STUDY INVOLVING METABOLISM OF 17-KETOSTEROIDS
AND 17-HYDROXYCORTICOSTEROIDS OF HEALTHY
YOUNG MEN DURING AMBULATION AND RECUMBENCY
A DISSERTATION
SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY IN NUTRITION
IN THE GRADUATE DIVISION OF THE
TEXAS WOI\IIAN 'S UNIVERSITY
COLLEGE OF HOUSEHOLD ARTS AND SCIENCES
BY
SANOBER QURESHI I B .Sc. I M.S.
DENTON I TEXAS
MAY I 1970 ACKNOWLEDGMENTS
The author wishes to express her sincere gratitude to those who assisted her with her research problem and with the preparation of this dissertation.
To Dr. Pauline Beery Mack, Director of the Texas Woman's
University Research Institute, for her invaluable assistance and gui dance during the author's entire graduate program, and for help in the preparation of this dissertation;
To the National Aeronautics and Space Administration for their support of the research project of which the author's study is a part;
To Dr. Elsa A. Dozier for directing the author's s tucly during
1969, and to Dr. Kathryn Montgomery beginning in early 1970, for serving as the immeclia te director of the author while she was working on the completion of the investic;ation and the preparation of this dis- sertation;
To Dr. Jessie Bateman, Dean of the College of Household Arts and Sciences, for her assistance in all aspects of the author's graduate program;
iii To Dr. Ralph Pyke and Mr. Walter Gilchrist 1 for their ass is
tance and generous kindness while the author's research program was
in progress;
To Mr. Eugene Van Hooser 1 for help during various parts of
her research program;
To Dr. Betty Bohon Alford, Dr. Roman J. Kutskyl Dr. James
Hardcastle, and Professor George P. Vose 1 for their invaluable in
struction and inspiration;
To Jessie Ashby 1 for coding and preparing data for statistical ana lysis;
To Reba Fry 1 Martin Cucldl and Lenoir 0 'Rear I for their as- sistance in this investigation;
To Bill Stover I for the drafting of graphs and figures included in the report;
To Dorothy Jones 1 for the typing of this dissertation with pre- cis ion;
To my brother 1 Dr. As mat Ullah Qureshi I and to my sisters I
Dr. Razia Matin and Miss Shehla Qureshi I for their devotion and
iv encouragement, which inspired the author throughout the pursuance of her graduate study;
Finally, the author would like to dedicate this dissertation to her parents, Mr. Hifazat Ullah Qureshi and Mrs. Saida Qureshi, who placed educational achievements high in the values of life, and made many sacrifices in order that the author might engage in graduate study.
v TABLE OF CONTENTS
INTRODUCTION 1
REVIEW 0 F LITERATURE 4
CHEl'v1ISTRY OF STEROID HORMONES 6
Steroisomeris m 10
BIOSYNTHESIS OF STEROIDS 13
Enzyme System 18
THE HYDROXYSTEROID DEHYDROGENASE 19
Hydroxylases and Lyases 19
METABOLISM 21
PHYSIOLOGIC CONSIDERATION OF ANDROGENS 25
Anubolic Effect . 25
Effect on Bone Metabolism 29
Effect on Lipid Me tubolis m 33
GLUCOCORTICOIDS 34
Physiological Considerations 34
Carbohydrate Metabolism 34
Fat Metabolism 37
Protein Metabolism 38
Electrolyte Iviet3.bolis m 39
vi Bone Metabolism . 41
17-KETOSTEROID AND 17 -HYDROXYCORTICOSTEROID
EXCRETION 43
17- Ketos teroids 43
17-Hydroxycorticos teroids 47
Stress and Steroid Hormones 48
EFFECT OF IMMOBILIZATION . 52
EXERCISE AND ALTITUDE EFFECTS . 57
THE CIRCADIAN RHYTHM PHENOMENON 59
PLAN OF PROCEDURE 66
PERIODS 0 F THE STUDY DURING THE 196 8 INVESTIGATION 66
SUBJECTS OF THE STUDY. 67
EQUILIBRATION PERIOD . G7
BED REST PERIOD NUMBER ONE G8
INTERIM AMBULATORY PERIOD 69
BED REST PERIOD NUMBER TWO G9
POST-BED REST PERIOD . 70
D IA GRAM I . EXERCISE PROGRi\M FOLLOWED IN BED REST
II 0 F THIS INVESTIGATION 72
PERIODS OF THE STUDY DURING THE 1969 INVESTIGATION 74
SUBJECTS OF THE STUDY 74
EQUILIBRATION PERIOD . 75
vii GENERAL PROCEDURE USED IN THE STUDY 76
METHODOLOGY 76
PROCEDURE FOR THE DETERMINATION OF URINARY 17 -KETO
STEROIDS 76
REAGENTS 77
PROCEDURE 78
CALCULATION 79
PROCEDURE FOR THE DETERMINATION OF URINARY 17 -HY-
DROXYCORTICOSTEROIDS 80
CALCULATION 82
STANDARDIZATION 8 2
REAGENTS 83
PRESENT AT I 0 N 0 F FINDINGS WITH DISCUSS I 0 N 84
PART I OF THE STUDY . 84
Comparison of Urinary 17-Ketosteroid Excretion
during the Initial Equilibration and the Bed
Rest I Periods 85
SUBJECT BB 85
SUBJECT FF 85
SUBJECT HH 86
SUBJECTS BB, FF, HH (Exercising Regularly) 86
viii Comparison of Urinary 17 -Ketosteroid Excretion
during the Initial Equilibration and the Bed
Rest I Periods 86
SUBJECT AA 86
SUBJECT EE 86
SUBJECT GG 87
SUBJECTS M 1 EE 1 GG (Subjects who Exercised "at
Will") 87
ALL SUBJECTS • 87
Comparison of Ambulatory Periods with Each
Other 88
SUBJECT BB 88
SUBJECT FF 91
SUBJECT HH 91
SUBJECTS BB 1 FF 1 HI-I (Regular Exercisers) 91
SUBJECT AA 92
SUBJECT EE 92
SUBJECT GG 92
SUBJECTS AA 1 EE 1 GG ("at \Nill" Exercisers) 92
ALL SUBJECTS • 93
COMPARISON OF BED REST I WITH BED REST II • 94
ix SUBJECT BB 94
SUBJECT FF 94
SUBJECT HH 94
SUBJECTS BB 1 FF 1 HH (Who Exercised Regularly) • 97
SUBJECT AA 97
SUBJECT EE 97
SUBJECT GG 97
SUBJECTS AA 1 EE 1 GG (Who Exercised "at Will") • 97
ALL SUBJECTS • 98
COMPARISON Or URINARY EXCRETION OF 17 -KETOSTEROIDS
DURING BED REST I AND BED REST II 99
CIRCADIAN PATTERN or URINARY 17-KETOSTEROID EXCRETION
DURING BED REST I 101
SUBJECT BB 101
SUBJECT Fr 102
SUBJECT HH 102
SUBJECT AA 105
SUBJECT EE 105
SUBJECT GG 106
ALL SUBJECTS • 106
COMPARISON OF 17 -KETOSTEROIDS WITH INDEPENDENT
VARIABLES. 109
X CORRElATION BETWEEN 17-KETOSTEROIDS AND CREATI
NINE 109
CORRElATION BETWEEN 17 -KETOSTEROIDS AND HY
DROXYPRO LINE 110
CORRElATION BETWEEN 17-KETOSTEROIDS AND TOTAL
CALCIUM 110
CORRELATION BETWEEN 17-KETOSTEROIDS AND URI
NARY CALCIUM 110
CORRElATION BETWEEN 17-KETOSTEROIDS AND TOTAL
NITROGEN 111
CORRElATION BETWEEN 17-KETOSTEROIDS AND 17-
l-IYDROXYCORTICOSTEROIDS • 111
CORRElATION BETWEEN 17-KETOSTEROIDS AND CREA
TINE 111
COMPARISON OF 17-l-IYDROXYCORTICOSTEROIDS WITH INDE
PENDENT VARIABLES 112
CORRElATION BETWEEN 17-HYDROXYCORTICOSTEROIDS
AND CREATININE • 112
CORRELATION BETWEEN 17-HYDROXYCORTICOSTEROIDS
AND HYDROXYPROLINE • 112
CORRElATION BETWEEN 17-HYDROXYCORTICOSTEROIDS
AND TOTAL CALCIUM 112
xi CORRELATION BETWEEN 17 -HYDROXYCORTICOSTEROIDS
AND URINARY CALCIUM 113
CORRELATION BETWEEN 17 -HYDROXYCORTICOSTEROIDS
AND TOTAL NITROGEN • 113
CORRELATION BETWEEN 17 -HYDROXYCORTICOSTEROIDS
AND CREATINE 113
PART II OF THE STUDY. 114
CIRCADIAN RHYTHM PATTERN OF 17-HYDROXYCORTICOSTER
OIDS. 114
SUBJECT 1A 117
SUBJECT 2A 117
SUBJECT 3A 117
SUBJECT 4A 118
SUBJECT 6A 118
SUBJECT 7A 118
SUBJECT SA 119
SUBJECT 9A 119
ALL SUBJECTS • 119
SUMMARY AND CONCLUSIONS 124
B I B L I 0 G RA P H Y 127
APPENDIX 143
xii LIST OF TABLES
TABLES FOR PART I OF THE STUDY
Including Excretion of 17-Ketosteroids by Six Subjects Participating in Two 28-Day Bed Rest and Preliminary
and Final Periods in 19 6 8 (Subjects AA I BB I EE I FF I GGI and HH) • 144
TABLE I. URINARY EXCRETION OF 17 -KETOSTEROIDS PER DAY
PART A. SUBJECT BB 145 PART B. SUBJECT FF 146 PART C. SUBJECT HH 147 PART D. SUBJECT AA 148 PARTE. SUBJECT EE 149 PART F. SUBJECT GG 150
TABLE II. URINARY 17-KETOSTEROID EXCRETION DURING THREE DAILY PERIODS WHILE BED REST I WAS IN PROGRESS
PART A. SUBJECTS BB AND FF 151 PART B. SUBJECTS HH AND AA 152 PART C. SUBJECTS EE AND GG 153
TABLE III. STATISTICAL COMPARISON OF URINARY 17 -KETO STEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS 0 F THE STUDY
PART A. SUBJECT BB 154 PART B. SUBJECT FF 155 PARTC. SUBJECTHH 156 PART D. ALL SUBJECTS EXERCISING REGUlARLY
(BB 1 FF 1 HH) 157
xiii TABLE III. (CONTINUED) STATISTICAL COMPARISON OF URI NARY 17-KETOSTEROIDS BE1WEEN PAIRS OF THE DIFFERENT PERIODS OF THE STUDY
PART E. SUBJECT AA 158 PART F. SUBJECT EE 159 PART G. SUBJECT GG 160 PART H. SUBJECTS EXERCISING "AT WILL"
(AA I EE I GG) 161 PART I. ALL SUBJECTS 162
TABLE IV. STATISTICAL COMPARISON 0 F URINARY 17-KETO STEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS BY GROUPS OF SUBJECTS EXERCISING
REGULARLY AND "AT WILL II 163
TABLE V. STATISTICAL ANALYSIS OF URINARY 17 -KETOSTER OIDS BE1WEEN GROUPS OF SUBJECTS EXERCISING REGULARLY AND "AT WILL" DURING BED REST ONE AND BED REST 1WO 164
TABLE VI. STATISTICAL COMPARISON OF URINARY 17 -KETO STEROID EXCRETION AT DIFFERENT TIMES OF THE DAY
PART A. SUBJECTS EXERCISING REGULARLY
(BB I FF I AND HH) 165 PART B. SUBJECTS EXERCISING "AT WILL"
(AA I EE I AND GG) 166 PART C. ALL SUBJECTS 167
TABLE VII. CORRELATION COEFFICIENT DERIVED BETWEEN 17-KETOSTEROID EXCRETION AND SEVEN INDE PENDENT VARIABLES (ALL SUBJECTS)
PART A. PRE-BED REST 168 PART B. BED REST ONE • 169
xiv TABLE VIII. CORRELATION COEFFICIENT DERIVED BETWEEN 17 -HYDROXYCORTICOSTEROID EXCRETION AND SEVEN INDEPENDENT VARIABLES (ALL SUBJECTS)
PART A. PRE-BED REST 170 PARTB. BEDRESTONE 171
TABLES FOR PART II OF THE STUDY
Including Excretion of 17-Hydroxycorticosteroids During Pre-Bed Rest Period by Eight Subjects Participating in a 56-
Day Bed Rest Study Conducted in 1969 (Subjects 1A I 2A 1 3AI 4A, 6A, ?A, SA, and 9A) 172
TABLE IX. URINARY 17 -HYDROXYCORTICOSTEROID EXCRETION DURING PRE-BED REST
PART A. SUBJECTS 1A, 2A 1 3A, AND 4A • 173 PART B. SUBJECTS 6A, ?A, SA, AND 9A. 174
TABLE X. URINARY EXCRETION OF 17-HYDROXYCORTICO STEROIDS DURING PRE -BED REST AT DIFFERENT PERIODS 0 F THE DAY
PART A. SUBJECT 1A 175 PART B. SUBJECT 2A 175 PART C. SUBJECT 3A 176 PART D. SUBJECT 4A 176 PARTE. SUBJECT 6A 177 PART F. SUBJECT 7 A 177 PART G. SUBJECT SA 178 PART H. SUBJECT 9A 178
TABLE XI. STATISTICAL COMPARISON OF URINARY 17-HY DROXYCORTICOSTEROID EXCRETION AT DIFFERENT PERIODS 0 F THE DAY DURING PRE- BED REST
PART A. SUBJECTS 1A, 2A, 3A 179 PART B. SUBJECTS 4A, 6A, ?A 1SO PART C. SUBJECTS SA, 9A, ALL SUBJECTS 181
XV LIST OF FIGURES
FIGURE 1. STRUCTURAL CHARACTERISTICS OF THE STEROIDS AS SHOWN BY TURNER (3) 8
FIGURE 2. CONFORMATION OF STEROID NUCLEUS AS SHOWN BY DORFMAN (4) 11
FIGURE 3. CONFIGURATION OF STEROID NUCLEUS AS SHOWN BY WEST AND TODD (6) • 11
FIGURE 4. PATHWAYS OF CORTICOSTEROID BIOSYNTHESIS AS SHOWN BY TURNER (3) 16
FIGURE 5. PATHWAYS OF ANDROGEN BIOSYNTHESIS IN THE ADRENAL CORTEX AS SHOWN BY TURNER (3) • 17
FIGURE 6. SHIFT OF DOUBLE BOND FROM ~5 TO~ 6 POSI- TION AS SHOWN BY EWALD (19) 20
FIGURE 7. METABOLISM OF CORTISONE AND HYDROCORTI- SONE AS SHOWN BY DORFMAN (4) • 2 3
FIGURE 8. METABOLISM OF TESTOSTERONE AND~4-ANDRO- STENE-3, 17-DIONE AS SHOWN BY DORFMAN (4) • 24
FIGURE 9. 17-KETOSTEROID EXCRETION PRODUCTS OF AN- DROGENS AS SHOWN BY TURNER (3) 46
FIGURE 10. MAN EXERCISING WHILE RECUMBENT 73
FIGURE 11. COMPARISON OF MEAN 17-KETOSTEROID EXCRE TION DURING DIFFERENT PERIODS OF THE STUDY WITH THE DATA FOR ALL SUBJECTS POOLED TO- GETHER 89
FIGURE 12. COMPARISON OF MEAN DAILY EXCRETION OF 17- KETOSTEROIDS DURING BED REST I (NO EXERCISE) AND BED REST II (EXERCISE) 95
xvi FIGURE 13. RHYTHMICITY OF 17-KETOSTEROID EXCRETION 103
FIGURE 14. TOTAL 24-HOUR 17-KETOSTEROID EXCRETION (ALL SUBJECTS COMBINED) 107
FIGURE 15. RHYTHMICITY OF URINARY EXCRETION OF 17 -HY DROXYCORTICOS TEROIDS DURING THE PRE -BED REST PERIOD 115
FIGURE 16. URINARY 17 -HYDROXYCORTICOID EXCRETION DUR- ING 24 HOURS MEAN OF ALL SUBJECTS • 121
xvii INTRODUCTION
Numerous physiologic changes similar to those said to be
experienced during the phenomenon of weightlessness have been found
in the two recumbency studies during which the author's investigation
was conducted. These have included to a different extent in different
individuals lack of coordination, loss of appetite, excessive urine
flow, poor muscular coordination, and reduction in bone density. In
two bed rest studies conducted, respectively, in 1968 and 1969, the author participated by running analyses of 17-ketosteroids and 17- hydroxycorticosteroids as part of an overall more extensive investi-
l!iltion reluted to the effects of recumbency on metabolism and retention of bone density on healthy young adult males.
In recent years many investigations have been conducted in order to determine whether or not the adrenal cortex has a cyclic uri nary excretion pattern within a 24 hour period. By rescheduling the activities of normal subjects, it has been possible to change the hours at which adrenal steroid production rises and falls. Pincus (1) was the first investigator to show a circadian periodicity occurring in the urinary excretion of 17-ketosteroids. According to Frazier, Rummel, and Lipscomb (2), confinement stress can lead to alteration in
1 2 circadian rhythmicity; even when the physical environment and activity schedule are held reasonably constant.
The present study was undertaken to observe the effect of ex ercise on both the total 24 hour excretion and the periodicity of adrenal hormone secretions by measuring the urinary levels both of 17-keto- s teroids and 17 -hydroxycorticosteroids of immobilized subjects.
The major objectives of the study have been the following:
1. To analyze the urinary excretion samples for 17-keto-
steroid values of six youn cumbency study; 2. To compare the vulues obtained for the 17-ketosteroid urinary excretion with the values of 17-hydroxycortico steroid urinary excretion samples obtained in this same study; 3. To determine the existence of a circadian rhythm in adrenal hormone urinary excretions; 4. To investigate the effect of programmed exercise on the urinary excretory levels of 17-ketosteroicls; 5. To compare the excretion levels of 17-ketosteroids with the excretion levels of creatine, creatinine, hydroxy proline, calcium (urinary and total) and total nitrogen; and 3 6. To analyze the urinary samples of eight young men engaged in a subsequent study for the 17-hydroxycorticosteroid con tent during an ambulatory phase of the study. REVIEW OF LITERATURE In 1563, Eustachius first gave a clear anatomic description of the adrenal glands, which are among the most highly vascularized organs of the body. These glands, which produce a number of hor mones, are composed of two distinct regions, including an outer cortex and an inner medulla. The cortex secretes hormones which are essential for life and are referred to as corticosteroids. The adrena ls play an important role in the adaptation of the human body to conditions of stress , although the exact mechanism of ,,'1drena l hormone participation in these complex events is not fully de termined. Adrenalectomized animals are known to be extremely sensi tive to trauma, and they hemorrhage and experience other characteristic behavior patterns, such as weakness, easy fatigability, hypertension and a variety of gastrointestinal disturbances. When the adrenal cortex is stimulated by adrenocorticotrophin hormone (ACTH) it releases various steroid hormones which produce numerous physiological conditions. The adrena lcorticoids are ca te gorized as: (a) glucocorticoids, which are associated with organic metabolism, (b) mineralcorticoids, which are related to water and electrolyte metabolism, and (c) sex hormones (androgens and estro gens), the exact function of which is still not fully determined. 4 5 The end products of many of the androgens 1 such as testosterone and dehydroepiandrosterone are 17-ketosteroids, which are eliminated in urine as conjugates of sulfuric acid and/or glucoronic acid. The concentration of 17 -ketosteroids in urine is an important and useful index of adrenal function. In various diseases, such as Addison's disease, the level of 17-ketosteroids decreases, whereas there is an increase in urinary 17-ketosteroid excretion in Cushing's disease. It is believed that most of the 17-ketosteroids found in urine are of adrenal origin, such as gonadal androgens 1 which metabolize differently. Cortisol, the main glucocorticosteroid in humans 1 normally metabolizes to produce 17 -hydroxycorticosteroids which are found in plasma and urine although some cortisol may be converted to 17-keto steroids. The measurement of 17-hydroxycorticosteroids in urine is indicative of the function of the adrenal cortex. An increased excretion of 17 -hydroxycorticosteroids is paralleled with an increase in 17 -keto steroid excretion in certain diseases such as Cushing's syndrome 1 whereas both metabolites decrease in Addison's disease. Under normal conditions 1 a 2:1 ratio exists between the urinary excretion of 17-keto s teroids and 17 -hydroxycorticos teroids • Both 17 -hydroxycorticos teroids and 17-ketosteroids exhibit a circadian rhythm with a minimum excretion occurring shortly after mid night followed by a sudden rise which attains a maximum between 8 A.M. 6 and 11 A.M. after which a gradual decline is noted during the day. Ex ternal factors such as stress and disease are known to disturb this diurnal variation. CHEMISTRY OF STEROID HORMONES The first steroid hormone, estrone, was isolated in 1929. At that time the structure of the steroid nucleus was not yet known. Sig nificant work on steroid metabolism started in the 1930's, with the use of the classical technique followed, such as that of administering large doses of known crystalline steroids to human subjects and to experi mental animals and isolating crystalline metabolites from urine. Now, many steroids with varying degrees of biological activities have been isolated from tissue sources. There are four steroid producing tissues, namely- the testis, the ovary, the adrenal cortex, and the placenta of pregnancy. The steroids comprise a group of biologically active organic compounds which include such substances as cholesterol, ergosterol and the bile acids, as well as the adrenal corticoids I androgens, estrogens, and progestogens. The steroid structure characteristically contains the perhydrocyclopentanophenanthrene nucleus I consisting of a fully hydrogenated phenanthrene (rings A, Band C) to which is fused a five -carbon cyclopentane ring (D). 7 The carbon atoms represented in the nucleus are of three types: (a) those that are linked to two adjacent carbon atoms and carry two hydrogen atoms 1 (b) those that are common to two different rings and carry only one hydrogen atom (number 13 and 14) 1 and (c) those that are linked to four carbon atoms and carry no hydrogen atoms. The naturally occurring steroids may be visualized as essentially flat structures with some substituents projecting below it and others pro jecting above it. The methyl groups containing carbon 18 and 19 fre quently are called angular methyl groups and are represented by a solid line between the respective rings. According to Turner (3) and Dorf man (4), the angular methyl groups are important for reference and may be regarded as arising from the nucleus and projecting above it. The structural characteristics of the steroids are illustrated in Figure 1. 11~:::'-~~ ,_-__~,:_·---,;.-.·~~~· .. -~ .. 7 ' -·''"·,.·_ ·''.'. '""'""''·'' ·.~,·- '"'·""' """·:.·,-,·.·,,."".:.::.<7.: ,,,t,,.,._,c-:c::-.~~.·.l ,. i! ::, I; c j ~ ! i ' H i li/-'...... _"_...... \·., i '·i I~ j i r: .'1 l ; • !j I I' L, L :·) ~...... _..,_ ...... , _ _;/--···-' - • il), b: '. I ,) I ~~;) ; '' '. ~ •. ;·'. ',; \ t' ('',1:=J (./-...... ,_/. o • I l...... __"/':"'J ~,j ij l\' ! n:, " L'l· cj ,· IJ .. ~>l /'c:'~[__ __ ; ~""-_) Fic;ure l. STRUCTUR4L CHARI\CTERISTICS OF THE STEROIDS 9 When substituent groups are connected to the nucleus by solid lines, this means that they exhibit a beta configuration and lie on the same side of the molecule as the angular methyl group. If a substituent group is connected to the nucleus in a dotted line, it means that the group is below the methyl group and has an alpha configuration. Groups that project in the sa me direction from the plane of the nucleus are said to be cis to each other; if they project in opposite directions they are trans to each other. All steroids of androgenic, estrogenic, adrenocortica 1, and pro gestational series muy be considered to be derived from certain busic hydrocarbons which are saturated, and which end with the suffix "ane". ·when these steroids become unsaturated through the loss of hydrogen atoms, this is indicuted in the structure of the nucleus by employing double bonds and in the nomenclature by changing the suffix "ane" to "ene". If the nucleus contains tvvo double bonds, the suffix becomes "cliene "; three double bonds, "triene "; etc. In naming the unsaturated compounds, the position of the double bonds is indica ted. These basic hydrocarbons may be modified further by substituting oxygen for hydro gen. The suffix "ol" is used to indicate an alcohol or hydroxyl group 1 whereas ketone groups are indicated through the usc of the suffix "one". All of the naturally occurring steroids in the sex hormone and adreno cortical hormone series have side chains of the beta configuration. 10 Steroisomeris m: The steroid nucleus has three cyclohexane rings and one cyclo pentane ring. As the six carbon atoms of the cyclohexane ring are not fixed, they can assume various conformations. The six membered cyclohexane structure can exist in two forms, the boat and chair shape, as ill us tra ted in Figure 2 • The chair shape, which most often is encountered in steroids, is the most stable thermodynamically. The valencies of the carbon a toms of the cyclohexane ring can exist in the general plane of the ring and are called equatorial (e). Six others are at right angles to the general plane of the ring and are called axial (a). In depicting the equatorial or axial bonds as lying parallel or perpendicular to the plane of the ring, the convention and significance of writing the bonds extending above the plane of the ring as a solid line (B-configuration) and those below the plane of the ring as broken line (alpha configuration) is still retained, as shown in Figure 3. ll 1,~,-,, .. ,·,.~·~· .. ==-"4•~··""'""'--'-·=-···--·~~~-~~----"-... ·-..--:..lo~· _... _,..._,.,._...~ ...... ------...._... , ... -~...... , .. (. . \, .. . ''-._\ j//' I -~ ____ .; /. . ' \ . ' ...... --·------1' .. t' ' '.' '. t ~ Fiqur.-:; ?. •. CONTOIUVI/\TION OF STE!\OlD NUCILUS AS SllOWN BY DORFMAI-J (-1) ------ CH 3 r~~~ ( ~:::/': ~- ..) H II Ill '-----~------~-• r------ Figure 3. CON FIGURATION 0 F STEROID NUCLEUS AS SHO\VN BY WEST l\ND TODD (G) 12 Barton (5) first developed the modern stereochemistry of cyclohexanes which has contributed tremendously towards the understanding of the chemical behavior of the steroids. According to West 1 Todd 1 Mason 1 and Van Bruggen (6) 1 another descriptive criterion of the three-dimensional structure of steroids is the manner of ring fusion. When two fully substituted cyclohexane rings are joined together 1 it is necessary to indicate the direction of the substituent on carbons 5 and 10. By convention the methyl group attached to carbon 10 is pictured as beta oriented 1 axial (a). In the cis configuration the substituent group (H) at carbon 5 is attached by solid lines whereas in the trans formation the substituent group (H) is attached in broken lines 1 as shown in Figure 2, already cited. The steroid hormones may huve the same molecular and structural formulas I but different spatial arrangemGnts. The attachment of substituGnts in thG cis or trans position produce diffGrences in the molecular shapes. Isomerism at carbon 3 1 51 11 and 17 is VGIY common among the steroid hormones. Among the adrenocortical steroids 1 a hydroxyl group fre quently is found at carbon 11. This generally is cis to the angular methyl groups and is there fore of the beta configuration. When a hy clroxyl group is presented at carbon 17 it may take either the alpha or beta form. The alpha configuration exists if an ethyl group is present in the side chain. The beta form 1 however I exists when the ethyl group is not present. 