PYRIDINE NUCLEOTIDE CONCENTRATION AND RATIOS I IN RAT MUSCLE, HEART, AND LIVER IN RESPONSE TO ACUTE AND CHRONIC EXERCISE Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY D. w. EDINGTON 1963 ‘ Lt LIBRARY Michigan State University This is to certify that the thesis entitled Pyridine Nucleotide Concentrations And Ratios In Rat Muscle. Heart, And Liver In Response To Acute And Chronic Exercise presented by Dee H. Edington has been accepted towards fulfillment of the requirements for Ph.D. Physical Education degree in a £24.” 75/ W Major professor Date /“’€/0/ /7(;/ i / 0-169 ABSTRACT PYRIDINE NUCLEOTIDE CONCENTRATION AND RATIOS IN RAT MUSCLE, HEART, AND LIVER IN RESPONSE TO ACUTE AND CHRONIC EXERCISE by D. W. Edington The purpose of this study was to investigate the con- centrations and various ratios of pyridine nucleotides in trained and sedentary male albino rats in response to a severe acute-exercise stress. Eighteen of thirty-six Sprague.Dawley rats were forced to swim for two hours twice a day, five days per week, for six weeks in thirty—two degree centigrade water. A weight equivalent to up to three percent of the animal's body weight was attached to its tail during the exercise sessions. At the end of the six-week experimental period, each animal was lightly anesthetized with ether and the left achilles tendon-was.severed.'.The distal end of the tendon was attached to a spring steel plate, upon which was mounted a strain guage, and later to a linear variable differential transformer, for static and dynamic muscle recordings respectively. D. W. Edington Muscle contractions were induced by direct stimula- tion with a current of two milliamps. Pre and post- exercise blood samples were taken for the determination of blood lactic acid. At the end of a ten—minute con— traction period, the muscle was frozen between two aluminum plates, pre-cooled in liquid nitrogen. The heart and liver were removed and frozen in liquid nitrogen. Modifications of the extraction and assay procedures of Klingenberg were used to measure the levels of the four pyridine nucleotides. The muscle of the sedentary animals performed the same absolute amount of work (2715.4 millimeter grams), with the isolated muscle preparation, as the trained animals (2645.4 millimeter grams). The muscles of the trained animals performed more work per gram of body weight (9.94 and 8.25 respectively). They also had a higher post-exercise blood lactic acid concentration (21.40 and 19.04 milligram percents). The trained ani— mals were observed to have a higher pyridine nucleotide oxidized to reduced ratio in skeletal muscles (7.3 and 4.4), a lower total DPN to total TPN ratio in the hearts (24.1 and 35.8), and a positive correlation between muscle pyridine nucleotide oxidized to reduced ratio and liver pyridine nucleotide oxidized to reduced ratios (r = .78). From the data in this study it can be concluded that the training routine did not produce a difference between the two groups in resting blood lactic acid concentration, D. W. Edington but the ten-minute direct muscle stimulation brought about a higher blood lactic acid concentration in the trained animal. It further can be concluded that the mean, total pyridine nucleotides in.skeletal muscle, heart, and liver were not different between the two treatment groups. The pyridine nucleotide oxidized to reduced ratio of the muscles suggest that total work performance may be limited by the ability of the cell to minimize the decrease in the oxidized to reduced ratio within the cell. PYRIDINE NUCLEOTIDE CONCENTRATION AND RATIOS IN RAT MUSCLE, HEART, AND LIVER IN RESPONSE TO ACUTE AND CHRONIC EXERCISE By D? W. Edington A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Health, Physical Education, and Recreation 1968 KR DEDICATION To Marilyn Edington whose encouragement and under- standing provided a happy environment for the writing of this thesis. ii ACKNOWLEDGMENTS The assistance of Dr. W. C. Deal of the Michigan State University Biochemistry Department, without whose loaning of equipment and laboratory space, and investment of time this study would not have been completed, is gratefully acknowledged. Acknowledgment is extended to Dr. W. W. Heusner of the Michigan State University Human Energy Research Labora- tory for giving three years of guidance, for providing research experience, and for exhibiting high standards of excellence. Fl. Flo H TABLE OF CONTENTS DEDICATION . . . . . . ACKNOWLEDGMENTS. LIST OF TABLES LIST OF FIGURES. LIST OF APPENDICES. Chapter I. INTRODUCTION . . . . Statement of the Problem Limitations of the Study Definition of Terms II. RELATED LITERATURE Control of Pyridine Nucleotide Bio- synthesis and the Responses to Various Stimuli. Levels of Coenzymes and the Various Ratios III. EXPERIMENTAL METHOD. Experimental Design Training Routine Sacrifice Procedures. ‘Work Performance and Methods of Taking the Tissues Pyridine Nucleotide Extraction Procedures. Pyridine Nucleotide Assay Procedures . Calculations and Statistical Treatment of the Data . . . . . . . IV. RESULTS AND DISCUSSION. Body Weight. . Blood Lactic Acid. . Static Strength and Dynamic Work Performance . Pyridine Nucleotides. iv Page ii iii vi vii viii O\ U124: F4 10 12 l2 16 16 19 19 23 24 24 25 26 28 Chapter Page Frozen Tissue Over Time. . . . . . . 29 Assays Over Time . . . . . . . . . 30 Percent Recovery . . . . 3O Tissue Concentrations in Trained and Sedentary Animals Following Acute Exercise . . . . . . . 31 Interrelationships . 36 Pyridine Nucleotides and Blood Lactic Acid 36 Pyridine Nucleotides and Work Performance. 41 Intercorrelations between Pyridine Nucleotides and Body Weight. . . . . 42 V. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS. . 47 Summary . . . . . . . . . . . . 48 Conclusions. . . . . . . . . . . 49 Recommendations . . . . . . . . . 51 LITERATURE CITED . . . . . . . . . . . . 52 APPENDICES . . . . . . . . . . . . . . 58 Table \ooowoxm 10. ll. 12. LIST OF TABLES Values Found in the Literature for Pyridine Nucleotides . . . . . . . . Blood Lactic Acid Concentrations. Static Strength and Work Performance Test-Retest Frozen Tissue Over Time. . . . Assays Over Time Percent Recovery of Pyridine Nucleotides Maximum Percent Error Pyridine Nucleotides Concentrations. Pyridine Nucleotides vs Blood Lactic Acid. Pyridine Nucleotide Correlations: Sedentary Group . . . Pyridine Nucleotide Correlations: Trained Group . . . vi Page 11 26 27 28 29 3O 31 32 33 39 44 45 Figure LIST OF FIGURES Experimental Design. Muscle Temperature Response to the Quick Freeze Technique. Flow Chart for Tissue Extractions Assay Methods for Oxidized Pyridine Nucleotides . . . . Assay Methods for Reduced Pyridine Nucleotides . . . Weight Profile of the Two Groups of Animals. vii Page 15 18 2O 21 22 25 Appendix A. LIST OF APPENDICES Revolutions Run by Each Animal During the Pre—Experimental Period . Body Weight of Each Animal for Each Week F- Statistics and Probabilities for One-Way AOV . . . . . Pyridine Nucleotides Concentrations for Each Animal Test- Retest and Recovery of the Pyridine Nucleotides . . . . . . . . Extractions of Frozen Tissues Over Time. Assay of Sample Over Time Correlation Coefficients. viii Page 59 6O 61 62 63 65 66 68 CHAPTER I INTRODUCTION As a social organization progresses towards a more mechanized form of living, there is a trend towards a decrease in the level of physical activity of the indi- vidual members of the society. Evidence is accumulating that persons who lead sedentary lives are more prone to diseases (45, 51, 52, 53, 61). It seems clear that this "exercise" situation is one that must be understood if the health needs of future generations are to be met. A study of the organism's adaptation to chronic exercise should allow an elucidation of these changes. An understanding of the exercise-induced adaptation eventually will permit a definition to be made of the minimal exercise level needed either to bring about the desirable changes which chronic exercise produces, or to prevent those changes associated with extreme sedentary existence. It is well known that the trained athlete can under- go sustained exertion for a greater length of time than the untrained individual. Studies of adaptation of the cell to chronic exercise indicate the following cellular biochemical changes: (a) increased myoglobin and increased cytochrome enzymes in chronically active muscles as opposed to inactive muscles(4l,.42, 48),(b) increased mitochon- drial density and increasedumitochondrial.activity in active as opposed to relatively inactive muscles (50), and (c) increased pyruvate oxidation, total mitochondrial protein, myoglobin, cytochrome c, succinate dehydrogenase, DPNH dehydrogenase, DPNH cytochrome c reductase, succinate oxidase, aconitase, mitochondrial malate and isocitrate dehydrogenase in active muscles as opposed to inactive muscles (31). Glickchas.shown.that acute-exercise acti- vates the dehydrogenase steps in the liver by elevation of the intramitochondrial concentration of usable DPN (16). These studies indicate that the changes associated with exercise may involve major changes in the oxidation- reduction capacity of the cell. ‘The critical oxidation- reduction steps in energy metabolism suggest an important role for the pyridine nucleotides in adaptations to exer- cise. The availability.or lack of availability of the pyridine nucleotides would effect energy metabolism through the activity of the dehydrogenases. Pyridine nucleotides are important coenzymes in nearly every major metabolic pathway known to exist in