, were as great as the variations within the group; and, f o r other in d i v i d u a l s , they claimed that the day-to-day v a r i a -tions were s i g n i f i c a n t l y l e s s , s t a t i s t i c a l l y , than the v a r i a t i o n within the group. The authors also concluded that the v a r i a -tions within a given i n d i v i d u a l may, l n some instances, be as large as the entire range of values found i n the normal population. 20. Moller (5) has claimed that the variations from day-to-day f o r any given subject were smaller than the v a r i a t i o n s between subjects i n his study. Moller, however, had compared the mean i n t r a - i n d i v i d u a l range of a group of 10 subjects, f o r the pH and Pcog parameters, with a range of twice the standard deviation of a control group of 100 subjects. Moller also concluded that, ". . . the i n d i v i d u a l patient often had s u r p r i s i n g l y constant values f o r a considerable length of time." (5:65) Twenty-four Hour Variations Cullen and Earle (8) found evidence that the acid-base condition of blood that had been established at the end of a period of sleep remained f a i r l y stable throughout the next few hours, and that early morning a c t i v i t y did not s i g n i f i c a n t l y a f f e c t i t . The changes i n pH that they found were a r i s e i n pH of 0.01 to 0.07 pH units during the day, and a s l i g h t f a l l i n pH during a period of sleep. Shock and Hastings (9) were unable to observe the r i s i n g pH phenomenon throughout the day i n t h e i r group. Some of t h e i r subjects exhibited a r i s e l n pH, others exhibited a f a l l i n pH. These authors (9) a l s o concluded that the v a r i a t i o n s i n the acid-base status of the blood during the day was not as great as the v a r i a t i o n s from day to day. Moller (5) has claimed that i n a l l his subjects the v a r i a t i o n s i n the pH and Pco2 parameters over twenty-four hours within the subjects were s i g n i f i c a n t l y lower than the va r i a t i o n s between the subjects. However, Moller had compared the mean 21. i n t r a - i n d i v i d u a l range of his group with the mean of the d i f f e r -ences between the subjects i n the group. ^Also, inspection of his subjects indicates that 4 were "normal," and 6 were "patients." Segregation of the data re s u l t s i n the conclusion that, f o r both.pH and Pco2, the mean variations between the subjects and the mean var i a t i o n s within the subjects were very s i m i l a r f o r the "normal" subjects, and were very d i f f e r e n t f o r the "patients." The consequence was that i n the combined r e s u l t s , upon which Moller based his conclu-sions, the re s u l t s from the "normal" subjects were masked by those of the "patients." Moller's conclusions must, therefore, be considered as being questionable.) 22. REFERENCES 1. Ad Hoc Committee on Methodology, S.R. Gambino, Chairman, Annals of the New York Academy of Science, 133, A r t . 1, 1966, p. 259-2. Hasselbalch, K.A., "Neutralitatsregulation und Reizbarkeit des Atemzentrums i n ihren Wirkungen auf die Kchlensaurespannung des Blutes," Blochemlsche  Z e l t s c h r l f t , 46, 1912, p. 403. 3- Hasselbalch, K.A., Gammeltoft, S.A., "Die N e u t r a l i t a t s -regulation des Graviden Organismus, Blochemlsche  Z e l t s c h r l f t , 68, 1915. P« 206. 4. Siggaard-Andersen, 0., The Acid-Base Status of the Blood, 2nd Ed., Williams and Wilkins Co., Baltimore, 1964. 5. Moller, B., "The Hydrogen Ion Concentration i n A r t e r i a l Blood," Acta Medica Scandinavica, suppl. 348, 1959. P- 1. 6. Lundbaek, K., "Fasting Values of Blood Sugar, R.Q., and Alveolar Carbon Dioxide Tension on High and Low Carbohydrate Diets," Acta Physiologlca Scandinavica, 7, 1944, p. 29. 7. Jansen, W.H., Karbaum, H.J., "Zur frage der Regulation des Saure - basen - gleichgewichts beim Normalen Menschen," Deutsches Archlv f u r Klinlsche Medlzln, 153, 1926, p. 84. 8. Cullen, G.E., Earle, I.P., "Studies of the Acid-Base Condition of the Blood, I I , H Journal of B i o l o g i c a l  Chemistry, 83, 1929, p. 5^5-9. Shock, N.W., Hastings, A.B., "Studies of the Acid-Base Balance of the Blood, I I I , " Journal of B i o l o g i c a l  Chemistry, 104, 1934, p. 585-10. Higgins, H.L., "The Influence of Food, Posture and Other Factors on the Alveolar Carbon Dioxide Tension i n Man," American Journal of Physiology, 34, 1914, p. 114. 11. Eldahl, A.,"Brintloncentrationen 1 blodet hos raske menneskar i f o r s k e l l i g e aldre og hos patienter med cancer og med hypertoni," Nordlsk Medlcln, 3, 1939, p. 2938. 12. Shock, N.W., Yiengst, M.J., "Age Changes l n the Acid-Base Equilibrium of the Blood of Males," Journal of  Gerontology, 5, 1950, p. 1. 2 3 . 1 3 . Shock, N.W., "Age Changes and Sex Differences i n Alveolar Carbon Dioxide Tension," American Journal of Physiology, 133, 1941, p. 610. 14. Singer, R.B., i n Altman, P.L., Gibson, J.F., Wang, C.C, Handbook of Respiration, W.B. Saunders Co., Philadelphia, 1958, p. 68 . 1 5 . Cournand, A., Riley, R.L., Bradley, S.E., Breed, E.S., Noble, R.P., Lauson, H.D., Cregersen, H.I., Richards, D.W., "Studies of the C i r c u l a t i o n i n C l i n i c a l Shock," Surgery, 13 , 1943, p. 964. 16 . Rahn, H., i n Comroe, J.H. J r . , Methods i n Medical Research, Volume 2, Chicago, 1950, p. 223. l ? . Holmgren, A., Mcllroy, M.B., "Effect of Temperature on A r t e r i a l Blood Gas Tensions and pH During Exercise," Journal of Applied Physiology, 19, 1964, p. 243. 1 8 . Teraslinna, P., McLeod, D.F., "The Effe c t of Exercise on Blood pH and P C 0 2 , Serum Glucose, Cholesterol, N.E.P.A. and Ketones," Journal of Sports Medicine, 6, 1966, 1, P. 235 . 1 9 . Freyschuss, U., Strandell, T., "Limb C i r c u l a t i o n During Arm and Leg Exercise i n the Supine P o s i t i o n , " Journal of  Applied Physiology, 2 3 , 1967, p. 163. 20 . Hansen, J.E., S t e l t e r , G.P., Vogel, J.A., " A r t e r i a l Pyruvate, Lactate, pH, and P C 0 2 During Work at Sea Level and High A l t i t u d e , " Journal of Applied  Physiology, 23, 1967, p. 523-2 1 . Bouhuys, A., Pool, J., Binkhorst, R.A., Van Leeuwen, P., "Metabolic Acidosis of Exercise i n Healthy Males," Journal of Applied Physiology. 21 , 1966, p. 1040. 22 . Delanne, R., Barnes, J.R., Brouha, L., Massark, F., "Changes i n Acid-Base Balance and Blood Gases During Muscular A c t i v i t y and Recovery," Journal of Applied Physiology, 14, 1959, P- 328 . 2 3 . Holmgren, A., Svanborg, N., "Venous Admixture During Exercise i n the S i t t i n g P o s i t i o n , " Scandinavian Journal  of C l i n i c a l and Laboratory Investigation, 17, 1965. p. 209. 24. Barr, D.P., Himwich, H.E., Green, R.P., "Studies i n the Physiology of Muscular Exercise, I," Journal of  B i o l o g i c a l Chemistry. 55 , 1923, p. 495 . 24. 2 5 . Baldwin, E. deF., Cournand, A., Richards, D.W., "Pulmonary Insufficiency, I," Medicine, 2 7 , 1948, p. 243. 2 6 . D i l l , D.B., Ta l l b o t t , J.R., Consolazio, W.V., "Blood as a Physiochemical System, XI, Man at High A l t i t u d e , " Journal of B i o l o g i c a l Chemistry, 1 1 8 , 1 9 3 7 , p. 649. 2 7 . L a h i r i , S., Mllledge, J.S., Chattopadhyay, H.P., Bhattacharyya, A.K., Slnha, A.K., "Respiration and Heart Rate of Sherpa Highlanders During Exercise," Journal of Applied Physiology, 2 3 , 1 9 6 7 , p. 5 4 5 . 28. Cain, S.M., Dunn, J.E., "Low Doses of Acetazolamide to Aid Accomodation of Men to A l t i t u d e , " Journal of Applied  Physiology, 2 1 , 1 9 6 6 , p. 1 1 9 5 . 2 9 . Senay, L.C., Chrlstensen, M.L., "Variations of Certain Blood Constituents During Acute Heat Exposure," Journal  of Applied Physiology, 24, 1 9 6 8 , p. 3 0 2 . 3 0 . Siggaard-Andersen, 0 . , "Sampling and Storing of Blood f o r Determination of Acid-Base Status," Scandinavian  Journal of C l i n i c a l and Laboratory Investigation, 1 3 , 1 9 6 1 , p. 1 9 6 . 3 1 . Torjussen, W., Nitter-Lauge, S., "pH and P C 0 2 i n C a p i l l a r y Blood from the Ear Lobe," Scandinavian Journal of  C l i n i c a l and Laboratory Investigation, 19, 1 9 6 7 . P. 7 9 . 3 2 . Maas, A.H.J., Van Hei j s t , A.N.P., "A Comparison of the pH of A r t e r i a l Blood with A r t e r i a l i s e d Blood from.the Ear Lobe with Astrup's Micro Glan Electrode," C l i n l c a  Chimlca Acta.,- 6 , 1 9 6 1 , p. 3 1 « 3 3 . Koch, G., "Comparison of P c© 2 , pH and Standard Bicarbonate i n C a p i l l a r y Blood and i n A r t e r i a l Blood," Scandinavian  Journal of C l i n i c a l and Laboratory Investigation. 1 7 , 1 9 6 5 , p. 2 2 3 . 3 4 . Gambino, S.R., "Comparisons of pH i n Human A r t e r i a l , Venous and C a p i l l a r y Blood," American Journal of C l i n i c a l  Pathology, 32, 1 9 5 9 , p. 2 9 8 . 3 5 . Genbelle, F., Nicolas-Goldstein, M., "Comparaison entre l e CO2 et l e pH du sang c a p i l l a i r e et du sang a r t e r i e l , " C l l n i c a Chimlca Acta.. 3 , 1 9 5 8 , p. 480. 3 6 . Rosenthal, T.B., "The Effe c t of Temperature on the pH of Blood and Plasma In V i t r o , " Journal of B i o l o g i c a l  Chemistry, 1 7 3 . 1 9 4 8 , p. 2 5 . 25 . 37- Astrup, P., Schroder, S., "Apparatus f o r Anaerobic Determination of the Blood at 38°C," Scandinavian  Journal of C l i n i c a l and Laboratory Investigation, 8, 1956, p. 30 . 38. Adamsons, K., Da n i e l l , S.S., Gandy, G., James, L.S., "Influence of Temperature on Blood pH of the Human Adult and Newborn," Journal of Applied Physiology, 19, 1964, p. 897-39. Bergman, N.A., "Temperature Coefficients f o r P C 0 2 and P 0 2 i n Blood with Varying Acid-Base Status," Journal of  Applied Physiology, 24, 1968, p. 225 . 40. Berglund, E., Malmberg, R., Stenhagen, S., "Determination of Carbon Dioxide Tension of Whole Blood by pH Measurements and Interpolation," Scandinavian Journal of C l i n i c a l and Laboratory Investigation, 16, 1964, p. 185-41. Siggaard-Andersen, 0 . , "Factors A f f e c t i n g the Li q u i d -junction Potential i n Electrometric Blood pH Measure-ment," Scandinavian Journal of C l i n i c a l and Laboratory  Investigation, 13, 1961, p. 205. 42. Maas, A.H.J., Van Heljst, A.N.P., "The Accuracy of the Micro-determination of the Pcoo of Blood from the Ear Lobe," C l l n l o a Chimlea Acta., 6, 1961, p. 34. 