13 BIOSYNTHESIS OF STEROIDS The main sequence of biosynthetic steps from acetate through cholesterol and pregnenolone to the adrenocortical and gonadal hor mones can be traced. Turner (3) states that the important concept has emerged from the early work on specific enzymes and alternative path ways that all of the organs that synthesize steroid hormones, viz, adrenal cortices, testis, ovaries, and placenta possess the same enzyme systems. It has been demonstrated by both in vivo and in vitro techniques that c1 4 -lobeled acetate and cholesterol both are precursors of the steroid hormones with cholesterol being the more efficient pre cursor. It was shown by Zaffaroni et al (7) in 1951 that rudioactive 17- hydroxycorticos terone a ncl corticosterone both were produced from either acetate c14 or c14 cholesterol by bovine adrenal glands profused with ACTH. The conversion of acetu te and cholesterol to adrenocortical hor mones by profused adrenals has been demonstrated by Betcher et al (8). Caspi et al (9) demonstrated in vivo the biosynthetic origin of individual carbon atoms of steroid hormones, adrenal cholesterol, and cholesterol. They reported the isolation of carbon atoms 3, and 4, and 6 and 7 of cholesterol c 14. In the carbons isolated from cortisol and from two samples of cholesterol, the distribution of "methyl" and "carboxyl" carbons was identical with that of the cholesterol c 14 bio- synthesized in vitro and in vivo from acetate 1-c14 These results suggest very strongly that corticosteroids are biosynthesized via 14 cholesterol. There is some possibility, however, that steroid hormone biosynthesis may proceed through a steroid intermediate similar to cho lesterol. Pregnenolone is the first c 21 compound to be produced from cho lesterol, and it is oxidized to progesterone. It was shown by Shimizu et al (10) that 20""( hydroxycholesterol could be cleaved at the side chain to give isoca proic acid 1 which converts to pregnenolone. Cons ta ntopoulos et al (11) confirmed further the formation of caproic acid and pregnenolone by the combined action of two enzyme fractions obtained from mitochon drial extracts. They also found that TPNH is the only required co-factor. Ch,,udhry et al (12) demonstrated the converstion of 22-keto and 22-hy droxycholesterol-3~ I-r 3 to prec;nenolone using an adrenal enzyme prepa- ration. PrecJnenolone cun form cortisol going through 17-< hydroxypreg nenolone. The 17 -hyclroxyla ted compound can convert immediately to 17-(hydroxyprogesterone 1 which may be 21-hydroxylated. This com pound is converted to cortisol by 11 fJ hydroxylation. Three enzyme systems ll,a hydroxylase, 17e.{ hydroxylase and 21-h)'droxylase, catalyse the reactions leading to cortisol and corticosterone. Aldo sterone also may arise from cholesterol, progesterone, deoxycortico sterone or corticosterone I hydroxylation being accomplished at carbon atom 18. Samuel and Greenberg (13) show that the conversion of 15 androgens to estrogen requires hydroxylation at carbon 19. Progesterone is converted readily into androstenedione and testosterone by testis tis sue I into cortisol and corticosterone by adrenal perfusion and to estro gen in vivo. Testosterone will be converted to estradiol and andro stenedione to estrone in placental, ovarian 1 and testicular tissue. This shows that pregnenolone and pregesterone 1 like cholesterol can give rise to every steroid thus far identified. The detailed Biosynthetic Path ways are shown in Figure 4 and Figure 5. !:'\\!.:;<) I, \1/J.:-H,' .- .. l' Figur·~ -1. FATH\VAYS OF CORTICOS.TI.:i\OlD GIOSYN'I'EESIS AS SHOV-/N DY TURI\JE: R (3) ( .'};~· 1J~i .!, ~) \; ,,': /~ ·.-(!: (l; .. ::.! ,·() 1 -~ fjgure 5. PJ\THWAYS OF ANDROGEN BIOSYI·\n·!ESIS IN THE ]1,DR~~N:\L CORTEX l\S SHO\VN E\Y Tl.IRNER (3) 18 Enzyme System There is a common course of reactions by which acetate synthe sizes cholesterol 1 and by which cholesterol is converted to pregneno lone and progesterone. The adrenal gland has not been shown to carry out any of the reactions of biosynthetic pathways of steroid hormones except possibly the conversion of androstenedione to testosterone. \Vith the exception of llp hydroxylase 1 which thus far is only demon strated clearly in the adrenal cortex and in interstitial cell tumors of the testis 1 the differences between the enzymic activities of different tissues secreting steroid hormones are quantitative rather than qualita tive. The qualitative differences, in most cases, consist of the factors which give the indi vidual steroid-forming organs their dis tinct hormona 1 character. Rabinowitz (14) found that, in various species, the testis can form small amounts of 174 estradiol and estrone. Under normal con ditions, however, the amounts secreted are too small to playa signifi cant role. Dominguez et al (15) demonstrated that, like a number of other tissues, the testis of small species possess 20-dehydrogenase activity in addition to having low levels of 21-dehydroxylase, the en- zyme which normally is present in mammalian testis. According to Samuels (16), most of the enzyme systems required in the steroid synthetic pathway fall into three major classes: the dehy drogenase or reductase, the hydroxylase, and the ketosteroid isomerase. 19 THE HYDROXYSTEROID DEHYDROGENASE These enzymes are found either in microsomes or in solution. NADP+ is the preferred hydrogen acceptor 1 and the hydrogen trans fer is directas follows: RCHOH + NADP+ RCO + NADPH + H+ Hydroxylases and Lyases. Although a separate hydroxyla ting system appears to be present for each position 1 all require molecular oxygen and NADPH as the co-factor. Lyases are grouped with hydroxy- lases since they require molecular oxygen and utilize NADPH as the co-factor. The specific function of the lyases is to split the side ch:11ns: Hydroxylase: RC -I-I + NADPH + H+ + 0 2 ~ RCOH + NADP+ + H20 Lyase: R-C -C R + NADPH + I-rt- + 0 2 ~ R-C + RCO + NADP+ + H20 Samuels (16) in reporting on recent work with mitochondrial frac- tions has indicated that the 11 beta-hydroxylase system is made up of an organized complex of several enzymes very similar to the electron transfer system of the mitochondrion. Estabrook et al (17) revealed this system to be: (a) associated with microsomes; (b) non-inhibited by cyanide; and (c) dependent on a supply of atmospheric oxygen andre- duced triphosphopyridine nucleotide (TPN). Garfinkel (18) discovered a co-combining pigment of unknown function in rat liver microsomes and designated it as cytochrome P-450 since its absorption maximum occurred 20 \:l-1.. 450 rnu. lt seems tbut microsornes of te"te;:> 1 oclrcnul~ and probably 0Llv.~r hydroxylating tissur::!s contain cytochrome P-4SO in much greater d.hundui!Ce thr:in cytochrorne bs. lt <1ppodrs then that hyclroxylases ond Jy.:1scs in general are multiple cnzymr:.~ systems and tcrmin:Jl steps in- vol.VL! a cytochrome ox!cluse of the P-450 type. These enzymes ca taly~;e the shifL of thc double bond in their rc:spectivc substrates from thc.L\ 5 (G) to A '1 (5) position as shown in figurE! 6. f(''1:-:! :-:::>::t:.. ~-~~:.:::...,~;..":.J\""':~::..'("~•~.f..I:''%:C£'!:•.' • .;..:0. ."i:':'"_:.:.:.:..:..:!'!:::~':i::C~~'":':.12':( ...·.;-,:;..:t-:r.;.;;-s..:_:.7.'C'..\.O::".::.;"!':""''~ r: i ,; I I l 1 /'IV /"-/ '·,' l I l ) -="·:=-~-=-~ i : ! I ' . ), )_. '\ ) ; : tr '-/ '- r.• ·,. "-.' , I ! . . ·. ; . ' ;,r{!"'t.l;r."~:;.":'..:!-:i!.':'.. ~;·:nr-l~~;:::·;..l:;..-~~~~i::::.~-~"\o;'".:l:F-!".--.:sr..!:.~~-f!'...::i.:lo".:"\.---::":·..:!""__-....:.."':~::.'C...!".;.:::::o.-.·.:.~ Figure 6. SHH'T Or DOU!l.l.E DOND f'l\Oivlfl S TO /J. 6 POSITION AS SHOWN DY EVv ALD (19) Ewald et al (19) in 1965 found that bovine cdrcnal cortex contains c:tt least thr·3e distinctlt 5-3-ketosteroid isomerases: 17-hydroxypreg- ncnedione isomerase I pregnenedione isomerase I and androstenedione isomerase. These isomerases appeur to be unstable and are destroyed easily by freezing in the purified state. 21 METABOLISM There are always two aspects of metabolic research-- the search for the intermediate and final products of metabolism, and the study of the regulating mechanisms that control the formation and breakdown of these products. The hypothalamus, hypophysis, and steroid hormone producing organs form a functional unit for the homeostatic regulation of the plasma levels of hormones. The steroid hormones are rapidly catabolized in the organism and excreted in urine and feces as conjugates of glucuronic acid or sul furic acid. The liver is the main site of steroid hormone catabolism, although the kidney and other tissues may function in this capacity to some extent. In hepactomized or eviscerated animals very little ring A reduction or conjugation occurs. The steroids of hormonal interest usually are 18 to 21 carbon compounds with varying substituent groups. Dorfman (4) divides the steroid hormones into two categories-- neutral and phenolic. Neutral steroids are androst 4-ene 3, 17 diane and progesterone. These com pounds contain the~ 4 -3-ketone grouping in ring A and an additional ketone group at position 17 or 2 0. These compounds which are the key metabolites of those steroids produced by the testis and adrenal cortex and are found in the neutral fraction. The 17 -ketosteroids regarded as neutral are androstane derivatives or urinary androgens. The phenolic 22 compounds have an aromatic ring A and a 17 -ketone group. The key phenolic compounds having only one ketone group can form 17 alpha and 17 beta hydroxyl derivatives. The group of carbon 21 steroids derived from the adrenal cortex and their carbon 21 metabolites are designated collectively as cortico steroids. Grollmun (20) classifies the corticosteroids in two chief groups, one of which exerts its principa 1 action on organic metabolism and thus is called glucocorticoids. Compounds possessing -0 or -OH groups at C1 7 (cortisone and cortisol) are glucocorticoids. In man the chi.ef glucocorticoid is cortisol and in the rodent it is corticosterone. Th::: complete metabolism of cortisone is shown in Figure 7. The metabolites of 17 -hydroxycorticos teroids ure excreted into the intestine with bile, where partial reabsorption occurs through enterohepatic cir culation as well us in the tubules of the kidney. Those substances which are not reabsorbed are excreted in the urine either in the free state or in conjugated form. Androgens produced by the adrenal cortex and testes include andrenosterone ,A 4 androstene-3, 17 dione and other c 19 steroids. Androstenedione is a potent androgen which under certain conditions is an important precursor of the urinary 17 -ketosteroids. The male hormone testosterone is metabolized entirely to the 17 -ketosteroids. The complete system of metabolism of testosterone and other andro- gens is shown in Figure 8. Figure 7. METABOLIS l'v1 OF CORTISONE 1\ND HYDROCOR.T'ISONE AS SHOWN f3Y DORFMAN (.:1) 24 II Et (ocholc:.ne -3, 17··dione Andl-oslcrone [tibchoLonc- 3o(-o[-17-onc J Ettocholanc-3c.<, 17(3-dLol Figure 8. IviETABOLISivi OF TESTOSTEK.ONE P.NDt. 4-i\.NDROSTENE-3, 17-·DIONE AS SHOV/N BY DORFJ\Li\.N (4) 25 PHYSIOLOGIC CONSIDERATION OF ANDROGENS Anabolic Effect. In man, significant phosphorus, calcium, po tassium, and nitrogen retention follows treatment with testosterone, methyltestosterone, and certain related steroids. In a study using cas trated rodents, Dorfman and Kincle (21) demonstrated that testosterone and relu.ted steroids stimulate body growth, retention of nitrogen, and an increase in the mass of certain muscles. The ability of compounds to influence nitrogen retention and relu.ted nonsexual functions is termed anabolic activity. As all anabolic steroids are chemically re lated to testosterone, each exhibits, to varying degrees, the andro genic effect of this hormone. Boris and colleagues (2 2) compared the effects of testosterone and nine other hormones on the testis, seminu.l vesicle, ventral prostu.te and levu. tor muscle, as well as the weights of immature rats, u.nd found testosterone to be effective on u.ll four para meters. All of the other compounds showed stimulation of leva tor and weight gain at dosages lower than those required to produce in creases in weights of the seminal vesicles and the ventral prostate. These findings indicate that chemical alterations in structures of andro genic steroids can result in compounds which differ significantly in their relative peripheral effects on androgenic u.nd anabolic parameters. Retention of nitrogen is the principal anabolic action of andro gens . It was noted by Kocha kian (2 3) that castration did not produce a 26 decrease in the rate of incorporation of amino acids in vitro by the cytoplasmic fraction of the adult mouse kidney until after two to four days. Androgens stimulated the earliest detectable increase in the rate of incorporation of the amino acids after 24 hours. There was a parallel increase in the concentration of RNA with the changes in amino acid incorporation, evidenced by an increase in the weight of the kidney and other target tissues. In another investigation conducted by Kochakian (24) it was determined that the protein anabolic action of the androgens is expressed in many tissues in addition to the accessory sex orgo.ns. The responsiveness of the nonsexual tissues varies remarkably among specirc:s with mouse kidney tissue proving most responsive. The changes occurring in the kidney are a ppa ren tly incl uced by the androgens through a regulation of the RNA-protein biosynthctic system. Avdalovic (25) found that the incorporation of all of the nucle otides in RNA was decreased by castration and restored to normal by testosterone propionate. Castration of adult male mice produced a sharp decrease in the RNA polymerase activity observed in the isola ted nuclei of the kidney. The administration of testosterone propionate re versed the effect. Adrenalectomy further decreased the RNA polymerase activity which was not only restored but also increased to the same levels as in non-adrenalectomized castrated mice following the ad ministration of testosterone propionate. Similar results were recorded by Widnell and Tata (26) in 1964. The effect of androgen on RNA 27 polymerase is not media ted through the ad rena ls . These glands, how ever, are responsible for maintaining part of the normal polymerase activity. This could be due to various corticoids or to the adrenal androgens. Since orotic acid is a known specific precursor of the pyrimidines, the cl4_labeled compound has been used by Kochakian and Hill (27) to provide further information concerning the details of the mechanism of regulation of RNA synthesis in the mouse kidney by androgens. Following castration, the rate of orotic acid-6-c 14 incorporution into the RNA of mouse kidney rapidly increased to a maximum value which was opproxi- matelv twice the normal rate. Within seven days following the udminis- tration of testosterone propionate to these same animals, the rate of incorporation was restored to normal. At the same time the concentration of the endogenous pool of RNA precursors , uricline (U), cytosine (C), guanine (G), and adenine (A), remained the same, indicating that re moval of androgen results in greater metabolism of the RNA precursors both in anabolic and catabolic phases. This could be due to a decrease in protein synthesis for the formation of ribonucleoproteins and mem branes for attachment of the RNA. It is believed by many investigators that androgens stimulate RNA synthesis at a selective site in the gen- orne. Fuji and Villee (28) confirm former findings that testosterone stimulates RNA metabolism within two hours after subcutaneous 28 administration. In 1969 Kochakian et al (29) studied the regulation of mouse kidney ribosomes. They found that castration produced a de crease in the concentration of polysomes to a constant minimum level within seven days. Testosterone restored the concentration of the ribosomes. The available data indicates that androgenic stimulation of protein biosynthesis in target tissue requires the synthesis of RNA and induction of other essential factors. The effect of an androgenic steroid on hemoglobin levels has been usually ascribed to an indirect effect of these hormones. Earlier s tudi.es of the in vitro effect of androgen on bone marrow cells provided conflicting evidence for a direct effect upon erythroid cell proliferation or hemoglobin synthesis. McCullagh (30) reported that prolonged therapy with testosterone corrected mild anemia in eunuchs indicating that androgens can also stimulate erythropoiesis in humans. The mechanism by which androgens influence erytheropoiesis remains un known, although testosterone has been shown by Fried and Gurney (31) and Mirand et al (32) to increase erythropoietin production in rodents and lower primates. The results of studies conducted by Reisner (33) and Rishpon-Meyerstein et al (34) on patients with various types of anemias suggest that testosterone can increase erythropoietic levels in humans. 29 Kappas and Garnick (35) reported stimulation of heme synthesis, in embroynic chick liver and erythropoietic tissue using certain meta- bolic derivatives of androgens steroids. Certain metalloporphyrin, in- eluding iron-protoporphyrin or heme, inhibit the steroid induction process. The ability of heme to block steroid induction supports the idea that heme and natural steroids may compete for a binding site on a hypothetical repressor protein which ultimately regulates the activity of the structural genes. Necheles (36) has recently confirmed these findings and demonstrutecl a similur in vitro effect using 5-beta H metabolites of testosterone on adult human erythropoietic tissue. Son- chez-Medal et ol (37) successfully employed testosterone theropeuti- cally both in children and adults sufferin9 from aplastic anemiu. Effect on Bone Metabolism Estrogen aclministra tion has been found to cause narrowing of the endochondra 1 os sificu tion zone in epiphyseal cartilage I inc rea sed calcification of bone and loosening and vacularity of the intra -carti- laginous ground substance. Rokkanin et al (38) evaluated the changes caused by estrogen, androgen, growth hormone, cortisone and para- thyroid hormone in the long bone and vertebra 1 of 2 3 mongrel dogs, four and six months old. Estrogen, and to a lesser extent androgen, was found to cause changes in the ground substance of epiphyseal cartilage, also in the cartilaginous zones of the acetabulum, and less frequently 1 in the vertebrae where cyst -like formations developed. 30 Howard (39) demonstrated that the administration of adrenal androgen advances the rate of skeletal maturation in infant mice. De hydroepiandrosterone (DHA), which appears to be of adrenal origin, is present in considerable amounts (chiefly as sulfates) in the plasma of young adults of both sexes, but declines markedly after the third decade. It is noted by Wilkins(40) that, although the thyroid hormone is more active than DHA or testosterone in accelerating skeletal matura tion, it is notable that an abnormal advance in skeletal age has been described frequently in children afflicted with the adreno -genita 1 syn dromc; I while hyperthyroidism as a cause of advanced skeletal a~re appears to be more rare. Howurd (41) found DHA and testosterone both to be equally active in promoting bone development in intact prepuberal female mice • Kowalewski (42) studied the effect of anabolic androgen by rating changes occurring in rat cartilage and bone by employing the s 35 labeled sulfate uptake procedure. It is known that the selective deposition of radio sulfate in certain tissues is due to the utilization of sulfate ions in the synthesis of the chondroitin sulfate present in connective tissue, ground substance and collagen. Hormones effect the brea kclown and synthesis of collagen. Young and Kowalewski (43) have shown that corticoids inhibit synthesis, and 31 under certain conditions anabolic hormones can prevent the pathological action of corticoids on connective tissue and collagen. It also was shown by s35 uptake studies that anabolic steroids stimulated, while cortisone inhibited the production of certain mucopolysaccharides which are essential in fibrillogenesis, and which specifically incor porate labeled sulfate • Puche and Romano (44) found increased synthesis of osteoid tissue when the frontal explants from 12 -day old chick embryos were treated with DHA as well as increased cellular density of the baso philia . Histological findings showed a significant increase in hy droxyproline and a net increase in DNA and RNA. Similar results were obtained when these implants were treated with testosterone with a concurrent increase in alkaline phosphatase activity. This suggests that testosterone acts directly on bone. Recently Puche and Romano (45) showed that differences in cell population seem to be the reason for the increase in osteoid synthesis which is noted following ad minis tra tion of testosterone and dehydroepiandros terone sulfate. Chick embryos • frontal bones at 12 and 13 days of development which were cultivated in vitro exhibited different patterns of glucose utilization, periosteal cellular density, and calcium and hydroxyproline content. When treated with dehydroepiandrosterone sulfate for 12 days , fronta ls engaged primarily in osteoid tissue synthesis while 13 day frontals calcified at a significantly greater rate than controls. Explants treated 32 with lmM dehydroepiandrosterone sulfate accumulated calcium at twice the rate of controls. It seems that overall conformation afforded by these experiments suggests that the hormone helps to provide more free energy to the cells, thereby modifying their metabolic behavior. Keele (46) using oxymetholone showed u significant increase in the linear growth and weight of a group of small children. Studies con ducted by Ray et al (47) (48) revealed the finding that, when mongoloid children were treated with oxandrolone, a significant increase in height was noted, although there was no statistically significant acceleration in skeletal age. Recently Keele and Vose (49), working with bedfast institutionalized mentally retarded children, found that the use both of fluoride and oxymetholone increased skeletal density. After the administration ofanclroqens the volume of urine climin ishes and the losses of sodium, chloride, potassium, and inorganic phosphorus, as well as nitrogen I are reduced. The retention of nitro gen, potassium, and phosphorus probably is reluted to the increased anabolism of tissue proteins. The capacity of androgens to promote the retention of sodium chloride resembles that of the adrenal cortical steroids. Androgens are much weaker in this respect 1 however, than are the adrenal steroids. 