mammalian biochemistry. It can be hypothesized that the several possible ratios of the.pyridine nucleotides can regulate control mechanisms in these pathways and thus partially determine the direction of synthesis and degrada- tion of the.various metabolic products. Evidence by Dietrich and Muniz (ll) and by Deal (10) suggests that pyridine nucleotide biosynthesis is inhibited strongly by DPN, but DPNH exhibits only a weak inhibitory effect. Thus, if the DPN to.DPNH ratio of the cell is decreased, then a net increased synthesis of pyridine nucleotides would be expected., The question of whether a relative short period of daily.exercise will "turn on" synthesis or "turn off" inhibition cannot be answered by the current investigation. A discussion of this form of feedback control as well as of other forms of biosynthetic control by the pituitary gland, thyroxine, and adrenal secretions are presented in the chapter on Related Literature. In this study, two extreme biological conditions were imposed upon two groups of animals. The first condi- tion was a training routine which could be expected to produce increased DPN or alterations in the various , pyridine-nucleotide ratios due to an increased energy demand. The second condition.was a sedentary existence which could be expected to produce increased TPN or altera- tions in the various Pyridineqnucleotide ratios due to an increased reductive biosynthesis. In the former condi- tion, an increase in muscle and.heart total DPN would be expected; in the latter, an increase in the liver total TPN would be expected. The above hypotheses are based upon the generally accepted fact that the diphosphopyridine nucleotides are related to energy metabolism while the triphosphopyridine nucleotides.are involved primarily with reductive biosynthesis. Statement of the Problem The purpose of this study was to investigate the concentrations and various ratios of pyridine nucleotides in the plantar-flexor muscles, hearts, and livers of chronically trained and sedentary male albino rats in response to a locally severe acute-exercise stress. It was hypothesized that the chronic-exercise program which consisted of swimming the animals four hours a day, five days a week, for six consecutive weeks would: (a) result in an increased total DPN to total TPN ratio in the liver and (b) bring about an increase in the total pyridine nucleotide level in skeletal and heart muscle. It was further hypothesized that in the trained as compared to the sedentary animals, the acute—exercise stress would result in: .(a) a greater oxidized to reduced pyridine-nucleotide ratio in skeletal muscle for a given amount of work and (2) a higher blood lactic acid concen- tration for any given muscle oxidized to reduced ratio. Limitations of the Study l. The results of this study are applicable only to male, Sprague-Dawley rats of a similar age and spontaneous- activity levels. 2. The results of this study are confined to the treatment regimens and the time span used in this study. 3. A more or less strenuous acute-exercise stressor might produce dissimilar results- 4. The training program and the acute-exercise procedure were not the same type of exercise. 5. No attempt was made to measure or control food intake of the rats. Definition of Terms The following abbreviations have been used through- out this study: (a) DPN—-the oxidized form of diphosphopy- ridine nucleotide, (b) DPNH--the reduced form of diphos- phopyridine nucleotide, (c) TPN--the oxidized form of triphosphopyridine nucleotide, (d) TPNH--the reduced form of triphosphopyridine nucleotide, (e) total DPN--the sum of DPN plus DPNH, (f) total TPN--the sum of TPN plus TPNH, and (g) oxidized to reduced ratio--the sum of DPN and TPN divided by the sum of DPNH and TPNH. CHAPTER II RELATED LITERATURE Control of Pyridine Nucleotide Biosynthesis and the ReSponses to VariOuS‘Stimuli Dietrich and Muniz (ll, 12) have reported evidence that rat liver nicotinamide mononucleotide pyrophosphory- lase is subject to strong.inhibitory feedback control by DPN. DPNH was reported by these same authors to have only a weak inhibitory effect on the enzyme. Deal (10) has suggested that the same type of control may exist in yeast. Ten minutes of tissue ischemia has been shown by Burch and Von Dippe (4) to bring about a 100 percent increase in DPNH and a twenty percent decrease in DPN. These data might suggest that the DPNH concentration is one-fifth that of DPN. But these same authors show the DPN to DPNH ratio to be between ten and twenty, not five. They also report data that indicate the TPN to TPNH ratio may be involved in the ischemic reaction. As the TPN con— centration decreases by sixty percent, the TPNH concen- tration increases by twenty—five percent. Thus, the TPN concentration would seem to equal about half of the TPNH concentration. Other data show the TPN to TPNH ratio to approximate one. These data.by.Burch and Von Dippe illus- trate the possible importance.of the trans hydrogenases and the kinases in the intraconversions of the pyridine nucleotides. Control of these two types of enzymes could theoretically bring about.shifts in the pyridine nucleotide pool and, therefore, the acute adjustments that the immediate environment requires. Giacobina and.Grasso (15) suggested that a phos- phatase may be involved when they showed that stimulation of a nerve brings about a decrease of both the DPN to DPNH and TPN to TPNH ratios in.the nerve. The TPN to TPNH ratio shift was shown to be the result of a decrease in TPN—~suggesting a TPN to DPN conversion. The DPN to DPNH ratio shift was found.to be due to a decrease in DPN and an increase in DPNH--suggesting the high-energy activity that would be expected. That is, during low—energy and high-synthesis requirements, the pyridine nucleotides could be shifted to the TPN form; in the case of high- energy and low-synthesis requirements, they could be shifted to the DPN form. In 1956 Kaplan (38) proposed that transhydrogenase activity may function as a device for regulating the flow of electrons between the two reduced pyridine nucleotides and, therefore, for regulating the energy production and reductive biosynthesis in the cell. Thus, a relatively stable absolute number of molecules could perform in the several physiological roles involved in the cell. Although the transhydrogenases, kinases, or phospha- tases discussed above,were not.investigated in the current study, it was felt that a short review would serve to illus- trate the interrelationships between the pyridine nucleotides. Indirect confirmation that pyridine nucleotides are involved in metabolically active tissues has been offered by Franz and'Franz.(l4)i‘_They showed that all of the pyridine nucleotides decreased in the pathologically nephrotic kidney.~ Since exercise would require a highly active cell, it could follow from these data that a long- term exercise program would produce the opposite effects to those found by Franz and Franz. Starvation would seem to be, in some ways, similar to prolonged exercise, in that.glucose would be limited and very little reductive biosynthesis would take place. Under conditions of starvation, Glock and McLean (20, 21) and Lardy (40) showed that the liver DPN to DPNH ratio decreases while the TPN to TPNH ratio remains relatively stable or increases."uPande.e§;al. (46) confirmed the work of Lardy and showed.an.increase in the TPN to TPNH ratio with starvation.< These authors found an increase in TPN and DPNH. Refeeding experiments by Pande demon— strated a decrease in the TPN to TPNH ratio. In summary, fasting causes an increase in DPNH and TPN and no change in DPN and TPNH. Hormonal actions almost certainly play some part in any exercise—induced effects on the pyridine nucleotide levels (13, 21, 25, 26). It has been shown by Greengard gg_§1. (24, 25, 26) that adrenalectomy, hypophysectomy, and thyroidectomy can bring about an increase in the total DPN level. .Administration of growth hormone, corticos- terone, and thyroxine caused a decrease.in total DPN levels in rat liver.‘ Thus, the.pituitary hormones exert a pro— nounced effect on DPN levels.in rat liver. Lee and Lardy (43) report a decrease in.DPNH.with thyroxine administra- tion, but this decrease was due to an increase in alpha- glycerophosphate dehydrogenase (alpha-CPD) which facili- tates the oxidation of DPNH through the alpha-GPD cycle of Bucher and Klingenberg. The increase in alpha-GPD serves to increase the DPN to DPNH.ratio.by increasing the DPNH availability to the mitochondria. In part, this accounts for the increased oxygen utilization brought about by thyroxine administrationtv The results of Click (16) are in agreement. He shows that acute-exercise activates the dehydrogenase steps in liver by elevating the intra- mitochondrial concentration of usable DPNH. Assuming the control model of Dietrich and Muniz and of Deal, where DPN inhibits strongly but DPNH does so only minimally, if there is to be an exercise—induced change in the absolute levels of the pyridine nucleotides, the exercise stress must be of sufficient rigor and 10 duration to push the cell into the reduced state and to hold it there long enough for the DPN inhibitory action to be removed. Levels of Coenzymes and the VariouS‘Ratios Table 1 gives a summary of the various tissue levels of pyridine nucleotides that.have been reported in the literature.‘ The great variations in the reported values are almost.certainly due to the varying conditions of the animals at the times of analysis. 