4 3 . Naeraa, N., "The Accuracy of Pco 2 Determinations with the Micro - pH Method Using One-point E q u i l i b r a t i o n , " Scandinavian Journal of C l i n i c a l and Laboratory  Investigation, 16, 1964, p. 195. CHAPTER III METHODS AND PROCEDURES Introduction This study may be considered as having been carried out i n three d i s t i n c t phases. Phase one consisted of work done i n developing estimations of the differences-between-days and of the r e l i a b i l i t i e s ; the second phase concerned the errors inherent i n the instruments that were used; and the t h i r d phase was devoted to the inves t i g a t i o n of the temporal aspect of the c o l l e c t i o n of the blood. ;1) The R e l i a b i l i t i e s and Differences-Between-Days  Outline A group of 30 volunteer male subjects provided blood samples f o r t h i s phase of the study. Blood was obtained from each subject on three consecutive mornings, under standard conditions, (1) while the subject was at rest, (2) immediately following a sub-maximal exercise task. The samples were then analyzed by means of the Astrup micro equipment. S t a t i s t i c a l analyses of the measurement values of the parameters of a r t e r i a l blood were designed to Indicate the existence of differences-between-days and to produce estimates of the r e l i a b i l i t i e s of the values of the parameters. 27. The Group The subjects used i n t h i s study were 3 0 volunteer male students attending the University of B r i t i s h Columbia. The group was made up from members of a class of Physical Education majors together with students from other f a c u l t i e s . The Timetable Each subject was tested on three consecutive mornings, with the i d e n t i c a l t e s t i n g procedure being used on each morning. The time i n t e r v a l between waking and r i s i n g and a r r i v i n g at the Laboratory was maintained as constant as"" possible, and no more than one hour's deviation i n t h i s time i n t e r v a l was allowed over the three day period. Instructions were given that the subjects were not to take breakfast or any beverage between waking and the laboratory session, and they were not to deviate any more than was necessary from t h e i r usual motor a c t i v i t i e s . Procedures The weights and the ages of the subjects were recorded on the f i r s t day of t e s t i n g . On each of the three mornings the barometric pressure and the room temperature were recorded, both p r i o r to and immediately a f t e r the t e s t . The o r a l temperature of the subject was measured during the i n i t i a l rest period, and the subject was questioned about his a c t i v i t i e s that morning, the time of r i s i n g , the amount of sleep the previous night, and the time and nature of his l a s t meal. The subject had an i n i t i a l ten minutes rest i n an upright s i t t i n g position, during which time the heart rate was obtained 28. by auscultation over a t h i r t y second period. A hot towel was applied to the subject's ear f o r the l a s t three minutes, a f t e r which blood was drawn from a puncture i n the fleshy lobe of the warmed ear into three heparinized c a p i l l a r y tubes / Radiometer D 551 . These were then sealed so that the blood was i n an anaerobic condition, and s t i r r e d with a s t i r r i n g rod and magnet to mix i n the heparin anticoagulent. The blood sampling was followed by a sub-maximal work task, consisting of a six minute ride on a Monark bicycle ergometer at a work rate of f i f t y cycles per minute with a lo a -ding of two kiloponds. During the f i n a l two minutes of the ride a hot towel was applied to the same ear as before, and the heart rate was taken by auscultation. Upon the cessation of the task, three more tubes of blood were immediately taken within a period of three to four minutes from the subject, who was, once again, i n an upright s i t t i n g p o s i t i o n . The purpose of the work task was simply to displace the acid-base status of the blood from Its resting l e v e l . It was judged that the work task set of 600 kilopond-meters per minute f o r six minutes was adequate f o r t h i s purpose, being neither too high nor too low f o r the range of f i t n e s s that could have been expected from the subjects. Analysis of the Blood A l l the analyses of the blood were carried out using the Astrup Radiometer micro equipment Radiometer AME 1 and the blood acid-base nomogram constructed by Siggaard-Andersen and 29 . Engel (1 ) , and revised by Siggaard-Andersen ( 2 ) . The micro equipment included a temperature-controlled mlcrotonometer Radiometer AMT 1 allowing f o r the simultaneous e q u i l i b r a t i o n of two samples at both high and low carbon-dioxide tensions, a temperature-controlled electrode system consisting of a replaceable c a p i l l a r y glass electrode Radiometer G 297 and a calomel reference electrode Radiometer K 4-97 . and a pH meter Radiometer PHM 22 . Carbon dioxide - oxygen gas mixtures used f o r the e q u i l i b r a t i o n were fed into the tonometer v i a temperature-controlled humidifiers. By means of the mlcrotonometer the samples of heparinized blood were equilibrated at known carbon dioxide tensions, namely 4. % carbon dioxide, and 8. % carbon dioxide. The pH of the blood was measured following the attainment of equilibrium, and the corresponding Pco2 and pH values were plotted on the Siggaard'-Andersen revised nomogram, a log.Pco2/pH diagram. The po s i t i o n of the Pco2/pH l i n e defined the acid-base parameters of the blood, other than that of the actual Pc©2 of the blood which was read d i r e c t l y off the l i n e at the point corresponding to the actual pH of the blood. The micro equipment, at a thermostatically-controlled temperature of 38°C, was accurately calibrated at the beginning of each week using the pr e c i s i o n buffer solutions, Radiometer S 1500 , pH 6.840 at 38°C, and Radiometer S 1 5 1 0 , pH 7.381 at 38°C,. A stock of buffer solution, pH 7.190 at 38°C, was used f o r the routine c a l i b r a t i o n s of the instrument. The majority of the tubes of blood were analyzed 30 . immediately a f t e r the te s t i n g period, but i n some cases i t was necessary to store the tubes f o r up to t h i r t y minutes i n a cold, wet towel. Of the three tubes of blood, the f i r s t tube obtained was used f o r the actual pH measurement, the other two were equilibrated with the high and low tension carbon dioxide gas mixtures i n the microtonometer, and were used to derive the Pc©2/ pH l i n e . The values of the parameters that were obtained at 38°C were converted to t h e i r appropriate values at 37°C using the established and conventional correction f a c t o r s . (4) Precautions In order to esta b l i s h standardized conditions f o r the estimation of the differences-between-days and the r e l i a b i l i -t i e s , the subjects were i n as consistent a physical condition as possible on each of the tes t i n g sessions. The precautions adhered to were, as mentioned previously, a consistency i n the time i n t e r v a l between waking and testing, an abstinence from food or beverage on the morning of the test, and the adherence to the normal routine of morning motor a c t i v i t i e s . S t a t i s t i c a l Treatments The data were analyzed primarily by an analysis of variance f o r a two fact o r design (days x subjects) with repeated measures on the subject factor, to indicate the variables i n which a difference-between-days occurred. (3) For those variables that had s i g n i f i c a n t F values, t tests were done on the differences between the means on adjacent days. 31. The t o t a l variance, the i n t e r - i n d i v i d u a l differences, and the i n t r a - i n d l v i d u a l differences, were obtained from the appro-priate r e l i a b i l i t y c o e f f i c i e n t s , which were computed as a r a t i o of i n t e r - i n d i v i d u a l variance to t o t a l variance. The possible e f f e c t of dlfference-between-days upon the r e l i a b i l i t y c o e f f i -cients was investigated by t e s t i n g the sig n i f i c a n c e of the difference between cor r e l a t i o n s . This was accomplished by the use of the t test on the difference between two c o r r e l a t i o n c o e f f i c i e n t s f o r nonindependent samples. (5) The ef f e c t of the exercise task upon the values of the r e l i a b i l i t y c o e f f i c i e n t s was tested by the z test f o r the s i g n i -ficance of the difference between two correlations c o e f f i c i e n t s f o r independent samples. There i s some question whether or not the samples are independent i n t h i s instance. The z test was, however, the only s t a t i s t i c a l test available, and so i t was necessary to assume that the samples were independent. 2) The Instrument Errors  Outline The errors inherent i n the Astrup micro equipment pH meter 22, and i n the Siggaard-Andersen revised nomogram, were investigated by means of comparisons of two readings from the same tube of blood, and by comparisons of the parameter values that the two readings produced. Procedure f o r the pH Meter Blood from a single heparinized c a p i l l a r y tube was equilibrated i n the mlcrotonometer. Following the attainment of 32. equilibrium, two measurements of pH were made on thi s blood. In t h i s way 216 pairs of readings were obtained, and the differences between the readings were taken to represent an error reading of the pH meter. The mean and the standard deviation of the error readings represented estimates of the constant error and the variable error, respectively, of the pH meter f o r the pH parameter of blood. Using t h i s same procedure f o r two tubes of blood taken from the same person at the same time, with one tube e q u i l i b r a -ted at the high carbon dioxide tension, and the other tube equilibrated at the low carbon dioxide tension, i t was possible to compare the ef f e c t that the error readings of the pH meter had on the parameters read off the Siggaard-Andersen revised nomogram. The f i r s t readings of the pH of the equilibrated blood, at both high and low tensions, were used as a pair f o r the nomogram purpose, and the second readings were s i m i l a r l y used as a p a i r . By t h i s method, 105 pairs of readings f o r each of the other parameters of blood were obtained, and the differences between the readings were taken to represent error readings f o r these parameters due to the error of the pH meter. The means and the standard deviations of the error readings were computed as estimates of the constant errors and the variable errors, respectively, of the pH meter f o r the Pco2, the Standard bicarbonate, the Base excess, and the Buffer base, concentra-tions of blood. 