33 Androgens 1 by virtue of having anabolic activity I influence the enzyme concentrations in plasma of various systems. Clifton (50) has reviewed the literature in this regard in detail. Effect on Lipid Metabolism The lower rate of ischemic heart disease among women often is ascribed to the "hypocholesterolemic" effect of estrogen. Do androgens contribute in some way to the high vulnerability of the male? Using healthy adults as subjects, Furman (51) found that sex and age differ- ences are evident in serum lipid concentrations and in the a mount of lipid present in the major lipoprotein fractions. In another study con ducted by Furman et al (52) healthy men and postmenopausal women were found to have significantly higher triqlycericle concentrations and greater umounls of cholesterol and phospholipids than do premenopausal women. Androgens characteristically have un effect on serum lipids and lipoproteins , which is opposite to the situation with estrogens . Andro gens diminish the a mount of serum lipid trans ported as alpha and very low-density lipoproteins. Beta lipoproteins are increased by androgen administration. It was shown by Howard and Furman (53) that, when testosterone derivatives were administered intramuscularly, a remark- able reduction of hyperglycericlemia and hypercholesterolemia occurred in three hypothyroid subjects, with the gonadal hormones also, chang ing the a mount of lipid circulating in the form of a lipoprotein particle, 34 Androgen increases the cholesteroyProtein ratio of a lipoprotein particle and a similar, but lesser, change occurs in the beta lipoproteins. GLUCOCORTICOIDS Physiological Considerations The carbon 21 steroid derivatives from the adrenu.l cortex, as well as their carbon 21 metabolites are designated collectively as corti costeroids. One group of these steroids exerts its principal action on organic metabolism and is referred to as glucocorticoids. Adrenalectomy results in an increased release of adrenocorticotrophin hormone (ACTI-I) by the pituitary. Also in hypophysectomized animals, the aclrena l out put of qlucocorticoicls is reduced to a very low level. The pituitary releases increasing amounts of ACTH when blood levels of glucocorti coids are low, and diminishes the output as the plasma glucocorticoicls are elevated. There is general agreement that a negative feed back mechanism operates to control the levels of glucocorticoids in the blood. The concentration of these hormones in the blood depends on how rapidly they are inactivated and eliminated from the system. Carbohydrate Metabolism In fasted, adrenalectomized animals, severe depletion of liver glycogen, low blood glucose levels and decreased intestinal absorption of glucose occur. Muscle glycogen is lost during the terminal stages of cortical insufficiency. Both adrenalectomy and hypophysectomy 35 alleviate the symptoms of pancreatic diabetes 1 with blood sugar lowered and the urinary excretion of glucose 1 ketone bodies and nitrogen de creased. The two fundamental defects in carbohydrate metabolism in adrenalectomized animals are: (a) excessive oxidation of glucose and (b) decreased gluconeogenesis from body proteins. Landau et al (54) found that cortisone significantly increased the incorporation of carbon dioxide and pyruvate into both glycogen and glucose. Weber et al (55) and Greengard et al (56) report that the in- creased gluconeogenesis induced by cortisone entails an increase in the synthesis of gluconeogenic enzymes--glucose -6 -phos pha ta se I a l dolase 1 fructose 1 1 6-diphosphatase 1 and lu.ctic acid dehydrogenase. Weber et a l (57) also found that there wu. s an elevation of gluconeo genic enzyme synthesis in starvation. Extensive evidence has been accumulating 1 which shows that uny increu.se in the four gluconeogcnic enzyme activities which is inclucecl by glucocorticoids can be prevented or blocked by inhibition of protein synthesis. Both Eisenstein (58) and Weber et a l (59) have determined that there occurs a rise in nitrogen excretion 1 which rna kes it apparent that new carbohydrates have been formed from protein in corticoid stimulated animals . Bella my and Leonard (60) I as well as Hornbrook et al (61) have presented evidence that any action of glucocorticoids is directed more specifically or earlier to glycogenesis than to gluconeogenesis. Oji and Shreeve (62) have found evidence of a greater percentage of incorporation of tracer 36 (either c14 or H3) into glycogen than into blood glucose after cortisone treatment. Taronwski et al (63) have cited a delay in liver glycogen accumulation. These observations indicate an early effect of hydro cortisone on glycogenesis from a hexose precursor. Despite the wealth of data 1 the site or sites of the ini.tial actions of steroids remain unknown. According to Landau (54) the initial action seems to be at one of five sites (a) carbon dioxide fixation by pyruvate; (b) inhibition of fructokinase activity; (c) inhibition of pyruvate con version to carbon dioxide and coenzyme A; (d) action at the amino acid level; ond (e) action at the enzyme synthetic level. Landau (64) su~rgests that the most likely sequence of events following glucocorticoicls administration seems to be: (a) a decrease occurring in hepatic glucose output und in the peripheral utilization of glucose; and (b) an increase occurring in the synthesis of liver enzymes associated with gluconeogenesis. Action on carbohydrate metabolism is heightened by the presence of a hydroxyl group at carbon 17. Corticoids having oxygen at carbon 11 either as a hydroxyl group or a keto group exert major activity in car bohydrate metabolism. Haag et al (65) found that I while the urinary excretion both of 17-ketosteroids and 17-hydroxycorticosteroids de- creases with fasting I both increase slightly but not to prefast levels, following glucose refeeding. 37 Fat Metabolism The conversion of carbohydrate to fat is markedly increased in adrenalectomized rats. The ad ministration of cortisol or cortisone to rats suppresses the ketosis that occurs during fasting or consequent to cold exposure. Adrenocorticotrophin and cortical steroids accentuate the diabetic state as these hormones enhance the degree of insulin re sistance. Adrenal steroids exertanaccelerating effect upon the forma tion of carbohydrate from fat. Kinsell et a l (66) confirmed the above findings in humans. Glenn et al (67) noted that shortly after the injection of hydro- cortisone to fasted adrenalectomized rats 1 there was a marked decrease in the rate of conversion of glucose and glycerol liver glycogen 1 and in the ra Le of oxida Lion of palmitate 1-C 14 . There also existed an in crease in incorporation of c 14 from palmitate l-c14, butyrate 1-c 14 1 and acetate 1-c14 into liver glycogen. According to L::lndau (64) 1 it may be that fatty acids 1 while not actually providing carbons for glycogen synthesis may have a sparing effect on amino acids. For example I the carbon of alanine 1 instead of going to Kreb's cycle, could be converted to glycogen. The increased mobilization of fatty acids would release glycerol from triglycerides, and this compound could be a source for additional glucose carbons. The evidence obtained by Munck and Koritz (68) suggests that in 38 glucose-fed rats, cortisol does not decrease the uptake of glucose by either muscle or liver within 3 to 5 hours, but may decrease the uptake by epididymal adipose tissue. Protein Metabolism Adrenal cortical hormones influence gluconeogenesis at some early stage, by facilitating the mobilization of a carbon source. In fasted rats, this source of carbon has to be, for the most part, amino acids. Based on evidence showing that adrenal cortical extracts en hanced the growth of livers of fasting, partially hepatectomized rats, it is hypothesized by Ray (69) that the primary action of glucocorticoids involves an increased mobilization of protein. For atrophy of any tissue to occur, protein catabolism must exceed protein synthesis. Thus a decrease in tissue protein may occur either by decreased protein syn thesis, by increased protein cCJ.tabolism, or by some combination of the two. Goldberg (70) examined the phenomenon of muscle wasting in re sponse to cortisone, and found that cortisone both increased protein degradation and decreased protein synthesis. Bethal et al (71) found an alteration in free amino acid patterns four hours after cortisone in jection resulting in an elevation of tyrosine and ketoglutarate trans- a minas e and tryptophan pyrro lase • Both Weber et al (72) and lardy et al (73) noted that, after cortisol treatment, there is a rise in the synthesis of gluconeogenic 39 enzymes which affects Rt'\fA metabolism by enhancing both RNA and pro tein synthesis. To find the initial effect of cortisone on protein syn thesis 1 one considers many biochemical parameters. Lang and Sekerkis (7 4) have found activation of RNA polymerase with cortisol. Both Pena et al (75) and Brink-Johnson (76) have observed that injection of cortisol resulted in a decrease in RNA polymerase activity of thymic homogenates with impaired amino acid incorporation into microsomal or ribosomal protein. Electrolyte Metabolism Although glucocorticoids are potent factors in the regulation of carbohyclra te metabolism I they also are capable of enhancing or inhib iting the excretion of water and sodium. Cortisol is needed to maintain a normal glomerular filtration rate. Mendelson et al (77) found that adrenalectomized patients 1 maintained with salt and cortisone 1 re- s ponded to withdrawal of cortisone by a drop in the glomerular filtration rate. Cortisone in a physiological dosage will facilitate sodium reab sorption. Adrenalectomized subjects cannot easily dispose of a high salt load in the absence of cortisol. It is well known that cortisol frequently promotes sodium retention and potassium excretion in man. Lutwa k (78) noted increased sodium retention with increased 17 -hy droxycorticosteroid excretion in men of the Gemini VII space flight. 40 Administration of large a mounts of cortisone have led to polyuria and polydipsia. There is poor water diuresis in the absence of cortisol. Dingman et al (79) observed that cortisone acted to alter the distribution of water between intra- and intercellular compartments 1 thus rna king more water a va ila b le for excretion . Hauger et al (80) have found that adrenocorticotrophin hormone produces a sustained rise in plasma renin level and glucocorticoids completely inhibit ACTH induced renin rise. The opposing effect of ACTH and glucocorticoids on plasma renin activity (PRA) may well ex- plain why 1 in normal subjects with responsive adrenals 1 little if any discernible rise in PRA occurs following ACTH udrninistration 1 especi ally when ACTI-I has produced elevation of corticoicls. The renul renin clecreuse noted after cortisone injection is consistent with the concept that expunsion of the plasrni1 and extracellular fluid volume leads to decreased activity of the renin angiotension system. The results of a recent study by Robb et ul (81) indicates that 1 during cortisone treat ment some rabbits retained sodium and water until death occurred 1 while others apparently escaped the sodium-retaining action of this sterol. The responsiveness of the kidney could be due either to (a) a direct action of cortisone on the renal tubular transport of sodium 1 or (b) some secondary consequence of the action of cortisone which usually affects a responsiveness of the renal tubules. 41 It was observed by Ingbar and Frienkel (82) that cortisone in fluences iodine uptake in human beings by reducing the rate at which thyroid hormone is released significantly. Schiller and Dorfman (83) found that incorporation of s 35 into chonodroitin sulfate was inhibited in the skin of intact rats injected with cortisone, while 17-hydroxycorticosterone injection resulted in a gradual decrease in the rate of turnover of chondroitin sulfate in skin. Adrenal steroids also have been shown to inhibit the incorporation of s35 in the cartilage of the normal rat. It was observed by Cox (84) that adrenoglucocorticoid hormones increase the uptake of zinc in certain mammalian cell cultures. This enhanced accumulation of zinc is specific I since the uptake of other cations is not a l tcred. Bone Metabolism Both 17-hydroxycorticosterone and cortisone have a marked ef fect on bone metabolism. It appears that all glucocorticoids reduce both chondrogenesis and osteogenesis I and that in particular I cortisone is known to slow the rate of s ke leta l maturation. The finding that the effect of corticosterone on bone maturation is opposite to that of andro- gens furnishes some support to the view that the gl ucocorticoids and the androgens of adrenal origin may be exerting opposite influences on the skeletal system. 42 Decreased adrenocortical activity is referred to as Addison's disease. Cushing's syndrome is a result of adrenocortical hyper activity. In Cushing's disease a negative calcium balance always exists and in most cases a marked increase in urinary calcium is ob served I indicating poor intestinal absorption due to excessive biliary excretion. Grollman (85) reported that corticoids have a depressing effect on calcium utilization and cited increased mobilization of bone calcium in nephrectomized dogs receiving large doses of cortisone. Clark et al (86) found that 17-hydroxycorticosterone administration inhibited the uptake of ca lei um4 5 by the femurs of norma 1 and ne phrectomized rats 1 although there was no indication of any mobiliza tion of total bone calcium. There is some indication that cortisone and 17 -hydroxycortico sterone promote a marked and rapid fall in the circulating phosphate levels. The muscles of adrenalectomized animals take up less phos phate as P32 than those of normal animals. Skeels (87) describes a 12 year old girl with severe osteoporosis resulting from hypercorti coidism in whom microscopic inspection revealed a diminution or possible total lack both of osteoblasts and osteoclasts I as well as a thinning of the bone trabeculae. Collins et al (88) report that 1 in some cases the pharmalogical doses of adrenocortical steroids alter calcium utilization by elevating the urinary and fecal calcium. Steroid induced alteration in bone is reversible. 43 17-KETOSTEROID AND 17-HYDROXY CORTICOSTEROID EXCRETION 17-Ketosteroids The determination of the neutral 17-ketosteroids in the urine often is used as a measure of adrenocortical and testicular function. The principal source of 17-ketosteroids is ll-hydroxyandrostenedione. Testosterone, the male hormone is metabolized entirely to the 17- ketosteroids. Adrenal androgens are believed to be the source of ap proximately 3/5 of the aggregate urinary androgen excretion product, the 17-ketosteroids. There is great variation in both sexes with ranges of five to 15 mg. of ketosteroids per day for females, u.nd s8ven to 20 mg. p8r day for males. It has been suggest8cl that the androgens of testiculur origin are less likely to appeur in urine as 17-k8tost8roids than are androgens of adrenul origin. During the first two years of life, children excrete only small amounts of 17-ketosteroicls. High values are obtained in diseases churacterizecl by adrenal or testicular hyperplu.sia. Hypofunction of the anterior hy·pophysis, testis, or adrenal cortices produce low 17 -ketosteroid values. The total neutral 17-ketosteroid excretion is approximately 33 per cent less in normal women than in men. The greater output by men is attributed to the fraction produced by the testes. Approximately 30 per cent of the urinary 17 -ketosteroids are produced by the testes, while the adrenal cortex contributes the remaining 7 0 per cent. 44 According to Henrotte eta (89) 1 there are indications that racia 1 and sex differences 1 as well as social and economic status in fluence 17-ketosteroids 1 which is concurrent with the reduction of body size prevalent among the underprivileged population. Mahesh et al (90) have reported on the use of 17-ketosteroids as diagnostic measurements in many diseases such as congenital adrenal hyperplasia 1 precocious puberty 1 and some ovarian tumors. Eleva ted levels of 17- ketosteroids are found in the above diseases whereas low levels are found in hypopituitarism 1 Simmond 's disease and myxedema. Kumaoka et al (91) found a significant difference in urinary 17-ketosteroids be tween rc·sponsive and unresponsive cases undergoing treatment for advanced breast cancer. Patients res paneling to the treatment secreted higher amounts of 17-ketosteroids than normal controls. There is an increase in 17-ketosteroid excretion following gonadotrophin hormone administration. According to Lloyd et al (92) I in women with hirustism of polycystic ovaries, determination of the urinary excretion of 17-ketosteroids aided in separating the subjects into two groups in which the excess androgens originated from either adrenal or ovarian sources. Nicholas et al (93) observed that 1 in iodopathic hirustism caused by excessive androgen I glucocorticoid administration caused a significant decrease in urinary 17-keto steroids I 17-hydroxycorticosteroids I and testosterone excretion. It 45 was noted by Jacobson et al (94) that a significant decline occurred in the mean excretion of 17-ketosteroids in obese girls when these young women were involved in a reducing regimen. The urinary excretion products of androgens are conjugated with glucoronic and sulfuric acid and are biologically inactive unless hydrolyzed. There are four principal urinary excretion products of testosterone shown in Figure 9. These involve androsterone 1 dehydro epiandrosterone, epiandrosterone 1 and etiocholanolone. The andro gens are degraded in the liver and other tissues by 17 -beta -de hydro genase-like enzymes to 17-ketosteroids of varying androgenicity. The 17-ketosteroids have a keto group at position 17 of the steroid nucleus. The keto group at position 17 is detectable by the Zimmerman Test (formation of a yellow color upon treatment with m -dinitrobenzene in alkaline sol uti on). ~""~.;:~~~~!:~\.~_,.~.. t~.~=-"'[%"~~~£-l.!'~~!!.."-=~.!·t.:.•r~~-:.:;;g~~-9 i 0 1\ n c; r o s ~ (· r o r1 e 0 II ~~ ~0/·~----J I ! I ! I 0 "y_<·;"'-._/ 1 fl I ,· (1 ,., I' ..., • "I (I - ,, F-: ,, i ( c!· l "'._ .. •I ' (••• •1 (' 'l1 (' P...:~ I I.• I . .._ I.,l'' '1 ",.,I. J' u ~~·· ~~'OIJ,"'fft.T.t.r'"~;:...~~~~"a.·~~~...... Figure 9. 17 -KETOSTEROID EXCRETION PRODUCTS OF ANDROGENS AS SHOWN BY TURNER (3) 47 1 7-Hydroxycorticos teroids 17 -hydroxycorticosteroid is the metabolite of cortisol and is a measurement of adrenal cortical activity. In cases of stress and in jury where hormonal output increases 1 the levels of 17 -hydroxycortico steroids in urine and plasma also increase. Van Derstrateen et al (95) report that the determination of the urinary excretion both of 17 -hy droxycorticosteroids and 17-ketosteroids gives a fairly accurate evalu ation of adrenal cortical function. The rate of secretion of cortico steroids may be measured by analysing the hormonal content of the gland and the rate of secretion of the hormones or their metabolites in the urine. Romanoff et al (96) observed that, as aging in man appears to decrease both adrenal and testicular activity 1 the excretory levels of 17 -hydroxycorticos teroids also decrease. Mig eon et al (97) have shown that I in pregnancy 1 even thouqh the cortisol level increases 1 17-hydroxycorticosteroids show decrease levels. Body weight and urinary 17-hydroxycorticosteroids excretion are positively correlated. As men are known to excrete more corticosteroids than women 1 it is suggested by Curtis et al (98) and by Streeten et al (99) that a sub- stantial part of the sex differences in corticosteroid excretion is clue to weight and body cell rna ss. The pattern of findings in men of large stature is qualitatively similar to those observed in men undergoing conditions of stress. According to Grant ei: al (100) 17-hydroxycortico- 48 steroid urinary levels tend to be lower after long term administration of estrogen. Adrenocortical steroids play an important part in the develop ment and maintenance of arterial hypertension, u.nd a grossly impaired renal clearance of conjugated 17 -hydroxycorticosteroids occurs in pa tients with essential hypertension. Kornel and Motohashi (101) have determined that lower levels of 17 -hydroxycorticos teroid gl ucuronides are found in hypertensive subjects than in normal subjects. Increased urinary excretion of 17-hyclroxycorticosteroids are found in Cushing's syndrome where excretion levels are elevated to more than 0.13 milligrams per kilogram of body weight per day. In hirus tis rn I the urinary excretion both of 17 -hyclroxycorticos teroids and 17-ketosteroids is elevu.ted. The 17-hydroxycorticosteroids are analyzed as Porter-Silber chromogens or as 17-ketogenic steroids. The side chains of the corticosteroids constitute a 17:21 dihydroxy- 20-keto (CH2 · OH ·CO ·COB) grouping which reacts with phenyl hydrazine and sulfuric acid to give a yellow color. These compounds are known as Porter -Silber chromogens. Stress and Steroid Hormones When intact animals are placed under stress I the pituitary adrenal axis is activated. The endocrine adjustment that occurs during stress aids the organism in its attempt to maintain homeostasis. Dif- ferences in adrenocortical activity are noted in patients during severe 49 depressive phases and during recovery. In one study conducted by Gibson (102) involving 15 hospitalized depressed patients, the mean cortisol secretion rate before therapy was 25.5 ± 2.4 milligrams per 24 hours, while after therapy it was 14.1 ± .09 milligrams per 24 hours. Depressive symptamology frequently is encountered in Cush ing's disease, in which the 17 -hydroxycorticos teroids excretion levels are markedly elevated. Rubin and Mandell (103) have found that an increase in the activity of the adrenal cortex occurs concomitantly with various types of depressive reactions, while in some anxiety or manic states lowered activity occurs. Clower et al (104) reported that the eleva ted urinary level of 17 -hydroxycorticos teroids noted in patients suffering from severe depression was decreased following ECT treat ment. A decrease in brain excitability also coulcl result in a decrease in hypothalmic activity which in turn would decrease pituitary secretion of cortisol. Studies by Wolf (105) and Wolf et al (106) have established that a relationship exists between adrenocortical activity and a psy chological state, and have suggested that certain stable personality characteristics may be used to predict the expected level of 17 -hy droxycorticos teroid excretion in individuals expos eel to chronically distressing situations. Rose et al (107) have found a significant cor relation between body size and psychological state in relation to the level of urinary 17-hydroxycorticosteroids excreted. The effects of 50 flight stress on urinary 17 -hydroxycorticos teroids levels was investi gated by Marchbank (1 08) 1 who found the 17 -hyc!roxycorticosteroid excretion of airborne pilots to increase from 11 to 2 0 mg. per 24 hours. Demos et al (109) observed the pilots of a Fl02 aircraft while they were engaged in unaccustomed flying missions 1 as well as during routine missions. During the training flight the subjects experienced both an increase in 17-hydroxycorticosteroid excretion and hypophos phouria. Lutwak et al (78) reported a decrease in urinary 17-hydroxy corticos teroid excretion during the Gemini VII space flight. It has been established that there is an important relationship between flsychological function and adrenal cortical activity in man. Experimental studies using animals have demonstrated that an increase ln anterior pituitary-adrenal cortical uctivity is a characteristic re- sponse of the animal subjected to stress. Pincus et al (110) found the elevated excretion of 17-ketoster oicls observed in flying pilots to be proportional to the flying time. Frost (111) conducted an experiment with automobile race drivers I and revealed a 50 per cent increase in the urinary excretion of 17-keto steroi.ds in six of seven drivers. Later it was observed by Hill et al (112) that the level of 17-ketosteroicls is a poor indicator of stress since the 17-ketosteroids are primarily gonadal hormones involving only fractional secretion by the adrenal cortex. They proposed that 51 the 17 -hydroxycorticosteroid level is much more reliable as an index of stress. Miller (113) has reviewed the effects of stress on the urinary excretion of 17 -hydroxycorticos teroids of a via tors extensively. A study conducted by Marchbank et al (114) revealed the finding that there is a significant increase in urinary 17 -hyclroxycorticosteroid excretion of pilots during transoceanic flight (supersonic FlOO, F104). Hale et al (115) also noted an increase in the 17-hydroxycorticoid excretion of nonpilots of F-4c aircra fl on an 18 -hour continuous flight. Increased aclrenucorticosleroicl secretion occurs in response to flight factors such as ellinger, cluralion of exposure 1 degree of responsi bility 1 and luck of ociC1pLalion (experience). It could be Lhat the 17 -ketos teroid/17 -hyclroxycorlicosteroid ratio may prove Lobe a reliable index of the meosurement of stress. It has alrc;acly been shown by Abbo (116) thaL this ratio has a direct physi- ological siCJnificancc. It c!Gmonslrates u nut:ural variation with a max- imum a ttainecl during the ovcrniqht periods of excretion a ncl a minimum occurring during the morning. This ratio dec lines with age. Further evidence of the physiological significance of the 17-ketosteroic!/17- hydroxycorticosteroid ratio is the lower 17-ketosteroid/! 7-hydroxy- corticosteroid ratio noted in patients suffering from Cushing's syn- drome and in patients receiving glucocorticoid therapy for diabetes 52 mellitus, hypertension, obesity, muscular wasting, thinning of the skin, osteoporosis, and arteriosclerosis. Patients with myocardial infarction have a lower ratio than their healthy controls. It has been suggested by Marmorston et al (117) that a lower 17-ketosteroid/17-hydroxycorti costeroid ratio through the preponderance of the anti-anabolic hormones might advance the aging process. On a biochemical level this ratio effect means that there could be a competitive inhibition of the effects of glucocorticoids by 17- ketosteroid and vice versa. Catabolism cannot occur except at the ex pense of anabolism, since the two processes are interrelated in a steady dynamic state intracellularly. 