11 Aamv .ew\mtaoz s mao.o :m.o eaomsz cam Aamv .ew\mmaoz : mm.o mw.o ae>aa cam Aamv .em\mtaoz s ewo.o :e.o steam cam 2ae Hmpoe 2am Haeoe Ammv .em\ms mm : ewfi mom steam cam Awmv .ewxw: m.o as on mea mam steam use A: v .mxxmeaoz e . m.m :.m ae>aq rem Amav .ewxms m.o . mom ma Ema mam ae>aq cam Amav .ew\w: _ mom m eom 0am Ee>fiq emm Aoov .. o.mmw . .am>fin cam Aomv .ewxms. mmm OOH mmm. Ee>aq pmm Ammv .ew\w: _ mam tween cam Amev .em\mmfloz 3e H.H _H.: m. a.m 3mm mew mm oom smear Lem Aoev o.a m.m Eteaq umm Amav .ew\meaoz as ea m om mm. eaoeare mom Ammv .ew\m: m.a ma m om Hmfi scapem cam Aeflv .em\mz Hm m mOH new Aeneas cam Lama .93 .memtaoz as A.H m.H m.H mm .mHH 03H mmfi 0mm cmesm moceaeeem made: .eem\.eaxo ame-e\2aoue mzae\zae. mzao\2ao mzae zae mzao zao mammfie .mcoapmpucoocoo oofipooaosz ocfiofipzm now mesam> empaoatmut.a mamas CHAPTER III EXPERIMENTAL METHOD Experimental Design One hundred and five male rats of the Sprague Dawley strain, twenty-three days of age, were purchased from the Hormone Assay Company of Chicago, Illinois. Since pyridine nucleotide levels may be influenced by activity and per- haps by unknown hereditary factors, it was decided that those animals with innately high or low spontaneous activ- ity levels should be deleted from the sample. The rats were placed into individual cages, with spontaneous-activity wheels attached, for five days. The activity wheels were 12.5 centimeters wide and 35 centimeters in diameter. The animals were allowed to run at any time, and a counter attached to each wheel recorded the revolutions run. The revolution count for each rat, over the final three days of this five—day period, was used to eliminate the extreme groups. A frequency distribution was made for each of these three days. Those animals, having revolution counts within the central-half of the distribution, were designated each day. Thirty of the required forty-eight animals were chosen at this step as each of these had revolution counts within the middle—half on all three days. The final 12 l3 eighteen animals had revolution counts within the middle- half of the distribution on two of the three days, and their revolution counts for the third day were not over plus (or minus) ten percent from the highest (or lowest) count in the central-half. (See Appendix A for the revo- lutions run by each of the chosen animals during the three days). The animals were ranked according to their three- day total revolutions run. The most active animal was assigned to group A, the next two to group B, the fourth and fifth to group A, the sixth and seventh to group B, etc. A flip of a coin decided which group would receive the physical training routine. After the groups were determined, the forty-eight animals were transferred to.individual sedentary cages. These cages were arranged so that twenty—four were on each side of a single rack. .Each sedentary cage measured 24 x 18 x 18 centimeters.. Each animal in the sedentary group had a sheet metal plate inserted into his individual cage so that the total volume of the cage was bisected from the upper left to the lower right. This was done to restrict activity as much as possible. The twenty—four cages on each side of the rack were distributed so that there were four levels of six cages. Three experimental animals and three control animals were placed in alter- nate cages on each level. Lighting in the animal room was maintained on a cycle of twelve hours of light and twelve hours of l4 darkness. The animal room was maintained at twenty-five degrees centigrade plus (or minus) one degree. The rats were fed Wayne Laboratory.Blocks ad libitum and had con- stant access to water. Body weight for each animal was recorded at the same hour on Thursday of each week, throughout the experimental period. A flow chart for the experimental design is shown in Figure l. TrainingrRoutine The swimming conditions were such that the experi- mental rats were in thirty to thirty—two degree centi— grade water, from three to five P.M. and from nine to eleven P.M., five days per week. No extra weights were attached to the animals? tails during the first week of training although miniature plastic Clothespins were placed on their tails during the swimming periods of the fourth and fifth days.- Two percent of each animal's body weight, as determined.after the first week of swimming, was added to the tail of each rat during each swimming period of the second week. For the.third week of training, the weights were increased to three percent of each animal's latest body weight. In all subsequent weeks, the weights were maintained at three percent of the animals' current body weights. All of the added weights were accurate to plus (or minus) ten milligrams. Each rat was swum 15 Animal's age 23 Days . . . . . . . 105 Animals Five days in voluntary cages 28 Da ys l I fl Lower group Middle group Upper group on the basis on the basis on the basis of spontan— of spontan- of spontan- eous activity eous activity eous actiVity (n=28) (n=48) (n=29) 28 Days . . I _j 24 animals in 24 animals in sedentary training treatment group treatment group 40 to 46 days of treatments 68 to 74 Days Sacrifice 1810f each treatment group Figure l.—-Experimental Design l6 individually in a twenty-five centimeter diameter cylinder immersed in 1.2 meters of water. After swimming, the animals were individually dried with towels, their weights were removed, and they were returned to their cages. Sacrifice Schedule At the end of the experimental period, eighteen animals were chosen randomly from each group for sacrifice. The order of random selection served as the sacrifice order. This sacrifice order was established so that three animals from each group could be sacrificed on each of six consecutive days. The animals not yet sacrificed were maintained on their normal treatment schedules. Work PerfOrmance and Methods of Takinggthe Tissues At the time of sacrifice, each animal was lightly anesthetized by ether in an anesthetic chamber and then maintained in this state by.the administration of ether through a nose cone. The femoral vein of the right leg was exposed and a one milliliter blood sample was taken for lactic acid determinations by the method of Barker and Summerson (2). The soleus muscle of the right leg was removed and used for histochemical analysis not reported in this thesis. The animal was placed on a muscle performance analyzer (44) for the measurement of the static and dynamic 17 work performance of the gastroenemius-plantaris muscle group.1 This muscle group was exposed in the animal's left leg and the distal.end of the muscle group was cut at the achilles tendon.. A clamp was attached to the cut tendon and served as the cathode for direct muscle stimula- tion. A hemostat attached near the proximal end of the muscle group served as the anode. A Grass stimulator, Model S4, provided a twenty—volt square wave input. A 10,000 ohm resistor, in series.with the muscle preparation, insured that the current acting upon the muscle group was two milliamps. The clamp on the achilles tendon was attached to a spring steel plate and later to a linear variable differential transformer for static and dynamic contraction recordings respectively. The force of a two—second static contraction was measured by a strain guage, mounted on the spring steel plate, and recorded on a Gilson Recorder. The stimulation frequency for the static contraction was one hundred per second with a duration of ten milliseconds. Next, ten minutes of dynamic contractions were measured by loading the muscle group with one.hundred grams and measuring the distance moved per contraction (two contractions per second) by monitoring the output of the differential transformer. 1The left soleus muscle was removed and used in bio- chemical analysis not reported in this thesis. 18 At the end of the ten-minute dynamic contraction cycle, a one-milliliter blood sample was taken from the left femoral vein and was used for.lactic acid determina— tion. The contracting muscle group was frozen with aluminum clamps pre-cooled in liquid nitrogen. The cooling response to this technique is shown in Figure 3. The response was measured by a thermocouple inserted into the center of the muscle group. The reference point was an ice-water bath, and the response was observed with a polaroid camera mounted on an oscilloscope which was placed in the thermo- couple circuit. The quick frozen muscle tissue was removed, crushed, placed in a pyrex culture tube, recooled in liquid nitrogen, and stored at minus twenty-five degrees 504 A 25 1 O 3, 0 . .5, -25. E (D 4") -50 1 3 o '75 " U) . {5-100 . \ o A) -125 . N _150 p kPre-cooled clamp applied to muficle 1 W l l r I l 1 t 2 l 0 l 2 3 4 5 15 Seconds Figure 2.--Muscle Temperature Response to the Quick Freeze Technique. l9 centigrade. The liver and heart were removed within two minutes, out into small sections, placed.in pyrex culture tubes, quick frozen in liquid nitrogen, and stored at minus twenty-five degress centigrade. All tissues were stored for periods ranging from two days to three weeks before being assayed. Pyridine Nucleotide Extraction Procedures A flow chart for the extraction procedures can be seen in Figure 3. These methods are essentially those of Klingenberg (39). The tissue extractions were all per- formed between two days and three weeks after being frozen in liquid nitrogen. Pyridine NuCleotide Assay Procedures The assay methods were spectrOphotometric procedures, and the reactions are depicted in Figures 4 and 5. The assays are essentially those as described by Klingenberg (39). Optical density recordings were made at a wave length of 340 millimicrons using a Bechman Model DU Spec- trophotometer with a Gilford Recorder. Enzyme activity was verified at the beginning of each assay period by the addition of the specific pyridine nucleotide to be assayed at any one time. LDH and the substrates used in this study were purchased from Sigma Chemical Company. The other three enzymes were purchased from Boehringer Corp., West Germany. 