33. Procedure f o r the Nomogram The p l o t t i n g of the same set of Pco2 and pH values on two separate nomogram diagrams produced two sets of readings f o r the Pco2, the Standard bicarbonate, the Base excess, and the Buffer base, concentrations of blood. The differences between the readings f o r each p a i r were taken to represent error readings of the nomogram. The means and standard deviations of the error readings were computed, f o r each of the parameters, as estimates of the constant errors and the variable errors, respectively, of the nomogram. 3) The Temporal Aspect of the Col l e c t i o n of Blood  Outline The inte r e s t In t h i s aspect of the c o l l e c t i o n of blood stemmed from the methodology i n blood analysis of using three tubes of blood. One of the three tubes - i n t h i s study the f i r s t tube obtained - i s analyzed f o r the actual pH of the blood, the other two tubes are equilibrated with the C02/O2 gas mixtures to esta b l i s h the Pco2/pH l i n e on the nomogram. The rationale f o r the methodology assumes that the actual pH of the f i r s t tube i s representative of the pH's of the other two tubes. In addition, the error of measurement possible i n the res t i n g condition was considered l i k e l y to be increased In value i n the post-exercise condition. The pH a f t e r the exercise would tend to be f a l l i n g back to the resting l e v e l , and during the immediate post-exercise period the f a l l would tend to be at i t s greatest. This section of the chapter describes the 34. i n v e s t i g a t i o n of the methodological rationale and the ef f e c t of exercise upon i t . Procedure The measurement of the pH of the blood i n two tubes was made.. The tubes were taken from the same subject, one immedi-ate l y a f t e r the other. The difference between the two readings was taken as an estimate of the error of measurement due to the time i n t e r v a l i n sampling. F i f t y pairs of readings were obtained i n t h i s way i n the re s t i n g condition, and 10 pairs of readings were obtained following the previously s p e c i f i e d sub-maximal exercise task. The mean and standard deviation of the differences between readings were computed, and these were regarded as estimates of the constant error and the variable error, respectively, due to the temporal aspect of the c o l l e c t i o n of blood. 4) S t a t i s t i c a l Significance The l e v e l s of pr o b a b i l i t y that are commonly accepted as being s t a t i s t i c a l l y s i g n i f i c a n t are the 0.05 and the 0.01 l e v e l s . This study had no hypothesis, consequently the le v e l s of s t a t i s t i c a l s i g nificance that occurred were simply reported as being at either the 0.05 l e v e l or at the 0.01 l e v e l of pr o b a b i l i t y . 3 5 . REFERENCES 1. Siggaard-Andersen, 0., Engel, K., "A New Acid-Base Nomogram. An Improved Method f o r the Calculation of the Relevant Blood Acid-Base Data," Scandinavian Journal of C l i n i c a l  and Laboratory Investigation, 12, I960, p. 177. 2. Siggaard-Andersen, 0., "The pH, Log P C 0 2 Blood Acid-Base Nomogram Revised," Scandinavian Journal of C l i n i c a l and  Laboratory Investigation, 14, 1962, p. 598. 3. Guilford, J.P., Fundamental S t a t i s t i c s l n Psychology and Education, McGraw H i l l Co., New York, 1942. 4. Ad Hoc Committee on Methodology, S.R. Gambino, Chairman, Annals of the New York Academy of Science, 133. 1. 1966, P. 259-5- Ferguson, G.A., S t a t i s t i c a l Analysis l n Psychology and  Education. McGraw H i l l Co., New York, 1959. CHAPTER IV RESULTS AND DISCUSSION 1) Environmental and Group Characteristics The measurable c h a r a c t e r i s t i c s of the group and of the environment have been summarized In Table I I . The barometric pressure fluctuated throughout a wide range during the two and one-half month tes t i n g period; however, the changes i n the baro-metric pressure throughout the test i n g program of any given subject lay well within the l i m i t s of one standard deviation. TABLE II GENERAL CHARACTERISTICS OF THE GROUP, AND OF THE ENVIRONMENT DURING THE TESTING PERIOD Variable Mean Range N 30 — Age (Years) 22.8 ± 2.2 20 - 29 Weight (lbs.) 173-7 i 18.6 138 - 230 Oral (°C) Temperature — 36.4 - 36.8 Barometric Pressure (mm.Hg.) 759-3 ± 6.3 740.2 - 763.4 Room (°C) Temperature 20.5 18.8 - 21.7 37. The subjects were asked to comply with c e r t a i n i n s t r u c -tions on the three mornings of the experiment; f o r example, an abstinence from food, an adherence to t h e i r usual motor routine. Unfortunately, checks were not able to be made to ensure that the subjects did indeed adhere to the instructions, beyond the questioning period given each morning of the experiment. The judgments to the extent to which the instructions were carr i e d out, judgments made on the basis of the questioning periods, were therefore subject to a cer t a i n amount of scepticism. Nevertheless, there were no apparent reasons why the subjects should have f a l s i f i e d t h e i r answers; i n consequence, the answers were accepted at t h e i r face value. The length of the time period between waking and r i s i n g and a r r i v i n g at the Laboratory d i d not vary f o r any Individual subject by more than one hour, and f o r the majority of subjects i t varied by not more than t h i r t y minutes. The time i n the morning at which the t e s t i n g took place did not vary by more than one hour f o r any given i n d i v i d u a l subject. The subjects, except f o r s i x instances, had not eaten f o r at least eight hours p r i o r to the test; the six exceptions were instances when the subject ate a l i g h t supper between s i x and eight hours p r i o r to the t e s t . The depth and extent of the previous night's sleep was reported as more or less consistent. Occasionally, there were reports of restlessness but the data associated with these did not appear to be aberrant. The physical condition at a r r i -v a l i n the Laboratory was generally uniform as f a r as could be estimated. The immediately previous a c t i v i t y was a walk from 38. the parking l o t to the Laboratory. The conclusion can be drawn then that the subjects were i n as consistent a physical condition over the three day tes t i n g period as was f e a s i b l y possible. S i m i l a r l y , the environment can be considered as being consistent f o r any given subject. On th i s basis, the contribution of these overt factors must be considered minimal to the effects of the d i f f e r e n t days on the values of the acid-base parameters. This aspect w i l l be discussed l a t e r under the "differences-between-days" and " r e l i a b i l i t i e s " t o pics. 2) The Mean Values of the Parameters The mean values, and the standard deviations, of the acid-base measurements of the a r t e r i a l blood have been summar-ized i n terms both of the values f o r each day of test i n g f o r the 30 subjects and of the o v e r a l l values, including the res u l t s of a l l three days. These values, i n the rest i n g condition and i n the post-exercise condition, have been i l l u s t r a t e d i n Tables III and IV, respectively. The r e s t i n g mean acid-base values were e s s e n t i a l l y the same as the established "normal" values which were summarized previously, i n Table I. The rest i n g mean values f e l l within the "normal range" and, although the mean values of the pH, the base excess, and the buffer base, were i n the lower part of the range, t h i s could be attributed to sampling differences. 3) The E f f e c t of Exercise l n the Variables The comparison of pre- and post-exercise mean values of 39. TABLE I I I MEANS AND STANDARD DEVIATIONS OP THE ACID-BASE PARAMETERS OF ARTERIAL BLOOD, AT REST (AT 37°C) Blood Acid-Base Parameter Overall Value Day 1 Value Day 2 Value Day 3 Value PH + 7.381 0.034 + 7-375 0.035 + 7.388 0.034 + 7.380 0.033 Pco2 (mm.Hg.) + 39.28 3.83 + 38.76 3.84 + 39.19 3.75 + 39.90 3.90 Standard Bicarbonate (m.equ./L.) + 22.51 1.40 + 22.26 1.51 + 22.77 1.61 + 22.49 1.08 Base Excess (m.equ./L.) + 1.77 1.80 + 2.13 1.95 + 1.39 2.00 + 1.79 1.44 Buffer Base (m.equ./L.) + 46.69 2.36 + 45.97 3.83 + 47.06 2.59 + 47.04 2.98 Heart Rate (beats/min.) + 62.7 9-5 + 63.9 9.8 + 60.7 9.5 + 63.4 9.1 40. TABLE IV MEANS AND STANDARD DEVIATIONS OF THE ACID-BASE PARAMETERS OF ARTERIAL BLOOD, AFTER EXERCISE (AT 37°C) Blood Acid-Base Parameters Overall Value Day 1 Value Day 2 Value Day 3 Value pH 7.378 t O.O36 7.371 + 0.032 + 7.384 0.042 + 7-380 0.033 Pco2 (mm.Hg.) 38.82 ± 3.74 38.85 + 4.01 + 38.47 3.83 + 39.13 3-35 Standard Bicarbonate (m.equ./L.) 22.04 ± 1.62 21.83 ± 1.72 + 22.16 1.77 •+ 22.13 I .38 Base Excess (m.equ./L.) - 2.44 ± 2.27 - 2.69 + 2.24 + 2.29 2.31 + 2.34 1.81 Buffer Base (m.equ./L.) 45.79 ± 3.29 45.41 + 4.40 + 46.01 2.93 - + 45.95 2.53 Heart Rate (beat/min.) 117.8 ± 14.4 119-8 ± 15.6 115.4 ± 13.8 + 118.2 13.8 the variables, taken from Tables II and III, showed that (Table XIX) the post-exercise mean values were not s t a t i s t i c a l l y d i f f e r e n t from the re s t i n g mean values, except f o r the buffer base and heart rate values. These re s u l t s indicate that any apparent effects of the exercise upon the variables were, i n general, not s t a t i s t i c a l l y r e l i a b l e . 41. TABLE V DIFFERENCES BETWEEN THE RESTING AND POST-EXERCISE MEAN VALUES OF THE PARAMETERS OF BLOOD PH Pco2 Standard Bicarbonate Base Excess Buffer Base Heart Rate t Ratio value 0.4? 