17-ketosteroids are known to counteract the catabolic action of glucocorticoids • Total l 7-ketos teroids do not necessarily measure anabolic activity, since the composition of the 17-ketosteroids varies among individuals. While this ratio might prove useful as an index in a population study where average effects are in volved, it may not be useful in examining individual subjects. EFFECT OF IMMOBILIZATION There has been a rapidly growing interest in recent years in the effects of immobilization upon various metabolic and physiologic functions of the normal human body. This interest may be due to the realization of space flight, during which crew members are subjected to prolonged immobilization. The results of animal and manned orbital 53 space flights to date suggest that weightlessness itself is tolerable 1 perhaps even pleasant 1 for some hours. The most important problem is that of the cumulative 1 time dependent and adaptive changes of pro longed exposure. It has been suggested by McCully and Graveline (118) that prolonged exposure to zero gravity will produce decondition ing of the physiological system through disuse 1 thereby seriously im pairing man's ability to tolerate normal or increased gravitational {g) forces. As a consequence of the assessment of the physiological sys tern of primates 1 extensive data are available on parameters such as mineral metabolism. Studies conducted by Bruce et al (119) and Pyke et al (120) have revealed that loss of calcium and phosphorus occurs clue to immobilization. Hoffman et al (121) found that nitrogen and creatine excretion is increased when primates are placed in restraint. Investigations by Mack et al (122) (123) have established the fact that immobilization causes a significant decrease in bone density. Recently studies conducted at Texas Woman's University Research Institute by Mayer (124) 1 Montgomery (125) 1 and Van Zanclt (126) have confirmed the above findings in human beings. It is believed that paralleling the changes in these various parameters which have been cited that there also is an alteration of endocrine metabolism in human subjects. Specifically 1 mineral and 54 nitrogen changes can influence the secretion of adrenal steroid hor mones. Also hormonal secretion can influence the changes observed in these metabolites. Immobilization causes a condition of stress which definitely affects the secretion of the steroid hormones of the adrenal cortex. Results of a study conducted by Deitrick et al (127) in 1948 revealed the fact that 17-ketosteroid excretions differed in individuals, with a small increase appearing only in two subjects during restraint periods, with a gradual return to normal occurring after recovery. Later in 1949 Whedon et al (128) found that the average values of 17-keto steroid excretion over the entire period of immobilization were not sig nificantly lower than those reported for the control and recovery periods. These investigators also found a slight and questionably significant re duction in 17-ketosteroid excretion at the end of the immobilization period. A number of studies on the effect of immobilization on 17-keto steroid excretion have been conducted in recent years in the Nelda Childers Stark Laboratory for Human Nutrition Research at Texas Wom an 1 S University. In this laboratory in a study using five adult healthy males as subjects, Wu (129) found that, during periods of immobiliza tion, two of the subjects showed a significantly higher urinary 17- ketosteroid excretion than in the ambulatory period, while two of the subjects showed a slight decrease in 17-ketosteroid excretion during immobilization when compared with a previous ambulatory period. All 55 subjects showed a sharp decrease in 17-ketosteroid excretion during the recovery period. Another study conducted by Clifton (50) in 1967 at the same laboratory showed that, during ambulatory periods, the mean values tended to fall within or slightly below the normal average for adult males. Urinary excretion of 17-ketosteroicls increased in all subjects during the first bed rest period, while a sharp decrease in 17-kctos teroicl excretion was noted during the interim equilibration period in all subjects. Enhanced adrenal corticill activity following immobilization of animals has been ascertained from the estim::1tion of the corticoid levels of peripheral blood. 1\n experiment conducted by Suzuki et al (130) showed that conscious dogs after immobilization showed u. marked increase in the rote of secretion of 17-hydroxycorticosteroids when they were excited and led into a strugc;le. The level of 17-hy droxycorticosteroicl secretion remuined unchanged in anesthetized im mobilized clogs. A study conducted by Burnstein I Bhavanani I and Kimball (131) revealed a threefold increase in the excretion of alpha hydroxycortisol and cortisol in inFno!:lilized guinea pigs over the co:1- trols. They also noticed a clay to clay variability in the excretion of 17 -hyclroxycorticosteroids. Knigge et al (132) observed similar patterns of corticoid excretion and in addition they also observed an initia 1 biphasic response consisting of a brief elevated secretion of this hor- mone followed by a period of lower sus ta inecl rate of hormone secretion. 56 This might stem from the inability of the pituitary to release ACTH, due either to cessation of the initial hypothalmic stimulation or to a neutral mechanism which temporarily suppresses or inhibits the secretion of this factor. Lin (133) in 1967 at Texas Woman's University's Nelda Childers Stark Laboratory for Human Nutrition Research conducted an investiga tion into the urinary excretion of 17 -hydroxycorticos teroids a ncl 17- ketosteroids of human subjects during immobilization. The results of this study showed irregular variations in the urinary excrc:tions of total 17-ketosteroids a nc! 17 -hyclroxycorticos teroids for each individual subject. In a similarly patterned study Hutton (134) found a slight, but nonsignificant, increase in the urinary excretion of 17-hyclroxy corticosteroids during a first bed rest as comp:ued to an initial equili bration ambulatory period. During a second bed rest period, the uri nary excretion of 17 -hydroxycorticos teroids W3 s significantly greater than during the post-bed rest ambulatory p2rioc!. The average excretion of 17-hydroxycorticosteroids during Bed Rest I and Bed Rest II was 9.19 mg. per 24 hours and 9.85 mg. per 24 hours, respectively. Ir- regular variations in the urinary excretion of total 17-hydroxycortico steroids for individual subjects also were observed by Liu (135), LBe (136), and Hsu (137). 57 EXERCISE AND ALTITUDE EFFECTS Effects of muscular exercise on the adrenocortical secretory activity has been evaluated in the past by indirect methods such as de pletion of plasma ascorbic acid and cholesterol. Suzuki et al (138) investigated the effect of muscular exercise on 17-hydroxycorticosteroid secretion in dogs. They noticed a marked increase in 17 -hydroxycorticosteroid secretion in completely exhausted dogs. In dogs which became somewhat tired, but were not completely exhausted/ the adrenal 17-hydroxycorticosteroid secretion rate in creased to a level slightly above the normal range of physiological variation. In animals which did not show any sign of fati~JUe after ex ercise the adrenal 17-hydroxycorticosteroid secretion rates were within the range of physiological variation established for the resting state. When sea level residents are exposed to conditions of high altitude r they experience a variety of physiological changes including an elevation in urinary steroids. Monchoa (139) found no difference to exist between the urinary excretion of 17-ketosteroids and 17-hy droxycorticosteroids of sea level and high altitude natives r with the catabolism of cortisol observed to be the sa me in both groups . In creased urinary excretion of 17-ketosteroic:l in humans and accelerated adrenocortical secretion of 17 -hydroxycorticosteroid have been ob served during hypoxia induced at ground levels by breathing a gas 58 mixture low in oxygen content. Marotta 1 Hirai 1 and Alkins (14 0) found markedly eleva ted levels of 17 -hydroxycorticos teroids in both conscious and anesthetized dogs when their dorsal roots had been severed and a glass cannula inserted in the lumbo-adrenal vein with decompression to 171000 feet simulated altitude for two hours while breathing ambient air (P 02 78 mm Hg). There is evidence that continued exposure to the sa me hypoxic stimulus is accompanied by a return of adrenal cortical function. Horn bein (141) evaluated the effect of altitude on adrenal cortical response by measuring 17-hydroxycorticosteroids in 10 members of the American Pakistun Karakoram Expedition during their ascent of 251GGO feet to Himalaya's Peak and comparing these values to those subsequently obtained at sea levels. In a study conducted by Biddulph et al (142) I no significant difference in the 17-hydroxycorticosteroid output was observed 1 indicating that response of the adrenal cortex to hypoxia is only temporary. Lau and M secretion concurrent with changes in pH I PC02 I P02. 59 It was observed by Lin (133) and by Clifton (50) that, durin9 bed rest with exercise the urinary excretion both of 17-ketosteroids and of 17 -hydroxycorticosteroids is lower than during bed rest with no exer- cise. THE CIRCADIAN RHYTHM PHENOMENON It has been established that animu.ls and plu.nts perform certain activities at fairly fixed times of the day. As early as 1729 De Marian, an astronomer, described experiments in which plants were seen to maintain a diurnal rhythm of leaf movement. Although several names have been given to this physiological behavior of animals and plants such as diurnal variation, circadian rhythm and biological clock, the term circadian rhythm (ciru-a.bout, dies-a da.y) best describes this bio logical rhythm. Since animals rarely display a periodicity of exactly 24 hours. Pittendrich (144) suggests that biological clocks be divided into three categories: a) continuously consulted clock, b) internal timers, and c) clocks controlling "pure rhythm." Once the phase of a circadian rhythm has been set by an en vironmental stimulus, the rhythm will remain constant under favorable conditions for a long time. Circadian rhythm is affected by such para meters as light, temperature and previous environment. According to Aschoff (145), continuous light causes a decrease in the period of rhythm of diurnal anim:ds and an increase in the period of nocturnal 60 animals. Bennet-Clark et al (146) have found that previous environment affects the length of free running periods. Van Zandt (126) has reviewed this subject in considerable detail for calcium and phosphorus. The phenomenon of different cellulur activities being restricted to particular times of the day provides an example of these uniquely interesting biological control system, although the molecular mechanism of circadiun rhythm rem-:-tins an unsolved question. Schmeiger et al (147) demonstruted thut the nucleus is cupable of determining u circadiun rhythm which is loc.:1lizccl in the cytoplus rn. Since horm::mes [)lu y u m:1 jor role in coorclina tion, u nd in view of the u!Jiquity of physioloqicul rhythm, it Wl)Uld be expected that hor- monJ l E>ecre tion would commonly occur as u rhythrnica l phenomenon. Several studies h:1ve prnviclccl evidence of the fuct th excretion of 17 -hycln>xyc 1 r·tic.'s ter:--ids. According to Silverberg ar1=l Krie·:::er (1.:±8), once sufficient cen- tral nervous system mJ:urit:y is attained anci nyctohe:neral periodicity of plas rna 17 -hydroxycorticos reroid level is present, ~his periodicity 61 becomes an inherent feature of the organism and is unaffected by any alteration in central nervous function. laatika. inen and Vihko (14 9) observed that the lowest concentrations of plasma 17-hydroxycortico steroids were seen at midnight. In addition they found th.:1t solvo lyzable steroid conjugates were following the same pattern. These in vestigators found that the concentration of 17 -hydroxycorticosteroids is highest in the early morning and diminishes during the day 1 while the solvolyzable steroids follow the opposite pattern during this same period. In man I changes in the habitual day and night rhythm results in changes of the diurnal rhythm in the plasma corticoids. Nicholas et al (150) have su]gested that rhythm is dependent on cyclic varia tions in the release of ACTH. Accordincr to Liddle (151) 1 the m.:1jor rhythm in adrenal steroid secretion appears to be related in a fixed way to external environment and the concept that there is an adrenal cycle with an inherent periodicity of about 24 hours cannot be fully accepted. Bowmann et al (152) (153) found that plasnn 17 -hydroxycorti costeroids exhibited a signifkant circadian rhythm in rhesus monkeys during the first week of life. Plasma 17-hydroxycorticosteroid levels avera g e d 3 5 J-1 g . per 1 0 0 m l . at 8 - 9 A . M . I and dec lin eel to 1eve 1s of 29 J-Ig. per 100 ml. at 9:00P.M. and 24 )lCJ. per 100 ml. at midnight. At the same time I the infant monkey 1 altlnugh having a polyphasic 24 hours sleep pattern 1 also exhibited a tendency to sleep more at 62 1:00 P.M. and 3:00A.M. This suggested a relationship existing be tween sleep -activity cycles and steroid eye les in the infant monkey similar to that observed in the adult monkey. There seems to be a stability of pituitary-adrenocortical function which is not a function of age. Both the cardiovascular system and adrenal activity participate directly or indirectly in the rhythmic shifts of the neurovegetative tonus. As adrena lee to my eliminates the mitotic eye le 1 the sensitivity of tis sues may vary from time to time. Bennet-Clark et al (146) suggest that the periodicity in the relative specific activity of phospholipid phos phorus is obliterated by adrenalectomy 1 indicating a dependency upon the periodic secretion of adrenal hormones. According to Klien et al (154) 1 two aspects seem to be of high significance in t:he estimatio;1 of the influence of circadian rhythm on human efficiency 1 "the range of oscillation" 1 or the "position of the peaks". Failure of rhyth:ns to have their peaks about 12 hours apart is termed as dissociation. DissCJciation in human beinqs may lead to loss of health. Increased rhythm dissociation may occur with aging. Pincus (1) was the first iiwestigator to sh::>'N a circadian rhythm periodicity on the basis of estim2tion of urinary 17 - 1<:etosteroid in ali- quots taken ever)' six hours over a five to nine day period 1 using seve!1 male subjects. Mills (155) states that the rate of urinary excretion of 63 corticosteroids lags behind the plasma concentration by two or three hours. Urinary estimation 1 however 1 has the advantage of avoiding the potentially stressful procedure of vena puncture. Psychiatric disorders sometimes are associated with changes in physiological rhythms. Conrey et al (156) showed that a subgroup of effective psychotics had higher plasma 11-hydroxycorticosteroid values as compared with schizophrenic and other psychotic groups 1 and somewhat less regular rhythm. An accentuated fall and rise in plasma 11-hydroxycorticosteroid values in night and early morning samples respectively was observed in schizophrenic subgroups. The dependence of adrenal rhythm on social contacts suggests that a higher level of the nervous system may be involved. According to Aschoff (145) I rhythm is known to be absent in diseases associated with an altered state of conscious or an abnormal sleep pattern. It was shown by Doe et al (157) that plasma corticoid rhythm is absent in Cushing's syndrome. Krieger and Krieger (158) found that patients suffering from diseases of the pituitary and central nervous system differed significantly from normal subjects in the existence of a plasma 17-hydroxycorticosteroid variation. Cade et al (159) studied the diurnal variation of plasma cortisol concentration in a group of patients with hypertensio:-1 resulting from renal artery obstruction. The results of this study indicated the 64 existence of a grossly abnormal diurnal variation in plasma cortisol concentration. Kornel and Takeda (160) obtained similar results upon examining urinary excretions of 17-hydroxycorticosteroids. Confine ment stress has been shown by Frazier et al (2) to lead to alteration of circadian rhythmicity, even when phys ica 1 environment and activity schedules are held highly constant. Rhythmically oscillating biological variables may be either endogenous, independent of any rhythm in habit or environment, or exogenous, dependent upon external rhythm, or they may result from an interaction of both. Mills (161) recorded the circadian rhythm of a man spending 105 days in solitude underground. He noticed that he fell asleep and woke a little later day by day following a sleep/wake cycle of approximo.tely 24 1/2 hours. For eight weeks, excretion of sodium chloride followed a rhythm similar to that of potassium, but thereafter they became dissociated and increasingly irregular. Perkoff et al (162) found that an eight-hour shift in the timing of the cortisol cycle was somehow controlled by the sleep/wake activity of the subject, rather than by other daily changes in the en vironment. Orth et al (163) found17-hydroxycorticosteroid and 17- ketosteroid values to be at a minimum during early hours of sleep, with a rapid regular increase observed during sleep, and maximal concentrations found near the time of awakening. During the period of 65 awakefulness, the 17-hydroxycorticosteroids fell irregularly to reach minimal values again shortly after the subjects went to sleep. Several individuals on 19 and 33 hour sleep/wake schedules appeared to exhibit two 17-hydroxycorticosteroid and 17-ketosteroid cycles for each sleep/ wake period. These experiments show the pituitary adrenal cycle is not necessarily 24 hours in length but I rather 1 is a function of the c us tomary duration of a subject's sleep/wake cycle. Reinberg et al (164) determined the rhythmic excretion of 17-ketos teroicls I 17 -hydroxycorti cos teroids and potassium usirHJ eight a sthnB tics a ncl four apparently healthy young men as controls subjects staying in bed at night. The excretion of these substances in both groups reached a minimum at night and a m3.ximum in the morning. In all eiqht subjects I asthma attacks occurred at a time when adrenal and potassium excretions vvere low. PLAN OF PROCEDURE The data presented in this dissertation were obtained during three bed rest studies conducted in the Nelda Childers Stark Laboratory for Human Nutrition Research 1 a component part of the Texas Woman's University Research Institute 1 as part of an extensive investigation sponsored by the National Aeronautics and Space Administration to study various metabolic reactions during immobilization with and with- out accompanying exercise. These studies are Pc1rt of an extensive research program designed to examine the response of healthy adult male subjects to conditions which closely resemble those encountered by astronauts during space flight. PERIODS OF THE STUDY DURING THE 1968 INVESTIGATION This study consisted of two bed rest periods accompanied by ambulatory periods as follows: Equilibration Period, 29 days, June 3- July 3, 1968 Bed Rest Number One, 28 days, July 3 - July 30, 1968 Interim Ambulatory Period, 14 clays, July 31 -August 14, 1968 66 67 Bed Rest Number Two, 28 days, August 14- September ll, 1968 Post-Bed Rest Period, 14 days, September ll -September 24, 1968 A record was made of height and weight changes in the subjects throughout the study. Male orderlies attended to the hygienic needs of the subjects during immobilization. During these periods they were spoon-fed by the three dietitians on the study. SUBJECTS ---OF THE STUDY Name Age Height Weight Occupation (years) (inches) (pounds) Initial Final AA 25 70 114 152.5 147 University s tuc!en t BB 22 72 152 149.5 University student EE 22 74 1/2 181 176.25 University s tuc!en t FF 20 69 1/2 155 149 University student GG 24 70 1/4 170 164.75 University student HH 21 69 170 165.5 University student EQUILIBMTION PERIOD This period lasted for 29 days. The six subjects led a normal life while engaged at various tasks in the laboratory, together with moderate exercise, for eight hours each day. They were required to 68 be in bed at l 0:3 0, with the lights out and the bed cubicles darkened from ll : 0 0 P • M • until 7: 0 0 A. M • BED REST PERIOD NUMBER ONE During this phase of the study, the subjects were completely immobilized for a period of 28 days. Urine and serum samples were collected six times on the first clay of immobilization. The second day samples were collected four times at three-hour intervals. There- after, urine samples were collected at 8 A.M., 12 Noon, and 8 P.M. daily, since Circadian Rhythms were to be investigated. The radio- active isotope, 47calcium in the form of 47cacl3 was ingested in 40 milliliter~,; of milk on the first moming of immobilization. Throughout immobilization, the men u.ss umecl a horizontu.l position on a single bed equipped with one pillow. They were encour- aged not to lift their heads, although very limited movement of the arms and legs was allowed. Reading was clone with the aiel of glu.sses equipped with prismatic lenses, and television was watched on hospi- tal type television sets. During this period of immobilization, trained male orderlies were present around-the-clock to attend to the hygi- enic needs of the subjects u.nd to safeguard their movement. Each subject was spoon-fed by one of three dietitians, and a record was kept of their individual daily food consumption. 69 During immobilization, x-rays were taken each day. Each man was lifted by three or four men from the bed to a stretcher cart for transportation across a hall to the X-ray Laboratory, where they were carefully lifted onto the x-ray table. The x-ray table was the same height as the mobile stretcher, which in turn was the same height as the beds. INTERIM AMBULATORY PERIOD During this 14-day period of ambulation, the six subjects en gaged in compulsory physical activity. They worked in the laboratory during the periods when they were not engaged in the physical exer cise program. Meals were consumed in the metabolic ward under the supervision of the dietitians. Urine samples for this period of the study were analyzed on a 24 -hour basis. BED REST PERIOD NUMBER TWO The variable in this 28-day bed rest was the introduction of an exercise program using the Exer-Genie and Exer-Grip Exercisers (Fig ure 10). Measurements of foot and hand action, squeeze, and iso metrics were made with the Lufkin Anthropometric (woven) Tape with a Gurlick Spring Attachment (3176ME). The measurements of hand and foot action were started on the second day of immobilization, while the squeeze exercise was introduced on the third day and isometrics 70 were begun on the fourth day of immobilization. Subjects AA, EE, and GG were allowed to exercise at will, while Subjects BB, FF, and HH exercised according to a firm schedule which was closely supervised, The exercise program employed is given in Diagram I 1 with an illustra tion of a man exercising while recumbent shown in Figure 10. The exercise periods called for by the "At Will" group also were supervised, but this group did not exercise unless they desired to do so. As in the previous bed rest, the same level of 47 caClz was in corpora ted into the milk during the first morning of recumbency. In this study, each subject's daily urinary output was collected individ ually whenever he voided. A 24 -hour sample was accumulated for each subject for the purpose of 17-ketosteroid analysis. This procedure was continued through the remainder of the study. In order to standardize the 24-hour analyses, each subject was encouraged to have a 12 noon sample. POST-BED REST PERIOD This final period of the study lasted for 14 days. During this time 1 the subjects engaged in supervised physical activity as much as their physical condition permitted. They also performed limited duties in the laboratory whenever they were not exercising. They re entered the university at the beginning of the 1968-69 term. During 71 this period, analyses of 17-ketosteroids also were made on a 24-hour bas is • D IA GRAM I EXERCISE PROGRAM FOLLOWED IN BED REST II OF THIS INVESTIGATION Instruments employed: Exer-Genie and Hand Gripper; Setting of Exer-Genie during bed rest 1 8 pounds; Position of bed rest subjects throughout the study 1 including the exercise period: subjects in horizontal position 1 completely recumbent and lying on the back; Steps in the Exercise Program: Time 1. Isometric exercise with strap of Exer Cenie pulled up to the chest I with subject stretching ... l 0 seconds 2 . Leg exercise (Exer-Genie) . 