20 Frozen tissue in powered form oxidized 2 ml HClO3- (.6N) weigh add HClO to get a 1-6 dilution 3——1 centrifuge for ten minutes at 3.86 x 103 X S 2 mlrof sediment supernatant ~—-.2 ml K2HPOu ———4 drops of KOH (6N) per ml of HClO3 precipatate ————.2 ml K2HP0u ___.KOH to pH 7.3 to assay reduced 2 ml alcoholic KOH at 70°C 90 seconds into ice bath weigh .5 ml PO,4 buffer precipate e—add PO buffer to pH 7.8 centrifuge for twenty minuEes at 3.2 x 10 x g I 1 sediment to assay Figure 3.--Flow Chart for Tissue Extractions 21 Ethanol + DPN< >Acetaldehyde + DPNH Alcohol Dehydrogenase Assay procedure Assay chafiacteristics 1. add .15 ml extract K7=lO‘ 2. add .15 ml Po, buffer (.lM) pH 7.5 with semicarbazide added 26°C 3. add .02 ml ethanol (absolute) O.D. read at 340mu 4. read 0. D. 5. add .02 ml ADH (1.2 mg per ml) 6. after 3 minutes read 0. D. Glucose-6-Phosphate + TPN<;————€>6-Phosphogluconate + TPNH Glucose-6-Phosphate Dehydrogenase Assay procedure Assay chgracteristics 1. add .3 ml extract K7=10 add .04 m1 G—6—P (.lM) H 7 2 read O.D. 9 ° 26°C add .02 ml G-6-P Dh O.D. read at 3ND mu (1_mg per ml) after 3 minutes read O.D. U'I JI'LUM Figure 4.--Assay Methods for Oxidized Pyridine Nucleotides 22 Calgplations'angStatistical Treatment.of the Data The calculational formulas used for each of the four pyridine nucleotides are those of Klingenberg (39). Pyruvate + DPNHe >Lactate + DPN Lactate Dehydrogenase (K7 = 2.9 x 10’5) o-Keto glutarate + NH“ + TPNH<+—————+>Glutamate + TPN Glutamate Dehydrogenase _ 6 (K7 - lO ) Assay procedure Assay characteristics add .4 ml extract pH 7.7 add .005 ml pyruvate (.3M) O.D. read at 340 mu read O.D. 26° C add .005 ml LDH (10 mg per ml) after 3 min. read O.D. add .005 ml a-keto glutarate (.5M) add. 005 ml ammonium chloride (1M) read O.D. add .005 ml Gl-Dh (5 mg per ml) after 3 minutes read O.D. OKOmNmUlerNH }_J Figure 5.--Assay Methods for Reduced Pyridine Nucleotides 23 Calculations and Statistical Treatment of the Data The calculational formulas used for each of the four pyridine nucleotides are those of Klingenberg (39). Statistical calculations, using the UNEQl routine for the one—way analyses of variance and the BASTAT rou- tine for the correlational analyses were performed on the Michigan State University Control Data 3600 Computer. Pilot work, completed three months prior to the experimental period of the present study, provided the basis for: (a) subjectively determining that mean dif—- ferences as small as one-half of one standard deviation should be detected as statistically significant and (b) estimating the necessary and sufficient sample sizes (n = n2 = 18) required to hold the probability of a 1 type I error at .05 and that of a type II error at .20. Equations employed for these determinations are found in Walker and Lev (58). CHAPTER IV RESULTS AND DISCUSSION Body_Weight The mean weekly body weights for the trained and sedentary groups are shown in Figure 6. It can be seen that the trained group fell below the sedentary group and that the mean group differences were significant beginning at the end of the first week of treatments. The F-value and the probability level for each mean difference are given in Appendix C. The weekly body weights for each animal are contained in Appendix B. The body-weight data might.be very critical. Since the sedentary group grew faster, perhaps the mean differ- ences observed in pyridine nucleotide concentrations, rela- tive work performance, and blood lactic acid concentrations were differential growth rather.than treatment effects. Van Russ (58) has observed that after forced-exercise treatments are terminated, trained rats "catch up" to sedentary rats in terms of body weights and body composi- tion data. 24 25 350 ‘ '———* sedentary group *——-* trained rou 300 . g P 2 wr~ 250 . ".32 3:2 200 1 €39 150. o m 100 . 50 I 0 F—*——'Treatment Period-—-——:;i T 20 25 3O 35 40 45 50 55 60 65 70 75 Age of Animals (days) Figure 6.-—Weight Profile of the Two Groups of Ani- mals. Differences between the groups are significant (p<.001) beginning at the end of the first week of treatments. Blood‘Lactic.Acid Eighteenl of the animals were measured for plasma lactic acid concentration before and after direct muscle stimulation. The chroniceexercise program did not alter the resting plasma lactic acid level. In response to the acute-exercise stress, the.trained animals had a signifi- cantlygreater increase in plasmalactic acid, as shown in Table 2. The F-value and the probability level for the mean differences are given in Appendix C. The post— exercise mean differences, shown in Table 2, could come 1Blood samples were taken on all thirty—six ani- mals but lactic acid was not determined until the final eighteen animals. 26 Table 2.--Blood Lactic Acid Concentrations Before and After Ten Minutes of Direct Muscle Stimula- tion. Table values are mg. % :_S.E.M. —--- ——- — ~— ~—— -———- —-~-- ~ ---— ul— Pre-exercise Post—exercise Group Blood Lactic Blood Lactic Increment Acid Acid Trained 14.78 i .39 *21.40 i .67 *6.62 i .69 Sedentary 14.81 i .56 19.04 :_.76 4.42 i .77 *p < .05 about by three mechanisms: (a) more lactic acid might have been produced in the muscle of the trained animal (assuming the blood lactic acid was at a steady state with the muscle lactic acid concentration), (b) a greater percentage of lactic acid may have been removed from the trained muscle by the circulation, and/or (0) other tissues, chiefly the liver, could have extracted a smaller percentage of the lactic acid that was produced. Further discussion of this blood lactic acid data can be found in the section on Pyridine Nucleotides and Blood Lactic Acid as well as in Sarenac (56). Static Strength and Dynamic Work Performance No differences were found in the absolute static contraction strength or in the absolute total work done by the two groups. The sedentary group performed signifi- cantly more work during the first minute of the dynamic work cycle. These results are shown in Table 3. The mo.v Qt 27 He. + mo. + .mo. + mm.m . m:.0 22.0 Hm. H mo. H mo. H 20.0 mm.0 m0.0 mo. +. mo. + mo. + . me. + mo. + . mo. +. co. + so. + co. + 0:10 mm.0 . wm.o 00.0 .mm.0 . no.0 m:.H 00.m Hm.H mumpcooom mo. H mo. H .mo. H co. H so. H Ho. H mo. H co. H mo. H 50.0 N>.o . m>.o m~.o mm.o mH.H mm.H MH.N om.m UOCHMLB AmouscHEv panoz zoom no Echo Loo xnoz OHEMCHQ 99: H. 0.2 H in? H ASH H NEH H 93 H 93H m.: H mu: H 3.2 H TS H .3: H e.mHHm m.oeH m.H:H o.mmH o.HeH. m.mmH m.oom m.Hem 0.0mm o.moe m.»~c c.mmm Hemcecccm m.meH H o.mH H e.mH H H.MH.H m.MH H o.MH.H m.mH.H H.5H H H.mH H m.om H m.mm H a.Hm.H .:.mecm m.omH m.mcH e.mHH m.mmH m.mmH, o.mHm 0.0mm 0.:om H.Hm: c.oem m.om: ccchce Hmcoe 0H m m s c m z m m H HmmpscHEv xpoz OHEHCHQ . . spwcoeum eHcccm. cacao mosHm> oHnt .ozogu oHomsz .mepwtpopoEHHHHE :H one chmpcmHmtmsHEocOoupmwo on» no mocmscomnom xcoz OHEmcmo one camcoppm OHHMHmII.m mqmHH upmmm mHomsz .ozmmHu no emnwOHHx nod moHoe opoHE wH mum mosam> mHome ..m:oHumLucoocoo ochooHosz ocHochmmtt.m mgmHq 5m. mm.- 0H. 00. MH.. mo. 0m. H0. 8000600 eoochcmccco 00. 00.- H0. Hm. mH.u 0m.: 00.: 00.- 6000600 coochcmoa mm.t 0H.- 0:. mm. 00.: 00.: mm.. HH.- cccccmq coochmEa . Humor H0.- 00. 0:., 0H. Hm. 5H. m0. 00. :00. 00. *m0. .00. cccccmn cooHctcwcmc0 0m.t 50. m0. HH. 0:. NH. *m5. m0.. 0:. mm.. H0. 0m.t ccccemn coochcmoa H0. 00.: mm. 50.- . 5H. 50.- NH. 00.: 00.- 00.- HH.- 00.- 0000600 cooHeucaa oHomjz CHHLH .Uom .CHHLB .oom .CHHLB .Uom .cngh .06@ .chLB .mom .chLE .nom mcwHoooHozz ZEH Hmcce za0_H0000 .caa H6000 cae\210 rzi0\za0 eceseem\0echHx0 .moUHpooHozz ocHUHL m0 new UHoq 0Huomq wooHr somepom mucoHOHcgooo CoprHQLLOQII.OH mum<8 40 since the trained animals were smaller than the sedentary animals, the livers of the trained animals would extract a lessor absolute amount of.lactic acid from the blood.3 However, the correlation of.r = .79, between the change in blood lactic acid and the oxidized to reduced ratio within the livers of the trained rats, indicates that the livers of these animals were able to convert the increased lactic acid to pyruvate and thus were forming large amounts of DPN. The non significant correlation of r = .21, for the sedentary animals, indicates that the livers of these animals were not adapted to decrease the exercise—induced blood lactic acid to the same extent as were the livers of the trained animals.. As.was the case with muscle, membrane permeability, enzyme structural alterations, and enzyme concentration may have been involved in this ability to increase the oxidized.to reduced ratio in response to an increase in blood lactic acid. A positive correlation of r = .77, between the change in blood lactic acid and the total DPN to total TPN ratio, was found for the livers of.the trained rats. A non significant negative correlation of r = .35 was found for the livers of the sedentary.rats. The contrast between these two correlation coefficients is another indication 3From previous work in the Human Energy Research Laboratory at Michigan State University, it can be cal- culated that the body weight (x) and liver (y) weights are correlated at r = .81. The regression line y = .9698 + .0283 x is significant at p < .005. 