0.66 1.78 1.88 2.00* 24.9** *Si g n l f l e a n t at P = <0.05 *Sig n l f l e a n t at P = <0.01 4) Dlfferenoes-Between-Days Analyses of variance were performed to indicate the exis-tence of s t a t i s t i c a l l y s i g n i f i c a n t differences i n the values of the parameters from the three days of t e s t i n g . The r e s u l t s of the F test have been summarized i n Table VI. The F r a t i o values were s t a t i s t i c a l l y s i g n i f i c a n t at the 0.05 l e v e l of p r o b a b i l i t y f o r the r e s t i n g condition pH, standard bicarbonate and the post-exercise heart rate, and at the 0.01 l e v e l of p r o b a b i l i t y f o r the base excess. The re s u l t s of the t tests between adjacent days upon those variables that had been shown to have s i g n i f i c a n t F values have been summarized i n Table VII. In the re s t i n g condition, the pH, the standard bicarbonate and the base excess were shown to have mean values on Day 1 that were s t a t i s t i c a l l y d i f f e r e n t from the corresponding values on Day 2 of t e s t i n g . The mean values on Day 2, f o r these parameters, were shown not to be d i f f e r e n t s t a t i s t i c a l l y from those on Day J. The t r a t i o values were s t a t i s t i c a l l y s i g n i f i c a n t between Days 1 and 2 and between Days 2 and 3 f o r the post-exercise heart rate mean values. 42. TABLE VI DIFFERENCES-BETWEEN-DAYS OVER. THE THREE DAY TESTING PERIOD IN THE VARIABLES OF THE BLOOD pH Pc©2 Standard Bicarbonate Base Excess Buffer Base Heart Rate F Ratio Value Resting 3.25* 1.78 3.34* 5.13** 1.40 3.00 Post Exercise 2.40 0.45 1.65 1.43 0.50 4.17* *Significance at P = <0.05 *Signlficance at P =<0.01 TABLE VII RESULTS OF t TESTS ON THE VARIABLES HAVING SIGNIFICANT F RATIO VALUES Rest PH Rest Standard Bicarbonate Rest Base Excess Post-Exercise Heart Rate t Ratio \felues Days 1-2 - 2 . 3 2 * - 2 . 8 3 * 3.96* 3.12* Days 2-3 1.55 1.25 -1.44 2.15* * S i g n i f l e a n t at P = <0.05 The t tests on the differences between the adjacent days enabled the i d e n t i f i c a t i o n of the s p e c i f i c day whose effects were s i g n i f i c a n t l y d i f f e r e n t from the effects of the others. The results showed that the e f f e c t of Day 1 f o r the pH, the standard bicarbonate, and the base excess, at rest was s i g n i f i c a n t l y d i f f e r e n t from the effects of Day 2 and Day 3. 43-This e f f e c t could not be a t t r i b u t a b l e to d i f f e r i n g environmental conditions because these conditions were approximately constant over the three day tes t i n g period. This might tend to suggest an "apprehension e f f e c t " on Day 1, possibly due to the strangeness of the environment combined with an apprehension towards the tes t i n g procedure. White et a l . (1) has commented that anxiety or stress conditions causing nervous stimulation r e s u l t s i n epinephrine release, which, i n turn, causes an increase i n l a c t i c a c i d con-centration. Taylor et a l . (2) advocates that, with an excitable or nervous subject, a usual procedure be adopted of repeating the work task with the subject on successive days to obtain controlled conditions before the actual measurements are taken. It i s curious that there were only 3 out of the 6 parameters exhibiting t h i s e f f e c t , and that the heart rate at rest, which i s often reported as susceptible to i n i t i a l t r a i n i n g or learning e f f e c t s ( 3 . 4 ) , d i d not show signs of t h i s e f f e c t . The conclusion might be drawn that the r e p r o d u c i b i l i t y of measurements taken of these three parameters may well be improved by a p r e - f a m i l i a r i z a t i o n with the t e s t i n g environment and the t e s t i n g experiences. The post-exercise mean values of a l l of the acid-base parameters on the three days were shown, by the F r a t i o , to be not s i g n i f i c a n t l y d i f f e r e n t . The d i f f e r e n t i a l e f f e c t of the days observed i n the res t i n g values had, then, been modified or n u l l i f i e d by the e f f e c t of the standard exercise. It may-be concluded that the administration of a standard exercise task to 44 a given i n d i v i d u a l on a number of days would enhance the repro-d u c a b i l i t y on those days of measurements taken of the acid-base parameters of his blood. iS) The Between-Days R e l i a b i l i t i e s The correlations between the values of the acid-base parameters on the Day 1 and Day 2 of testing, and between the values of the parameters on the Day 2 and Day 3 of testing, have been summarized i n Table VIII. The values of the between-days r e l i a b i l i t i e s were not of the same order f o r a l l of the parameters i n any given c o r r e l a -t i o n s e r i e s . For example, i n the r e s t i n g condition Day 1 - Day 2 c o r r e l a t i o n series, the values of the correlations varied from 0.870, f o r the base excess concentration, to 0.418 f o r the buffer base concentration. The values of the between-days r e l i a b i l i t i e s were, with the exception of the buffer base values i n both of the re s t i n g condition correlations, s t a t i s t i c a l l y s i g n i f i c a n t at the 0.01 l e v e l of p r o b a b i l i t y . The non-significance of the two buffer base r e l i a b i l i t y c o e f f i c i e n t values indicates that they cannot be considered s t a t i s t i c a l l y d i f f e r e n t from zero. The values f o r the parameter on any of the three days cannot, therefore, be considered as representative of the values on the other two days. The l e v e l of r e l i a b i l i t y f o r a variable that may be acceptable as the basis f o r inferences or conclusions must, i n the f i n a l instance, depend upon the requirements of the 4 5 -TABLE VIII BETWEEN-DAYS RELIABILITIES OF THE VALUES OF THE PARAMETERS OF ARTERIAL BLOOD Acid-Base Parameters of Blood At Rest A f t e r Exercise Day 1 vs. Day 2 Day 2 vs. Day 3 Day 1 vs. Day 2 Day 2 vs. Day 3 PH 0 . 6 0 4 * 0 . 6 5 3 * 0 . 7 2 6 * 0 . 5 5 5 * P C 0 2 0 . 5 3 6 * 0 . 5 5 7 * 0 . 5 6 9 * 0 . 4 9 0 * Standard Bicarbonate 0 . 8 0 6 * 0 . 6 6 1 * 0.808* 0 . 8 1 0 * Base Excess 0 . 8 7 0 * 0 . 6 9 1 * 0 . 8 1 3 * 0 . 8 0 9 * Buffer Base 0.418 0 . 2 3 2 0 . 4 9 8 * 0,667* Heart Rate 0 . 6 3 9 * 0 . 8 0 4 * 0 . 8 7 3 * 0 . 8 7 1 * * S i g n l f l e a n t at P = < 0 . 0 1 researcher, and upon the circumstances of the investigation. The si z e of the at - r e s t r e l i a b i l i t y values of the pH and P C 0 2 parameters i n t h i s study suggests doubts about the v a l i d i t y of accepting any one single estimate of the pH or P C 0 2 as being representative of the true re s t i n g pH and P c o 2 values of the in d i v i d u a l . The r e l a t i v e l y higher values of the between-days correlations f o r the standard bicarbonate and base excess, at rest, suggest that they are more r e l i a b l e values. Since a l l measurements over the three day period were made under s i m i l a r conditions, the differences i n the sizes of the r e l i a b i l i t y c o e f f i c i e n t s could not be attributed to d i f f e r -ing environmental conditions but must be at t r i b u t a b l e to the 46. natures of the variables themselves. The only r e l i a b i l i t y values previously reported were by Shook and Yiengst (5), yet i t i s hardly r e a l i s t i c to compare t h e i r values with the values from t h i s study. Shock and Yiengst used a two to three week i n t e r - t e s t i n t e r v a l , whereas t h i s study used only a twenty-four hour Interval. In the early parts of t h i s chapter, i t was established that there was an e f f e c t due to days upon the mean values of the var i a b l e s . In order to investigate the possible e f f e c t of t h i s difference-between-days upon the r e l i a b i l i t y c o e f f i c i e n t values, t tests were done on the differences between the values of the co r r e l a t i o n s e r i e s . In both the re s t i n g and post-exercise conditions the r e s u l t s , summarized i n Table IX , of the t tests between the Day 1 - Day 2 series and the Day 2 - Day 3 series, were not s i g n i f i c a n t at the 0.05 l e v e l of p r o b a b i l i t y . These r e s u l t s indicate that the previously suggested "apprehension e f f e c t " d i d not have a s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t upon the r e l i a b i l i t y c o e f f i c i e n t s . Consequently, i t may be concluded that the.overall r e l i a b i l i t y of the values of the variables did not increase with repeated measurements. Although there appeared to be a common trend of Increased r e l i a b i l i t y values i n the post-exercise condition, the e f f e c t of the exercise task upon the values of the r e l i a b i l i t y c o e f f i -cients was shown (Table IX) to be not s t a t i s t i c a l l y s i g n i f i c a n t by use of the z t e s t . In t h i s instance, a s t a t i s t i c a l l y r e l i a b l e difference between r e l i a b i l i t y c o e f f i c i e n t s had to be approximately 0.4; t h i s i s due to the size of the sample. 47-TABLE IX RESULTS OP t AND z TESTS ON THE DIFFERENCES BETWEEN CORRELATION SERIES Post Series Series Rest Exercise 1-2 2-3 (1-2) - (2-3) (1-2) - (2-3) Rest-Ex. Rest-Ex. C r i t i c a l Ratio 0.45 0.12 -0.51 -0.79 Value 6) The Variances . The t o t a l variance (TOVAR), the inter-Individual variance (COVAR) and the i n t r a - i n d i v i d u a l variance (WIVAR), values are summarized i n Tables X, XI and XII, respectively. These variances are related to the between-days r e l i a b i l i t y i n the following manner: ''bet. days = ggY^I = COVAR TOVAR COVAR + WIVAR (6 ,7) NOTE: The WIVAR i n t h i s relationship, and i n any reference to th i s r e l a t i o n s h i p , w i l l always be assumed to Include the measurement error variance. A l t e r a t i o n s i n the value of the between-days r e l i a b i l i t y , i n the same variable and f o r the same group of subjects, can be explained i n terms of t h i s r a t i o and i t s variance components. An increase i n the value of the between-days r e l i a b i l i t y , f o r example, represents an increase l n the r a t i o of COVAR to TOVAR; and t h i s Increase must be due to a r e l a t i v e increase i n the COVAR component, or to a r e l a t i v e decrease l n the TOVAR component. 