6 minutes 3. Rest . 2 minutes 4. Hand - finr;ers squeezin9 gripper 1 minute 5. Rest 2 minutes 6 . Isometric exercise (Exer-Genie) 10 seconds 7. Arm exercise (Exer-Genie) 6 minutes 8 . Rest . 2 minutes 9. Hand FinCJers squeezing gripper 1 minute 10. Rest . . 2 minutes 11. Isometric exercise (Exer-Genie) 10 seconds 12. Leg exercise (Exer-Genie) 6 minutes To ta 1 isometric exercise 30 seconds Total isotonic exercise 2 0 minutes 74 PERIODS OF THE STUDY DURING THE 1969 INVESTIGATION This study consisted of one bed rest period accompanied by a pre-bed rest and a post-bed rest ambulatory period as follows: Equilibration Period, 17 cL:ws, July 7 -July 24, 1969 Bee! Rest Period, 56 clays, July 24- September 17, 1969 Post-Bee! Rest Period, 15 clays, September 17- October 1, 19G9 The cluta of the ,,uthor included in this dissertation covers only the equilibration period of Lhe 1969 InvesLil]Cltion. SUBJECTS OF THE S'J'UDY PurticiputinCJ in this study were seven university students be- tween 20 and 25 years of uqe, one! one older mun 40 yeurs old. The initial and final weiCJhls nf the subjects were as follows: 75 Name Height Weight (inches) (pounds) Initia 1 Final 1A 70 157 153 2A 70 142 146 1/2 3A 72 147 146 4A 69 159 157 6A 73 183 178 7A 71 175 176 SA 74 199 196 9A 70 131 137 EQUILIBRATION PERIOD This period lasted for 17 days. The eight subjects led a norma 1 life while engaged at various tasks in the laboratory. They also participated in moderate exercise each clay. They were required to be in bed at 10:30 P.M, 1 with the lights out and the bed cubicles darkened from 11:00 P.M. until 7:00A.M. During this period the diet was planned to contain 2600 cal- aries including 1.0 gram calcium and 100 grams protein. 76 GENERAL PROCEDURE USED IN THE STUDY Throughout the entire study, the subjects were housed and fed in the metabolic ward of the Nelda Childers Stark Laboratory for Human Nutrition Research at the Texas Woman's University Research Institute. Specially trained dietitians supervised the preparation of the meals which were optimum in all major nutrients. The food fed during the Pre-Bed Rest Equilibration Period was regular food chiefly purchased through the University Purchasing Department. That fed during the Bed Rest and the Post-Bed Rest Periods was astronaut food furnished by Manned Spacecraft Center 1 Houston. The daily food intake of each subject was recorded throughout the study by individual foods. This study was conducted under close medica 1 supervision. A record was made of height and weight changes throughout the study. Male orderlies attended to the hygienic needs of the subjects during immobilization 1 and safeguarded the movements of the subjects. METHODOLOGY PROCEDURE FOR THE DETERMINATION OF URINARY 17- KETOSTEROIDS The method used by the author for the determination of tota 1 17-ketosteroids is based on the Zimmerman reaction (165) 1 which in- eludes three main steps: (a) Hydrolysis to free the 17-ketosteroids 77 from the sulfate and glucuronic bonds; (b) Purification to remove inter ferring substances which react with the Zimmerman reagent to give color; and (c) Use of the Zimmerman reagent to produce the charac teristic red -purple color. Extraction is done with carbon tetrachloride because of the lack of inflammability and high solvent power for ster oids of this reagent. REAGENTS 1 . 1% m-Dinitrobenzene: Grade V. Specially prepured for the Zimmermun Reuction in steroid determinations. Prepare quantity needed in absolute alcohol. Make up fresh for each run. Keep in clark when not in use. 2. Dehydroisoandrosterone Standard: 10 mg/100 ml absolute alcohol 3. 8N Potassium Hydroxide: Use highest purity (electrolytic pellets, if possible) potus sium hydroxide. Store in clark brown bottle. Stable for about one month. 4. Carbon Tetrachloride: Reagent grade 5. 0.85% saline: 6. Sodium Hydroxide: Pellets 7. Hydrochloric Acid: Concentrated (s p. gr. 1.18-1.19) 8. Absolute Ethanol: 78 9. 70% Ethanol: Prepare fresh as needed PROCEDURE 1. Set up 50 ml. screw-cap pyrex centrifuge tubes: Unknown(s) Standard Reagentblank Urine 10.0 ml Standurd l.O rnl 0. 85% Su line 9.0 ml 10.0 ml Cone. HCl 3.0 ml 3.0 ml 3.0 ml 2. Mix thoroucJhly unci hydrolyze in u boiling wuter bath for 10 minutes. Coul immediately. 3. 1\ccurotcly udd 10 rnl. curbon tetrachloride into each tube. 4. SLoppc~r ond shokc un ,l mechunicul shaker for 10-12 minutes. S. l\ftcr phc1sc:; helve :;cpurc1Lecl, carefully suclion off the upper aqueous layer. G. Add about 15-20 pellets uf sodium hydroxide t_o each tube. Shake u(_}ain us before. Suctinn c1ff any uqucnus luy·cr which might re- rna in from s le p 5 . 7. Immeclia tely filter the curbnn tetrachloride extruct through qualitative filter paper. (!\~)~Jrc_>ximately 8 or 9 ml. of extrac[ will be recoveree!). 8. Pipet 3 mi. aliquocs .'f each filtrate into each of 2 graduated centrifuge tubes and e\'aporate to c!r'y"ness in a 90-95° C. ·water bath. (One tube will be used as the test; the ocher a blank). 79 9. After the tubes have cooled to room temperature 1 add: Reagent Blank Standard Unknown(s) Test Blank Test Blank Test Blank Absolute Ethanol 0. 5 ml 0. 5 ml 0.5 ml 1% m -dinitrobenzene 0.5 ml 0.5 ml 0.5 ml 8 N KOH 0.2 ml 0. 2 ml 0.2 ml 0.2 ml 0.2 ml 0.2 ml 10. Mix thoroughly and let stand in the clark exactly 30 minutes to develop color. 11. Forcefully but carefully 1 add 5 rnl. of 70% ethanol to each tube in such a manner as to produce adequate mixing. 12. Set Coleman Jr. Spectrophotometer at a wave length of 520 mJl. Read test reagent blank against blank reagent blank as zero. Record. 13. Read standard against standard blank as zero. Record. 14. Read unknown(s) against unknown blank(s) as zero. Record. CALCUlATION OD Unknown - OD Reagent Blank x 0 . 1 x 24 hour vol. (ml) = OD Standard - OD Reagent Blank l 0 mg. 17 -Ketosteroids/24 hours 80 PROCEDURE FOR THE DETERMINATION OF URINARY 17-HYDROXYCORTICOSTEROIDS The method employed at the 1WU laboratories for the analysis of urinary 17 -hydroxycorticosteroids is adapted from the procedure out- lined by Porter and Silber ( 16 6). l. Urine collection and storage. A urine specimen is collected in a plastic bottle. Record the volume and keep in the frozen state un- til ready for extraction. 2. If the specimen is frozen, remove this from the deep freeze and permit it to thaw. Thoroughly mix specimen and adjust pH to 2.4 with sulfuric acid by means of a pH meter. 3. For extraction, the screw cap test tubes are set up as fol- lows: Reagent Unknown Standard Blank Urine, pH adjusted 8 ml. Working Standard 1 10 pg/ml. 8 ml. Distilled Water 8 ml. n -Butanol 4 ml. 4 ml. 4 ml. 4. The sample is shaken for l 0 minutes on a mechanical shaker 1 and then is centrifuged at 3000 RPM for 10 minutes. 81 5. The supernatant (butanol) layer is transferred to a set of the clean screw cap test tubes by means of "serum lifter". 6. A second similar extraction is made, and the butanol ex- tracts are combined. The aqueous phase may be discarded. 7. This butanol extract then is shaken with 1 ml. of 10 per cent potassium carbonate on a mechanical shaker for 30 seconds and is centrifuged for 10 minutes at 3000 RPM. 8. The butanol extract again is transferred to a new set of screw cap test tubes by means of "serum lifter". 9. Immediately one gram of anhydrous sodium sulfurate is added to remove any water reserved. The mixture then is shaken on a mechanical shaker for 30 seconds and is centrifuged for 10 minutes at 3000 RPM. 10. Finally, the butanol extract is decanted into a clean cen- trifuge tube, and is stored in the refrigerator until ready to continue. ll. Prepare tubes for color development. For each butanol ex- tract, set up two tubes as indica ted below. Then place in refrigerator until needed. A. tube (sample) --- 4 ml. phenylhydrazine sulfuric acid reagent. B. tube (sample blank)--- 4 ml. 18N sulfuric acid. 82 12. Add 2 ml. of butanol alcohol extract to both the A tube and B tube. 13. The tubes are mixed with a mixer. Incubate in a water bath at a temperature of 60° C for exactly 30 minutes. 14. At end of incubation transfer to ice-water bath for 5 minutes. 15. Read in spectrophotometer at 410 m)l wave length. Set zero OD with tube B (sample blank) and read tube A (sample) against it. CALCULATION: OD Unknown - OD Reagent Blank X l. 10 = mcg./ml. OD Standard - OD Reagent Blank Total Volume X meg ./ml. 2 · 1000 = mg ./24 hours STANDARDIZATION: Stock Hydrocortisone Standard (2 00 ).lg ./ml.) Dissolve exactly 2 0 mg. of hydrocortisone (free alcohol) in about 1/2 ml. of alcohol and dilute to exactly 100 ml. with distilled water. Working Hydrocortisone Standard (1 0 ).lg ./ml.) Dilute 5 ml. of stock standard to 100 ml. with distilled water. 83 REAGENTS: (l) n-Butanol ---Reagent grade, obtainable from City Chem ical Corporation, New York, or Distilled Industrial Products, East man Kodak. The reagent must be checked with the phenylhydrazine and sulfuric acid reagent to give a low blank reading. (2) l8N Sulfuric Acid --- Carefully add 12 7 ml. of concen trated sulfuric acid (with cooling) into 100 ml. of distilled water. (3) Phenylhydrazine-Sulfuric Reagent--- Dissolve 49 mg. of recrystallized phenylhydrazine in 75 ml. of l8N sulfuric acid. Recrystallized Phenylhydrazine--- Dissolve phenylhydrazine hydrochloride in a minimal amount of absolute alcohol by heating. Allow to cool at room temperature. Then place in a refrigerator ot least for one hour. Filter through a sintered glass filter. Transfer crystals, which should be peach colored, to a clean container and Place in a des sica tor. (4) 10% Potassium Carbonate--- Dissolve 10 gm. of potassium carbonate in l 00 ml. of distilled water. (5) 2N Sulfuric Acid--- lu1 approximate dilution of l to 10 may be made of the l8N sulfuric acid. (6) Sodium Sulfate, Anhydrous --- Rea gent grade. PRESENTATION OF FINDINGS WITH DISCUSSION PART I OF THE STUDY Six healthy adult male subjects participated in a 113 day Bed Rest study which consisted of five different periods- Pre-Bed Rest, Bed Rest I (no exercise), Interim Ambulatory, Bed Rest II (exercise), and Post-Ambulatory. The study using radioactive Ca 47 as a tracer was designed primarily to observe changes in bone density clue to immobilization and the effect of exercise as a possible a meliorating measure. Also included in this study was a detailed observation of various metabolites throughout all periods of the investigation in order to determine both the effects of different levels of activity and the existence of a circadian periodicity in relation to these metabo- lites. The daily urinary excretion data on 17-ketosteroicls are re- corded in the Appendix in Tables I and II. The statistical comparisons of 17 -ketosteroid excretions during various periods of the study are reported in Table III. The statistical data relating to exercise are presented in Table IV and Table V. The cia ta recorded in Table VI show the statistical comparison on circadian rhythm. Table VII 84 85 contains the data on coefficient of correlation values between 17-keto- steroid excretion and seven independent variables, while Table VIII contains the comparison of values between 17 -hydroxycorticos teroid excretion and seven independent variables. Comparison of Urinary 17-Ketos teroicl Excretion during the Initial Equilibration and the Bed Rest I Periods Statistical analysis of the 17-ketosteroid urinary excretion is shown in Table III {Appendix) unci is depicted graphically in Figure 11. SUBJECT 88 The average doily urinary excretion of 17-ketosteroids by this subject during Pre-Bed Rest unci Bed Rest I are presented in Table III, Part A. The amount of urinary 17-ketosteroids excreted by this subject during Bed Rest I is appreci2bly greater in quuntity than during Pre- Bed Rest by a difference which was highly significant (P< 0. 001). SUBJECT FF When the amount of urinary 17-ketosteroids excreted by this subject during the Pre-Bed Rest Period was compared 'INith that ex- creted during Bed Rest I, the greater quantity excreted during the Bed Rest Period was highly significant (P< 0.001). See Table III, Part B. 86 SUBJECT HH During Bed Rest I 1 the urinary 17-ketosteroid excretion by this subject was greater than during the Pre-Bed Rest Period. This difference was highly significant (P< 0. 001) as shown in Table III 1 Part C. SUBJECTS BB 1 FF, HH (Exercising Regularly) As shown in Table III 1 Part D 1 when the data for the subjects who exercised regularly during the second Bed Rest were pooled to- gether for comparison of urinary 17-ketosteroids during Pre-Bed Rest and Bed Rest I 1 the a mount excreted during Bed Rest I was significantly higher than that during the Equilibration Period, the difference being highly significant (P< 0.001). Comparison of Urinary 17-Ketosteroid Excretion during the Initial Equilibration and the Bed Rest I Periods SUBJECT AA A higher level of 17-ketosteroids was excreted by Subject M during Bed Rest I than during the Initial Equilibration Period. The dif- ference was highly significant (P< 0. 001). See Table III 1 Part E. SUBJECT~ The excretion of urinary 17-ketosteroids was significantly 87 higher (P< 0.01) during Bed Rest I than during the Initial Equilibration Period by Subject EE. See Table III, Part F. SUBJECT GG A comparison of the 17 -ketos teroids excreted in the urine of Subject GG during the Initial Ambulatory Period with that of Bed Rest I shows that the quantity during Bed Rest I was higher than during the Pre-Bed Rest Period by a difference which was s ta tistica lly significant (P<0.05). See Table II, Part G. SUBJECTS AA, EE, GG (Subjects who Exercised "at Will") When the data of the "at will" exercise group were pooled, the urinary excretion of 17-ketosteroids durinc; the recumbency period of Bed Rest I, when no exercise wos taken, surpassed that of the Initial Ambulatory Period by a highly siqnificont difference (P< 0. 001). See Table III, Part H. ALL SUBJECTS When the data for all six subjects were pooled together for the comparison of urinary l 7- ketosteroid excretion during the Initial Equili bration Period and that of Bed Rest I, Table III, Part I shows that the Bed Rest Period was significantly higher (P< 0. 001) than that during the Pre-Bed Rest Period. Figure 11 compares t:hese periods graphically for all subjects. 88 The results of this study show that 1 during the Bed Rest Period 1 the urinary excretion level of 17-ketosteroids was significantly higher for all subjects than during the Pre-Bed Rest Period. These findings are consistent with those of Clifton (50) 1 Lin (133) 1 and Burnstein et al (131). Immobilization causes a condition of stress which enhances the secretion of adrenal steroid hormones, Increase in the activity of the adrenal cortex occurs concomitantly with various types of depressive reactions. Comparison of Ambulatory Periods with Each Other See the same series of parts of Table III as was cited in the previous comparisons 1 as well as Figure 11. SUBJECT BB There was a distinctly significant difference (P< 0.02) in the urinary excretion of 17-ketosteroids exhibited by this subject when the Pre-Bed Rest Period was compared with the Interim Ambulatory Period I the latter showing the higher excretion of urinary 17-ketosteroids. No significant difference was noted in the excretion of urinary 17-keto- steroids between the Initial and Final Periods or between the Interim and the Fin a 1 Ambulatory Periods • 89 Figure ll. COMPARISON OF MEAN 17-KETOSTEROID EXCRETION DURING DIFFERENT PERIODS OF THE STUDY WITH THE DATA FOR ALL SUBJECTS POOLED TOGETHER Ke; D Pre-Bed Rest ~ Bed Rest I Comparision Of Mean 17-Ketosteriod Excretion During Interim Ambulatory Different Per1ods Of Study ru Period (J) 0~ - {j) Bed Rest II Ocr: D a:::> 13 wo 12 Post-Bed Rest I-I ~ (J)'i" I I 0(\J 10 f-er:w WCL 9 '::L{j) 8 I~ ['-"""_q 7 6 6:~ 5 <(_J z~ 4 -2 a:~ 3 :::J 2 I AA BB EE FF GG H H SUBJECTS OF THE STUDY <.0 0 91 SUBJECT FF There was no significant difference observed in the excretion of urinary 17-ketosteroids by Subject FF during the Initial and Ambulatory Periods, the Initial and Final Periods, or the Interim Ambulatory and Fina 1 Ambulatory Periods of the study. SUBJECT HH The difference in the excretion of urinary 17-ketosteroids during the Initial Equilibration Period and the Interim Period was not statistical ly significant for Subject HH. This also was true when the Initial Period was compared with the Final Ambulatory Period, and when the Interim Period wus compared with the Final Ambulatory Period. SUBJECTS BB, FF, HH (Regular Exercisers) When the data for the three subjects in the regular exercise group were pooled in order to compare the pairs of the Ambulatory Periods with one another, there was a distinctly significant difference (P< 0. 05) between the Initial and Interim Periods, the Initial Period be ing higher, with a slightly significant difference (P< 0.10) between the Interim and Final Ambulatory Periods with values higher during the Final Period than during the Interim Period. No significant difference was observed when a comparison was made between the Initial and Final Ambulatory Periods. 92 SUBJECT AA There was no significant difference found between any of the pairs of the three Ambulatory Periods for Subject M throughout the study. SUBJECT EE In analyzing the data for Subject EE no statistically significant difference was observed between the Initial and Interim Periods 1 the Initial and Final Periods 1 or the Interim and Final Periods. SUBJECT GG This subject showed a decrcuse in urinury 17-ketosteroid ex cretion occurring durinc; the Interim Ambulu tory Period. When this was compared with the Initial Period 1 the difference was shovvn to be only very slightly significant (P< 0.10). There was no siC}nificant differ ence between the urinary excretion of 17-ketosteroids during the Initial and Final Periods nor between the Interim and Final Periods of the study for this subject. SUBJECTS AA 1 EE 1 GG ("At Will" Exercisers) When the data of all three subjects who exercised "at will" were pooled together 1 no s ta tis tica lly significant difference was found between any of the pairs of the three Ambulatory Periods . 93 ALL SUBJECTS When the pooled data for all six subjects were compared sta tistically 1 it was noted that the excretion of urinary 17-ketosteroids during the Pre -Bed Rest Period was significantly higher (P < 0. 05) than during the Interim Ambulatory Period. During the Interim Ambulatory Period the 17-ketosteroid excretion was higher than during the Post Bed Rest Period by a difference which was only slightly significant (P < 0.1 0). No significant difference was found between the Pre-Bed Rest and Post-Bed Rest Periods • All subjects 1 except Subject AA I excreted higher amounts of urinary 17-ke tos teroids during the Pre-Bed Rest Period . During the Interim Ambulatory Periods the lowest levels of 17-ketosteroid ex cretions of the entire study were recorded for all subjects. Similar findings were noted by Clifton (50) 1 Lin (133) 1 and Whedon et a l (128). There seems to be a tendency for the urinary excretion levels of 17-ketosteroids to fall rapidly following immobilization. These dif ferences could be explained on the basis of the fact that somewhat more vigorous exercise was taken during the Lnterim Period than during the Initial and Final Periods. 94 COMPARISON OF BED REST I WITH BED REST II The results of the statistical analysis of the data from the two Bed Rest Periods are given in Parts A through I of Table III (Appendix) and in Figure 12. SUBJECT BB The quantity of urinary 17-ketosteroids excreted by this sub- ject during Bed Rest I was higher than during Bed Rest II, the statisti- cal significance being (P< 0.01). SUBJECT FF The excretions of this subject showed a slightly significant difference (P< 0.1 0) between the two Bed Rest Periods, the value be- ing higher for Bed Rest I than for Bed Rest II. SUBJECT HH When a comparison was made between the two periods of Bed Rest for Subject HH the quantity of urinary 17-ketos teroids excreted during Bed Rest I was higher than that during Bed Rest II, the difference being barely significant (P< 0.1 0). 95 Figure 12. COMPARISON OF MEAN DAILY EXCRETION OF 17-KETOSTEROIDS DURING BED REST I (NO EXERCISE) AND BED REST II (EXERCISE) Mean Daily Excretion Of 17- Ketosteroids (./) During Bed Rest 0 ('.'liTH NID ,'/ITHOUT EXeRCISE) Ul - 0:: 0 :::) 0:: 0 Key w I 12 1- ~ Rest I (No Exercise) (./) N fl ~Bed 0 a:,o 1- w. D Bed Rest n (With Exercise) (L w 9 ::s:::: Ul I 2 8 ['- tD CJ) 97 SUBJECTS BB, FF, HH (Who Exercised Regularly) The pooled data for the three subjects who exercised regularly during Bed Rest II showed a highly significant difference (P < 0. 001) in the excretion values of 17 -ketosteroids between the two Bed Rest Periods, the values being greater for Bed Rest I, when no exercise was taken. SUBJECT AA A comparison of Bed Rest Periods for Subject M indicated that a significant decrease (P< 0. 01) had occurred in urinary 17 -ketosteroid excretion during Bed Rest II in comparison with that observed in Bed Rest I. SUBJECT EE The urinary 17-ketosteroid excretion of this subject showed a highly significant decrease during 13ed Rest II in compctrison with Bed Rest I, the difference being distinctly significant (P< 0.001). SUBJECT GG This subject excreted a higher amount of urinary 17-ketosteroids during Bed Rest I than during Bed Rest II, with a difference which was distinctly significant (P < 0. 02). SUBJECTS AA, EE, GG (vVho Exercised "At \Vill") The statistical analysis of the pooled data of these three 98 subjects revealed the finding that there was a significantly greater amount of urinary 17 -ketosteroids excreted in Bed Rest I with no exer cise than during Bed Rest II 1 when regular supervised exercise was taken I the difference being highly significant (P< 0. 001). ALL SUBJECTS When the data on the total urinary excretion of 17-ketos teroids for all six participants in the study were combined, the "t" test showed that the quantity of 17-ketosteroids excreted during Bed Rest I with no exercise surpassed that during Bed Rest II, with exercise, by a highly significant difference (P< 0. 001). In all cases during Bed Rest I, the excretion values of urinary 17-ketosteroids were significantly higher than those observed during Bed Rest II. This finding probably could be attributed to the introduc tion of exercise as a variable during Bed Rest II, when all subjects were engaged in programmed exercise either on a routine or on an "at Will" basis 1 whereas, during Bed Rest I, there was no exercise taken by these subjects. This is consistent with the findings of Knigge et al (132). 99 COMPARISON OF URINARY EXCRETION OF 17 -KETOSTEROIDS DURING BED REST I AND BED REST II The statistical data pertaining to the level of exercise is found in Table IV and Table V (Appendix). Table IV shows the comparison of urinary 17-ketosteroid ex- cretion during the two Bed Rest Periods by the regular and the "at will" exercise groups. There was a highly significant difference (P< 0.01) found between the regular exercise group and the "at will" exercise group during Bed Rest I, the "at will" group being higher. No significant difference was observed between the two groups clurinq Bed Rest II when the routine exercise was introduced to the subjects. Upon comparing Bed Rest I and Bed Rest II for the "at will" exercisers, it was noted that there was a highly significant difference (P< 0.001) in the amount of urinary 17-ketosteroid excretion, with the greater excretion occurring during Bed Rest I. Similarly when the two Bed Rest Periods were compared as to the urinary excretion of 17-keto- steroids by the group exercising regularly, the values for Bed Rest I were noted to surpass those of Bed Rest II, by a difference which was highly significant (P< 0.001). 