41 of the differences which existed in the energy metabolism of the trained as opposed; o the sedentary rats. The liver DPN to TPN ratio has been discussed earlier. In this context, it indicates.that an increase in the blood lactic acid concentration can.be.handled better by the liver of the trained rat than by that of the sedentary rat. One other possibility, which is specific to this investigation, should be mentioned. The lower blood lac- tic acid concentrations in.the.larger sedentary animals might be explained partially by a greater dilution of the lactic acid in the blood of those animals--assuming that a larger animal has a greater volume of blood than a smaller animal. No attempt was made to check this possi— bility; however, the muscle and.liver results would indicate that a blood.dilution did not account for much, if any, of the difference observed in blood lactic acid concentrations. In summary, it appears that, in the trained animals as compared to the sedentary animals: (a) there was less muscular production of lactic acid, (b) the blood trans- port of lactic acid was much more efficient, and (c) the liver extraction of lactic acid from the blood was more efficient. Pyridine Nucleotides.and Work Performance It can be seen from the regression lines relating the oxidized to reduced ratio (x) and the total work divided 42 by body weight (y), that.for any given work performance level, the muscle of the.trained animal (x = 80.73 - 7.38y) had a higher oxidized.to.reduced ratio than the muscle of the sedentary animal (x.= 63.21 - 7.13y). Although the slopes of the regression lines.were statistically non sifnificant these data tend to support one of the initial hypotheses of this study, that for any given amount of work, the muscle of the trained animal will have a greater ozidized to reduced level than that of the sedentary animal. Thus, to do more work, the cell must be capable of maintaining a more oxidized environment or be able to tolerate a more reduced state. The critical oxidized to reduced ratio. is a function of the muscle cell; and from other data presented.previously, it appears that this ratio can be altered by training. Within each treatment.group no statistically signifi— cant correlations were found between the muscle pyridine nucleotides and any of the.work.parameters. Intercorrelations Between Pyridine Nucleotides and Body Weight By examining Tables 11 and 12, it can be observed that, within the sedentary group, body weight had a dis- tinct relationship to total pyridine nucleotides in the muscle and heart. The trained.group did not show this relationship. These results could be expected, as it may be possible to think of the body weights of the trained group as being controlled by the external stress of the 43 four-hour daily swim. Thus, food intake in the trained animal was converted to energy storage, and then to energy utilization, during the training sessions. The body weights of the sedentary animals were controlled from within, as food intake.was used only for energy storage and the normal body functions. In the sedentary animals, heart total pyridine nucleotides correlated.with muscle total pyridine nucleo- tides (r = .52) and with body weight 1r = —.52). The negative correlation with body weight suggests that the total pyridine nucleotides (especially in the heart) become diluted within the tissues in these sedentary animals. That is, as the animal increased in body weight, the tissue concentration decreases. This could lead, possibly, to a pathological condition if the dilution becomes critical. These relationships were not observed in the trained animals. The trained animals showed simultaneous changes between muscle and liver pyridine nucleotides. Altera- tions in the oxidized to reduced ratio, the DPN to DPNH ratio, and the total DPN to total TPN ratio were in the same direction (r > .59). As the muscle worked, and thus became more reduced, the liver also became more reduced. The mechanism for this increased reduced state (decrease in the oxidized to reduced ratio) in the liver of the trained animals was not investigated by the present study. No hypothesis can be offered to explain this 44 .m 00.H 00. 0H. 00. 50. .00.: 00. 00.: 00.: 0:.: 0H. 00.: 0H.: 000.: HH.: ccmHoz 50cm 00.H 00.: H0.: 00. H0.: 0H.: 0H. 50. 0H.: 00. 00. 0H.: 50.: 0H. .60: .EHQ Hacoe 00.H .H5.. 00. 00. 00.: 0H. 5H. 00.: 0H. 00.: 0H.: 00.: 0H.: :09 Hccoe\200 H0000 00> 00.H #00. 00.: 00.: 0H. em. 50.: 50. 0H.: 0H.: 00.: 0H.: couscomxcoNHcon H0 00.H H0.:. 00.: 50. 0H. 00.: 00. 00.: 00.: 5H.: 0H.: 0200\200 00.H 0H.: 00. 0H. *00. 00.: 00.: mm. 00:. 00. .83: .000 H0060 00.H 0H. 00. 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L 4 . ,0 H “#000. m! .0 .. .. ward LABEL «ravapkf... :6“... HMUOLE 00.H 0...: *02. .mm. nomnrtmeEMHmec .-;-..H ,5..-.l .I 4.: : t ) i. r . . 1: ELI-Art rw.«o._i I-drxmjuh .|,. H... at. . .3. D C M .1. .2. r0. .(5 \ H4 PM 1,..— i .dnoam pochLB org 0. szHmH >000 020 mvoHpocHosm ECHUHLHL cmmzuor mucoHoHumoor cowumHoLLootl.mH samqb U6 phenomenon but one or more of the following events could account for this change: (a) the first step in the extrac- tion of lactic acid from the blood, the conversion to pyruvate, (b) oxidation of.fatty acids, and/or (c) an ischemic environment in.the liver cell. Since parallel changes were not observed in the sedentary animals, perhaps these.rats were not "adapted" to exercise. That is, their systems seemed to operate more independently of.one another. The coordination of metabolic systems of different tissues may be a crucial factor in any "adaptation" to training. This concept merits further investigation. CHAPTER V SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS Summary The purpose of this study was to examine the pyridine nucleotide concentrations in.skeletal muscle, heart, and liver of trained and sedentary male albino rats in response to a ten-minute acute in-situ, exercise stress. It was also the purpose of this investigation to assess the total pyridine nucleotide concentrations of the two groups as a result of the treatments applied over the six-week experimental period. The general concept underlying the experimental hypothesis was that the diphosphopyridine nucleotides (DPN) are involved in energy metabolism, while the triphosphopyridine nucleotides (TPN) are uti- lized, primarily, in reductive biosynthesis. Two groups of eighteen-young male albino rats (Sprague-Dawley strain) were assigned randomly to treat- ments: (a) sedentary.housing, and (b) sedentary housing with two two-hour swim sessions per day, five days per week. The animal that was forced to swim had an addi- tional weight of up to three percent of its body weight attached to the tip of its tail. 147 H8 The cage housings for the trained group were standard 2“ x 18 x 18 centimeter.smalleanimal cages. The sedentary animals lived in this same type of cage but a sheet metal plate was inserted to diagonally bisect the total volume of the cage. During the six-week experimental period, the trained animals were forced to swim from three P.M. to five P.M. and from nine P.M. to eleven P.M. five days per week. Body weights for all the animals were recorded on Thursday mornings of each week. At the end of the six—week training period, six rats per day were lightly anesthetized with ether, the right femoral vein exposed and a blood sample taken for the pre-exercise blood lactic acid determination. The distal end of the gastrocnemius—plantaris muscle group was exposed by severing the achilles tendon. A clamp attached to the cut tendons served as the cathode for direct muscle stimulation. The clamp was connected to a spring steel plate, upon which was mounted a strain guage, for muscle static work recordings.. Following the static recordings the clamp was connected to a linear variable differential transformer for dynamic work.recordings. The stimulation frequency was two per second and the current was two milliamps. At the end of the ten—minute stimulation period a blood sample was taken from the exposed left femoral vein U9 and used for the post—exercise blood lactic acid deter— minations. The muscle was than clamped between two aluminum plates, pre—cooled in liquid nitrogen. The heart and liver were removed within two minutes and frozen in liquid nitro- gen. These tissues were stored at minus twenty-five: degrees until the pyridine nucleotides were extracted. Extraction procedures and.assays are essentially as those described by Klingenberg.(39). Significant differences between the trained and sed- entary groups were found in muscle DPNH (124.9 and 262.8 micro moles per kilogram of muscle), muscle pyridine nucleo- tide oxidized to reduced ratio (7.3 and H.H), and heart DPN to TPN ratio (24.1 and 35.8 respectively). Significant correlations were found in the relationship between: (a) body weight and muscle DPN in the sedentary animals (r = -.H8), (b) body weight and heart total pyridine nucleotides in the sedentary animals (r = -.52), (c) elevation in blood lactic acid and muscle oxidized to reduced ratio in the trained animals (r = .83), (d) elevation in blood lactic acid and liver oxidized to reduced ratio in the trained animals (r = .79), and (e) oxidized to reduced ratio in muscle to the oxidized to reduced ratio in the liver of the trained animal (r = .78). Conclusions l. The six-week training stress was severe enough to result in lower body weights in the trained animals. 