48. TABLE X THE TOVAE COMPONENTS OF THE BETWEEN-DAY RELIABILITIES OF THE ACID-BASE PARAMETERS OF BLOOD Acid-Base Rest Post-Exercise Parameters 1-2 2-3 1-2 2-3 PH 11.35 10.57 12.74 12.84 Pco 2 13.95 14.17 14.84 12.41 Standard Bicarbonate 2.357 1.689 2.939 2.364 Base Excess 3-763 2.783 5.011 4 .054 Buffer Base 9.596 7.489 12.445 8 . I 6 3 Heart Rate 90.57 84.27 208.66 184.47 TABLE XI THE COVAR COMPONENT OF THE ACID-BASE OF THE BETWEEN-PARAMETERS OF -DAYS RELIABILITIES ARTERIAL BLOOD Acid-Base Parameters of Blood Rest Post-Exercise 1-2 2-3 1-2 2-3 PH (xlO" 4) 6.86 6.92 9.24 7.13 Pco 2 7.475 7.896 8.439 6.081 Standard Bicarbonate , 1.899 1.117 2.375 1.915 Base Excess 3.276 1.924 4.072 3.280 Buffer Base 4.008 1.729 6.200 4.783 Heart Rate 57.87 67.75 182.09 160.66 49-TABLE XII THE VALUES OF THE WIVAR PLUS MEASUREMENT ERROR COMPONENT OF THE BETWEEN-DAYS RELIABILITIES OF THE PARAMETERS OF BLOOD Acid-Base Rest Post' -Exercise Parameter 1-2 2-3 1-2 2-3 PH (x lo- 4 ) 4.49 3.65 3.50 5.71 Pco 2 6.477 6.275 6.401 6.330 Standard Bicarbonate 0.458 0.572 0.564 0.449 Base Excess 0.487 0.859 0.939 0.774 Buffer Base 5-588 5.760 6.245 3.380 Heart Rate 32.70 16.52 26.57 23.81 The COVAR i s an in t e g r a l part of the TOVAR. Thus, f o r any increase i n the COVAR as the numerator, there i s also an increase i n the COVAR as part of the denominator. The d i r e c t i o n that, and the extent to which, the r a t i o changes depends upon the extent and d i r e c t i o n of changes i n the COVAR and WIVAR components, r e l a t i v e to each other and to t h e i r o r i g i n a l values. For example, a 20% increase i n the COVAR may not correspond to a 20% increase i n the TOVAR, unless the WIVAR increases by 20$ a l s o . An increase i n the value of the between-days r e l i a b i l i -t i e s indicates that there i s , (1) a decrease i n the value of the WIVAR while the COVAR remains the same or increases, or (2) an increase i n the WIVAR value less than an increase i n the COVAR value, or (3) a decrease i n the WIVAR value greater than a decrease i n the COVAR value. •50. Any change, or absence of change, i n the value of the between-days r e l i a b i l i t y i s , then, the re s u l t of the int e r a c t i o n of the COVAR and WIVAR components i n the COVAR to TOVAR r a t i o . It was not supposed that the consideration of thi s r e l a -tionship would lead to conclusions that would be methodologically valuable. Yet It was considered of Interest to c l a r i f y the sources of changes i n r e l i a b i l i t y values i n these physiological variables, following i n p r i n c i p l e the work of Henry (6,7) on strength v a r i a b l e s . It was possible to accomplish t h i s objective f o r the resting versus post-exercise comparisons. It was shown, i n Table XIII, that the changes i n the r e l i a b i l i t y values were due predominantly to changes l n the COVAR values, f o r both the Day 1 - Day 2 and the Day 2 - Day 3 s e r i e s . The increased values of the COVAR i n the post-exercise condition indicates an increase of the i n t e r - i n d i v i d u a l differences i n the parameters. This appears to be a fi n d i n g s i m i l a r to those f o r the heart rate and work capacity parameters that have led to the use of the Astrand (8) and Sjostrand (9) work capacity tests f o r the separation and c l a s s i f i c a t i o n of groups. The WIVAR values i n the re s t i n g - post-exercise compari-sons did not exhibit any tendencies, either to uniformly increase, or to decrease. It may be concluded that the eff e c t of the exercise task was to increase the differences between individuals i n the values of the acid-base parameters, and that i t had no apparent e f f e c t upon the v a r i a b i l i t y i n the values within the i n d i v i d u a l . 51-TABLE XIII THE CHANGES IN THE COVAR AND WIVAR VALUES IN THE COMPARISON OF THE RESTING AND POST-EXERCISE CONDITIONS, AS PERCENTAGES OF THEIR ORIGINAL VALUES Acid-Base Day 1 - Day 2 Series Day 2 - Day 3 Series Parameter COVAR WIVAR COVAR WIVAR PH + 34.7 - 22.0 + 3.0 + 56.4 Pc©2 + 12.8 - 1.2 - 23.0 + 0.9 Standard Bicarbonate + 25.6 + 23.1 + 70.6 - 21.5 Base Excess + 24.3 + 92.8 + 69.8 - 9.9 Buffer Base + 54.7 + 11.8 + 176.6 - 58.7 Heart Rate + 123.2 - 18.7 + 137.1 + 44.1 TABLE XIV THE CHANGES IN THE COVAR AND WIVAR VALUES IN THE COMPARISON OF THE DAY 1 - DAY 2 CORRELATION SERIES WITH THE DAY 2 - DAY 3 CORRELATION SERIES, AS PERCENTAGES OF THEIR ORIGINAL VALUES Acid-Base Resting Post -Exercise Parameter COVAR WIVAR COVAR WIVAR pH + 0.9 - 18.7 - 22.8 + 63.I Pco 2 + 5.6 - 3.1 - 28.0 - 1.1 Standard Bicarbonate - 41.2 + 24.9 - 19.4 - 20.4 Base Excess - 41.3 + 76.4 - 19.4 - 1?.6 Buffer Base - 56.9 + 3.3 - 22.9 - 62.0 Heart Rate + 17.1 - 49.5 - 10.8 - 10.4 52 . There was no evidence, i n Table XIV, that the changes i n the r e l i a b i l i t y values between the Day 1 - Day 2 series and the Day 2 - Day 3 series were predominantly due to changes i n either variance. The "between-Individuals 1 1 s t a t i s t i c used by Shock and Hastings (10 ) , and by Moller (11) , i n t h e i r work do not corres-pond to the between-indivlduals variance (COVAR) s t a t i s t i c used i n t h i s study. These authors had used the standard deviation of the group as an estimate of the between-individual v a r i a t i o n . This s t a t i s t i c , however, includes i n t r a - i n d i v i d u a l v a r i a t i o n and measurement error together with the i n t e r - i n d i v i d u a l v a r i a t i o n , whereas COVAR i s the true i n t e r - i n d i v i d u a l variance only. The i n t r a - i n d i v i d u a l v a r i a t i o n s t a t i s t i c s used by Shock and Hastings (10 ) , and Moller (11 ) , did not correspond to the WIVAR used i n t h i s study. Moller used the mean range of the values of the in d i v i d u a l subjects as his s t a t i s t i c , and Shock and Hastings used the standard deviation of repeated measure-ments made on a given Individual as the i n t r a - i n d i v i d u a l v a r i a -t i o n f o r that i n d i v i d u a l . The WIVAR, i n t h i s study, was the standard deviation of the differences between pairs of values of a given parameter f o r the individuals of the group. •7) The Errors of Measurement The pH Meter Measurement Error The pH measurement errors, which are summarized i n Table XV, included, f o r the pH parameter, the true pH meter measurement error and the error of reading the pH meter scale. The reading 53. error i s Intimately involved i n the measurement error and has not been p a r t i a l l e d out. The pH meter measurement errors f o r the other four acid-base parameters includes the true pH meter measurement error, the reading error of the pH meter, the true measurement error of the Siggaard-Andersen revised nomogram, and the reading error of the nomogram. The observed pH meter measurement error i s the sum of the errors of two sets of measurements, and therefore as an estimate of the pH meter measurement error f o r a single test one-half of the observed measurement error was used. TABLE XV THE pH METER MEASUREMENT ERRORS POR THE ACID-BASE PARAMETERS PH Pc©2 Standard Bicarbonate Base Excess Buffer Base N 216 105 105 105 105 Constant Error - 0.00026 - 0.300 - 0.237 + 0.045 -0.173 Measurement Error Variance 1.28 x IO"* 1.426 0.168 0.449 3.11 Single Test Measurement Error Variance 0.64 x IO"* 0.713 0.084 0.225 1.56 Henry (7) has described how the influence of the measure-ment error on the r e l i a b i l i t y c o e f f i c i e n t can be assessed. By the removal of the measurement error variance from the denominator of the COVAR to TOVAR r a t i o , the adjusted r e l i a b i l i -t i e s i s an i n d i c a t i o n of the eff e c t of the measurement error. 54 . The removal of the single test measurement error variance from the values of the TOVAR i n thi s study, as shown i n Tables XVI and XVII, indicates that the measurement error had a d i f f e r -ent e f f e c t upon the r e l i a b i l i t y values of the d i f f e r e n t v a r i a b l e s . The pH, P c© 2 , standard bicarbonate, and base excess showed very small increases i n the values of the r e l i a b i l i t y c o e f f i c i e n t s i n d i c a t i n g that the measurement error variances f o r the single test may be Ignored provided they remain at the values shown i n t h i s study. The buffer base exhibited increases of the order of 0 . 1 ; which seemed to be too large not to be taken into account; therefore, as long as the measurement error variance Is of the order of I . 5 6 , then the error must be taken into account i n the assessment of the representativeness of the recorded values of the parameter. TABLE XVI THE ADJUSTED VALUES OF THE BETWEEN-DAYS CORRELATION BY THE REMOVAL OF MEASUREMENT ERROR VARIANCE FROM THE DENOMINATOR IN THE COVAR TOVAR/RATIO Acid-Base Parameters of Blood Rest Post -Exercise 1-2 2-3 1-2 2-3 pH 0.641 0.697 0.764 0.584 P C 0 2 0.565 0.587 0.597 0.521 Standard Bicarbonate O.836 0.696 O.832 0.840 Base Excess 0.926 0.752 0.851 0.857 Buffer Base 0.570 0.292 0.569 0.724 55-TABLE XVII THE DIFFERENCES BETWEEN THE ORIGINAL VALUES OF THE BETWEEN-DAYS CORRELATIONS AND THE ADJUSTED VALUES FOR THE ACID-BASE PARAMETERS Acid-Base Parameters of Blood Rest Post -Exercise 1-2 2-3 1-2 2-3 PH + 0.037 + 0.044 + 0.038 + 0.029 Pco2 + 0.029 + 0.030 + 0.028 + 0.031 Standard Bicarbonate + 0.030 + 0.035 + 0.024 + 0.030 Base Excess + 0.056 + 0.061 + 0.038 + 0.048 Buffer Base + 0.152 + 0.060 + 0.071 + 0.057 The Measurement Errors of the Nomogram The Siggaard-Andersen revised nomogram measurement errors, which are summarized i n Table XVIII, includes the true measure-ment error of the nomogram plus the reading error of the nomogram. The comparison of the nomogram measurement error variance values with the values f o r the pH meter measurement error variances indicates that the error of measurement of the nomogram was a n e g l i g i b l e part of the t o t a l measurement error and as such may be ignored. The Temporal Measurement Error Table XIX summarizes the re s u l t s of t tests on the d i f -ferences between the mean values of the f i r s t tube of blood collected and the mean values of the second tube c o l l e c t e d . The 56. TABLE XVIII THE MEASUREMENT ERRORS OP THE SIGGAARD-ANDERSEN REVISED NOMOGRAM Pco2 Standard Bicarbonate Base Buffer Excess Base N 40 40 40 40 Constant Error 0.0 0.0 0.0 0.0 Measurement Error Variance 0.081 0.036 0.036 0.049 Single Test Measurement Error Variance 0.040 0.018 0.018 0.025 TABLE XIX RESULTS OF TWO TUBES OF t TEST ON BLOOD DUE THE DIFFERENCE BETWEEN THE pH OF TO THE TEMPORAL ERROR OF COLLECTION Rest Post-Exercise Tube 1 Mean 7.390 7-358 Tube 2 Mean 7.393 7.379 Mean Difference Between Tubes - 0.003 - 0.021 Standard Deviation of Differences t 0.010 t 0.005 Standard Error of Differences 0.0014 0.0015 t Test Value 2.14* 14.00* *S i g n i f l e a n t at P = <0.05 57. r e s u l t s showed that there was a s i g n i f i c a n t difference between the mean values of the two tubes i n the res t i n g condition at the 0 . 