100 When a comparison was made between the regular exercise group and the "at will" exercise group during Bed Rest I there was a significantly (P< 0.01) higher rate of excretion by the "at will" group than by the regular group. When these sa me groups were compared during Bed Rest II when actual exercise was introduced 1 a considerable decline was noted in the excretion rate of 17 -ketosteroids in both groups 1 although excretion levels of 17-ketosteroids remained higher in the "at will" group than in the regular group. Subject EE had the highest rate of 17-ketosteroid excretion of all of the subjects participating in this study. As a member of the "at will" group during Bed Rest II 1 he was observed to exercise the least amount of time. In reviewing the statistical information I it should be considered that the initial excretory rates of 17 -ketostcroicls were higher in the "at will" group. This is in accordance with the find ings of Dietrick et al (127) 1 W11edon et al (128) 1 Clifton (50) I and Wu (129) where individual differences in the normal excretory levels of 17-ketosteroids and other steroid hormones were quite apparent. When the excretory levels of the groups exercising regularly and those exercising "at will" were compared betvveen Bed Rest I and Bed Rest II 1 a highly significant decline (P< 0. 001) was observed in the excretory levels of 17-ketosteroids during Bed Rest II in both groups . This indicates that participation in exercise tends to lmA.rer 101 the urinary excretion of 17 -ketosteroids. Lin (133) has reported the sa me phenomenon. CIRCADIAN PATTERN OF URINARY 17-KETOSTEROID EXCRETION DURING BED REST I As it was noted earlier in this report, in order to determine whether or not there was any degree of rhythmicity in the urinary ex- cretion of 17-ketosteroids , the urine excreted during the 2 8 day Bed Rest was collected in three aliquotsduring a 24 hour period. For analytical purposes, the intermediate urinary voids were pooled for each individual at 8 A.M., 12 Noon, and 8 P.M. daily. The statistical information regarding the content of 17-keto- steroids in these collections is found in the Appendix in Table VI and Figures 13 and 14. SUBJECT BB When pairs of the three collection periods for urine were com- pared for quantities of excreted 17-ketosteroids by means of the "t" test, the amount excreted by Subject BB during the period from 12 Noon to 8 P.M. was higher than that excreted between 8 P.M. and 8 A.M. , but the difference was not significant. The 17-ketosteroid excretion from 8 A.M. until 12 Noon was significantly higher (P< 0. 01) than the excretion from 12 Noon to 8 P.M. There also was a highly significant 102 increase (P< 0.01) in the 17-ketosteroid excretion between 8 A.M. and 12 Noon, as compared to the excretion occurring between 8 P.M. and 8 A.M. During the sub-period from 8 A.M. until 12 Noon the highest level of this steroid hormone was excreted. SUBJECT FF The quantity of urinary 17 -ketosteroids excreted by Subject FF from 8 A.M. to 12 Noon was greater than that from 8 P.M. to 8 A.M. by a significant difference (P< 0. 05). The 17 -ketosteroid excretion from 8 A.M. to 12 Noon also surpassed that from 12 Noon to 8 P.M. by a significant difference (P< 0.05). There was no statistically signifi cant difference in the 17-ketosteroid excretion from 8 P.M. to 8 A.M. and that from 12 Noon to 8 P.M. The 8 A.M. to 12 Noon samples con tained the highest aggregate value of 17-ketosteroid excretion. SUBJECT HH The statistics for this subject revealed the finding that the uri nary 17-ketosteroid excretion from 8 A.M. to 12 Noon greatly exceeded the excretion from 8 P.M. until 8 A.M., the level of significance of the difference being (P< 0. 0 01). The 8 A.M. to 12 Noon collection also markedly surpassed that from 12 Noon to 8 P.M. (P< 0.01). The period from 8 P.M. to 8 A.M. was not significantly different from that of 12 Noon to 8 P.M. 103 Figure 13. RHYTHMIC liT 0 F 17-KETOSTEROID EXCRETION w r.J)Ul Key: ou 1.3 Rhythmicity Of 17-Ketosteroid Excretion -a:~ ow a: 1.2 D 8PM-8AM o:::x:) ww$ 1.1 ~ 8AM-12NOON 1-0~ 1.0 r.J)Zw Qio_ 0.9 1<:1 f\.JOON-8 PM 1-~Ul 0.8 ws2 0.7 ::s:::\-<1: l(j)a: 0.6 ['-W~ -a:::i 0.5 >-o_j o:w:;; 0.4 <(CD~ 0.3 z~ 1-' C> .+:> 105 SUBJECT AA This subject excreted the largest amount of urinary 17-keto steroids during the period from 8 A.M. until 12 Noon. The quantity of 17-ketosteroids excreted during this period markedly surpassed that excreted from 8 P.M. until 8 A.M. (P< 0.001). Similarly, the morn ing value exceeded that from 12 Noon until 8 P.M. by a highly signifi cant difference (P< 0.01). No significant difference was noted, how ever, between the periods 12 Noon to 8 P.M. and 8 P.M. to 8 A.M. SUBJECT EE This subject showed a higher amount of excretion of urinary 17-ketosteroids during the 8 A.M. to 12 Noon period than did any other subject, as shown in FiCJure 13. The amount of 17-ketosteroicls excreted from 8 A.M. until 12 Noon surpassed the amount excreted from 8 P.M. until 8 A.M. by a difference which was highly significant (P< 0.001). The quantity of 17-ketosteroids excreted during the same period which ended at noon surpassed that from 12 Noon until 8 P.M. by a highly significant difference (P< 0. 001). This subject also showed a very slight increase (P< 0.10) in the excretion of 17-k.eto steroicls during the 12 Noon until8 P.M. collection as compared to the 8 P.M. to A.M. collection. 106 SUBJECT GG This subject also excreted the highest level of urinary 17-keto steroids from 8 A.M. to 12 Noon. In comparing the excretion values for this period with the periods from 12 Noon to 8 P .M, and from 8 P ,M, to 8 A.M., the difference was determined to be statistically significant in both cases (P< 0.01). Again, the period from 8 P.M. to 8 A.M. was not significantly different from the period from 12 Noon to 8 P.M. ALL SUBJECTS When the data for all subjects were pooled together for Bed Rest I, the sub-period from 8 A.M. to 12 Noon showed the highest level of 17-ketosteroid excretion. The excretion level of 17-ketosteroicls during this time, when compared with that from 12 Noon until 8 P.M. was barely significant (P< 0.10). When it was compared with that from 8 P.M. to 8 A.M., the difference Wi1s only moc!erC1tely significant (P< 0.05). No significant difference was found between the 12 Noon to 8 P.M. and the 8 P.M. to 8 A.M. collection periods. The period from 8 P.M. to 8 A.M. ranked lowest in excretory levels of 17-ketosteroicls. This is shown graphically in Figure 14. 107 Figure 14. TOTAL 24-HOUR 17-KETOSTEROID EXCRETION (ALL SUBJECTS COMBINED) 108 I -+- "'~ r-- ~ -~ !,_') L U ' :J >< _() ::: (J) o .____ _, rt: rc w,A _-)-) I .,... ___.n,) . r c/lT IJ ....--~Q~ rr: '""""'"'~""'"'>l uJ (L I'- 10 lJ] -.;! <'1 (\J - 0 ci c._) ci c5 '~·i c5 c5 (t!ilOH t!Jd S~"'J\f~.L iliW-l NVJv'J) JSI~)U]X3 ON HJ..IM .. LSJCJCJ8 0NICJilO SOOitlJlSOlJ'>-1-LI Atl'v'NIC:ln 109 Although some individual variations were observed in the peri odicities of the subjects of the study 1 the pooled data for Bed Rest I indicated the existence of a marked characteristic circadian cycle in 17 -ketosteroid excretion. The highest levels of excretion by all sub jects were attained during the morning hours (8 A.M. to 12 Noon). Most subjects excreted the least amount of 17-ketosteroids during the 24 hour period from 8 P.M. to 8 A.M. COMPARISON OF 17 -KETOSTEROIDS WITH INDEPENDENT VARIABLES The statistical information regarding the correlation between 17-ketosteroids and other independent variables found in the same uri nary samples is recorded in the Appendix in Table VII 1 Part A and Part B. CORRELATION BETWEEN 17-KETO STEROIDS AND CREATININE A positive correlation was noted to exist between 17-ketoster oids and creatinine during the Pre-Bed Rest Period. The level of sig nificance was (P< 0.10). No correlation was found, however, between these two substances during Bed Rest I. 110 CORRELATION BETWEEN 17-KETO STEROIDS AND HYDROXYPROLINE When the data for all of the six subjects were pooled, a cor relation between the two factors was found at a highly significant level (P< 0. 01) between the excretion of these two substances during Pre Bed Rest. The correlation between these two metabolites, however, was not significant during Bed Rest I. CORRELATION BETWEEN 17-KETO STEROIDS AND TOTAL CALCIUM When these two variables were compared statistically, a sig nificant correlation was not apparent during Pre-Bed Rest or during Bed Rest I. CORRElATION BETWEEN 17 -KETO STEROIDS AND URINARY CALCIUM Urinary calcium showed the same trend as total calcium, with no significant correlation evident between 17-ketosteroid urinary ex cretion and urinary calcium excretion during both the Pre-Bed Rest and the Bed Rest Periods • 111 CORRElATION BETWEEN 17-KETO STEROIDS AND TOTAL NITROGEN Excretion levels of nitrogen in urine did not show any significant correlation when compared with the excretion levels of 17 -ketosteroids during both the Pre-Bed Rest and the Bed Rest I Periods o CORRElATION BETWEEN 17-KETO STEROIDS AND 17-HYDROXYCORTI COSTEROIDS There was a slightly significant correlation between the urinary excretory levels of these two metabolites during the Pre-Bed Rest Period o The correlation coefficient (r) was 0 o1687, and the level of significance was (P< 0 o1 0) 0 The level of correlation was not significant when these two factors were compared for Bed Rest I o CORRElATION BETWEEN 17-KETO STEROIDS AND CREATINE There was no significant correlation between these two variables during either the Pre -Bed Rest or the Bed Rest Periods o 112 COMPARISON OF 17 -HYDROXYCORTICOSTEROIDS WITH INDEPENDENT VARIABLES Table VII 1 Parts A and B (Appendix) 1 contains the statistical data relating to the correlation coefficient for all variables. CORRELATION BETWEEN 17-HYDROXY CORTICOSTEROIDS AND CREATININE There was no significant correlation found between these two variables during the Pre-Bed Rest Period 1 although/ during Bed Rest I, the amount of 17-hydroxycorticosteroid excreted in the urine of all sub jects was positively correlated with the amount of urinary creatinine ex creted. The level of significance was (P< 0.01). This finding is in accordance with the results reported by Hutton (134). CORRELATION BETWEEN 17-HYDROXY CORTICOSTEROIDS AND :HYDROXYPROLINE Upon pooling the data for all six subjects 1 there was no evi dence of a significant correlation existing between these two substances during the Pre-Bed Rest or during Bed Rest I. CORRELATION BETWEEN 17-HYDROXY CORTICOSTEROIDS AND TOTAL CALCIUM During the Pre-Bed Rest no statistical significant correlation was found between these substances. The level of total calcium excretion 113 for all six subjects was distinctly correlated with the urinary excretion levels of 17-hydroxycorticosteroids during Bed Rest I (P< 0.01). CORRElATION BETWEEN 17-HYDROXYCORTI COSTEROIDS AND URINARY CALCIUM In comparing the data on urinary calcium excretion with urinary excretion of 17-hydroxycorticosteroids 1 no significant correlation was evident for either Pre-Bed Rest or Bed Rest I. CORRElATION BETWEEN 17-HYDROXYCORTI COSTEROIDS AND TOTAL NITROGEN No significant correlation was found between these two sub stances either during the Pre-Bed Rest Period of the study or during the first Bed Rest. CORRElATION BETWEEN 17-HYDROXYCORTI COS TEROIDS AND CREATINE The excretory levels of creatine were not correlated significantly with the urinary excretory levels of 17 -hydroxycorticosteroids during the Pre -Bed Rest Period. When these variables were compared during Bed Rest I 1 however 1 a distinctly significant level of correlation was noted (P< 0. 05). 114 PART II OF THE STUDY This portion of the study covers the Pre-Bed Rest Period of a subsequent investigation in which eight healthy adult males participated in a 56 day Bed Rest period designed primarily to observe the circadian rhythm pattern of certain metabolites. During the Pre -Bed Rest portion of the study, the 17 -hydroxycorticosteroid excretions were ana lysed and observed by the author. The statistical data pertaining to the urinary 17-hydroxycortico- steroid excretion for this portion of the study are recorded in the Ap- pendix in Tables IX, X, and XI and in Figures 15 and 16. CIRCADIAN RHYTHM PATTERN OF 17 -HYDROXYCORTICOSTEROIDS In order to study the diurnal variation of 17-hydroxycorticostcr- oids, the urine excreted at different times during the 24 hour period was collected into three aliquots which ended respectively at 8 A.M., 12 Noon, and 8 P.M. The statistical data regarding the excretion pat- tern are given in Tables X and XI (Appendix) and in Figures 15 and 16, as noted. 115 Figure 15. RHYTHMICITY OF URINARY EXCRETION OF 17-HYDROXYCORTICOSTEROIDS DURING THE PRE-BED REST PERIOD "'"-'".cs.o.-=-~----~-=>·,..,...c"-"'~-,__...... -=. ,..c-·~ (J) 0 0 0:: w f (J) 0 u RHYTHM !CITY OF 17-HYDROXYCORTICOSTEROID f-n::: URINARY EXCRC:TION D U R \ N G 0::=> ?RE-BED REST 0 ~ 1.1 U o:: 10 ~ BAM 12 NOON >- Lu X Q.. 0.9 D f'IOON B PM 0 2 08 0:: - z 1%1 >- I-' I-' 01 117 SUBJECT 1A In a statistical analysis of the data, it was shown that the quan tity of 17 -hydroxycorticosteroids excreted during the three periods of the day by Subject 1A were not significantly different from each other. As shown in Figure 15, however, the highest mean value was for the period 8 A.M. to 12 Noon. SUBJECT 2A An analysis of the data pertaining to Subject 2A showed that, when the different pairs of the three collection periods were compared by means of the "t" test, the amount of 17 -hydroxycorticosteroids ex creted in the urine by this subject from 8 A.M. to 12 Noon surpassed the amount excreted between 8 P.M. and 8 A.M. and between 12 Noon and 8 P.M., by a difference which was slightly significant (P< 0.10). When the 17 -hydroxycorticosteroid excretion from 12 Noon to 8 P.M. was compared with that from 8 P.M. to 8 A.M. no statistically sig nificant difference was found. SUBJECT 3A An analysis of the Pre-Bed Rest data on this subject showed that there were no statistically significant differences in urinary 17- hydroxycorticosteroid excretion between any of the pairs of three col lection periods. Figure 15 shows that the urine collected from 8 Ao M • 118 to 12 Noon I however 1 contained the highest amount of 17 -hydroxycorti costeroids 1 with the lowest amount of this metabolite appearing in the collection taken from 8 P.M. to 8 A.M. SUBJECT 4A The 17 -hydroxycorticosteroids excreted by Subject 4A from 12 Noon to 8 P.M. surpassed in total amount that which was excreted from 8 P.M. to 8 A.M. by a difference which was significant (P < 0. OS). The period from 8 A.M. to 12 Noon was not different statist1c:dly from the periods from 8 P.M. to 8 A.M. or from 12 Noon to 8 P.M. SUBJECT 6A For this subject there was no significant difference in the uri nary 17 -hydroxycorticosteroid excretion between any of the three col lection periods • As shown in Figure 15 1 this subject excreted the high est a mount of urinary 17 -hydroxycorticosteroids from 8 A.M. to 12 Noon I with the lowest level of excretion of this metabolite occurring between 8 P.M. and 8 A.M. SUBJECT 7A An analysis of the data pertaining to Subject 7AI showed that the a mount of 17 -hydroxycorticosteroids excreted by this subject from 8 A.M. to 12 Noon was higher by a slightly significant difference (P < 0.1 0) than that excreted between 8 P.M. and 8 A.M. On the other hand 1 there was no significant difference in the amount of 17-hydroxy- 119 corticosteroids present in the urinary collections from 12 Noon to 8 P.M. as compared to the levels observed in the other two periods. SUBJECT 8A The quantity of 17 -hydroxycorticosteroids excreted by Subject SA from 8 P.M. to 8 A.M. was somewhat greater statistically (P< 0.10) than the amount of this metabolite excreted between 8 A.M. and 12 Noon. There was no significant difference found when comparing the period from 8 A.M. to 12 Noon with the period from 8 P.M. to 8 A.M. Similarly, the period from 8 P.M. to 8 A.M. wus not significantly dif ferent from that from 12 Noon to 8 P.M. SUBJECT 9A Subject 9A displayed no significant difference in urinary 17- hydroxycorticosteroid excretion between ony of lhe pairs of the three collection periods. It is of note 1 however 1 that during the period from 8 A.M. to 12 Noon the highest amount of 17-hyclroxycorticosteroids was excreted by this subject I with Lhe lowest level of excretion of this metabolite occurring during the overnight period as shown in Figure 15. M.!: SUBJECTS When the data were pooled for all of the eight subjects 1 an analysis of the data showed that the level of 17-hy·ciroxycorticosteroids excreted during the period from 8 A. M · to 12 Noon was distinctly higher than that from 8 p.M. to 8 A.M. 1 by a difference vvhich vva s highly 12 0 significant (P< 0,001). The 17-hydroxycorticosteroid excretion from 8 A.M. to 12 Noon surpassed that from 12 Noon to 8 P.M. by a highly significant difference (P< 0.01), The quantity of 17-hydroxycortico steroids excreted from 12 Noon to 8 P .M, was significantly greater (P< 0.05) than the amount excreted from 8 P.M. to 8 A.M. as shown in Figure 16, The individual subjects varied as to the comparison of the ex cretion of the 17 -hydroxycorticosteroids during the three sub -periods of the 24 hour day. Most subjects showed an increase in the amount of urinary 17-hydroxycorticosteroids excreted during the morning hours from 8 A.M. to 12 Noon. Subjects 4Aand 8A, however, showed a slight decline during these hours. The lowest excretory levels of 17 -hydroxycorticosteroids was observed in all subjects (Subject 8A excepted) between the hours of 8 P.M. and 8 A.M. On pooling the data for all subjects together, a significant increase was noted in the a mount of 17 -hydroxycorticoster oids excreted in the urine during the period from 8 A.M. to 12 Noon with the lowest rates of excretion being observed in the overnight col lection. 121 Figure 16. URINARY 17-HYDROXYCORTICOID EXCRETION DURING 24 HOURS MEAN OF ALL SUBJECTS 122 lf) n_ () ld 0 0 ...._ a: 0 w m rL <( 1- -"' (\1 OJ OJ lf) 0 I ~ 0 u "---· (f) -~ 0 u I- <( 0 rL ~ 1- u "' OJ OJ Ill 'L"' n::: } m 0 } u (f) >- _j ~DO X _j <~ 0 ,/) 0:: IJ 1-- n .I >- rL :J -: } I m I rr: . .J >- ~ ) '------1 J IL 0 ._; I -t ~~:---c--~~"-..,j, '"""? -'t L (\j 0:: ~-- ~ - 0 0\ 0~ t'-: '~ Li) '1'. 0) (\1 (' __: __: 0 o o o ~5 o 0 c> c-i (CJnOH C:I::Jd SI':VC:J'Jillll~ 1\'\'JI'~) SC lOt:! 31 SO'J I H:JO'J;\ XOtl 0 ;\ H- Ll ;\tl \:"I~ I C:! n 123 These findings suggest the existence of a definite circadian rhythm pattern for urinary 17 -hydroxycorticosteroid excretion. These observations are in accord with those of Lee (136) 1 Bowman et al (152) 1 and Orth et al (163) who show a similar pattern of excretion for this metabolite. SUMMARY AND CONCLUSIONS A number of metabolic investigations have been conducted at the Nelda Childers Stark laboratory for Human Nutrition Research at the Texas Woman's University Research Institute as a project sponsored by the National Aeronautics and Space Administration. This report is con cerned: (a) with the 17-ketosteroid metabolism of six healthy adult male subjects participating in one portion of this investigation which was conducted in the summer of 1968 1 and (b) with the 17-hydroxy corticosteroid metabolism of eight healthy adult males who participated in a subsequent study which was conducted in the summer of 1969. Urinary 17 -ketosteroids were analyzed by the Zimmerman (165) method and 17 -hydroxycorticosteroids were detected using the method of Porter and Silber (166). The first study consisted of two 28 -day horizontal Bed Rest Peri ods I with periods of Pre -Bed Rest Equilibration I Interim Ambulatory 1 and Post-Bed Rest Recovery. The urinary excretory levels of 17-keto steroids for all subjects were measured throughout the five periods. The total 24 hour urinary excretion of 17-ketosteroids of all subjects was distinctly higher during Bed Rest I than during Pre -Bed Rest 124 125 (P< 0. 001). Immobilization appears to increase the excretion levels of 17-ketosteroids, possibly because of stress. The circadian variation of 17 -ketosteroids was studied during Bed Rest I. A marked periodicity was noted in the excretory levels of 17-ketosteroids in all six subjects with the highest amount being ex creted during the morning hours from 8 A.M. to 12 Noon and the lowest excretion of this metabolite occurring overnight. During Bed Rest II the subjects were divided into two groups and a supervised exercise program was introduced as a variable. Three subjects were required to exercise routinely and three subjects were allowed to exercise ad-libitum. Both groups exhibited a highly sig nificant decrease in urinary 17 -ketosteroids during the Exercise Period as compared with Bed Rest I, with no exercise (P< 0.001). Although there was no statistically significant difference found when the group exercising regularly was compared with the group exercising "at will", it was of note that the 17-ketosteroicl excretory values of the subjects exercising regularly showed the greatest decline. A correlation study was conducted in which urinary 17-keto steroid excretion was correlated with that of creatinine, creatine, hy droxyproline, 17-hydroxycorticosteroids, total calcium, urinary cal cium, and total nitrogen during both the Pre-Bed Rest and the Bed Rest I Periods. Of these metabolites only creatinine, hydroxyproline, 126 and 17 -hydroxycorticosteroids showed a barely significant correlation (P< 0.10) with the 17-ketosteroid excretion, and this correlation was not found during Bed Rest I. The 17-hydroxycorticosteroid excretion was correlated with the same variables during both the Pre -Bed Rest and Bed Rest I. No sig nificant correlation existed during Pre-Bed Rest between the 17 -hy droxycorticosteroid excretion and any of these variables. During Bed Rest I, however, a significant correlation (P< 0.01) was noted be tween 17-hydroxycorticosteroid levels and creatinine and total cal cium. There also existed a moderate level of correlation between the amounts of 17-hydroxycorticosteroids and creatine excreted (P~ 0.05). The second part of this study consisted of a 56 day Bed Rest Period with a 17 day Equilibration Period and a 14 day Post-Bed Rest Period. In this portion of the study the rhythmicity of urinary 17 -hy droxycorticosteroids was noted during the Pre -Bed Rest Period. The pooled data for all eight subjects exhibited a distinct circadian rhythm pattern with the highest levels of 17 -hydroxycorticos teroids being ex creted during the morning between 8 A.M. and 12 Noon, and the lowest level being excreted during the period from 8 P.M. till 8 A.M. In re viewing the results of this study, it is interesting to note that the nor mal amounts of 17-hydroxycorticosteroids excreted in the urine of these subjects varied from individual to individual. BIBLIOGRAPHY 1. Pincus, G. A., Diurnal Rhythm in the Excretion of Urinary l..Z_ Ketosteroids by Young Men, Journal of Clinical Endocrinology and Metabolism, l..Z_:1150 (1957) 2. Frazier, T. W. , J. A. Rummel, and H. S. Lipscomb, Circadian Variability in Vigilance Performance, Aerospace Medicine, 39: 383 (1968)- - 3. Turner, C. D. , General Endocrinology, Fourth Edition, W. B. Saunders Company, Philadelphia (1966) 4. Dorfman, R. I. , and F. Unger, Metabolism of Steroid Hormones , Academic Press, New York (1965) - 5. Barton, D. H. R., The Stereochemistry of CycloHexane Deriva tives, Journal of American Chemical Society, J.2..: 102 7 (1953) 6. West, E. S., W. R. Todd, H. S. Mason, and J. T. Van Bruggen, TextBook of Biochemistry, The Macmillan Company, New York (1966) - 7. Zaffaroni, A., 0. Hechter, and G. A. Pincus, Adrenal Conver sion of c14 labeled Cholesterol and Acetate to Adrenal Corti------cal Hormones, Journal of American Chemical Society, .zl: 1390 (1951) 8. Hectcher, 0., M. M. Solomon, A. Zaffaroni, and G. A. Pincus, Transformation of Cholesterol and Acetate to Adrenal Cortical Hormones, Archives of Biochemistry and Biophysics, !