50 The training routine did not produce a difference between the two groups in resting blood lactic acid concentration. A ten-minute direct.muscle stimulation brought about a higher blood lactic acid concentration in the trained animal. The mean work performance was not different between the two experimental groups; but.work per unit of body weight was greater for the trained group. Correlations of work to blood lactic acid concentra— tion and work to oxidized to.reduced ratio of the mus- cle suggest that total work performance may be limited by the ability of the cell to minimize the decrease in the oxidized to reduced ratio within the cell. The ten-minute work stress resulted in a less reduced state in the muscle of the trained as compared to the sedentary animal. Mean, total pyridine nucleotides in skeletal muscle, heart, and liver were not different between the two treatment groups. In the trained animals there existed a significant correlation (r = .83) between the change in blood lactic acid and the muscle oxidized to reduced ratio. The decrease in muscle.oxidized to reduced ratio fol- lowing an acute-exercise stress was accompanied by a decrease in the liver oxidized to reduced ratio in the trained animals. 51 9. Heart total TPN was greater and thus the total DPN to TPN ratio was lower in the trained group. ReCOmmendations A considerable amount of research, at the cellular level, is needed in order to establish a workable model for the acute and long term.animal responses to the various types of exercise stressors. This future research should include the following recommendations. 1. Regulation of diet should be a major criteria in any experimental design. 2. The size of the individual muscle fiber in relation to the various muscle sizes should be established. 3. Not only enzyme concentrations, but more importantly the specific activities of.each enzyme in response to the various exercise stressors should be examined. U. Pyridine nucleotide concentrations should be separated as to their concentrations.within the various cellular compartments in relation to the several oxidized to reduced ratios. 5. The relationship should be established between muscle lactic acid and blood lactic acid and observations made of the alteration, if any, of this function with training. 6. Correlative studies should be carried out to link observable changes in the various exercise related parameters. u‘. L' NL' "Av (L'Hhfln V." I” ...-. 11. 12. LITERATURE CITED Altland, P. 0., Highman, B., and Nelson, B. D. Serum enzyme and tissue changes in rats exercised repeatedly at altitude: effects of training. Am. J. Physiol. 214: 28, 1968. Barker, S. 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Correlation of Cyclo- phorase activity and mitochondria in striated muscle. Proc. Soc.’Expt.'Bio. Med. 79: 352, 1952. Raab, w., Chaplin, J. P. and Bajusz, E. Myocardial necroses produced.in domesticated rats and in wild rats by sensory and emotional stresses. Proc. Soc. Egpt.'Bio. Med. 116: 665, 1964. Ratcliffe,.H. and Cronin, M. Changing frequency of arteriosclerosis in mammals and birds at the Phila- delphia Zoological Garden. Circul. 18: 41, 1958. Retzlaff, B., Fontaine, J., and Furuta, W. Effect of daily exercise on life span of albino rats. Geri- atrics 21: 171, 1966. Robinson, S. and Harmon, P. M. The lactic acid mech- anism and certain properties of the blood in relation to training. Am. J. Physiol. 132: 757, 1941. Robinson, 8., Edwards, H., and Dill, D. New records in human power. Science. 85: 409, 1937. Sarenac, B. Unpublished Master's Thesis, Michigan State University, 1968. Steinhaus, A. H. Chronic effects of exercise. Phy- Siol. Revs. 13: 103, 1933. Van Huss, W. D. Personal communication. Michigan State University, 1968. Walker, H. M. and Lev, J. Statistical Inference. New York: Holt. 1963, p. 165. Williamson, D. H., Land, P., and Krebs, H. A. The redox state of free nicotinamide adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem. J. 103: 514, 1967. Wolffe, J. B., Digilio, V., Dale, A., McGinnis, G., Donnelly, D., Plungian, M., Sprowls, J., James, F., Einhorn, C., and Weckheisa, G. Experimental athero- matosis and athero-hepatosis in Ducks and Geese: Its reversibility and its clinical implications. Am. Heart J. 38: 467, 1949. ’ 62. 57 Young, D. R., Shapira, J., Forrest, R., Adachi, R., Lim, R., and Pelligra, R. Model for evaluation of fatty acid metabolism for man during prolonged exer- cise. J. Appl. Physiol. 23: 716, 1967. APPENDICES 58 59 APPENDIX A.--Revolutions Run by Each Animal During the Pre— experimental Period. Animal Number First Day Second Day Third Day Total 1 1059 903 1034 2996 2 906 991 1085 2982 3 1151 1026 783 2960 4 1249 747 946 2937 5 818 871 1232 2921 6 520 997 1393 2910 7 788 902 1032 2722 8 598 641 1452 2691 9 606 986 1096 2688 10 1188 539 959 2686 11 1380 572 727 2679 12 1267 598 781 2646 13 957 909 743 2609 14 933 920 745 2598 15 835 549 1210 2594 16 638 740 1105 2483 17 625 654 1181 2460 18 916 936 603 2455 19 831 843 761 2435 20 954 825 611 2390 21 834 707 789 2330 22 910 734 660 2304 23 795 774 709 2278 24 771 556 948 2275 25 823 552 895 2270 26 763 626 878 2267 27 1303 505 446 2254 28 801 876 545 2222 29 781 591 779 2151 30 757 540 753 2050 31 851 774 422 2047 32 687 876 472 2035 33 462 659 893 2014 34 496 709 767 1972 35 442 582 846 1870 ' 36 406 606 827 1839 37 610 549 656 1815 38 645 565 545 1755 39 495 539 595 1629 40 551 585 466 1602 41 816 322 460 1598 42 540 511 525 1576 43 645 533 346 1524 44 337 511 620 1468 45 343 489 633 1465 46 528 471 431 1430 47 409 425 510 1344 48 548 355 422 1324 60 mmm mmm mmm HNH mma :NH M Nam oam NNN mam mma mmH M :mm wmm mam omH ova MNH m: mmm pom 0mm mma Hma mma N: wmm mam mom mza mma mHH m: 2mm mom 23m mwa me mHH m: 05m 5mm mmm Nma 03H mad :3 mmm mmm :mm pom HNH Hma mm mmm mmm mom Hma mma NHH mm Hmm owm now now 05H NNH mm mmm mam cam mma mza omH mm ozm mmm mom mom mma wma mm 2mm 0mm wmm mma bwa mmH mm mmm mam mum 0mm mma wma Hm mmm mmm mam mma Hwa NHH mm mmm mmm owm Nmm mma mmH om mom mam mum mma mwa HmH mm :om omm mmm ozm mma mma Fm :om 3mm mmm Nma :wa 53H :m mum mam wwm mmm mam omH mm :om mmm mzm 52H omH omH om mmm mam mmm mam :ma ::H mm mam mmm mom Nma msa mma NH mnm mam mmm wmm :mH 02H mm :om 3mm 0mm mma mma mmH ma mmm mom mmm mwa mwa :ma ma mom mum 3mm :NH mwa omH ma mnm mam owm Hmm owa :MH ma :wm mam :Hm omH NmH NNH NH mmm :Hm mmm mmm wwH H2H ma mmm mmm 0mm mma HNH omH m mmm mom owm mmm mma wad 3H mam mum 0mm mwa mwa omH m mmm :om mmm mmm mwa me Ha :mm mmm mmm Nza owH mma m mmm mmm 3mm wmm pom oma m 33m mzm com 00H mma moa a mam mmm mam mom 25H QMH m m m 2 m m H 909832 m m 2 m m H LmQEdz 9663 x663 9663 9663 9663 x663 Hmefic< 9663 9663 9663 9663 9663 x663 Hmefica .x663 :66m 666 Hmsfic< comm no pcwfi63 suomuu.m xHozmmma 61. APPENDIX C.--0ne Way Analysis of Variance With the Exercise Group as the Category Variable. Dependent Variable F-Value Probability Dependent Variable F-Value Probability Pyridine Nucleotides MDPN .23 ' .6} Total H--reduced . .04 ' -.83 MDPNH 6.25 .02 , Total oxid/reduced .00 .98 MTPN .37 .54 LDPN/LDPNH .36 . .55 MTPNH 1.29 - .26 LTPN/LTPNH ‘ 1.51. , .23 HDPN .21 .65 1 Total LDPN 5 1.98 .17 HDPNH . . 1 .18 ,.67 Total LTPN ' 1.20 .28 HTPN .63 .43 Total LDPN/LTPN 1.75 .19 HTPNH ~ 3.21 .08 Total L—pyr. nuc. .08 .78 LDPN 2.06 r'.16 Total L-okidized 1.96 .17 LDPNH ~ .00 .94 Total L—reduced 1.11 .30 LTPN .10 _ .75 Total oxid./reduced ' 1.62 .21 LTPN ' 1.23 ~ .28 Total Muscle wt. ‘49.35 <.0005. ‘ .1 MDPN/MDPNH 1.88‘ .18 DPN in Muscle .66 .42 f MTPN/MTPNH .59 - .45 DPNH in Muscle 8.19 .007 3 Total MDPN .37 .55 TPN in Muscle .11 .75 . Total MTPN . 1.15 .29 TPNH in Muscle 2.04 - .16 ' 7 Total MDPN/MTPN 2.01 .16 Total DPN in Muscle 4.08 _ .05 { Total M pyr. nuc. .68 ' .42 Total TPN in Muscle 1.95 .17 - A Total M-oxidized . .25 .62 - Total pyr. nuc.-muscle 4.85 .03 4 Total M-reduced 4.54 . .04 Total oxid. in muscle .64 > .43 . gi,s Total oxid./reduced 4.53 .04 Total reduc in Muscle 6.13 .02 HDPN/HDPNH ' .25 .62 HTPN/HTPNH 2.57 .12 Total HDPN . .03 .86 Total HTPN ‘ 2.98 .09 Total HDPN/HTPN , 3.61 .07 Total N pyr. nuc. .10 . . .76 Total H-oxidized . .23 .63 Body Weight week 1 ' 12.69 ' <.001 week 5 37.34 <.0005 Week 2 20.45 <.0005 Week 6 49.35 <.0005 Week 3 , 61.20 ‘ <.0005 Final 52.51 <.0005 Week 4 47.16 .<.0005 Blood Lactate Pre—lactate ' 0.00 .97 ‘ A-lactate 5.34 .03 -Postglactate 5.44 . .03 I Work C Static .78 .38 One/body wt. ’1.23 .27 One 14.84 <.0005 Two/body wt. 3.98 .05 Two 2.63 ' .11 Three/body wt. 4.41 . .04 Three .36 .55 ‘ Four/body wt. 7.60 . .009 Four .14 ' .71 Five/body wt. 6.14 , .02 Five .21 .65 Six/body wt. 5.10 .03 Six .14 .71 Seven/body wt. 8.18 .007 Seven .76 .39' . Eight/body wt. 6.32 .02 Eight ‘ .57 .45. Nine/body wt. 6.77 .01 Nine . .81 .37 Ten/body wt. 5.76 - .02 Ten .64 .43 3 First three min. 5.27 .028 rLast seven min. .45 h .51 Total work ‘ .ll - .73 Static/body wt. 12.66 .001 622 APPENDIX D.--Pyridlne Nucleotide Concentrations for Each Animal. Kilogram of Tissue. Values are in Micro—moles per Animal .Final Muscle Heart Liver Number Body _ , “eight DPN DPNH TPN TPNH DPN DPNH TPN TPNH DPN DPNH TPN TPNH Sedentary Group 2 322 807.4 142.9 5.5 72.0 1031.4 79.6 28.8 40.7 778.4 63.4 36.3 174.5 6 353 622.2 79.3 ' 5.4 18.2 633.3 97.3 .16.1 5.6 560.9 72.0 78.3 1179.0 11 '325 1156.0 454.9 5.0 274.6 684.0 1488.8 23.1 14.9 50.1 124.4 34.3 128.8 14 329 1325.9 566.6 3.6 49.6 1707.1 217.7 19.2 12.4 88.8 104.0 8.1 887.9 15 328 706.0 94.3 11.5 12.2 265.7 249.4 11.1 5.0 427.3 134.8 18.6. 