0 5 l e v e l of p r o b a b i l i t y . The mean value of the differences was so small ( - 0 . 0 0 3 pH u n i t ) , however, that i t s p r a c t i c a l s i g n i f i c a n c e , i n terms of the derivation of data from the nomogram, i s n e g l i g i b l e ; so that i t may be concluded that i n the resti n g condition there was no difference i n pH between the two sets of tubes of blood. The post-exercise comparison produced a t test value that was s i g n i f i c a n t at the 0 . 0 1 l e v e l of p r o b a b i l i t y . A difference of - 0 . 0 2 1 (the mean difference) i n pH could produce errors i n the data derived from the nomogram. It must be inferred that the e f f e c t of exercise was to produce the p o s s i b i l i t y of errors i n the estimation of the acid-base variables from the nomogram. 58 . REFERENCES White, A., Handler, P., Smith, E.L., DeWlttsletten. P r i n c i p l e s of Biochemistry. McGraw H i l l Co., New York, 1959-Taylor, H.L., Wang, Y., Rowell, L., Blomquist, G. "The Standardisation and Interpretation of Submaximal and Maximal Tests of Working Capacity." Pedi a t r i c s, 32, 4, 1963, 703 . Debienne, R.L. "Changes i n Body Fat, Physical Working Capacity, and Personality of Obese Women Undergoing Training." M.P.E. Thesis University of B r i t i s h Columbia, Vancouver, 1968. Zahar, W.E.D. " R e l i a b i l i t y and Improvement with Repeated Performance of the Sjostrand Work Capacity Test." Master's Thesis, U. Alberta, 1965. Shock, N.W., Yiengst, M.J. "Age Changes i n Acid-Base Equilibrium of the Blood of Males." J . Geront. 5, 1950, 1. Henry, F.M. " R e l i a b i l i t y , Measurement Error, and Intra-Indlvidual Differences." Res. Quart. 30, 1, 1959. 2 1 . Henry, F.M. "Influence of Measurement Error and Intra-i n d i v i d u a l V a r i a t i o n on the R e l i a b i l i t y of Muscle Strength and V e r t i c a l Jump Tests." Res. Quart. 30, 2, 1959. 155 . Astrand, P. Work Tests with the Bicycle Ergometer. Monark-Crescent AB., Sweden, 1967* C.A.H.P.E.R. Research Committee. The Physical Working  Capacity of Canadian Children. C.A.H.P.E.R., 1968. Shock, N.W., Hastings, A.B. "Studies of the Acid-Base Balance of the Blood, I I I . " J . B i o l . Chem. 104, 193^, 585-Moller, B. "The Hydrogen Ion Concentration i n A r t e r i a l Blood." Acta. Med. Scand. suppl. 3 4 8 , 1959. CHAPTER V SUMMARY AND CONCLUSIONS The Problem The pH, the Pco2, the standard bicarbonate concentration, the base excess concentration, and the buffer base concentration of blood are physiological parameters that are used extensively i n medicine. In order to use the measurements of the parameters i n research and diagnosis, i t i s necessary to know within what l i m i t s of accuracy the measures, that are made on samples of blood taken from an in d i v i d u a l under standardized conditions, can be accepted. The s p e c i f i c objectives of the study were: 1) The determination of the error Inherent i n the temporal aspect of blood c o l l e c t i o n . 2) The determination of the measurement error of the pH meter (Radiometer PHM 22). 3) The determination of the error involved i n the use of the Siggaard-Andersen revised nomogram. k) The determination of the presence of differences-between-days l n the values of the parameters of a r t e r i a l blood over the successive periods of te s t i n g . ,'5) The determination of the r e l i a b i l i t i e s of consecutive day-to-day measurements of the acid-base parameters 60 . of a r t e r i a l blood, at rest and a f t e r sub-maximal exercise. •6) The determination of the i n t r a - i n d i v i d u a l differences of the acid-base parameters of a r t e r i a l blood, at rest and a f t e r sub-maximal exercise. 7.) The determination of the i n t e r - i n d i v i d u a l differences of the acid-base parameters of a r t e r i a l blood, at. rest and a f t e r sub-maximal exercise. The problems were investigated by using a volunteer group of 30 male subjects. The f i r s t objective was investigated by i the comparison of two blood samples taken immediately one a f t e r the other from the same subject under the same conditions. The seeond and t h i r d objectives were investigated by the comparison of two readings from the same tube of blood under constant conditions, and the comparison of two sets of readings from the same tubes under constant conditions. Objectives 4 - 7 were investigated by the use of appropriate s t a t i s t i c a l analyses upon the re s u l t s of the analyses of a r t e r i a l blood samples taken from the group of subjects, each of whom was tested, under, standar-dized conditions, on three consecutive mornings, both at rest ,and a f t e r a sub-maximal exercise task. Conclusions 1) Estimates were produced of the temporal error of the c o l l e c t i o n of blood, at rest and post-exercise. There was no p r a c t i c a l difference between the pH of two tubes of blood taken at r e s t . There was a 61. s i g n i f i c a n t difference between the pH of two tubes of blood taken a f t e r exercise, such that errors were possible i n the derivation of data from the nomogram. 2) Estimates were produced of the measurement error of the Siggaard-Andersen revised nomogram. The sizes of the measurement error variances were so small that they were n e g l i g i b l e when compared with the t o t a l measurement error variance. 3) Estimates of the pH meter measurement error were produced. The sizes of the measurement error variances f o r the pH, Pco2, standard bicarbonate and base excess, were so small that t h e i r effects upon t h e i r respective r e l i a b i l i t y c o e f f i c i e n t s values were n e g l i g i b l e . The buffer base value of the measurement error variance was large enough to have a decided e f f e c t upon the r e l i a b i l i t y c o e f f i -cients of buffer base. 4) It was shown that f o r pH, standard bicarbonate and base excess, the e f f e c t of Day 1 of te s t i n g on the values of the parameters was d i f f e r e n t from the effects of Days 2 and 3 . This e f f e c t was a t t r i b u -ted to apprehension towards the te s t i n g experience, and to the strangeness of the tes t i n g environment. 5) Estimates of the r e l i a b i l i t y of the variables were produced. The standard bicarbonate and base excess 62. at rest, and the post-exercise heart rate, had the more r e l i a b l e of values; the pH and P C 0 2 had less r e l i a b l e values. The buffer base resting r e l i a -b i l i t y values were not s t a t i s t i c a l l y d i f f e r e n t from zero. There was no s t a t i s t i c a l difference between r e l i a b i l i t y values at rest and those a f t e r exercise. ;6) Estimates of the i n t e r - and l n t r a - i n d i v l d u a l variances were produced f o r the six v a r i a b l e s . Changes i n the values of the r e l i a b i l i t y c o e f f i -cients, from the r e s t i n g to the post-exercise condition, were explained by the predominantly increased COVAR values. BIBLIOGRAPHY Adamsons, K., D a n i e l l , S.S., Gandy, G., James, L.S. "Influence of Temperature on Blood pH of the Human Adult and New Born." J . Appl. Physiol., 19. 1964, 897 . Ad Hoc Committee on Methodology, S.R. Gambino, Chairman. Ann. N.Y. Acad. S c i . 133, 1. 1966, 259-A l b r i t t o n , E.C. Standard Values l n Blood. W.B. Saunders Co., Philadelphia, 1952. Astrup, P., Schroder, S. "Apparatus f o r Anaerobic Determination of the Blood at 38°C." Scand. J . C l i n . Lab. Invest., 8, 1956, 30 . Astrup, P. "A Simple Electro-Metric Technique f o r the Deter-mination of Carbon Dioxide Tension i n Blood and Plasma, Total Content of Carbon Dioxide i n Plasma, and Bicarbonate Content i n 'Separated' Plasma at Fixed Carbon Dioxide Tension (40 mm. Hg.)." Scand. J . C l i n . Lab. Invest., 8, 1956, 33-Astrup, P., Jorgensen, K., Siggaard-Andersen, 0., Engel, K. "The Acid-Base Metabolism: A New Approach." Lancet, May 14th, I960. Astrup, P., Engel, K., Siggaard-Andersen, 0., Jorgensen, K. "Definitions and Terminology i n Blood Acid-Base Chemistry." .. Ann. N.Y. Acad. S c i . , 133, 1, 1966, 5 9 . Astrand, P.- Work Tests with the Bicycle Ergometer. Monark-Crescent AB, Sweden, 1967-Baldwin, E.D., Cournand, A., Richards, D.W. "Pulmonary Insufficiency, I." Medicine, 27 , 1948, 243. Barr, D.P., Himwlch, H.E., Green, R.P. "Studies i n the Physiology of Muscular Exercise." J . B i o l . Chem.. 55 , 1923, 495 . Berglund, E., Malmberg, R., Stenhagen, S. "Determination of P C 0 2 of Whole Blood by pH Measurement and Interpolation." Scand. J . C l i n . Lab. Invest., 16, 1964, I85. Bergman, N.A. "Temperature Coefficients f o r Pco£ and P 0 2 i n Bloods with Varying Acid-Base Status." J . Appl. Physiol. 24, 1968, 225 . Bouhuys, A., Pool, J., Binkhorst, R.A., Van Leeuwen, P. "Metabolic Acidosis of Exercise i n Healthy Males." J . Appl. Physiol. 21 , 1966, 1040. Bradley, A.F., Stumpfel, M., Severinghans, J.W. "Effect of Temperature on Pco2 and P02 of Blood In V i t r o . " J . Appl. Physiol. 9. 1956, 201 . C.A.H.P.E.B. Research Committee. The Physical Working Capacity  of Canadian Children. C.A.H.P.E.R., 1968. Cain, S.M., Dunn, J.E. "Low Doses of Acetazolamide to Aid Accommodation of Men to A l t i t u d e . " J . Appl. Physiol. 