§..:201 (1953) 9. Cas pi, E., R. I. Dorfman, B. I. Khan, G. Rosenfeld, and W. Schmid, Degradation of Corticosteroids, (VI} Origin of the Carbon Atoms of Steroid Hormones Biosynthesized In Vitro in the Bovine AdrMal from Acetate c14 I Journal of Biological Chemistry, 237:2085(1962) - 127 128 10. Shimizu, K., M. Gul, and R.I. Dorfman, ~' ~ E-Dihydroxy Cholesterol an Intermediate in the Biosynthesis of Pregnenolone from Cholesterol, Journal of Biological Chemistry, 237:699 (1962) 11. Constantopoulos, G., and T. T. Techer, Cleavage of Cholesterol Side Chain by Adrenal Cortex. _!. Co-Factor Requirement and Product of Cleavage, Journal of Biological Chemistry, 236:65 (1961) 12. Cha udhuri, A. C., Y. Harada, K. Shimizu, M. Gul, and R. I. Dorfman, Biosynthesis of Pregnenolone from 22-Hydroxycholes terol, Journal of Biological Chemistry, 237:703 (1962) 13. Samuel, L. T., and D. M. Greenberg, Metabolic Pathways, Aca demic Press, New York (196 0) 14. Rabinowitz, J. L. , The Biosynthesis of Radioactive 17B -Estradiol II. Synthesis of Testicular and Ovarian Homogenates, Archives of Biochemistry~nd Biophysics, §.!:285 (1956) 15. Dominguez, 0. V., H. F. Acevedo, R. A. Husely, and L. T. Sam uels, Steroid 21-Hydroxylase in Normal Testis and Malignant Interstitial Cell Tumors, Journal of Biological Chemistry, 235: 2608 (1960)-- -- 16. Samuels, L. T., and K. B. Eik-Nes, MetabolismofSteroid Hor mones, Metabolic Pathways, Edited by D. M. Greenberg, Aca demic Press, New York (1968) 17. Estabrook, R. W. , D. Y. Cooper, and 0. Rosenthal, The Light Reversible Carbon Monoxide Inhibition of the Steroid g2l Hydroxylase System of the Adrenal Cortex, Biochemistry Zeitschrift, 338:741 0963) 18. Garfinkel, D., Studies on !lei Liver Microsome I. Enzymic and Pigment Composition of Different Microsomal Fractions, Ar chives of Biochemistryand Biophysics, .z.z_:493 (1958) 19. Ewald, W., H. Werben, and I. L. Chaikoff, Evidence for the Presence of 17-Hydroxy Pregnenolone Isomerase in Beef Adrenal Cortex, Biochemistry Et Biophysics Acta , 111:3 06 (1965) 2 0. Groll man, A., Clinical Endrocrinology and its Physiologic Basis, J. B. Lippincott Company, Philadelphia (19 64) 129 21 . Dorfman, R. I. , and Fred B. Kincle, Methods in Hormones Re search, Volume IV, Academic Press, New York (1965) 22. Borris, A., R. H. Stevenson, and T. Trmal, Comparative Andro genic, Myotrophic and Antigonadotrophic Properties of Some Anabolic Steroids, Steroids, ..!2_:61 (1970) 2 3. Kocha kian, C. D., Early Effects of Androgens on the Biosynthesis of Protein and RNA by the Post mitochondria 1 Fraction of the Mouse Kid~, Steroid~l.±:77 (1969) --- 24. Kochakian, C. D., and J. Hill, Effect of Androgen on the Incor poration -of Orotic Acid-- 6 -C 14 ------into the RNA and Free Nuc~ tides of Mouse Kidney, Biochemistry, _?_: 1696 (1966) 25. Avdalovic, N., and C. D. Kochakian, Androgen Regulation of RNA Polymerase Activity in Isolated Mouse Kidney Nuclei, Biochem istry et Biophysics Acta, 182:382 (1969) 2 6. Widnell, C. C. , and J. R. Ta ta, Additive Effect of Thyroid Hor mone, Growth Hormone and Testosterone on DNA-Dependent RNA Polymerase in Rat Liver Nuclei, Biochemical Journal, 98: 621 (1964) ---- - 27. Kochakian, C. D., M. Nishada, and T. Hirone, Ribosomes of Mouse Kidney: Regulation by Androgens, American Journal of Physiology, 217:383 (1969) 28. Fuji, T., and C. Villee, Effect of Testosterone on RNA Metabolism in the Prostate, Seminal Vesicle, Liver and Thymus of Immature Rats, Endocrinology, .§l:463 (1968) 29. Kochakian, C. D., Intracellular Regulation of Nucleic Acid of Mouse Kidney by Androgens, General Comparative Endocrinology, _!l: 14 6 ( 1 9 6 9) - 30. McCullagh, E. P., Effects of Androgens on Blood Count of Men, Journal of Clinical Endocrinology, ~:243 (1942) 31. Ftied, W., and C. W. Gurney, Erythropoietic Effect of Plasma from Mice Receiving Testosterone, Nature, 206:1160 (1965) 32. Mirand, E. A. , A. S. Gordon, and J. Wenig, Mechanism of Tes to sterone Action on Erythropoiesis, Nature, 206:270 (1965) 130 33. Reisner, E. H., Tissue Culture of Bone Marrow II. Effect of Ster oid Hormones on Hematopoiesis In Vitro, Blood, Q:460 (1966) 34. Rishpon-Meyerstein I N. I T. Kilbridge I J. Simone I and vV. Fried I The Effect of Testosterone on Erythropoietin Levels in Anemic Patients, Blood, l.!_:453 (1968) 35. Kappas, A., and S. Garnick, Steroid Induction of Porphyrin Syn thesis in Liver Cell Cultures, Journal of Biological Chemistry, 243:346 (1968) 36. Necheles, T. F., and U. S. Rai, Studies on the Control of Hemo globin Synthesis: The In Vitro Stimulating Effect of~ 2_ B-H Steroid Metabolites on Heme Formation in Bone Marrow Cell~, Blood, .H_:380 (1969) 37. Sanchez-Medal, L., A. Gomez-Leal, L. Duarte, and M. Gradua l upe Rico, Anabolic Androgenic Steroids in the Treatment of Ac quired Aplastic Anemia, Blood, l:!_:283 (1969) 38. Rokkanin, P., S. Paatsama, and P. Rissmon, Hormone Induced Changes in the Bones of Young Dogs, Calcified Tissue Research, Supplement 2:95 (1968) -- 39. Howard, E., Steroids and Bone Maturution in Infant Mice. Relative Activities of D8hydroepiandrosterone and Testosteron8, Endocri- nology, 70:131 (1962) - 4 0. Wilkins, L. I The Dia qnosis and Tr8a tment of Endocrine Disorders in Childhood and Adolescence, Charles C. Thomas, Springfield, Illinois (1957) 41. Howard I E., Effects of Steroids on Epiphysioclia physia 1 Union in Prepuberal Mice 1 Endocrinology I 7.1_: 11 (1963) 42. Kowaleswski, K., Uptake of Radio Sulfate in Growing Bones of Cockerels Treated with Cortisone and Certain Anabolic -Andro genic Steroids, Endocrinology, §1:759 (1958) 43. Young, S., and K. Kowalewski, Dermal Collag8n Thermal Injury in Normal and in Steroid Hormone Treated Rats I The Canadian Journal of Surgery, _22:342 (1969) 44. Puc he, R. C., and M. C. Romano I The Effect of Dehy·droepian drosterone Sulfate and Testosterone on the Development of Chick Embryo Frontal Bones In Vitro, Calcified Tissue Research, 2:133 (1969) 131 45. Puche, R. C., and M. C. Romano, Effect of Dehydroepiandro sterone Sulfate on the Mineral Acceleration of Chick. Embryo Frontal Bones Cultivated In Vitro, Calcified Tissue Research, !:39 (1969) --- 46. Keele, D. K., and J. W. Worely, Study of an Ar1abolic Steroid, Certain Effects of Oxymetholone in Small Children, American Journal of Diseases of Children, 113:422 (1967) 47. Ray, C. G., J. F. Kirschvink., S. H. Waxman, and V. C. Kelley, Studies of Anabolic Steroids, III. The Effect of Oxandrolone on Height and Skeletal Maturation in Mongoloid Children, Ameri can Journal of Diseases of Children, 110:618 (1963) 48. Ray, C. G., J. F. Kirschvink., S. H. Waxman, and V. C. Kelley, Studies of Anabolic Steroids, II. The Effect of Oxandrolone on Height and Skeletal Maturation in Mongoloid Children, Ameri can Journal of Diseases of Children, 106:375 (1963) 49. Keele, D. K., and G. P. Vase I f2 Study of Bone Density Compari son of the Effects of Sodium Fluoride, Inorganic Phosphate and an Anabolic Steroid (Oxymetholone) on Dimineralized Bone, American Journal of Diseases of Children, 118:759 (1969) SO. Clifton, M. B., Changes in Urinary 17-Ketosteroids by Four Healthy Males During Ambulation and Horizontal Bed Rest Re cumbency, Master's Thesis, Texas Woman's University, Den ton, Texas (1967) 51. Furman, R. H., Are Gonadal Hormones (Estrogens and An:lrogens) of Significance in the Development of Ischemic Heart Disease I New York. Academycl Science Annal~ 149:822 (1968) 52. Furman, R. H., P.R. Howard, K. Tak.shami, and L. N. Norcia, The Serum Lipids and Lipoproteins in Normal and Hyperlipidem ic Subjects as Determined by Preparative Ultracentrifugation Effects of Dietary and Therapeutic Measures. Changes Induced by In Vitro Exposure of Serum to Sonic Forces, American Journal ;£clinical Nutrition:-~:73 (1961) 53. Howard, R. P. , and R. H. Furman, Estrogens, Androgens and Serum Lipids: The Enigmatic Triad of Atherogenesis, Annals of Internal Medicine, ~:668 (l~- 54. Landau, B. R. , Adrenal Steroids and Carbohydrate Metabolism, Vitamins and Hormones, 23:2 (1965) 132 55. Weber, G., L. R. Singhal, and N. B. Stamm, Actinomycin: In hibition of Cortisone-Induced Synthesis of Hepatic Gluconeo genic En;ymes, Science, 142:390 (1963)- 56. Greengard, 0., G. Weber, and R. L. Singhal, Glycogen Deposi tion in the Liver Induced by Cortisone Dependence on Enzyme, Science, 141:160 (1963) 57. Weber, G., R. L. Singhal, and I. K. Sirvastana, EffectofNu tritional State on Hormonal Regulation of Liver Enzymes, Cana dian Journal of Biochemistry, .:!1_:1549 (1965) 58. Eisenstein, A. B., Adrenocortical Hormones and Carbohydrates Synthesis in Liver, Advances in Enzyme Regulation, 3:121 (1965) 59. Weber, G., R. L. Singhal, and S. K. Sirvastava, Action of Glu cocorticoids as Inducer and Insulin as Suppresser of Biosyn thesis of Hepatic Cluconeoqenic Enzymes, Advances in Enzyme Regula lion, l: 43 (1965) 60. Bellamy, D., and R. A. Leonard, The Effect of Cortisol on the Activity of GluLnmate-Pyruvate Transaminnse <:1ncl the Formation of Glycoqcn and Uren in Starved R1ts, Biochemico.L Journo.l, 93: 331 (1964) ----- 61. Hornbrook:., K. R., H. 13. Burch, and 0. H. Lowry, Cho.nqes in Substrate Levels~ Liver Durinq Glycoqen Synthesis Induced by La eta te and Hydrocortisone, Biochemistry Biophysics Resee1 reb Communication, ~:206 (1965) 62. Oji, N., und W. W. Shreeve, Gluconeo(Jenesis from C14 ~.!::):d 3 Labeled Substrute in Normol and Cortisone-Tree1tecl Rats, Endo crinology, ~:765 (1966) 63. Taronwski, W., i\:I. Kittler, and H. Hilz, Die \Virkunq Von Cortsol auf Die Umwandlung Von HexosePhosphaten in Glykogen, Bio chemistry Zeitschrift, 341:45 (1964) 64. landau, B. R., Adrenal Steroid and Carbohydrate i\Ietabolism, Vitamins and Hormones, 23:2 (1965) 65. Haag, B. L., M. M. Reidberg, C. R. Shuman, and B. J, Channick, Aldosterone, 17-Hydroxycorticosteroid, 17-Ketosteroid, and Fluid and Electrolyte Response to Starvc.tio:1 c.nd Selective Re feeding, American Journal of Medical Science, 254:653 (1967) 133 66. Kinsell, L. W,, G. D. Michaels, S. Morgan, J, Vv. Partridge, L. Boling, and H. E. Balch, The Case for Cortical Steroid Hor mone Acceleration of Neoglucogenesis from Fat in Diabetic Sub jects. A Summary of Five Years Investigative Work, Journal of Clinical-Endocrinology and Metabolism, _!_!: 161 (1954) 67. Glenn, E. M., B. J, Bowman, R. B. Bayer, and C. E. Meyer, Hydrocortisone and Some of Its Effects in Intermediary Metabo lism In Vivo Studies, Endocrinology, ~:386 (1961) 68. Munck, A. S., and B. Koritz, Studies on the Mode of Action of Glucocorticoids in Rats. I. Early Effects of Cortisol on Blood Glucose and on Glucose Entry into Muscle, Liver and Adipose Tissue, Biochemical et Biophysical Acta, 2Z_:310 (1962) 69. Ray, P. D., Adrenal Glucocorticoids and Gluconeogenesis, Func tion of the Adrenal Cortex, lancet, ~:1137 (1968) 7 0. Goldberg, A. L. , Protein Turnover in Skeleta 1 Muse le. II. Effects of Denervation and Cortisone on Protein Catabolism in Skeletal Muscle, Journal of Biological Chemistry, 244:3223 (l969) 71. Bethal, J. J., M. Feigelson, and P. Feigelson, The Differential Effects of Glucocorticoids on Tissue and Plasma Amino Acid Levels ,Biochemical et Biophysical Acta, 104:92 (1965) 72. Weber, G., S. K. Sribastava, and R. L. Singhal, Role of Enzyme Homeostasis Early Effect of Corticosteroid Hormone on Hepatic Gluconeogenic Enzymes, RNA Metabolism and Amino Acid Levels, Journal of Biological Chemistry, 240:750 (196--sr- 73. lardy, H. A., D. 0. Foster, E. Shrago, and P. D. Ray, Metabolic and Hormonal Regulation of Phosphopyruvate Synthesis, Ad vances in Enzyme Regulation, ~:39 (1964) 74. Lang, N., and C. E. Serkerkis, Stimulation of RNA-Polymerase Activity in Rat Liver by Cortisol, Life Science, l_:391 (1964) 75. Pena, A,, B, Dvorkin, and A. White, Effect of~ Single Injection of Cortisol on Amino Acid Incorporation Activities of Rat Liver and Thymic Preparation In Vitro, Journal of Biological Chemistry, 241:2144 (1966) - -- 134 76. Brink-Johnson, T., and T. F. Dougherty, Studies on the Effect of Cortisol and Corticotrophin on Incorporation of Ade~e-8-cl4 into the Lymphatic Tissue Nucleic Acid, Acta Endocrinologia (Kobenhaven), ~:471 (1965) 77. Mendelsohn, M. L., and 0. H. Pearson, Alteration in Water and Salt Metabolism After Bilateral Adrenalectomy in Man, Journal of Clinical Endocrinology and Metabolism, ]2_:409(1955) 78. Lutwak, L., D. G. Whedon, P. A. LaChance, J. M. Reid, and H. S. Lipscomb, Mineral, Electrolyte and_ Nitrogen Balance Studies of the Gemini VII, 14 Day Orbital Space Flight, Journal of Clinical Endocrinology, 29:1140 (1969) 79. Dingman, J. F. , D. H. P. Streeten, and G. W. Thorn, Effect of Cortisone --on the Abnormal Distribution of Intravascular Water in Adrenal Cortical Insufficiency in Man, Journal of Laboratory and Clinical Medicine, 49:7 (1957) 8 0. Hauger, J. H. , H. Brown, and N. Fleischer, AC TH Stimulation a ncl Glucocorticoids Inhibition of Renin Release in the Rat, Proceed ings of Society of Experimental Biology and Medicine, 131:539 (1969) 81. Robb, C. A., J. 0. Davis, C. I. Johnston, and P.M. Hartroft, Effects of Cortisone on Renal Sodium Excretion in Rabbits, Endo- - -- - crinology, _§l:1200 (1968) 82 · Ingbar, S. H., and N. Frienkel, ACTH, Cortisone and the Me tab olism of Iodine, Metabolism, Clinical and Experimental, 2:652 (1956)- 83 · Schiller, S., and A. Dorfman, The Metabolism of Muccopolysac charide in Animals· the Effect of Cortisone and Hydrocortisone - '- - -- on Rat Skin, Endocrinology, §_Q: 376 (195 7) 84. Cox, R. P., Hormonal Induction of Increased Zinc Uptake in Mam malian Cell Cultures: Require;8nt for RNA and Protein Syn- thesis, Science, 165:196 (1968) -- 85 · Grollman, A., Effect of Cortisone on Serum Calcium, Magnesium, Phosphate Levels i~Nephrecto~ed Dogs, Proceedings of Society of Experimental Biology and Medicine, §2.:582 (1954) 86 · Clark, I., R. Geoffroy, and N. Bomers, Effect of Adrenalcortical Steroids on Calcium Metabolism, Endocrinology, §!:849 (1959) 135 87. Skeels, R. F., The Reversibility of Osteoporosis ~n Cushing's Syndrome, f2 Case Report, Journal of Clinical Endocrinology and Metabolism, 1:..§_:61 (1958) 88. Collins, E. J., R. E. Garrot, and R. L. Johnston, Effect of Adrenal Steroids on Radiocalcium Metabolism in Dogs, Metabolism, 11: 716 (1962) --- 89. Henrotte, J. G., S. Subrahmanyan, A.M. Ramnathan, and M.P. Satyanarayan, Urinary Excretion of 17 -Ketosteroids in South Indian Population, lancet, _l:84 (1965) - 90. Mahesh, V. B., R. B. Greenblatt, and R. F. ConifC Adrenal Hy perplasia: f2 Case Report of Delayed Onset of the Congenital Form or Acquired Form, Journal of Clinical Endocrinology and Metabolism, ~:619 (1968) 91. Kumaoka, S., N. Sakaauchi, 0. Abe, M. Kusama, and 0. Taka tani, Urinary 17-Ketosteroid Excretion of Women with Adva need Breast Cancer, Journal of Clinical Endocrinology and Metabo lism, ~:667 (1968) 92. Lloyd, S. W., J, Lobotsky, E. J. Seger, T. Kobayashi, M. L. Taymur, and R. E. Batt, Plasma Testosterone and Urinary lZ_ Ketosteroids in Women with Hirustism and Polycystic Ovaries, Journal of Clinical Endocrinology, ~::314(1966) 93. Nicholas, T., C. A. Nugent, and F. H. Tyler, Glucocorticoids Suppression of Urinary Testosterone Excretion in Patients with Iodopathic Hirustism, Journal of Clinical Endocrinology, ~:79 (1966) 94. Jacobson, G., C. C. Seltzer, P. K. Bondy, and J. Mayer, Im portance of Body Characteristic in Excretion of 17-Ketos teroids and 17-Ketogenic Steroids in Obesity, New England Journal of Medicine, 271:651 (1964) 95. Vanderstraten, M., A. Vermeulen, N. Ori, and P. Regniers, On Value of Para meters of Adrenocortical Glucocorticoids Function, Acta Endocrinology, 44:499 (1963) 96. Romanoff, L. P., K. K. Malhotra, M. N. Baxter, A. W. Thomas, and G. Pincus , Metabolism of 17'{ Hydroxypregnenolone J....q H3 and 17 -9"Hydroxyprogesterone .:!_- _g14 in Young and Elderly Men, Journal of Clinical Endocrinology and Metabolism, ~:836 (1968) 136 97. Migeon, 0. N., F. M. Kenny, and F. H. Taylor, Cortisol Pro duction Rate. VIII. Pregnancy, Journal of Clinical Endocrinology and Metabolism, 28:661 (1968) 98. Curtis, G. C., M. L. Fogel, D. McEvoy, and C. Zerati, Effects of Weight, Sex and Diurnal Variation on the Excretion of ..!2_ Hydroxycorticosteroids, Journal of Clinical Endocrinology, 28: 711 (1968) - 99. Streeten 1 D. H.P. 1 C. T. Stevenson, T. G. Delakos, J. J. Nicho las 1 L. G. Den nick 1 and H. Fellerman 1 The Diagnosis of Hy percortilism Biochemical Criterea Differentiating Patient from Lean and Obese Normal Subjects from Females on Ora 1 Contra ceptive 1 Journal of Clinical Endocrinology and Metabolism, ~: 1191 (1969) 100. Grant, D. S., F. Partatos, and P. H. Forsham, Effects of Estrogen Therapy on Cortisol Metabolism, Journal of Clinical Endocrinol ogy, ~:l057 (1965) 101. Kornel, L., and K. Motohashi, Corticosteroids in Human Blood II. Free and Conjugated 17-Hydroxycorticosteroid in Essential HypertM~, Journa 1 of Clinical Endocrinology, 25:904 (1965) 102. Gibson, J. L., Cortisol Secretion Rate in Depressive Illness, Archives of General Psychiatry, 10:572 (1964) 103. Rubin, R. T., and A. J. Mandell, Adrenal Cortical Activity in Pathological Emotional States, [2 Review, American Journal of Psychiatry, 12 3:38 7 (1966) 104. Clower, C. G. 1 and c. J. Migeon, Psychoendocrine Aspects of Depression and ECT, Johns Hopkins Medical Journal, 121:227 (1967) -- lOS. Wolf, C. T., Relationship Between Psychological Defense and Mean Urinary 17-Hydroxycorticosteroid Excretion Rates. I. A Predictive Study of Parents of Fatally Ill Children, Psychoso- matic Medicine, 26:575 (1964) - 106. Wolf, C. T., M.A. Hofer, and J. M. Mason, Relationship Be tween Psychological Defenses and Mean Urinary 17 -Hydroxy corticosteroid Excretion Rates. II. Methodological and Theo retical Consideration, Psychosomatic Medicine, .?..§_: 592 (1964) 137 107. Rose, R. M., R. D. Poe, and J. W. Mason, Psychological State and Body Size ~Determinant of 17-Hydroxycorticosteroid Ex cretion, Archives of Internal Medicine, 121:406 (1968) 108. Marchbank, H. V., Effects of Flying Stress on Urinary 17-Hy droxycorticos teroid Levels Observation During ~ 2 2 • 5 Hour Mission, Journal of Aviation Medicine, ~:676 (1958) 109. Demos, G. T., B. H. Hale, and E. W. Williams, Anticipatory Stress ~nd Flight Stress in F-102 Pilots, Aerospace Medicine, iQ_: 3 8 5 ( 19 6 9) 110. Pincus, G., The Urinary Ketosteroids. Report of~ Conference, Journal of Clinical Endocrinology, l: 30 (1943) 111. Frost, J. W., R. L. Dryer, and K. G. Kohlsteadt, Stress Studies on Auto Race Drivers, Jouma l of La bora tory und Clinical Medi cine, l.§_:523 (1951) 112. Hills, S. R., F. C. Goets, H. M. Fox, B. J. Murowsk, L. J, Krakauer, R.N., R. W. Rcifenstein, S. J. Gray, W. J. Reddy, S. E. Hedberg, J. R. March, and G. W. Thorn, Studies on Adrenocortical and Psycholoqical Response to Stress in Mon, Archives of Intern<:ll Medicine, ~:269 (1956) 113. Miller, R. G., Secretion of 17-Hyclroxycorticos teroid ~ Mililary Aviators as an Index of Response to Stress, [2 Review, Aero space Medicine, 39:498 (1968) 114. Marchbank, V. H., I-I. B. Hale, one! J.P. Ellis, Stress Response of Pilots FlyinC) 6-Hour Over Wotor Missions in F-100 and_[_- 104 Aircraft, l\erospuce Medicine, 34:15 (1963) 115. I-Iale, H. B., J.P. Ellis, unci E. W. Williams, Endocrine I\!Ieta bolic Changes During 12-Hour Simulated Flight, /~erospace Medicine, 36:717 (1965) 116. Abbo, F. E., The 17-Ketos teroicl/17-Hyclroxycorticos teroicl Ru tios ~~Useful Measure of the Physioloqica lAce of the Human Adrenal Cortex, Journal of Gerontology, ~:112 (1966) 117. Marmorston, J., J. J. Lewis, J. L. Burnstein, I-I. Sobel, 0. Ku zuma , R. Alexander, 0. rvra giclson, a ncl F. J. ivioore, Excretion of Urinary Steroids b;{ Men a ncl Women with l\Iyocardia l Infarc tion, Geriatrics, _!1_:297 (1957) 138 118. McCully, C. P., and D. E. Graveline, Physiologic Aspects of Prolonged Weightlessness, Body Fluid Distribution and the Car diovascular System, New England Journal of Medicine, 269:5 08 (1963) 119. Bruce, D., and J. E. Wiebers, Mineral Dynamics During Hiber nation and Chronic Immobility: A Review, Aerospace Medicine, 4 0:855 (1969) - 120. Pyke, R. E., P. B. Mack, R. A. Hoffman, W. W. Gilchrist, W. N. Hood, and G. P. George, Physiologic and Metabolic Changes in Macaca Nemestrina on Two Types of Diets During Restraint and Non-Restraint: III. Excretion of Calcium -----and Phos- phorus, Aerospace Medicine, ~:704 (1968) 121. Hoffman, R. A., E. A. Dozier, P. B. Mack, W. N. Hood, and M. W. Parrott, Physiologic and Metabolic Changes in Macaca Nemestrina on Two Types of Diets During Restraint and Non-Re straint: I. Body Weight Changes, Food Consumption and Urinary Excretion of Nitrogen, Creatine and Creatinine, Aerospace Medi cine, ~:693 (1968) 122. Mack, P. B., R. A. Hoffman, and A. N. Al-Shawi, Physiologic and Metabolic Changes in Macaca Nemestrina on Two Types of Diets During Restraint and Non -Restraint: II. Bone Density Changes, Aerospace Medicine, ~: 698 (1968) 123. Mack, P. B., and P. A. LaChance, Effects of Recumbency and Space Flight on Bone Density, American Journal of Clinical Nu trition, ~:1194 (1967) 124. Mayer, H. J., Excretion of Nitrogen by Healthy Men During Two Horizontal Bed Rest Periods with and without Exercise, Master's Thesis, Texas Woman's University-;-Denton, Texas, June (1969) 125. Montgomery, K. B., Metabolism Including Circadian Rhythms of Creatine and Creatinine of Healthy Men During Recumbency and Ambulation ----with and without Exercise, Unpublished Doctor's Dis- sertation, College of Household Arts and Sciences, Texas Worn- an 's U ni vers ity ( 19 6 9) 12 6. Van Zandt, D. P., Circadian Rhythms of Urinary Calcium and Phos phorus in Healthy Adult Men During Recumbency and Ambulation, and the Effect of Exercise on these Excretions and Periodicity, Unpublished Doctor's Dissertation, College of Household Arts and Sciences, Texas Woman's University (1969) 139 127. Deitrick, J. E., G. D. Whedon, and E. Shorr, The Effect of Im mobilization upon Various Metabolic and Physiologic Functions of Normal Men, American Journal of Medicine, .:!