278.7 18 349 588.3 881.6 6.9 1489.4 742.6 479.1 23.7 47.9 431.3 96.8 192.5 915.6 19 288 1242.5 40.6 11.8 87.2 1717.2 446.5 56.0 95.8 15.9 77.2 78.5 1145.9 22 342 872.3 60.1 5.4 24.7 863.5 .216.9 14.8 15.1 283.0 766.6 22.8 285.4 23 333 1147.2 261.4 2.3 16.9 941.1 346.2 20.9 95.8 372.2 180.4 55.1 134.3 26 373 474.4 179.6 . .9 38.6 611.9 102.4 ‘2.7 5.8 481.7 42.2 17.3 769.2 27 304 . 1452.4 164.0 11.3 138.3 586.8 349.8 18.6 5.2 312.7: 237.2 68.2 272.8 30 353 525.9 172.3 6.5 18.8 966.1 403.3 25.2 16.6 527.7 144.0 .8 , 85.3 31 338 824.2 335.2 1.7 212.9 '385.8 488.6 5.5 8.5 221.9 70.4 67.2 665.2 35 340 1215.9 189.6 5.5 30.9 1062.7 332.5 25.2 59.7 716.4 43.2 .8 551.1 38 321 815.9. 42.7 6.2 81.6 1206.8’ 613.9 37.9 65.8 722.4 76.0 .7 365.7 39 295 3 1022.5 449.2 11.3 302.5 2262.3 230.2 51.5 4.6 421.9 266.4 '48.2 139.6 42 294 1183.8 232.7 16.4 29.2 815.9 29.5 19.4 5.9 358.9 340.0 29.2 1353.5 47 323 1491.4' 383.1 22.6 23.3 612.7 851.7 16.0 40.4 950.8 67.0 4.5 486.4 Trained Group 1 244 1152.5 186.7 29.8 18.4 846.2 308.8 44.1 66.3 502.6 52.2 49.0 281.7 48 257 1554.4 140.7 4.6 50.7 855.4 332.7 9.4 6.6' 766.1 141.4 59.7_ 471.8 5 284 1246.3 85.4 4.6 17.8 1282.6 292.4 13.8 46.5 566.0 63.6 38.0 580.6 8 245 1143.8 122.2 .8 45.5 1440.3 274.2 19.2 39.8 554.6 292.8 76.3 1318.3 9 296 757.5 74.1 .8 11.0 1252.3 483.1 19.1 16.9 354.6 319.2 5.8 159.5 12 264 1285.5 52.4 .9 5.2 543.4 110.9 24.6 11.0 1591.6 48.4 32.6 36.4 13 309 887.7 86.4 42.9 92.8 1164.6 569.6 22.9 23.4 19.7 82.4 28.7 144.9 16 304 851.0 120.8 18.8 '98.4 1180.4 311.5 33.8 55.9 469.4 147.4 20.5 63.3 17 248 1791.7 97.3 6.4 .39.8 1851.2 375.3 6.0 84.0 759.4 144.4 .9 144.2 20 304 _1064.3, 139.4 2.4 80.0 969.4 375.3 33.5 59.8 1082.0 98.0 12.5 327.0 24 304 1327.4 88.0 .8 54.3 1299.6 56.1 58.2 54.1 315.1 394.2 51.1 146.4 25 296 958.3 83.4 34.0 8.3 950.0 62.2 16.4 32.0 2303.2 121.4 82.9 484.3 29 229 151.0 25.8 .6 144.7 941.1 343.8 23.1 87.5 296.1 45.0 34.7 501.1 32 234 405.4 174.6 .9 5.8 838.9 '357.7 26.3 124.3. 827.4 350.9 58.8 1235.5 36 258 720.5 61.0 .9 176.9 811.8 778.4 69.0 70.0 364.4 94.8 31.5 510.8 37 229 1074.8 219.9 20.1 93.6 588.4 570.5 28.6 10.0 252.9 269.4 61.1 158.2 44 270 1095.1 390.2 12.0 249.4 752.7 487.9 19.5 52.8 147.6 110.0 18.3 591.9 45 245 1024.4 95.8 1.8 11.3 707.7 517.6 20.5 33.4 286.8 68.8 33.6 71.5 . FT:- 0. Inf-31.1.1; 9- _ I . 6:3 APPENDIX E.--Test-retest and Recovery of Pyridine Nucleotides. Values are in Micro—moles per Animal .First . Second . With 213.4 First - Second With 213.4 Number Extraction Extraction- . M added Extraction Extraction M added Muscle DPN Muscle TPN 2 - 807.4 ' 811.0 1001.4 5.6 4.6 .,209.4 18 ' 588.3 , 594.2' 804.5 '7.0 7.3 216.3 22 872.3 '843.1 1110.4 5.5 5.7 ,205.8 35 1215.9 1166.5 1421.4 - 5.5 5.9 208.1 8 1143.8- 1193.7 1328.8 .9 1.8 211.1 36 720.5 643.5 903.1 .9 1.3 203.4 27 1452.4 1328.4 1593.6 11.3' 9.8 213.7 '47 1491.4 1582.4 1683.8 27.7 28.5 228.6 29 151.0 143.2 357.4 .6 1.1 207.8 . Heart DPN - . , Heart TPN .6 633.3 566.2 16.2. 15.7 . 19 1717.2 1921.1 ‘ 56.1 266.7 38 ‘ 1206.8 1299.7 38.0 37.0 8 . 1440.3 1643.8 19.2 225.1 20 969.4 1029.2 ' 33.5 30.1 ‘- 37 . 588.4 801.1 ’. 28.6 221.8 26 ' 611.9 628.1 2.7 3.4 35 1062.7 1261.4 25.2 227.7 16 1180.4 ~1091.3 ' ' 33.8 34.4 Liver DPN Liver TPN 15 427.3 384.2 627.1‘ 18.6 16.9 230.1 22 283.0 270.9 481.4 22.9 18.7 209.8 31 . 221.9 262.4 , 426.1 67.2 73.9 , 268.4 48 . 766.1 759.9 968.4 54.8 61.4 261.6 24 ' 315.1 347.3 530.8 51.2 56.8 247.3 9 354.6 410.6 548.7 5.8 6.3 208.6 25 2303.2 '2200.1 2465.0 ‘ 82 9 89.3 300.1 26 . 481.7 434.9 701.8 17.3 19.7 221.8 9 354.6 370.5 557.6 5.8 4.9 215.3 611 Kilogram of Tissue. Animal First Second With 213.4 ' First Second With 213.4 Number Extraction Extraction u M added Extraction . Extraction p M added Muscle DPNH ' 4 Muscle TPNH 14 566.6 489.5 770.1 49.6 - 44.9 238.1 30 172.3 -200.9 371.5 18.8 17.4 - 201.9 42 232.7 223.6 431.8 ' 29.2 28.1 225.3 13 86.4 80.3 296.5 92.8 93.7 287.1 29 25.8 33.3 245.6 144.7 _ 154.2 331.6 44 390.2 351.2 607.4 249.4 193.0 447.8 6 79.3 84.1 283.4 18.2 19.8 207.1 24 88.0 93.1 301.4 54.3 51.1 251.4 9 74.1 69.4 272 4 11.0 12.9 198.8 Heart DPNH » ‘ Heart TPNH 14 217.7 192.0 ’ 12.4 13.3 27 349.8 547.8 5.2 206.8 12 110.9 115.6 ‘ 11.0 10.3 24 56.1 261.1 54.1 241.1 25 . 62.2 67.6 32.0 ' 37.1 45 517.6 ' , 714.9 ' 33.4 228.8 31. - 488.6 436.4 . 8.5 7.8 9 483.1 696.4 16.9 210.4 29 343.8 380.4 87.5 82.1 ' Liver DPNH Liver TPNH 22 766.6 846.2 908.8 285.4 254.8 472.4 23 180.4 171.4 376.7 134.3 127.6 341.7 42 340.0 297.9 523.8 1353.5 1261.9 1384.9 12 48.4 42.8 260.8 36.4 32.1 234.8 29 45.0 53.1 240.3 501 1 540.2 700.4 36 94.8 108.2 297.5 510 8 544.7 691.6 18 96.8 89.4 301.4 . 915 6 839.5 1170.2 27 237.2 208.7 426.1 272 8 294.5 450.3 44 110.0 117.5 313.9 591.9 650.1 774.3 nd davs L \ A ram of g -moles per Kilo 'n Hicro l are Values -25°C. OZGH at Fr 1, umber APPENDIX F.-Extractions After Davs Animal I”. I ‘I 65 r—i [\- r-«l O"\ O‘\ H :J’ r-4 (3\ "J\ 4‘0 ’30 C) on LI'\ Fr- . I I C O I O 0 O I O I O O O O O 0 k3 r-1 3' ’1') (‘3 TI‘ SI .fl ON CI) (\J (10 O Lf\ Ln ('1) 53' r-fi m m (\l (“J 1’ [\ :I H r—1 :1’ O\ \O r-i [\ CO (\1 (‘1 «--1 ( \J H 6'!) :5— .-—i O‘\ (3‘. 1f 1') If r". O\ ON (0 O (I) (\J KO C) 43’ O O O O C O O U 0 O O I O O O O O O C“ 7—4 :3 CO C) CD \~.) hi) 0\ (X) (\l (D C) Lfl [\ :3 O O\ (\l ('1 (\J (\I L! 5- (VW :T H H :3” O\ \O m [\ I‘- 1’ \J m H (\J . I." E {.11 B (I t~4 r4 CF\ (T\ (T) c3 .:r r4 ' (3\ :7 (*3 (0 r4 cu \1) (D .:r 0 I O C O 0 0 O 0 O O O O 0 O O O O (h r-i :1 n) C) «'3 (13 1 Q 0\ (I) :I no r“. L'\ t\ (I) C) O\ C J (‘7’) (“nl CJ t1’\ t»- ("‘1 :T r-i r-i >2? 0\ \D C) [‘~ f ~ (\1 1") H (\1 r! H . ON (\1 O C) (\J O :‘I :1” m [\- O m \0 O 0’) I a ‘0 I o o o o o o o I o a o o I C) 7-4 ~— 0 L) (‘J T) t» . C) 70 O CO C 1“» \D Lfl 0') ('0 r") (“W (“‘1 ("-1 L? k [‘r- m Lfl r—1 H :r C\ \O 0\ {“~ an) (\l 1") r-i < \J ,_4 .. \ (\1 ("1 Cu C) 1M (‘1 ( \J 4: .-—1 (\1 :3 r: ( \J (n ’r) .7 (11 O O O O I O O O I O O 0 O O O O I LN :7 10 r! C) K-) I\~ "'1 :1 ) N Lf\ m I‘ -— ‘D '70 m; m (\1 L4 \ Pd 2‘ ~ ("d {(1 —: Li ‘. ..) 3\ CT) (“'3 (‘0 [‘~ J C) Ch \I) Lfl r-1 ’ ” 4.1 ’ I) O‘. ’1’) UN :\~ :7 H m 0"» "3 C‘ 7—4 r—-‘l CD «1’ O") :J -I LN ‘4') (\1‘ «LT ST (\1 CO ‘0 r-i CD CD if (\J I O O 0 O O I O I I O O I O I C O 0 ix 1 0. LL“. 7~1 r—i IT . . r “3 .3 m Ln .1r '* C) CC) r \ j W') \1 1'3 .-—4 z 1“") 11' ‘. OW . £'\ ".0 1',“ \ 2 1:; (““1 4f) [\~ :1 C) ON KO L1”\ Pl 5)") Q ~. ’.' D C\ I) J“. [‘- - :T H WW “’3 (”I TV 7'" r-4 1272 {L1 Q .1 \ v—4 3' IT L) f _, (M -3 r—4 (*1 (\i :T ‘~.O r--4 0.: :I' E~ (\J O U 0 O I I O O l O O O I O O ' I O ( ‘J ’3 O ‘\ r-i m \f') 1 J ’3) O C) m (V ’7\ O 5» C) 31’ (\J .1 ((4 KL) (‘0 ‘1') If 7* (‘7— ‘J O\ m {\- 4T {—1 CD LIN r"'{ 0 T3'\ (3 C“ =7“) LIN E\ 113 H m (V) (Y) N r—i r”! KO :1" 0'3 C) H (7') (\1 (Y1 [\ H H r-i (I) N L{\ (\l O O O O O O I 0 O O O O I O O O O O [\- \O (Y) O H F J (‘J m (‘J \D H m r-i N 00 N N (\l :r r~1 LG 1..’\ 3 \ 1_, O\ O CO :1” 01') LO O 00 \O U“ r4 (0 O\ O O\ (I) Lfl t\- LO H m m m (\l r-l H "? S . I :2 2 pl d »J 5? E E I »J A p3 r-i 7—1 L;\ (\J m H CO OJ H H H Ln (\1 m H (\J H m (\J (‘4 m 663 APPENDIX G.--AsSay After Sample was Stored at 2°C. Values are in Micro-moles per Kilogram ' Hours sample remained at 2°C. 338:2; 0 12 24 ' ' 36 - ' o ‘ 12 24 36 Muscle DPN _- ‘ . . Muscle DPNH 31 865.3 821.2 838.4 ' 824.2 _ 351.3 2 334.2 338.8 335.2 45 1077.8 1017.3- 1024.4 1011.8 ' V 99.8 94.8 95.8 , -93.7 .39 1072.1. 1025.7 1036.4 1022.5 470.1 449.2 451.8 450.6 22 913.8 870.6 ~ 881.5 872.3 ' ' 63.3. _ 60.1 60.8 . 60.0 12 1351.9 1292.1 1299.8 1285.5 5.5 '> 5.2 5.3 , 5.2 6 651.4 622.2 618.7 635.5 _ . 83.1 _ 80.1 79.0 _ 79.3 29 158.2 151.0 '145.0 149.1 27.1 25.8 26.0 25.5 1 1207.1 1139.4 1159.9 _1152.5. 191.4 185.5 186.7 186.6 24 1388.4 1312.4 1328.8 1327.4 92.1 88.0 89.2 88.3 Heart DPN - ' Heart DPNH 12 570.8 543.4' 547.7 541.4- 116.8 110.9 111.4 111.2 5 1349.5 ' 1380.5 1397.4 1282.6 307.5 292.4 295.5 292.8 47 643.4 612.7 619.3 615.6 '901.8 860.2 870.1 851.7 22 907.8 862.1 871.8 863.5 227.2 216.9 221.3 216.9 37 - 616.4 584.4 588.4 588.4 600.0 - 570.5 570.5 570.5 25 ‘ 1007.4 958.4 950.0 950.0 65.8 62.2 63.0 62.8 11. 718.4 690.4 695.0 684.0 1561.1 1480.0 1488.8 1476.4 '9 1320.1 1252.3 1277.4 1252.3 510.1 475.5 483.1 480.4 45 742.0 707.7 717.7 704.8 543.1 514.1 517.6 517.6 [Liver DPN Liver DPNH 1 528.3 502.6 511.3 504.6 53.0 53.0 52.2 52.2 16 ' 486.1 469.4 469.4 469.4 153.2 147.4 149.0 147.4 32 865.1 827.4 845.1 827.4 367.2 358.8 358.8 350.4 24 330.8 315.1 324.4 315.1 413.2 392.1 399.8 394.2 47 2 988.4 950.8 950.8 958.8 69.8 66.1 67.0 66.8 18 460.1 431.3 439.9 431.3 99.1 95.4 96.8 94.“ 17 794.1 761.1 771.8 759.4. 150.1 143.8 144.4 143.8 29 308.8 293.8 298.8 . 296.1 ° 47.0 45.0 46.0 45.0 20 1131.6 1082.0 1082.0 1082.0 "103.1 98.0 98.0 98.0 637 of Tissue. 36 0 12 . 24 36 0 12 24 Muscle TPN ' Muscle TPNH 1.9 1.8 1.7 1.7 225.3' 213.6 212.9 211.5 1.9 1.8 1 8 1.8 11.7 11.3 11.5 11.4 11.9 11.3 11 4 11.3 322.2 309.8 307.5 306.5 5 8 5.5 5 6 5.5 25.6 24 4 24.8 24.7 .9 1.0 .9 5.4 5.2 5.3 '5.2 5 4 5.4 5.4 18.8 18.2 18.3 18.2 .6 .6 .5 .7 151.1 144.7 144.7 143.3 31.3 29.8 30.0 79.8 19.3 18.4 18.4 18.4 .8 8 .8 .8 56.7 54.1 54.3 54.3 Heart TPN . Heart TPNH 25.9 24.5 24.7 24.4 11.5 11.1 11.1 11.0 14.4 13 8 13.9 13.9 48.7 46.5 46.5 46.5 16.8 16 1 16.1 16.1 42.5 40.8 40.5 40.4 15 5 14.8 14.8 14.8 15.8 15.1 15.3 15.1 29 6 28.6 28.6 28.6 10.5 10.0 10.0 10.0 17.5 10.8 16.4 16.8 33.6 32.0 33.0 32.4 24.3 33.3 23.8 2 .1 15.6 14.9 14.9 14.9 20.1 19.6 19.8 19.2 17.7 16.9 17.2 16.9 21.5 20.8 21.2 20.5 35.1 33.4 33.4 33.4 Liver TPN Liver TPNH 51.6 49 1 49.9 49.1 292.3 281.7 287.1 281.7 2 .3 20.9 20.5 20.5 65.3 64.0 63.3 63.3 64.0 57 7 - 58.9 58.9 1291.7 1248.8 1264.8 1235.5 53.3 50 8 51.2 51.9 153.6 146.8 150.4 146.4 4.8 4 6 4.6 4.6 502.1 487.7 493.3 486.4 201.8 192 5 196.4 193.7 963.3 915.6 925.8 918.8 0.9 0 9 0.9 0.9 148.8 . 