21 , 1966, 1195. Cournand, A., Riley, R.L., Bradley, S.E., Breed, E.S., Noble, R.P., Lauson, H.D., Gregersen, H.I., Richards, D.W. "Studies of the C i r c u l a t i o n i n C l i n i c a l Shock." 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Quart., 30, 2, 1959. 155* Hlgglns, H.L. "The Influence of Food, Posture, and Other Factors on the Alveolar C02 Tension i n Man." Am. J . Physiol., 34, 1914, 114. Hlgglns, H.L., Peabody, F.W., F i t z , R. "A Study of Acidosis i n Three Normal Subjects with Incidental Observations of the Action of Alcohol as an Antiketogenic Agent." J . Med. Res., 34, 1916, 263. Holmgren, A., Mcllroy, M.B. "Effect of Temperature on A r t e r i a l Blood Gas Tensions and pH During Exercise." J . Appl. Physiol., 19, 1964, 243-Holmgren, A., Svanborg, N. "Venous Admixture During Exercise i n the S i t t i n g P o s i t i o n . " Scand. J . C l i n . Lab. Invest., 1?, 1965, 209. Jansen, W.H., Karbaum, H.J. "Zur frage der regulation des saure-basen-glelchgewichts beim normalen menschen." Deutches Arch. K l i n . Med., 153, 1926, 84. J a r r e t t , R.F., Henry, F.M. "The Relative Influence on E r r o r J o f Replicating Measurement i n Individuals." J . Psychol., 31, 1951, 175-Jorgensen, K., Astrup, P. "Standard Bicarbonate, Its C l i n i c a l Significance and a New Method f o r Its Determination." Scand. J . C l i n . Lab. Invest., 9. 1957. 122. Kildeberg, P. C l i n i c a l Acid-Base Physiology. Munksgaard, Copenhagen, 1968. Koch, G. "Comparison of P C 0 2 , pH and Standard Bicarbonate i n C a p i l l a r y Blood and i n A r t e r i a l Blood." Scand. J. C l i n . Lab. Invest., 17, 1965, 223 . L a h i r i , S., Milledge, J.S., Chattopadhyay, H.P., Bhattacharyya, A.K., Sinha, A.K. "Respiration and Heart Rate of Sherpa Highlanders During Exercise." J . Appl. Physiol. 23, 1967, 545. Lundbaek, K. "The pH of Blood During the Action of Large Doses of Insulin." Acta. Physiol. Scand., 7. 1944, 5. Maas, A.H.J., Van Heijst, A.N.P. "A Comparison of the pH of A r t e r i a l Blood with A r t e r i a l i s e d Blood from the Earlobe with Astrup's Microglasselectrode." C l i n . Chim. Acta., 6, 1961, 3 1 . Maas, A.H.J., Van H e i j s t , A.N.P. "The Accuracy of the Micro-Determination of the P C 0 2 of Blood from the Earlobe." C l i n . Chim. Acta., 6,. 1961, 34 . Moller, B. "The Hydrogen Ion Concentration i n A r t e r i a l Blood." Acta. Med. Scand.. Suppl. 348, 1959-Naeraa, N. "The Accuracy of P C 0 2 Determinations with the Micro pH Method Using One Point E q u i l i b r a t i o n . " Scand. J . C l i n . Lab. Invest., 17 , 1965, 209. Radiometer A/S. Introduction to pH Measurement. Instruction Manual. Rahn, H., i n Methods l n Medical Research. Comroe, J.H. (Ed.), V o l . 2, Chicago, 1950, pp. 223-224. Rispens, P., Van Assendelft, O.W., Brunsting, J.R., Z i j l s t r a , W.G., Van Kampen, E.J. "A Direct Method f o r the Determina-t i o n of the Bicarbonate Concentration as Total C0 2 i n Blood or Plasma." C l i n . Chim. Acta.. 14, 1966, 760. Robinson, J.R.- Fundamentals of Aold-Base Regulation. 2nd Ed., Blaekwell, Oxford, 1965. Rosenthal, T.B. "The Ef f e c t of Temperature on the pH of Blood and Plasma." J . B i o l . Chem. 173, 1948, 2 5 . Senay, L.C., Chrlstensen, M.L. "Variations of Certain Blood Constituents During Acute Heat Exposure." J . Appl. Physiol., 24, 1968, 302. Shock, N.W., Hastings, A.B. "Studies of the Acid-Base Balance of the Blood, I I I , V a r i a t i o n i n the Acid-Base Balance of Normal Individuals." J . B i o l . Chem.. 104, 1934, 585 . Shock, N.W. "Age Changes and Sex Differences i n Alveolar C02." Am. J . Physiol., 133. 1941, 610. Shock, N.W., Yiengst, M.J. "Age Changes i n Acid-Base E q u i l i -brium i n the Blood of Males." J . Geront., 5, 1950, 1. Siggaard-Andersen, 0 . , Engel, K., Jorgensen, K., Astrup, P. "A Micro-Method f o r the Determination of pH, Pco2, Base, Excess, and Standard Bicarbonate, i n Cap i l l a r y Blood." Scand. J . C l i n . Lab. Invest.. 12, i 9 6 0 , 172. Siggaard-Andersen, 0 . , Engel, K. "A New Acid-Base Nomogram." Scand. J . C l i n . Lab. Invest.. 12, i 9 6 0 , 177• Siggaard-Andersen, 0 . "Sampling and Storing of Blood f o r the Determination of Acid-Base Status." Scand. J . C l i n . Lab. Invest., 13 , 1961, 2. Siggaard-Andersen, 0 . "Factors A f f e c t i n g the Liquid-Junction Po t e n t i a l i n Electrometric Blood pH Measurement." Scand. J . C l i n . Lab. Invest.. 13 , 1961, 205-Siggaard-Andersen, 0 . The Acid-Base Status of Blood. 2nd Ed., Williams and Wllklns Co., Baltimore, 1964. Singer, R.B., Hastings, A.B. "An Improved C l i n i c a l Method f o r the Estimation of Disturbances of Acid-Base Balance of Human Blood." Medicine, 27 , 1948, 223 . Singer, R.B., i n Handbook of Respiration. Altman, P.L., Gibson, J.F., Wang, C.C. (Eds.), W.B. Saunders Co., Philadelphia, 1958, pp. 68-71. Taylor, H.L., Wang, Y., Rowell, L., Bromquist, G. "The Standardisation and Interpretation of Submaximal and Maximal Tests of Working Capacity." Pe d i a t r i c s, 32, 4, 1963, 703 . Terasllnna, P., MacLeod, D.F. "The Ef f e c t of Exercise on Blood pH and Pco2. Serum Glucose, Cholesterol, N.E.F.A., and Ketones." J . Sports Med., 6, 1966, 235 . Torjussen, W., Nitter-Lauge, S. "pH and PC02 i n C a p i l l a r y Blood from the Earlobe." Scand. J . C l i n . Lab. Invest., 19 , 1967, 7 9 . White, A., Handler, P., Smith, E.L., DeWittsletten. P r i n c i p l e s  of Biochemistry. McGraw H i l l Co., New York, 1959-Van Slyke, D.D. "Some Points of Acid-Base History i n Physiology and Medicine." Ann. N.Y. Acad. S c i . . 1 3 3 : , 1, 1966, 5. Zahar, W.E.D. " R e l i a b i l i t y and Improvement with Repeated Performance of the Sjostrand Work Capacity Test." Master's Thesis, U. Alberta, 1965. APPENDIX A STATISTICAL TREATMENTS STATISTICAL TREATMENTS The t Test a) C r i t i c a l Ratio = obtained difference between means S.E. difference between means b) S.E.,,^ -u 4. = S.E.? + S.E.f - 2 K 9 SJE- S.E.? ' difference bet. means 1 ^ 1.Z l ^ (1:137) where S.E. i s the S.E. of the mean. r 1 . 2 i s the r e l i a b i l i t y c o e f f i c i e n t between Variable 1 and 2. °) S ' E . mean = = 1 = (1:128) V N - l The t Test f o r the Difference Between Two R e l i a b i l i t y C o e f f i c i e n t s f o r Non-independent Samples. t = ( r 12 - r 23) (N- 3 ) ( 1 + r 1 3 ) (2:154) V 2 ( i - r122- r 2 3 2 - r 1 3 2 + 2 r 1 2 r 2 3 r 1 3 ) where t^2 i s the r e l i a b i l i t y coeff. between variable 1 & 2 r^ 3 i s the r e l i a b i l i t y coeff. between variable 2 & 3 |^ 3 i s the r e l i a b i l i t y coeff. between variable 1 & 3 The z Test f o r the Difference Between Two R e l i a b i l i t y C o e f f i c i e n t s f o r Independent Samples. z l - 2 2 (2:1-53) z = /(—1-) + (—i_) ^ - 3 ' N2-3' where z \ ~ f~i transformed into z terms Z 2 = ^ 2 transformed into z terms The R e l i a b i l i t y C o e f f i c i e n t N&Y - (£X £Y) (1:205) V ( N £ X 2 - (*X) 2)(N*Y 2 - (£Y) 2) where the Total Variance = V (N i X 2 - (£X) 2 )(N £ Y 2 - (£Y)2) where the Covariance = N $ XY - (*X£Y) where the Within-Individual Variance = V(N*X 2 - (£X) 2)(N*Y 2 - UY)27) - (N*XY- (*X*Y)) REFERENCES Guilford, J.P. Fundamental S t a t i s t i c s i n Psychology and  Education. McGraw H i l l Co., New York, 1942. Ferguson, G.A. S t a t i s t i c a l Analysis In Psychology and  Education. McGraw H i l l Co., New York, 1959* APPENDIX B DATA SHEET tJhtKb .. Pfrt tO«-|6rtr Dtr-r&. f u s t GltW T&NPtrrt-A-rvO-e . CZ3 s 1 1 — n — : 1 r , — : — r 6-vei2.Cf<,fr LO<*-O "Poart-r-.o^ T ? i 2 r t i N ^ c tor £Arr p?4 a* 4-7. ^Ofc J j 1 APPENDIX C RAW SCORES PH METER MEASUREMENT ERROR DATA PH VARIABLE 1 1- 024- 029- 0 2 1 - 0 1 8 - 026- 010- 0 1 3 - 0 1 2 - 004- 002 1 2- 0 1 2 - 010- 024- 0 0 5 - 014- 010- 007- 017- 0 0 3 - o n 1 3- 019- 002- 009- 0 0 8 - 003- 0 2 1 - 015- 005- 014- 018 1 4- 018- 003- 004- 0 0 2 - 003- 0 0 1 - 002- 0 0 2 - 026- 014 1 5- 006- 01 3 - 009- 0 0 3 - 001- 0 1 9 - 004- 006- UU7- 008 1 6- 004- 018- 020- 0 2 2 - 006- 0 0 2 - 009- 017- 016- 009 1 7- 014+ 016 + 010 + 013 + 031 000 + 013 + 001 + 013 + 001 1 8 + 003 + 010 + 004 + 006 + 001 + 006 000 + 017 + 005 + 004 1 9 + 003 000 + 007 + 008 + 011 + 016 + 009 + 011 + 002+ 016 110 + 013 + 018 + 016+' 018 + 013 + 004 + 001 + 014 + 011 + 012 111 + 005 + 016 + 008'- 007- 004- 02 3 - 005- 006- 006- 008 112- 0 0 5 - 002- 008- 010- 009- 002- 006- 0 0 2 - 008- 008 113- 005- 006- 020- 0 0 2 - 007- 0 1 3 - 0 3 1 - 0 0 3 - 006- 003 114- 0 1 3 - 0 0 1 - 018- 0 1 6 - 050- 0 1 0 - 0 1 5 - 004- 009- 010 1 15- 0 0 1 - 0 0 3 - 006- 017- 026- 010- 008 + 006 + 001 + 016 116 + 001 + 002 + 002 + 003 + 009 + 002 + 009 + 020 + 014+ 013 117 + 010 + 007 + 010 + 028 + 001 + 002 + 003 + 003 + 002 + 008 118 + 019 + 007 + 017 + 005 + 002 + 012 + 029 + 002 + 015 + 020 1 19 + 013 + 023 000 + 017 + 024 000 + 003 + 012 + 011 + 00 8 120 + 001 + 012 + O i l 000 + 010 000 + 005 + 003 + 007 000 1 2 1 - 009 + 010- 003 + 0 1 2 - 002- 009- 0 1 1 - 0 0 2 - 010 + 003 122 + 005 + 016 + 008- 003 + 004 + 006 PC02 VARIABLE 2 1- 25 - 18 - 13 - 34 - 29 - 03 - 05 - 09 - 11 - 07 2 2- 0 3 - 14 - 25 - 12 - 02 - 13 - 17 - 11 - 03 - 05 2 3- 1U - U6 - 05 - 13 - Ug - TPT" - 22 - 22 - 09 - 07 2 4- 21 - 06 - 03 - 21 - 08 - 19 - 56 - 18 - 25 - 04 2 5- 04 - 18 - 02 - 05 - 07 - 15 - 17 - 26 - 20 - 20 2 6- 04 - 07 - 07 + 11 + 14 + 49 0 00 + 11 + 19 + 05 2 7 + 14 + 07 + 03 + 04 + 03 + 05 + 10 + 04 + 10 + 15 2 8 + 04 0 00 +- 19 + 05 + 19 + 16 + 19 + U8 + 13 + 13 2 9 + ~Q~4 + 19 + 30 + 43 + 10 + o r +" 02 + 09 + 07 + 01 210 + 09 + 02 + 08 + 17 0 00 + 12 + 07 - 09 - 09 0 00 2 11 + 03 + 10 + 12 + 06 + 07 STANDARD BICARBONATE : VARIABLE 3 1- 11 — 09 — 08 - 12 06 _ 07 03 04 03 01 3 2- 03 - 10 - 03 - 07 - 06 - 02 - 02 - 01 - 03 - 15 3 3- 03 - 02 - 06 - 05 - 02 - 03 - 06 - 02 • - 02 - 05 3 4- 04 - 07 - 13 - 0 4 - 03 - 03 - 06 - 02 — 03 — 06 3 5- 07 - 09 - 04 - 01 - 07 - 04 - 03 - 02 0. 00 0 00 3 6 00 + 01 0 00 + 02 + 04 + 06 + 04 0 00 + 0 2' + 04 3 7+ 03 + 13 + 02 0 00 + 04 + 08 + 02 + 06 + 02 + 10 3 8+ O l + 01 + 02 0 00 0 00 + 03 + 01 + 0 4 + 06 + 01 3 9+ 0]. + 05 + 03 + 04 + 05 + 05 + 03 + 03 + 02 + 02 310+ O l + 06 + 09 + 03 + 0 3 + 10 - 04 + 05 0 00 0 00 3 1 1 - O3 - 04 + O l + 0 4 BASE EXCESS VARIABLE 4 1- 13 - 16 - 15 - 07 - 03 - 05 - 03 - 03 - 05 - 02 4 2- 02 - O l - 02 - 02 - 02 - 01 - 06 - 02 - 05 - 08 4 3- Ol - 02 - 07 - 03 - 05 - 07 - 07 — 07 — 01 — 04 4 4- 03 - O l - 09 - 05 - 01 - 04 - 05 - 01 - 01 - 01 4 . 5- 03 - 02 - 06 - 07 - 04 + 03 + 04 + 01 + 09 + 09 4 6 + 12 + O l + O l + 06 • + 10 + 06 + 04 0 00 + 05 + 01 4 7 00 + 10 + 05 + 0 4 + 16 + 10 + 15 + 02 + 04 + 04 4 8 + 09 + 07 + 08 + 05 0 00 + 03 + 04 0 00 + 02 + 07 4 9 + 04 + O l + 08 + 08 + 01 + 04 + 08 + 02 + 03 + 07 410 + 04 + 10 + 13 + 04 + 04 + 05 + 09 + 06 + 05 + 02 4 1 1 - 02 + 04 — 05 — 02 — 03 ' BJ FF ER BASE ' VARIAB LE 5 1- 23 _ 03 0 4 1 5 _ 07 __ 06 _ 06 _ 08 04 01 5 2- 25 - 13 - 20 - 1 5 - 09 - 09 - 21 - 31 - 07 - 03 5 3- 24 - 4 5 - 08 - 05 - 14 - 29 - 23 - 24 - 03 - 62 5 4- 13 - 25 - 14 - 23 - 35 - 07 - 08 - 37 50 - 06 5 5- 04 - 13 - 08 - 17 + 16 + 33 + 04 + 15 + 48 + 07 5 6 + 10 + 12 + 06. + 20 + 35 + 22 + 12 + 11 + 08 + 03 5 7 + 15 + 52 + 10 0 00 + 08 + 0 9 + 03 + 07 + 08 + 23 5 8 + 12 + 28 + 19 + 10 + 41 + 11 42 + 33 + 02 + 30 5 9 + 15 + 16 + 42 + 04 + 08 + 20 + 18 + 12 + 10 + 18 510 + 08 + 23 + 23 + 08 + 05 + 20 + 21 + 12 + 22 + 22 I + o I I—1 o + o RAW U I FFcsiE N C Q oCORcb FOR THE M E A i> O R LIVIti\lT ERROK .OF Trie o I A A R u ANUbRStN NOMOGRAM STAND B A i F bJFFER PC02 BICARB EXCEbb BASE 0 0 0 + 0 . 1 + 0.1 0 0 + 0 . 1 0 +0.1 0 + 0.1 + 0.1 0 - O . i 0 + 0. 1 + 0.1 - o . i 0 + 0.1 0 0 + 0 . 1 + 0.1 +0 . 1 0 0 0 0 + 0.1 0 0 + 0.1 + 0 . 1 -0.1 - o . i 0 0 0 0 +0.1 0 0 -0.2 0 0 -0.2 0 0 0 -0 . 1 - o . i 0 0 + 0.1 + 0.2 0 0 -0.1 0 - o . i + 0.1 0 + 0.1 0 0 -0.3 0 0 0 -0.2 + 0.2 0 0 -0.2 0 - o . i + 0.1 0 0 0 + 0.1 0 + 0.2 0 + 0.1 + 0 . 1 0 0 0 + 0 . 1 0 - o . i - o . i -0 . 1 + 0.1 + 0. 1 0 + 0 . 1 + 0.1 - o . i 0 s 0 -0.1 -0.1 0 0 + 0.2 0 0 0 + 0.1 0 0 0 0 -0.1 0 0 - o . i • 0 + 0.1 + 0.1 + 0.1 +0. 1 0 -0.1 + 0.2 0 + 0 . i + 0 . 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 RA'/v 5C0KEi> FOR THE TEMPORAL ERROR' IK BLOOD COLLECTION. Z R E S T I N G C O N D I T I O N TJBF I T U R F ? TuRF 1 TURF•2 7.380 7.392 7.387 7.380 7.392 7.373 7.409 7.41 4 7.401 7.381 7.386 7.387 7.381 7.380 7.410 7.4-0 6 " 7.380 7.393 7.383 7.381 7.375 7.381 ' 7.413 7.40 1 7.381 7.40.8 7.390 7.397 7 .399 7.410 7 .406 7 .415 7.410 7.405 7.378 7.368 7 .369 7.381 7.418 7.419 7.381 7.391 7.398 7.40 1 7.391 7.390' 7.391 7.395 7 . 390 7.400 7.405 7.398 • 7.401 7.411 7.398 7.40 4 7.411 7.421 7.398 7.394 7.348 7.355 7.401 7.40 3 7 .390 7.380 7.428 7.425 7.356 7.390 7. 36 8 7.369 7.355 7.349 7.349 7.361 7.381 7.3 89 7.389 7.3 96 7.385 7.391 7.355 7.392 7.396 7.398 7.400 7.392 7.390 7.389 7.410 7.40 7 7 . 393 7.389 7.403 7.405 7 .397 7.403 7 . 399 7.399 P O S T - E X E R C I S E C O N D I T I O N TOBE1 TUBE 2 —J —J —J OJ OJ OJ -{> 4> U i >l O —J —J —-J —i —I UJ OJ OJ VJ1 O -f> I—' CO CO -J OJ OJ OJ on -^ i 4> OJ O -vl OJ OJ OJ o a- o a o o OJ 4> 0- 4> OJ. - J C 1 — rvj vo BASE bXCLbb t X t K C I i t S 3 3b.63 2 9 11.68 & 2-v8-7 2 i-s-4-4 1—4-3 WON — SD 57.34 58 0.99 +J-UTTER BASE REST — — • — S 397.05 29 13.69 D 23.40 2 11.70 1.40 NON —6+3 4-8-e-s-5-f3 5-6 frr£9-BUFFER BASE EXERCISE —-S- 6^ -6-.-9-2 i-9 2-i-.-fe-2-D 6.40 2 3.20 0.50 NON SD 368.03 58 6.35 HEART RATE REST S 6160.99 29 212.45 & 1-7-5-.-3-6 2 8-ihr6-8 3-^-6-0- —N0M SD 169 3.97 58 29.21 -H-E-A+VT—R-A-T-E— E-X-&R-ei-&E -' S 16037.73 29 553.03 D 297.60 2 148.80 4.17 0.05 S-E> 2-Q-7-1-TT0-& 5-8 3-5nrfi -ANOVA DATA INDEX ! 5 CT NUMBER OF LEVELS 30 3 POPULATION S U E INF 3 3UM5QU. Vf ME ANSUU. F SIGN. PH REST S 0.073U 2^— 0.0025 : D .0.0026 2 0.0013 3.25 0.05 SD 0-.0259 58 0.0004 PH E X E R C I S E S 0 . 0 8 0 3 2 9 0 . 0 0 2 8 D 0 . 0 0 2 5 ~2 0 . 0 0 1 2 2 T 4 0 MOW SD 0 . 0 2 9 7 5 8 0 . 0 0 0 5 P C U 2 RE.ST ' ' S 9 6 5 . 8 7 2 9 3 3 . 0 0 D 1 9 . 7 6 2 9.88 1 . 7 8 NON — S O 3 2 2 . 1 2 Tt 5TT5S PC02 EXERCISE o /oo.uo D 6.65 SD 430.09 2 58 3.32 7.42 0.45 NON STANDARD BICARB S 142.09 . REST 29 4.90 D 31-86 '2 I . V3 3 * 34 0.05 SD 33.54 58 0.58 STANDRAD BICARB. EXERCISE S 196.08 29 6.76 D 2.02 2 1.01 1.65 NON —4rB 35*40 &-6 0*61 BASE EXCESS REST S 239.74 6*27 D 8.23 2 4.11 5.13 0.01 SD 46.51 58 0.80 s u B E B B SI X U H J R TC BC FB ER E D E AA AE FA AA C A 5 NR SS ES RT T Y T PH PC02 DB ES RE TE AGE WT 1 1 1 7397 373 244 + 05 474 69 138 24 1 2 1 7425 390 249 •+ 12 480 70 1 3 1 7432 343 236 - 04 516 58 2 1 1 7386 380 223 - 20 451 54 165 20 2 2 1 7390 424 . 240 + 02 487 46 2 3 1 7388 389 227 - 16 475 55 3 1 1 7433 337 230 - 11- 450 76 178 22 3 2 1 7409 370 231 - 10 462 74 3 3 1 7450 363 233 - 08 483 79 • 4 1 1 ' 7433 332 227 - 15 454 41 182 22 4 2 1 7410 345 201 - 22 44 5 46 4 3 1 7393 381 227 - 16 471 45 5 1 1 7376 412 228 - 14 49 0 ' 74 171 23 5 2 1 7402 393 236 - 04 488 78 5 3 1 7390 405 233 - 08 5 05 74 6 1 1 7365 379 212 - 37 48 3 50 185 23 6 2 1 7377 393 222 - 22 49 0 56 6 3 1 7340 413 211 - 36 . 448 57 7 1 1 7367 395 2 19 - 27 457 62 - 185 22 7 2 1 7355 404 215 - 31 45 8 55 7 3 1 7362 414 224 - 19 435 62 8 1 1 7360 440 230 - 12 476 82 175 2 4 8 2 1 7360 '464 . 2 31. - 10 498 78 8 3 1 7370 435 233 - 07 482 84 9 .1 1 7340 378 201 - 49 420 53 185 23 9 2 1 7364 375 210 - 38 444 60 9 3 1 7415 355 226 - 16 440 58 10 1 1 7366 352 204 - 48 438 80 230 21 10 2 1 7385 343 210 - 38 439 51 10 3 1 7380 3 84 221 - 22 462 50 11 1 1 7425 382 244 + 07 507 55 187 28 11 2 1 7392 43 8 246 + 09 508 53 1 1 3 1 7383 403 232 + 07 422 52 12 1 1 7402 368 226 - 17 457 67 168 23 13 1 1 7387 3_77 _R.2.2_.. — 2 2 465 . 60 160 20 13 2 1 7389 383 226 - 17 447 51 13 3 1 7347 429 2 19 - 26 462 58 14 1 1 7323 356 234 - 07 492 68 " 162 23 14 2 1 7425 383 2 45 + 07 487 67 14 3 1 7396 377 227 - 16 478 74 15 1 1 7310 404 195 - 56 399 62 189 22 15 2 1 7324 399 200 - 51 419 47 15 3 1 7330 449 215 - 29 459 54 16 1 1 7365 372 210 - 38 443 64 180 22 16 2 1 7393 366 224 - 20 472 68 16 3 1 7403 366 l i b - i / 4 7 5 l l 17 1 1 7323 500 233 - 07 462 68 161 29 1 7 2 1 7367 412 227 - 1 5 429 68 17 3 1 7324 488 220 - 25 485 66 18 1 1 7407 382 246 + 08 483 58 195 23 18 2 1 7423 395 249 + 12 502 61 -18 3 1 7437 398 236 - 04 450 57 19 1 1 7425 430 260 + 26 ' 550 66 170 25 19 2 1 7445 398 263 + 29 511 72 19 3 1 7430 418 264 + 30 534 72 20 1 1 7363 411 222 - 22 46 1 60 160 24 20 2 1 73 80 426 236 - 03 477 64 20 3 1 7353 430 220 - 25 488 72 21 1 1 7397 379 228 - 14 465 60 144 21 21 2 1 7387 398 229 - 13 483 50 2 1 3 1 7356 402 215 - 33 468 68 22 1 1 7405 380 233 - 08 49 0 57 175 21 22 2 1 7392 407 2 36 - 02 463 50 22 3 1 7366 430 232 - 08 453 57 23 1 1 7404 347 210 - 31 326 5 4 182 25 23 2 1 7423 364 237 - 02 478 60 23 3 1 7406 329 222 - 25 537 59 24 1 1 7315 448 207 - 42 450 56 165 22 24 2 1 7375 364 212 - 3 5 451 61 24 3 1 7343 436 222 - 22 48 8 62 25 1 1 7408 374 232 - 09 488 ' 62 160 26 25 2 1 . 7423 369 239 0 00 • 492 59 25 3 1 7363 418 220 - 24 44 3 59 ( 26 1 1 7375 337 205 - 4 7 47 5 86 165 20 26 2 1 7383 357 215 - 33 470 72 26 3 1 7367 390 217 - 29 455 66 27 1 1 7370 353 205 - 44 42 3 70 200 24 27 2 1 7347 380 204 - 48 449 66 . 27 3 1 7377 343 203 - 45 398 72 28 1 1 7335 382 208 - 40 446 70 150 20 ^28 2 1 7395 333 211 - 37 458 70 28 3 1 7380 348 210 - 39 464 71 29 1 1 7353 408 217 - 28 437 66 165 21 29 2 1 7337 500 231 - 10 49 8 56 29 3 1 7370 421 229 - 14 475 65 30 1 1 7340 460 224 - 20 479 66 190 20 30 2 1 ' 7314 445 211 - 35 426 52 30 3 1 7337 466 225 - 19 470 56 s u B E B B SI X U H J E TC BC FB ER E D X AA AE FA AA C A E NR SS ES RT T Y R PH PC02 DB ES RE TE 1 1 2 7337 308 175 - 84 378 158 1 2 2 7322 333 178 - 81 388 142 1 3 2 7325 338 182 - 77 398 146 2 1 2 7395 379 228 - 15 468 102 2 2 2 7420 353 231 - 10 487 102 2 3 2 7423 3 46 231 - 10 486 107 3 1 2 7423 383 244 + 06 480 138 3 2 2 7432 378 231 - 10 500 140 3 3 2 7446 3 74 231 - 10 470 136 4 1 2 7378 423 232 - 09 494 105 4 ~2~ 2 7417 347 222 - 20 411 99 4 3 2 7383 376 220 - 23 430 113 5 1 2 7386 3 8 6 _ 225 - 18 477 127 5 2 2 7415 3 92 2 43 + 04 470 130 5 3 2 7362 436 231 - 10 465 122 6 1 2 7358 425 221 - 24 496 94 6 2 2 7365 434 231 - 10 465 94 6 3 2 7387 351 214 - 34 463 94 7 1 2 7356 414 220 - 25 452 110 7 2 2 7370 367 212 - 37 487 105 7 3 2 7367 400 220 - 24 449 121 8 1 2 7352 429 223 - 20 450 132 8 2 2 7435 424 216 - .2 9 447 122 8 3 2 7349 428 220 - 25 459 125 9 1 2 7368 374 211 - 36 444 103 9 2 2 7363 366 208 - 42 461 96 9 3 2 7385 379 222 - 21 470 89 10 1 2 7373 354 207 - 41 431 134 10 2 2 7357 388 211 - 37 455 111 10 3 2 7370 390 219 - 28 489 108 1 1 1 2 7420 414 254 + 18 . 500 108 11 2 2 7460 357 253 + 17 485 94 1 1 3 2 7410 3 78 234 - 06 429 106 12 1 2 7396 318 203 - '47 418 128 13 1 2 7350 399 212 — 35 436 116 13 2- 2 7327 429 208 - 41 •4 5 3 123 13 3 2 7357 40 3 216 - 30 46 2 125 14 1 2 7366 391 217 - 30 472 119 14 2 2 7393 378 226 - 17 493 116 14 3 2 7396 354 214 - 33 462 128 15 1 2 7307 409 196 - 52 380 99 • • 15 2 2 7323 402 200 - 52 435 95 15 3 2 7352 427 220 - 25 458 99 16 1 2 7393 352 211 — 37 432 99 16 2 2 7405 334 215 - 31 453 106 16 3 2 7373 424 231 - 10 479 114 17 1 2 7322 496 228 - 14 472 1 26 17 2 2 7335 468 225 - 18 440 122 17 3 2 7343 437 223 - 20 434 127 18 1 2 7415 386 241 + 06 485 113 18 2 2 7426 373 241 + 02 48 2 115 18 3 2 7442 377 232 - 08 460 115 19 1 2 7430 331 236 - 04 584 129 19 2 2 ' 7445 388 260 + 2 5 516 131 19 3 2 7432 407 265 + 31 526 129 20 1 2 7360 367 204 - 42 __363_ 1 1 5 20 2 2 7366 433 230 - 11 479 110 20 3 2 7392 360 220 - 25 473 109 2 1 1 2 7384 358 214 - 32 431 142 2 1 2 2 7330 411 205 - 45 439 132 2 1 3 2 7373 357 210 - 39 46 8 134 22 1 2 7390 412 236 - 03 487 115 22 2 2 7383 407 232 - 09 462 n o 22 3 2 7387 397 229 - 13 479 115 23 1 2 7362 433 227 - 16 498 114 23 2 2 7433 346 234 - 05 470 120 23 3 2 74 02 359 224 - 19 452 122 24 1 2 7314 415 200 - 52 42 7 -115 24 2 2 7353 405 215 - 32 46 7 115 24 3 2 7435 422 223 - 28 477 118 25 1 2 7409 390 239 0 00 490 98 25 2 2 7414 392 243 + 04 48 8 102 25 3 2 ' 7373 411 228 14 461 105 26 1 2 7343 396 207 - 4 7 443 . 144 26 2 2 7381 354 211 3 7 43 8 134 26 3 2 7370 386 217 - 28 450 141 27 1 2 7386 321 201 - 49 427 129 27 2 2 7 3 9 0 311 201 - 50 438 107 27 3 2 7380 341 206 - 43 43 3 101 28 1 2 7350 362 195 - 58 410 135 28 2 2 7348 358 197 - 55 415 129 28 3 2 7327 386 196 - 55 403 122 29 1 2 7345 417 215 - 32 450 121 29 2 2 7343 412 214 - 32 429 113 29 3 2 7353 404 215 - 32 449 129 30 1 2 7370 413 227 - 15 44 9 126 30 2 2 7331 4 56 220 - 23 44 9 122 30 3 2 7349 478 230 - 12 477 112 

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