142.3 144.2 141.7 37.1 34 7 35.4 35.0 526.6 506.6 506.6 501.1 13.1 12.6 12.6 12.6 342.2 327.0 330.2 327.0 APPENDIX E 8 H m 3 >9 2 Q a. O Q m 1‘. 1.00 0.05 1.00 -0017 °.lu 1 0.28 0.01 0. -0.02 ”0.24 0. 0.27 0.32 -0. -o 19 -Q.26 0. 0.15 -0.24 -0. -O.23 -0.39 0. 0 11 0.15 -0. 0 01 .0.03 0. -0.30 -o.06 0. -0 38 —0.29 0. -0.10 0.25 0. 0 64 0.03 0. 0.57 -0 16 -0. 0.37 -0 24 -0. o 27 -0.17 0. 0.27 —0.04 0. 0 09 0.04 0. -0.03 0.00 0. 0.02 0.03 0. 0.12 -0.06 -0. 0.20 -0.12 -0. -0.49 0.20 .0. 0.01 0:02 0. 0.20 0.55 -O. 0.01 0.97 0. 0.02 -0.23 0. 0.16 0.58 -0. -0 08 0.16 -0. 0.02 0.94 0. 0.41 0.23 -0. 0.42 0.28 -0. -0.05 0.36 -0. 0.14 0.11 —0. 0.05 0.16 -0. 0.03 0.23 -0. 0.12 0.27 -0. -0.11 -0.03 -0. -0.05 -0.05 0. 0.34 -0.08 0. -0.15 ~0.10 0. 0.60 -0.12 —0. 0.15 -0.05 0. .1 2 H. MDPNH 3: z 2 2 m m m E-< [-4 D 2: E J— 1,00 0.02 1.00 -o.05 -0.18 1 00 0.01 0.50 -0 17 1. —0.05 0.28 -0 22 0. -0.23 0.16 0 2 0. 0.08 -0.45 -0 11 —0 -0.25 -0 20 0 26 -0. 0.20 -0.28 -0 24 —0. -0.24 0.03 0 07 —0. 0.03 0.05 '0 21 -0. 0.14 -0.08 0 28 -0. 0.30 -0.13 -o 01 —0. 0.26 -0.08 -0.04 0. 0.14 0.02 -0.12 0. 0.16 0.05 -0.13 0. 0.10 .0.02 -0.17 o. 0.01 0.04 '0.20 0. 0.14 0.00 -0.20 0. 0.08 0.01 -0.25 0- 0.17 -0.05 —0.? 0- -0.08 0.04 0.09 -0. —0 03 -0 10 0.15 -0- -0.20 -0.40 0.24 -0. 0.04 -0.13 0.25 -0- 0.21 0.98 -0.18 0. -0.14 —0.61 0.11 -0. -0 33 -0.55 -0 26 -0. 0.08 o, 2 0.2 —0. 0.03 -0.24 0.29 -0. 0.06 -o.33 0.32 —0. 0.03 -0.18 0.10 0 -0.06 0.11 0.84 0 0.00 -o.35 -0.32 -0 ~0.11 -o 31 -0 32 —0 -0.14 -o.32 -0 18 -o -0.09 -0.38 -0 01 —0 0.13 0.04 —0 25 0 0.18 -o 03 -0 09 O 0.10-—0 03 —0 26 0 0.25 -0.11 0.10 -0. 0.12 0 01 —0 2 o 4 5 6 68 -—Correlation Coefficients for HLPKH —O. —0. -0. —0. -O. —O. -O. -0. -O. -0. OOOOOOOOOOOOODOOOH OOOOO ETEN HTPHH LDPN OC‘OODOK’JOOOOOOOOH .3C)OC)*-‘ Z‘LHD ’73C E“J\.""\}‘ "J ‘43 H90C2Hrur -O -0. ~O. -O. -O. -O. —C. -O. -O. -O. -O. -O. —O. -O. -O. —O. -O. -0. -O. -O. -0. -o_ -0. -0.. -O. LTFN .00 .‘30 .27 .34 LTPSE -O. -O. —O. -O. «1 -O. -O. -0. -O. -O. -0. -0. -O. -O. -O. -0. -O. -O. -O. -O. -0. -0. -0. -0. -O.' -O. STATIC -O. -O. -O. -O. -O. -0. I OOOOOOOOOOOOOOOOOOOO OOH the Trained Group. OOOOOOOOOOOOOOOOOOOOOOOOOCOOOOH ONE THO I 8 l I I I OOOOOOOOOOOOOOOH -O. THREE 1.00 -0.25 —o.31 -0.24 -0.02 -0.34 -0.28 -o.24 0.08 0.01 -o.05 0.06 -0.27 —o.24 -0.14 _o.19 0.74 0.92 0.77 0.87 0.84 FOUR FIVE 1.00 SIX 1.00 0.95 0.94 0.90 -0.02 0.40 -O.31 0.10 -0.00 -0.26 -0.18 0.10 -0.08 -0.14 —0.08 -0.04 -O.lT -0.13 -0.03 -0.21 0.94 0.93 0.93 0.68 0.97 SEVEN EIGHT NINE 1.00 0.93 -0.07 0.27 -0.17 -0.06 0.03 -0.22 -0.17 -0.05 0.01 -0.05 -0.20 -0.12 —0.06 -0.03 0.04 —0.27 0.88 0.91 0.64 0.97 23 TEN 1.00 -0.20 0.18 -0.15 -0.14 —0.02 -0.18 —O.10 -0.14 -0.09 -0.13 -0.08 -0.13 -0.04 0.03 0.13 -O.28 0.77 0.88 0.82 0.66 0.91 24 STATIC/BODY WEIGHT 1.00 0.57 -0.07 0.24 0.03 -0.02 -0.07 0.25 0.07 0.12 0.19 —0.07 —0.00 -0.05 0.34 -0.03 -O.l3 0.10 -0.11 -0.12 25 HEIGHT ONE/BODY MDPN/MDPNH 1.00 0.41 —0.43 0.91 0.60 0.33 0.34 0.42 0.12 0.00 0.59 0.68 0.70 0.05 -0.35 -0.23 -0.35‘ -0.11 -0.27 27 TOTAL HDPN 1.00 -0.12 0.46 0.09 0.98 0.17 0.20 0_34 0.0 0.09 0.16 0.19 -0.03 0.00 -0.06 -0.06 -0.13 -0.02 28 TOTAL MTPN 1.00 -0.02 -0.60 0.04 -0.24 -0.30 —O.l7 0.10 -O.32 -0.32 -0.34 -0.38 0.07 0.00 -0.01 -0.05 0.03 29 H-OXIb/EEDUCED 0.16 - .20 0.71 0.77 0.77 0.20 -O.32 -0.23 -0.32 -O.10 -0.28 30 TCTAL EDEN/HTPN 1.00 -0.00 ‘ -0.0l -0.01 -0.18 -0.15 -0.10 -0.20 31 NUC. 70141 M-FYR. 1.00 0.13 0.15 0.31 0.09 0.04 0.10 -0.10 0.02 —0.07 -0.06 -0.14 -0.01 32 hLPN/ECPHE 1.00 0.90 -0.17 -0.09 0.14 0.00 0.11 0.30 -0.16 0.03 -0.17 0.20 -0.06 33 {EEDUCLL IL L—LI 1.00 -0.13 -0.10 0.26 0.16 0.20 0.40 -0.24 -0.02 -0.23 0.17 -0.13 34 69 L LLPh/ETFH ~12 («I 1.00 0.07 —O.10 -D.lJ -O.C0 -0.05 - .23 -0.11 -0.09 —0.31 -O.10 35 TOTAL L—PYE. -0, -0. _o_ -O. -0, -0. -0: LLPNE LLP3~ ‘CED _U “LL-.2 LLPN/LTEN ' l L-CXIL. TOTAL NU TOTAL L-FYE. FIVE/ECLI NT. L KORE 10TA HH./ED. WT. TOTRL FIRST TEREE WORK 1.00 0.60 0.92 43 LAST SEVEN WORK 1.00 0.70 44 1. 00 45 '7() Correlation Coefficients for the Sedentary Group E" 3: o H . g c) >‘ z 2 2 z 2 z z 2 2 Z Z Z 3* LIJ m Id § 8 E Q .93 E E: 2 53 S 3 3 S S S 5 6:3 :65 8 E 8 31' 1.00 L -o.66 1.00 ' 0.08 0.08 1.00 -o_52 3.50 0.02 1.00 _o_09 -o,1o 0.75 -0.09 1.00 -o.47 0.25 0.12 -0.00 0.14 1.00 . -0.14 0.32 0.29 0.22 0.34 -0.16 1.00 -0.64 0,22 -0.03 0.17 0.24 0.81 0.07 1.00 . —o.17 0,22 -0.08 -o.00 -0.05 0.26 0.12 0.49 1.00 0,23 -o,15 -0.16 0.33 -0.25 -0.18 -0.16 -0.08 0.10 1.00 -0.14 0.07 -0.16 0.05 -0.08 0.05 -0.15 -0.05 -0.24 -o.25 1.00 0,00 -0.19 0.51 -o.10 0.72 -0.07 -0.02 0.11 0.14 -0.25 -c.07 1.00 -o,05 0,01 0.09 0.11 -0.02 0.00 -0.23 -0.04 -0.00 —0.19 -0.15 0.33 1.00 —o.11 0,15 -o.o3 0.02 -0.10 -0.12. 0.09 -0.26 -0.43 -o.13 0.21 -0.34 —0.42 1.00 0,37 -0.19 -0.00 —o.42 0.03 -0.32 -0.11 -0.48 -o.16 -o.17 -0.05 0.04 -0.17 0.06 1.00 - 0,31 -0.06 -o.11 -0.26 0.04 -o.47 0.20 -o.49 -0.22 0.17 0.02 -0.14 -o.40 0.11 0.62 1.00 0.43 -o.07 0.14 -0.29 0.19 -0.35 0.15 -0.46 -0.10 0.17 0.09 0.00 -C.14 -o.39 0.46 0.85 1.00 0.54 -o.45 0.19 -0.36 0.25 -0.35 0.11 -0.43 -0.13 0.15 0.04 0.13 0.05 -o.16 0.42 0.60 0.79 1.00 0.55 -o.52 0.24 -0.35 0.22 -0.29 -0.14 -0.40 -0.11 0 16 0.02 0..h 1.00 -0.1' 0.38 0.44 0.63 0.95 1 00 0.3; -o,35 0.19 -o.25 0.27 -0.21 0.27 -0.20 -0.06 0.15 -0.04 0.34 -..13 -1.16 0.42 0.48 0.60 0.‘4 0'88 1 00 0,37 -o.37 0.31 -0.26 0.36 -o.21 0.24 -0.27 -0.11 0.10 -0 00 0.17 -0.03 -0.18 0.44 0.48 0.64 0.90 0:92 0:96 0,25 -o,27 0.25 -O.21 0.36 -0.12 0.30 -0.12 0.05 0.11 -0.01 0.18 —0.03 —0.:3 0.40 0.40 0.63 0.87 0.87 0.94 1'00 o 14 -0.16 0.21 -0.16 0.37 -0.06 0.36 -0.01 0.13 0.13 -o.09 0.18 :.00 -0.23 0.33 0.44 0 57 0 79 0 78 0 86 0'97 0.02 -0.07 0.04 -0.14 0.21 0.00 0.34 0.09 0.24 0.23 -0.19 0.03 -0.05 —0.13 0.2 0.36 0:43 0:63 0:64 0:77 0'90 .0.40 0,32 -o.03 0.18 -0.05 0.02 0.12 -0.05 -o.37 -0.18 0.23 -0.31 -0.37 0.95 -0.04 -0.00 -o.22 —o.31 -0.26 -0 24 0'76 —o.34 0.38 -0.07.-0.05 0.10 0.00 .0.01 -0.02 -0.04 _0.34 0.04 0.05 -0.13 0.12 0.74 0.36 0.15 0.01 -0.04 0'19 '0'28 -0.41 0.15 -0.60 0.10 -0.26 0.26 -0.03 0.53 0.51 -0.17 0.13 -0.0 0.19 —o.27 -0.12 -0.22 -0.24 —0.23 -0.27 -0213 0'15 09.48 0.84 0.61 0.41 0.32 0.27 0.42 0.16 0.13 -0.21 .0.03 0.12 0.05 0.1 -0.15 -0.11 0.01 -0.26 -o.28 -0.18 ‘0'20 -0.11 -o.08 0.75 -o.04 0.99 0.14 0.35 0.25 -0.05 -0.24 -0.08 0.72 -0.02 -0.10 0.01 0.03 0.17 0.2 0.20 0 26 “0'12 -o.22 0,11 -o.75 0.14 -0.55 0.01 -0.22 0.20 0.30 -0.02 0.42 -0.21 0.12 -0.12 -0.22 -o.15 -0.16 -o.24 -0 30 -0'26 0'35 0,15 0,23 —0.20 0.04 -0.65 -0.10 -0.21 -0.26 0.30 0.15 0.1 -0.33 -0.04 -0.05 -0.15 -0.12 -0.06 -0.17 -0:14 -o°27 ‘0'32 -o.45 0.70 0.74 0.34 0.56 0.27 0.46 0.21 0.09 -0.25 -0.05 0.31 0.04 0.05 -0.13 -0.09 0.06 -0.16 -0.19 -0:08 '0‘27 —o.06 —o.14 -o.06 -0.22 —0.0! 0.55 -o.58 0.28 -0.10 0.13 -o.00 -o.07 -0.00 0.15 -0.10 -0.34 -0.36 -0.25 -0.19 -o.24 ‘°'°° -o.06 -0.13 -o.01 -0.23 -0.05 0.62 —0.59 0.29 -0.20 0.06 0.04 -0.08 0.05 0.09 -0.11 -0.32 -0.32 -0.21 -o.17 -0.22 '0'20 0,25 —o.13 0.15 -o.25 0.05 -o.13 0.03 -0.49 -0.72 -0.38 0.03 -0.20 0.12 0.31 0.50 0.33 0.24 0.29 0.28 0.17 '0'16 -0.52 0.43 0.27 0.12 0.32 0.81 0.43 0.80 0.37 -0.24 —0.06 -0.06 —0.13 -0.08 -0.37 -0.31 -o.23 -0.25 -o.19 -0.03 0'25 0,37 -0.11 —o.15 0.11 —0.25 -0.18 -0.09 -0.15 0.18 0.85 -0.48 -0.27 -0.02 -0.17 -0.05 0.24 0.27 0.18 0.14 0.06 ‘0'06 0.07 —o.19 -o.19 0.09 —0.18 0.00 -o.11 0.14 0.19 0.72 —o.25 -0.27 -o.57 0.18 —0.09 -0.01 —0.20 -0.16 —o.04 0.02 0'0“ 0,11 -o.25 -0.22 0.00 —o.19 0.13 -0.10 -.16 -0.02 0.34 0.23 -0.34 -0.69 0.23 -0.11 -0.02 -0.23 -0.19 -0.06 -o.04 ’0'09 0,03 -0.07 -0.02 0.32 -0.13 -o.09 -0.38 —0.10 —0.02 0.27 0.09 0.23 0.80 -0.43 -0.38 —o.28 0.00 0.17 0.12 .0.04 "0'15 0.41 -o.44 0.24 -0.26 0.24 -0.23 0.19 -0.32 —0.09 0.16 0.06 0.04 -0.00 -0.07 0.31 0.40 0.61 0.93 0.98 0.90 0'05 0,41 -o.30 .0.17 -0.32 0.27 -o.29 0.24 -0.34 -0.06 0.15 -0.02 0.08 -0.11 -0.12 0.56 0.69 0.80 0.93 0.89 0.92 0'93 0,13 -o.12 0.15 -0.18 0.31 .0.17 0.31 -o.17 -o.o3 0.11 0.02 0.06 -0.13 -0.09 0.47 0.65 0.74 0.83 0.79 0.91 0'9“ 0.44 -o.12 0.00 -0.36 0.10 -o.44 0.10 -0.54 -0.18 0.08 0.03 -0.04 -0.28 0.03 0.77 0.95 0.89 0.70 0.55 0.57 0'91 0.34 -0.34 0.22 -0.26 0.31 -o.19 0.27 -0.21 -0.00 0.16 —0.04 0.12 -0.03 -0.18 0.40 0.50 0.66 0.91 0.92 0.96 8'33 1 '2 3 4 5 6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 EIGHT NINE 1.00 0.9u -0.26 0.01 -0.01 0.36 -0.21 —0.36 0.09 -0.11 -0.11 0.10 0.16 0.15 -0.04 -0.25 0.07 0.81 0.89 0.92 0.52 0.9“ 23 STATIC/BODY WEIGHT -0.26 0.1“ 0.17 -0.90 -0.19 -O.2U -0.13 -0.10 -0.27 ONE/BODY HEIGHT MDPN/MDPNH TOTAL MDPN -O.17 -0.25 -O.32 -0.07 -0.21 -0.15 -0.01 -0.09 -0.15 28 TOTAL MTPN -0.5“ -0.65 0.58 -0.09 -0.06 0.04 0.33 -0.25 -o.18 -0.19 -O.12 0.22 0.26 0.31 0.30 29 M-OXID/REDUCED TOTAL MDPN/MTPN 1.00 -O.ll -0.09 -0.09 -0.27 -O.2O 0.08 0.07 0.1“ 0.06 -0.16 -0.25 -O.3l -0.12 -0.27 31 NUC. TOTAL M-EYR. 1.00 -0.15 -0.11 -0.00 0.51 -o.22 -0.27 -0.3“ -0.09 -0.12 -0.05 0.07 -0.05 -0.04 32 71 Q ‘61.! U :3 L1; Q L5 L11 .1. L12 Q ‘\ In 63 \ H 2 >< 04 O Q I 2: :1: 1.00 0.97 1.00 0.05 0.16 0.15 0 2 0.15 0 07 0.3“ 0 19 0.16 0.12 0.06 0.09 -0.19 -0.17 -O.2U -0.22 -O.2u —O.23 -0.32 —0.29 -0.18 -0.17 .33 3“ TOTAL HDPN/HTFH TOTAL h-PYR. NUC. -0.21 —0.0“ 0.06 -O.30 -0.lO -O.l3 0.02 -0.3& -0.02 OOOOOOOOH LDPN/LDPNH -O -0. -0. -0. -0. -0. L-OXID/REDUCED .00 .20 07 ll 03 38 03. TOTAL LDPN/LTPN 1.00 _0.u0 ~0.06 NUC. 1. TOTAL L-PYR. 90 .12 -O.l7 -0.01 -O.15 -0.0H -0.1“ -0.21 --.10 39 0- 06 90 WT. FIVE/BODY 1.00 0.99 0.83 0.51 0.9“ 91 ‘2 OT L 1.00 0.95 0.78 0.96 “2 HT. TOTAL WK./BL. 1.00 0.71 0.93 “3 FIRST TEREE WORK 1.00 0.60 ‘44 1. WORK LAST SEVEN 00 “5 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIII IIII IIIIIIII III II III IIIIIIIIIIIIIIIIIIIIII 31