_: 3 (1948) 12 8 . Whedon, D. G. I J. E • Deitrick, and E. Shorr I Modifications of the Effects of Immobilization Upon Metabolic and Physiologic Functions of Normal Men by the Use of an Oscillating Bed, American Journa 1 of Medicine, §_: 6 84 (194 9) 129. Wu, Martha Chia-Tzu, 17-Ketosteroid Metabolism During Bed Rest Immobilization and During Ambulation, Master's Thesis 1 Texas Woman's University, Denton, Texas (1966) 130. Suzuki, T., R. Higashi, H. Tanigawa, H. Ikeda, and K. Tamura, Adrenal Cortical Response to Immobilization in Conscious and Anesthetized Dogs, Tohoku Journal of Experimenta 1 Medicine, ~:281 (1968) 131. Burstein, B., B. R. Bhavnani, and H. L. Kimball, Observation on Urinary Corticosteroid Excretion Patterns in Individual Guinea Pigs, Endocrinology, 22_:226 (1964) 132. Knigge I K. M., C. H. Penrod, and J. W. Schinder 1 ~1_ Vitro and In Vivo Adrenalcorticosteroid Secretion Following §tress, Ameri can Journal of Physiology, 196:579 (1959) 133. Lin, Sue Whei, Urinary Excretion of 17-Hydroxycorticos teroids and 17-Ketosteroids During~ Bed Rest §_tudy with and without Exercise, Master's Thesis, Texas Woman's University, Denton 1 Texas (1967) 134. Hutton, N. G. , Urinary 17-Hyclroxycorticosteroid Excretion, Cre atine and Creatinine Excretion as Influenced by Stress I Master's Thesi~exas Woman's University, Denton I Texas (1967) 135. Liu, Cecilia Li-Ju, 6_ Study on 17-Hydroxycorticos teroid Excretion During Horizontal Bed Rest 1 Master's Thesis I Texas Woman's University, Denton I Texas (1970) 136. Lee, Lil Ha, Circadian Rhythm Pattern of 17 -Hyclroxycorticosteroicl Urinary Excretion in Healthy Adult Human l'viales, Master's Thesis, College of Household Arts and Sciences, Texas Woman's University, Denton, Texas (1969) 140 137. Hsu, Catherine Wei-Shan, Changes in Urinary 17-Hydroxycortico steroids by Five Young Adult Males During Ambulation and Hori zontal Bed Rest Recumb~ and at Different Periods of the Day, Master's Thesis 1 College of Household Arts and Sciences, - Texas Woman's University, Denton, Texas (1968) 138. Suzuki, T., K. Otsuka, H. Matusui, S. Ohukuzi, K. Sakai, and Y. Harada, Effects of Muscular Exercise on Adrenal 17 -Hy droxycorticosteroids Secretion in Dogs, Endocrinology, 80:1148 (1967) --- - 139. Moncloa, F., E. Pretell, and J. Correa, Studies on Urinary Ster oids of Men Born and Living at High Altitudes, Proceedings of Society on Experimental Biology and Medicine, 108:336 (1961) 140. Marotta, S. F., K. Hirai, and G. Atkin, Secretion of 17-Hydroxy corticosteroids in Conscious and Anesthetized Dogs Exposed to Simulated Altitude, Proceedings of Society on Experimental Biol ogy and Medicine, 114:403 (1963) 141. Hornbein, T. F., Adrenal Cortical Response to Chronic Hypoxia, Journal of Applied Physiology, .!.Z_:246 (1962) 142. Biddulph, C., J. C. Finerty, and J. P. Ellis, Blood Corticoster oids and Anterior Pituitary ACTH and Cytology ~ Dogs Exposed to Hypocapnia and/or Hypoxemia 1 American Journal of Physi ology 1 197:126 (1959) 143. Lau, C. 1 and S. F. Marotta, Role of Peripheral Chemoreceptors on Adrenocortical Secretory Rates During Hypoxia, Aerospace Medicine 1 4 0:1065 (19 69) 144. Pittendrich, C. B., and V. G. Bruce, An Oscillator Model for Bio logical Clocks, Rhythmic and Synthetic Processes in Growth, Edited by Rudnick, Princeton University Press (1957) 145. Aschoff, J. 1 Circadian Clocks, North-Holland Publishing Com pany I Amsterdam (1965) 146. Bennet-Clark, T. A., G. Salt, and V. B. Wigglesworth, The Physiology of Diurnal Rhythms, University Press, Cambridge (1964) - 14 7. Schmeiger 1 E., H. G. Wallraff 1 and H. G. Schweiger, Endogenous Circadian Rhythms in Cytoplasma of Acetabularia: Influence of the Nucleus, Science 1 146:658 (1964) 141 148. Silverberg, A., F. Rizzo, and D. T. Krieger, Nytohemeral Peri odicity s:1_ Pla s rna 17-Hydroxycorticos teroid Levels in Elderly Subjects, Journal of Clinical Endocrinology, ~: 1661 (1968) 149. Iaatikainen, T., and R. Vihiko, Diurnal Variation in the Concen tration of Solvolyzable Steroids in Human Plasma, Journal of Clinical Endocrinology, ~:1350 (1968) 150. Nicholas, T., and F. H. Tyler, Glucocorticoids Suppressions of Urinary Testosterone Excretion in Patients with Iodopathic Hirustism, Journal of Clinical Endocrinology, 25:243 (1965) 151. Liddle, G. W., Analysis of Circadian Rhythm in Human Adreno cortica 1 Secretory Activity, Archives of Interna 1 Medicine, 117:739 (1966) 152. Bowman, R. E,, R. C. Wolf, and G. P, Sackett, Circadian Rhythm of Plasma 17-Hydroxycorticosteroids in the Infant Monkey, Pro ceedings of Society on Experimental Biology and Medicine, 133: 42 (1970) - 153. Bowman, R. E., and R. C. Wolf, Plasma 17-Hydroxycorticostcr oids Responses to ACTH in M Mulatta Dose, Age, Weight and Sex, Proceedings of Society on Experimental Biology and Medi cine, 130:61 (1969) 154. Klien, K. E., H. M. Wegman, and H. Bruner, Circadian Rhythm in Indices of Human Performance, Physical Fitness and Stress Resistance ~Aerospace Medicine, ~:512 (1968) -- 155. Mills, J, N., Human Circadian Rhythm, Physiological Review, 46: 128 (1966) 156. Conroy, R. T. W. L., B. D. Hughes, and J. N. Mills, Circadian Rhythm of Plasma 11-Hydroxycorticosteroids in Psychiatric Dis- orders, British Medical Journal, 1:405 (1968-) -- 157. Doe, R. P., J. A. Vennes, and E. B. Flinck, Diurnal Variations of 17-Hydroxycorticosteroids, Sodium, Potassium, Magnesium, and Creatinine in Normal Subjects and in Cases of Treated Adrenal Insufficiency and Cushing's Syndrome, Journal of Clini cal Endocrinology, ~:253 (1960) 158. Krieger, D. T., and H, P, Krieger, Circadian Variation of the Plasma 17-Hydroxycorticosteroids in Central Nervous System Diseases, Journal of Clinical Endocrinology, 26:929 (1966) 142 159. Cade, R., D. L. Sheres, M. V. Barrow, and W. C. Thomas, Jr., Abnormal Diurnal Variation of Plasma Cortisol in Patients with Renovascular Hypertension 1 Journal of Clinical Endocrinology I 27:800 (1967) 160. Kornel, L., and R. Takeda, Studies on Steroid Conjugates: V. Uri nary 17 -Hydroxycorticosteroids in Essential Hypertension, Journal of Clinical EndocrinologY: ~:233 (1967) 161. Mills, J. N., Circadian Rhythms During and After Three Months Solitude Underground, Journal of Physiology, 174:217 (1964) 162. Perkoff, G. T., T. K. Eik-Nes, C. A. Nugent, H. L. Fred, R. A. Nimer 1 L. Rush, L. T. Samuels, and F. H. Tyler, Studies of the Diurnal Variation of Plasma 17-Hydroxycorticosteroids in Man, Journal of Clinical Endocrinology, ..!2_:432 (1959) 163. Orth, D. N., D.P. Islan, and G. W. Liddle, Experimental Al teration of Circadian Rhythm in Plasma Cortisol (17-Hydroxy corticost-;(oids) Concentratio~in Men, Journal of Clinical Endo- crinology, ll:555 (1967) --- 164. Reinberg, A.,J.Ghata, and E. Sidi, Nocturnal Asthma Attacks, Their Relationship to the Circadian Adrenal Cycle, Journal of Allergy, 1!:323 (1963) 165. Zimmerman, Wilhelm, Colorimetrisch Bestimmung cler Keimclrusen Hermones, Hoppe -Seyler's Zeitschrift Physiologische Chemie, 245:47 (1936) 166. Porter, C. C., and R. H. Silber, The Determination of lZ_, ~, Dihydroxy 20-Ketosteroids in Urine and Plasma, Journal of Bio logical Chemistry, 210:505(1954) -- APPENDIX 143 TABLES FOR PART I OF THE STUDY Including Excretion of 17-Ketosteroids by Six Subjects Participating in Two 28 -Day Bed Rest and Preliminary and Final Periods in 1968 (Subjects M I BB I EE I FF I GG I and HH) 144 145 TABLE I URINARY EXCRETION OF 17-KETOSTEROIDS PER DAY (Milligrams per 24 hours) PART !2_. SUBJECT BB Initia 1 Interim Post Bed Equi libra tion Bed Rest l Ambulatory Bed Rest 2 Rest Period Period Ambulatory Period Day Mg/ Day Mg/ Day Mg/ Day Mg/ Day Mg/ 24 hr 24 hr 24 hr 24 hr 24 hr l 3.30 l X l 2.61 l 4.54 l 5. 51 2 6.80 2 X 2 4.90 2 1.40 2 7.20 3 X 3 X 3 4. 62 3 2.87 3 4. 51 4 X 4 6 .19 4 l. 30 4 5. 02 4 3.62 5 9.21 5 8.36 5 l. 25 5 3.42 5 6,43 6 3.90 6 6.46 6 l. 00 6 3.22 6 2.53 7 9.70 7 7.30 7 4.76 7 6.83 7 X 8 6.61 8 8.73 8 3.37 8 8.93 8 13.82 9 X 9 12.61 9 8.13 9 7 .16 9 X 10 2. 72 10 6.21 10 6.98 10 5.70 l 0 2. 03 11 5.70 11 5.35 11 l. 70 11 10.97 ll 4.40 12 2.91 12 7.46 12 4. 71 12 7.99 12 4.50 13 6.51 13 9.60 13 3.74 13 14.84 13 3.15 14 3.60 14 9.29 14 4.54 14 5 .l 0 14 2.74 15 6.4 0 15 7.91 X 15 6.73 X 16 3.72 16 8.55 X 16 5.89 X 17 5.40 17 11.05 X 17 7 .16 X 18 6.25 18 4.60 X 18 8. 32 X 19 5.11 19 11.11 X 19 6.65 X 20 3.43 20 12.44 X 20 2. 03 X 21 8.24 21 ll . 2 5 X 21 X X 22 X 22 7.59 X 22 8.75 X 23 3.20 23 8.84 X 23 6.54 X 24 X 24 ll. 08 X 24 X X 25 6.50 25 6.88 X 25 X X 26 7.33 26 9. 04 X 26 6.24 X 27 7.22 27 8.70 X 27 3.81 X 28 3.8 0 28 10.84 X 28 X X 29 9.92 X X X X Mean: 5.73 Mean: 8.70 Mean: 3.83 Mean: 6.25 Mean: 5. 04 146 TABLE 1_, CONTINUED URINARY EXCRETION OF 17 -KETOSTEROIDS PER DAY (Milligrams per 24 hours) PART~· SUBJECT FF Initial Interim Post Bed Equilibration Bed Rest 1 Ambulatory Bed Rest 2 Rest Period Period Ambulatory Period Day Mg/ Day Mg/ Day Mg/ Day Mgl Day Mg/ 24 hr 24 hr 24 hr 24 hr 24 hr 1 6.44 1 X 1 X 1 5.17 1 X 2 4.33 2 X 2 2.60 2 1.42 2 5.95 3 X 3 X 3 2.88 3 2 .15 3 3.86 4 7.60 4 2. 69 4 3.60 4 8.62 4 6.80 5 6.44 5 6.62 5 3.60 5 4. 06 5 6.22 6 5.68 6 10.27 6 3.45 6 6.50 6 4.20 7 X 7 6. 09 7 X 7 2.52 7 5.82 8 6.63 8 7.03 8 l. 80 8 l. 86 8 6.86 9 3.60 9 8.80 9 7.70 9 l. 00 9 5.82 10 1.44 10 5.88 10 8.90 10 7.90 1 0 6.28 ll 3.83 ll 5.55 ll 4.89 11 l. 74 11 4. 31 12 1.20 12 5. 51 12 4. 72 12 10.17 12 l. 70 13 X 13 6.49 13 2 .14 13 6.30 13 2.90 14 9.2 0 14 8. 06 14 X 14 12.20 14 2.23 15 X 15 8.29 X 15 6.20 X 16 3.30 16 5.48 X 16 10.60 X 17 3.30 17 6.97 X 17 5. 11 X 18 X 18 5.44 X 18 5 .15 X 19 7.04 19 4.50 X 19 X X 20 2.60 20 7 • 11 X 20 X X 21 X 21 11 • 04 X 21 6. 09 X 22 3. 8 0 22 9.46 X 22 6.90 X 23 4. 8 0 23 6.92 X 23 9.61 X 24 4. 04 24 7.49 X 24 X X 25 1.48 25 6.36 X 25 3.26 X 26 8. 2 0 26 3.00 X 26 9.40 X 27 3.8 0 27 12. 03 X 27 X X 28 5 .40 28 12.06 X 28 2.30 X 29 5. 2 0 X X X X Mean: 4.75 Mean: 7.17 Mean: 4.21 Mean: 5.68 Mean: 4.84 147 TABLE l_, CONTINUED URINARY EXCRETION OF 17 -KETOSTEROIDS PER DAY (Milligrams per 24 hours) PART _g. SUBJECT HH Initial Interim Post Bed Equilibration Bed Rest 1 Ambulatory Bed Rest 2 Rest Period Period Ambulatory Period Day Mg/ Day Mg/ Day Mg/ Day Mg/ Day Mg/ 24 hr 24 hr 24 hr 24 hr 24 hr 1 7.75 1 X 1 1.83 1 10.97 1 8.87 2 X 2 X 2 2.80 2 4.22 2 5.20 3 X 3 X 3 1.85 3 0.69 3 3.00 4 2 .8 0 4 1.20 4 3.26 4 2.27 4 X 5 7.48 5 5.95 5 3.40 5 10.36 5 tl . 6 0 6 2.90 6 5.85 6 2.00 6 4.99 6 l. 00 7 4.64 7 7. 04 7 l. 70 7 l. 27 7 7.28 8 4.23 8 8.44 8 6.69 8 5.64 8 2.00 9 l. 2 0 9 4 .18 9 l. 97 9 l. 70 9 4.31 10 3.39 10 5.14 10 0.76 10 5.24 10 3.45 11 1.90 ll 6.91 ll 5.90 ll 6.96 ll X 12 2.54 12 9.92 12 2.60 12 10.2 0 12 3.40 13 3.64 13 8.42 13 2.50 13 6.12 13 3.73 14 5.91 14 6.39 14 8. 08 14 8.80 14 1 . 22 15 4.20 15 3. 01 X 15 9.05 X 16 3.50 16 4.23 X 16 10.95 X 17 2.82 17 8.12 X 17 7.30 X 18 3.23 18 4.52 X 18 X X 19 7.36 19 9.49 X 19 8.10 X 20 2.60 20 3.86 X 20 3.50 X 21 X 21 7.99 X 21 4.42 X 22 6.60 22 7.93 X 22 6.34 X 23 X 23 11 . 12 X 23 2.12 X 24 2.99 24 10.87 X 24 X X 25 5. 02 25 7.84 X 25 3.50 X 26 3.2 0 26 8.57 X 26 3.68 X 27 1.22 27 6.96 X 27 4.86 X 28 X 28 10.79 X 28 l. 30 X 29 6.50 X X X X Mean: 4.07 Mean: 6.99 Mean: 3.24 Mean: 5.56 Mean: 4.00 148 TABLE I_, CONTINUED URINARY EXCRETION OF 17-KETOSTEROIDS PER DAY (Milligrams per 24 hours) PART D. SUBJECT AA Initial Interim Post Bed Equilibration Bed Rest l Ambulatory Bed Rest 2 Rest Period Period Ambulatory Period Day Mg/ Day Mg/ Day Mg/ Day Mg/ Day Mg/ 24 hr 24 hr 24 hr 24 hr 24 hr l 6.14 l X l 3.71 l 4.00 l 2.22 2 5.40 2 X 2 4. 06 2 2. 32 2 9. 02 3 X 3 X 3 5.91 3 4.80 3 3. 2 0 4 1.41 4 3.81 4 6.00 4 4.98 4 4 .14 5 X 5 7.26 5 7.34 5 11.85 5 5.94 6 5.71 6 5. 09 6 3.35 6 2.18 6 l. 35 7 7.18 7 5.43 7 4. 07 7 l. 06 7 3.00 8 7.04 8 4.17 8 5.90 8 l. 94 8 2.54 9 X 9 5.42 9 5.74 9 l. 51 9 4.00 10 1.86 10 5.72 10 2.83 10 4.97 10 4.57 ll 2.50 ll 9.07 ll 3.03 ll 4.23 ll 2. 05 12 X 12 7. 01 12 2.15 12 6.70 12 2.89 13 3.44 13 7.14 13 5.81 13 3.81 13 5. 05 14 2 .13 14 5. 72 14 X 14 6.23 14 2.33 15 2. 02 15 5.79 X 15 7.30 X 16 1.50 16 5.85 X 16 ll . 10 X 17 6.70 17 5.88 X 17 7.65 X 18 1.39 18 5 .13 X 18 X X 19 5.70 19 8.35 X 19 6.80 X 20 2.52 20 7.05 X 20 X X 21 X 21 13.90 X 21 4.68 X 22 5.61 22 8 .15 X 22 2.43 X 23 1.44 23 6. 38 X 23 3.90 X 24 4.70 24 ll. 96 X 24 X X 25 4.80 25 8.29 X 25 7.30 X 26 2.60 26 6.22 X 26 4.60 X 27 5 .13 27 6.65 X 27 1.85 X 28 X 28 13.28 X 28 X X 29 3.30 X X X X Mean: 3.92 Mean: 7.15 Mean: 4. 61 Mean: 4.92 Mean: 3.74 149 TABLE _l, CONTINUED URINARY EXCRETION OF 17-KETOSTEROIDS PER DAY (Milligrams per 2 4 hours) PART ~. SUBJECT EE Ini tia 1 Interim Post Bed Equilibration Bed Rest 1 Ambulatory Bed Rest 2 Rest Period Period Ambulatory Period Day Mg/ Day Mg/ Day Mg/ Day Mg/ Day Mg/ 24 hr 24 hr 24 hr 24 hr 24 hr 1 6.62 1 X 1 6.00 1 9.10 1 8.87 2 2 0.91 2 X 2 2.96 2 4.70 2 8.62 3 X 3 5.39 3 5.00 3 3.15 3 4.20 4 6.43 4 8. 71 4 6.91 4 1.66 4 7.47 5 14.20 5 19.39 5 9. 04 5 5. 2 0 5 8.57 6 X 6 13.66 6 11.62 6 3.31 6 4.65 7 X 7 15.96 7 5.24 7 l. 41 7 5.80 8 11.60 8 13.96 8 3.80 8 4.70 8 10.20 9 3.95 9 25.50 9 5.63 9 4. 04 9 X 10 7.90 10 12.13 10 10.70 10 2.86 10 8.11 11 1.80 11 6. 07 11 5.33 11 9.60 11 4.42 12 3.15 12 11.36 12 3.33 12 12.60 12 3 .1 0 13 4.30 13 12.15 13 2.40 13 9.60 13 8.89 14 2.92 14 10.70 14 X 14 X 14 16.24 15 3.50 15 16.15 X 15 13.50 X 16 X 16 9.83 X 16 8.21 X 17 5.80 17 5.14 X 17 9.61 X 18 X 18 9.26 X 18 8.76 X 19 X 19 10.15 X 19 X X 20 X 20 9.24 X 20 7.60 X 21 3.46 21 7.09 X 21 10.50 X 22 7.85 22 15.84 X 22 8.71 X 23 X 23 17.87 X 23 2.50 X 24 5.84 24 14.81 X 24 8.13 X 25 8.44 25 8.85 X 25 9.47 X 26 4.13 26 9.21 X 26 X X 27 4.07 27 7.88 X 27 14.03 X 28 17 .11 28 9.73 X 28 3.50 X 29 9.20 X X X X Mean: 7.29 Mean: 11.77 Mean: 6.00 Mean: 7.06 Mean: 7.63 150 TABLE l I CONTINUED URINARY EXCRETION OF 17-KETOSTEROIDS PER DAY (Milligrams per 24 hours) PART[. SUBJECT GG Initia 1 Interim Post Bed Equilibration Bed Rest 1 Ambulatory Bed Rest 2 Rest Period Period Ambulatory Period Day Mg/ Day Mg/ Day Mg/ Day Mg/ Day Mg/ 24 hr 24 hr 24 hr 24 hr 24 hr 1 9.00 1 X 1 3.96 1 X 1 8.51 2 X 2 X 2 4.16 2 4.57 2 X 3 12.64 3 X 3 2.23 3 2.41 3 X 4 8.30 4 8.64 4 l. 70 4 2.65 4 4.80 5 X 5 6. 08 5 X 5 12.59 5 5.48 6 1.2 0 6 6.24 6 8.55 6 8.80 6 6.53 7 14.89 7 6.78 7 6.64 7 4.00 7 X 8 X 8 8.35 8 2.00 8 3.00 8 4.86 9 7.74 9 8.61 9 8 .16 9 9.85 9 10.32 10 6.93 10 6.50 10 8. 18 10 3.34 10 X 11 2 .13 11 6.30 11 l. 40 11 5.70 11 4.86 12 X 12 4.47 12 l. 65 12 3.32 12 l. 98 13 3.42 13 5.54 13 4.51 13 8.80 13 3.06 14 5.63 14 5.89 14 X 14 X 14 6.30 15 X 15 11 .14 X 15 9.61 X 16 7.90 16 12.44 X 16 14.80 X 17 X 17 11 .83 X 17 7.89 X 18 X 18 5. 71 X 18 X X 19 l. 93 19 7. 16 X 19 9.00 X 20 1.27 20 8. 02 X 20 6.45 X 21 4.86 21 10.58 X 21 11.00 X 22 9. 21 22 8.58 X 22 3.00 X 23 6.54 23 14.86 X 23 5.40 X 24 7.30 24 6 .1 0 X 24 6.74 X 25 5.07 25 8. 52 X 25 3.41 X 26 X 26 13.65 X 26 X X 27 10.12 27 12.24 X 27 4.74 X 28 X 28 20.18 X 28 4. 08 X 29 6.97 X X X X Mean: 6.65 Mean: 8.98 Mean: 4.43 Mean: 6.46 Mean: 5.67 151 TABLE II URINARY 17-KETOSTEROID EXCRETION DURING THREE DAILY PERIODS WHILE BED REST I WAS IN PROGRESS (Milligrams per hour) Subject BB Subject FF Day Noon- 18 P.M.-~ 8 A.M.- Noon- 8 P.M.- 8 A.M.- 8 P.M. 8 A.M. Noon 8 P.M. 8 A.M. Noon 1 X X X X X X 2 X X X X X X 3 X X X X X X 4 0.220 0.299 0.210 0.234 0.056 0.038 5 0 .191 0.299 0.810 0 .19 7 0.269 0.453 6 0. 230 0.338 0.480 0.400 0.392 0.593 7 0.320 0. 27 0 0.380 0.376 0.130 0. 38 0 8 0.128 0.368 0.830 0.663 0.094 0 .15 0 9 0.730 0. 383 0.550 0.074 0.11 0 0.390 10 0.320 0.237 0.210 0. 28 0 0.210 0. 28 0 11 0.287 0 .1 71 0. 2 50 0.275 0.086 0. 58 0 12 0. 500 0.057 0.681 0.194 0.230 0.300 13 0. 300 0.570 0.100 0.305 0.2 00 0.413 14 0.358 0.299 0.710 0.314 0.320 0.428 15 0.292 0.295 0. 508 0. 38 0 0.138 0.900 16 0.288 0.213 0.926 0.019 0.317 0. 38 3 17 0.662 0. 208 0.788 0.300 0.324 0.170 18 0.159 0. 043 0. 703 0. 240 0.243 0. 15 0 19 0.2 04 0.489 0. 902 0.066 0. 2 03 0.384 20 0.656 0.413 0.558 0. 22 0 0.288 0.473 21 0.320 0.687 0.113 0.590 0.310 0.650 22 0.188 0.350 0.470 0.443 0.400 0. 28 0 23 0.130 0.470 0. 540 0.220 0. 280 0. 450 24 0.880 0. 230 0.320 0. 380 0.2 08 0.488 25 0.140 0.313 0.500 0.091 0.422 0.143 26 0.44 0 0. 260 0. 6 01 0 .14 3 0.068 0. 26 0 27 0.467 0.13 0 0.850 0.271 0. 704 0.353 28 0.421 0.189 1. 3 00 0.281 0. 6 51 0.501 152 TABLE D._, CONTINUED URINARY 17 -KETOSTEROID EXCRETION DURING THREE DAILY PERIODS WHILE BED REST l_ WAS IN PROGRESS (Milligrams per hour) PART B. SUBJECTS HH AND AA Subject HH Subject AA Day Noon- I 8 P.M.- 8 A.M.- Noon- 18 P.M.- 8 A.M.- 8 P.M. 8 A.M. Noon 8 P.M. 8 A.M. Noon 1 X X X X X X 2 X X X X X X 3 X X X X X X 4 0.060 0. 042 0.055 0.101 0.184 0.198 5 0.246 0.303 0.085 0.288 0.259 0.463 6 0.091 0.233 0. 580 0.124 0.259 0.248 7 0.295 0.177 0.640 0.280 0.198 0. 205 8 0.313 0.412 0.250 0.175 0.124 0. 32 0 9 0.200 0.134 0.243 0.251 0.179 0.315 10 0.103 0.277 0.250 0.175 0.167 0. 580 11 0.121 0.200 0.886 0.470 0.315 0. 38 3 12 0.681 0.256 0.350 0. 345 0.210 0.433 13 0. 220 0.305 0.750 0.495 0.192 0. 220 14 0.400 0.16 0 0.318 0.200 0.180 0.489 15 0.078 0.140 0.178 0.362 0.129 0.335 16 0.100 0.130 0.468 0.206 0.150 0.600 17 0.550 0.167 0.430 0.054 0.198 0. 770 18 0.066 0. 204 0.385 0.098 0.212 0.550 19 0.246 0.547 0.240 0. 420 0.160 0. 770 20 0.124 0.174 0.195 0.421 0.180 0. 380 21 0.054 0.490 0.420 0.230 0.560 1. 350 22 0.230 0.431 0.230 0.150 0.533 0.138 23 0.246 0.386 1 .130 0.288 0.290 0.150 24 0.318 0.480 0.643 0.389 0.460 0.833 25 0.230 0.230 0.810 0.521 0.250 0.290 26 0.104 0.441 0.612 0.210 0.123 0. 760 27 0.200 0.118 0.984 0.058 0.285 0.693 28 0.460 0.450 0.428 0.313 0.498 1. 2 00 153 TABLE .u I CONTINUED URINARY 17-KETOSTEROID EXCRETION DURING THREE DAILY PERIODS WHILE BED REST I WAS IN PROGRESS (Milligrams per hour) PART g. SUBJECTS EE AND GG Subject EE Subject GG Day Noon- I 8 P.M.-~8 A.M.- Noon- 8 P.M.- 8A.M.- 8 P.M. 8 A.M. Noon 8 P.M. 8 A.M. Noon 1 X X X X X X 2 X X X X X X 3 0.053 0.013 1 . 2 02 X X X 4 0.230 0.299 0.820 0.301 0.353 0.498 5 0.742 0. 720 1 . 2 02 0.196 0.353 0.070 6 0.594 0.299 1.330 0.18 0 0.18 3 0.660 7 0.483 0.308 2.100 0.14 0 0. 2 33 0.730 8 0.910 0.217 1. 02 0 0. 152 0. 268 0.982 9 0.988 0.983 1. 450 0.375 0.382 0.250 10 0.812 0.198 0.813 0.14 0 0.370 0.250 11 0.241 0. 083 0.785 0.12 0 0 .18 3 0.700 12 0.413 0.461 0.633 0. 13 0 0.17 0 0.3G3 13 0.382 0.357 1 . 2 02 0. 24 0 0. 2 35 0.210 14 0.429 0.299 0.920 0.186 0 .18 7 0.540 15 0.689 0. 387 1. 2 00 0. 64 0 0.353 0.452 16 0. 300 0.289 0.990 0.301 0.717 0. 4 00 17 0.188 0.058 0.732 0. 54 0 0.317 0.683 18 0.124 0.383 0.921 0. 151 0 .192 0.550 19 0.387 0.312 0.827 0.095 0. 4 50 0.250 20 0.291 0.130 1 . 35 0 0. 29 0 0. 301 0.681 21 0.288 0. 088 0.930 0. 4 3 0 0 . 5 0-4 0.230 22 0.370 0.100 2.920 0. 060 0. 48 0 0.730 23 1.060 0.440 1. 02 0 0.793 0.58::, 0.373 24 0. 38 0 0.111 2.620 0.13 0 0.341 0.250 25 0. 278 0.12 0 1 . 310 0.275 0.283 0. 725 26 0 .113 0.500 0. 6 00 0. 5-! 0 0. 56 0 0.670 27 0.282 0.295 0.520 0. 410 0.330 1 . 2 3 0 28 0.130 0.330 . l . 2 02 0.798 0.830 0.980 154 TABLE III STATISTICAL COMPARISON OF URINARY 17-KETOSTEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS OF THE STUDY PART fl. SUBJECT BB Standard "t II Population Compared Means Probability Deviation Value Pre-Bed Rest 5.73 2.15 4.6602 P< 0.001 Bed Rest One 8.70 2 .13 Pre-Bed Rest 5.73 2.15 2.5243 P< 0. 02 Interim Ambulatory 3.83 2.06 Pre-Bed Rest 5.73 2 .15 0.6847 N.S. Bed Rest Two 6.25 2.90 Pre-Bed Rest 5.73 2.15 0.7407 N.S. Post-Bed Rest 5. 04 3.04 Bed Rest One 8.70 2.13 6.5770 P< 0.001 Interim Ambulatory 3.83 2.06 Bed Rest One 8.70 2.13 3.2369 P< 0. 01 Bed Rest Two 6.25 2.90 Bed Rest One 8.70 2 .13 3.9897 P< 0.001 Post-Bed Rest 5. 04 3. 04 Interim Ambulatory 2.06 3.83 2.6124 P< 0. 01 Bed Rest Two 6.25 2.90 Interim Ambulatory 3.83 2. 06 1 .1 041 N .S. Post-Bed Rest 5. 04 3.04 Bed Rest Two 6.25 2.90 1.1035 N.S. Post-Bed Rest 5. 04 3.04 155 TABLE I I I I CONTINUED STATISTICAL COMPARISON OF URINARY 17-KETOSTEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS OF THE STUDY PART B. SUBJECT FF Standard lit II Probability Population Compared Means Deviation Value Pre-Bed Rest 4.75 2.12 3.5339 P<0.001 Bed Rest One 7.17 2.39 Pre-Bed Rest 4. 75 2.12 0.6592 N .S. Interim Ambulatory 4.21 2 .15 Pre -Bed Rest 4. 75 2.12 1 . 1 09 3 N .S. Bed Rest Two 5.68 3. 2 0 Pre-Bed Rest 4.75 2 .12 0.1213 N.S. Post-Bed Rest 4.84 1.69 Bed Rest One 7 .17 2.39 3.3377 P<0.001 Interim Ambulatory 4. 21 2.15 Bed Rest One 7 .17 2.39 1.7752 P< 0.10 Bed Rest Two 5.68 3.2 0 Bed Rest One 7.17 2.39 2.9690 P< 0. 01 Post-Bed Rest 4.84 1.69 Interim Ambulatory 4.21 2.15 1.3110 N.S. Bed Rest Two 5.68 3.2 0 Interim Ambulatory 4.21 2.15 0.7421 N.S. Post-Bed Rest 4.84 l. 69 Bed Rest Two 5.68 3. 2 0 0.8313 N .S. Post-Bed Rest 4.84 l. 69 156 TABLE I I I I CONTINUED STATISTICAL COMPARISON OF URINARY 17-KETOSTEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS OF THE STUDY PART C. SUBJECT HH Standard Itt II Population Compared l Means Deviation Value Probability Pre-Bed Rest 4. 07 l. 90 4.3832 P< 0.001 Bed Rest One 6.99 2.52 Pre-Bed Rest 4.07 l. 90 1.1957 N.S. Interim Ambulatory 3.24 2.06 Pre-Bed Rest 4.07 l. 90 1.9457 P< 0.10 Bed Rest Two 5.56 3 .11 Pre-Bed Rest 11. 07 1 . 9 0 0.0829 N.S. Post-Bed Rest 4.00 2. 21 Bed Rest One 6.99 2.52 4.5177 P<0.001 Interim Ambulatory 3.24 2. 06 Bed Rest One 6.99 2.52 Bed Rest Two 5.56 3 .11 1.7287 P<0.10 Bed Rest One 6.99 2.52 Post-Bed Rest 4.00 2. 21 3.3177 P<0.001 Interim Ambulatory 3.24 2. 06 2.3952 P< 0. 02 Bed Rest Two 5.56 3 .11 Interim Ambulatory 3.24 2. 06 0.8474 N.S. Post-Bed Rest 4.00 2. 21 Bed Rest Two 5.56 3.11 1 . 48 02 Post-Bed Rest 4.00 2. 21 N.S. 157 TABLE III, CONTINUED STATISTICAL COMPARISON OF URINARY 17-KETOSTEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS OF THE STUDY PART .Q. ALL SUBJECTS EXERCISING REGULARLY (BB, FF, HH) Standard "t II Population Compared IMeans I 0 0 Probability Dev1at1on Value Pre-Bed Rest 4.85 2.17 7.0697 P< 0.001 Bed Rest One 7.62 2.47 Pre-Bed Rest 4.85 2 .17 Interim Ambulatory 3. 72 2 .12 2.5816 P< 0. 05 Pre-Bed Rest 4. 85 2 .17 Bed Rest Two 5.82 3.09 2 .152 6 P< 0. 05 Pre -Bed Rest 4.85 2.17 Post-Bed Rest 4.63 2.41 0.4670 N .S. Bed Rest One 7.62 2.47 8.2224 P< 0. 001 Interim Ambulatory 3. 72 2 .12 Bed Rest One 7.62 2.4 7 3.8657 P< 0.001 Bed Rest Two 5.82 3.09 Bed Rest One 7.62 2.47 5.9524 P< 0.001 Post-Bed Rest 4.63 2.41 Interim Ambulatory 3. 72 2 .12 3.7358 P< 0.001 Bed Rest Two 5.82 3.09 Interim Ambulatory 3. 72 2.12 1 • 7 06 5 P< 0.10 Post-Bed Rest 4.63 2.41 Bed Rest Two 5.82 3.09 2.0152 Post-Bed Rest 4. 63 2 .41 P< 0. 05 158 TABLE I I I I CONTINUED STATISTICAL COMPARISON OF URINARY 17-KETOSTEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS OF THE STUDY PART~· SUBJECT M Standard "t II Population Compared Means ~ Probability I Deviation Value Pre-Bed Rest 3.92 1. 97 4.7051 P< 0.001 Bed Rest One 7.15 2.52 Pre -Bed Rest 3.92 1. 97 1 . 02 51 Interim Ambulatory 4.61 1. 52 N.S. Pre-Bed Rest 3.92 1. 97 1.3672 Bed Rest Two 4.92 2.76 N.S. Pre-Bed Rest 3.92 1. 97 0.2676 Post-Bed Rest 3. 74 1. 91 N .S. Bed Rest One 7. 15 2.52 3 .166 3 Interim Ambulatory 4.61 l. 52 P< 0. 01 Bed Rest One 7 .15 2.52 2.8278 p < 0. 01 Bed Rest Two 4. 92 2.76 Bed Rest One 7. 15 2.52 4.1856 Post-Bed Rest 3.74 1. 91 P< 0.001 Interim Ambulatory 4.61 1.52 0.3645 Bed Rest Two 4.92 2.76 N .S. Interim Ambulatory 4. 61 1 . 52 1.2078 N .S. Post-Bed Rest 3. 74 1. 91 Bed Rest Two 4. 92 2.76 Post-Bed Rest 3. 74 1.91 1.3535 N .S. 159 TABLE II I I CONTINUED STATISTICAL COMPARISON OF URINARY 17-KETOSTEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS OF THE STUDY PART f. SUBJECT EE Standard lit II Population Compared Means Probability Deviation Value Pre-Bed Rest 7.29 4.86 3.0850 P< 0. 01 Bed Rest One 11.77 4.63 Pre-Bed Rest 7.29 4.86 0.8295 N.S. Interim Ambulatory 6.00 2. 77 Pre-Bed Rest 7.29 4.86 0.0660 N.S. Bed Rest Two 7.06 3.67 Pre-Bed Rest 7.29 4.86 0.0236 N.S. Post- Bed Rest 7.63 3.34 Bed Rest One 11.77 4.63 3.9390 P< 0.001 Interim Ambulatory 6.00 2.77 Bed Rest One 11.77 4.63 3.6891 P< 0.001 Bed Rest Two 7. 06 3.67 Bed Rest One 11.77 1.63 3.0158 P< 0. 01 Post-Bed Rest 7.63 3.34 Interim Ambulatory 6.00 2. 77 0.9850 N.S. Bed Rest Two 7.06 3.67 Interim Ambulatory 6.00 2. 77 l . 04 07 Post-Bed Rest 7.63 3.34 N.S. Bed Rest Two 7.06 3.67 0.0994 Post-Bed Rest 7.63 3.34 N.S. 160 TABLE III I CONTINUED STATISTICAL COMPARISON OF URINARY 17-KETOSTEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS OF THE STUDY PART Q. SUBJECT GG Standard II t II Population Compared Means Probability Deviation Value Pre -Bed Rest 6.65 3.55 Bed Rest One 8.98 3.58 2.0756 p < 0. 05 Pre- Bed Rest 6.65 3.55 Interim Ambulatory 4.43 2.67 1.7613 P< 0.10 Pre -Bed Rest 6.65 3.55 Bed Rest Two 6.46 3.37 0.1715 N.S. Pre- Bee! !<.es L 6.65 3.55 PosL-I3ecl ResL 5.67 2. 31 0. 74-15 N .S. Bed Res L One 8.98 3.58 Interim flmbulzl Lory ·l. 43 2.67 3.7115 P< 0.001 Bed f<.es L One 8.98 3.58 Bed Res L Two G. -'lG 3.37 2.4251 P< 0. 02 Bed r<.es t One 8.98 3.58 2.56:25 P< 0. 02 Pos L -Bc;d r<.es t 5.67 2. 31 Interim J'\mbulu tory :J. 43 2.67 1.7291 P<0.10 Bed Res l Two G. -lG 3.37 Interim i\mbulatnry ·L--±3 2.67 1.0510 Post-Bed Resl 5.67 2.31 N.S. Bed Rest Two 6.--±6 3.37 0.6':!--±3 Post-Bed Rest 5.67 2.31 N.S. 161 TABLE II I I CONTINUED STATISTICAL COMPARISON OF URINARY 17-KETOSTEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS OF THE STUDY PART B. SUBJECTS EXERCISING "AT WILL II (AA, EE, GG) 1Standard "t II Population Compared I Means Deviation Value Probability Pre-Bed Rest 5.88 3.91 4.9516 P<0.001 Bed Rest One 9.33 4.16 Pre-Bed Rest 5.88 3.91 l .1888 N .S. Interim Ambulatory 5.03 2.48 Pre-Bed Rest 5.88 3.91 0.4959 N .S. Bed Rest Two 6.16 3.42 Pre-Bed Rest 5.88 3.91 0.4144 N .S. Post-Bed Rest 5.57 3. 04 Bed Rest One 9.33 4.16 5.7832 P< 0.001 Interim Ambulatory 5.03 2.48 Bed Rest One 9.33 4.16 4. 9231 P< 0.001 Bed Rest Two 6.16 3.42 Bed Rest One 9.33 4.16 4.8700 P< 0.001 Post-Bed Rest 5.57 3.04 Interim Ambulatory 5.03 2.48 Bed Rest Two 6.16 3.42 1. 8361 P Interim Ambulatory 5. 03 2.48 0.8301 N .S. Post-Bed Rest 5.57 3. 04 Bed Rest Two 6.16 3.42 0.9351 N .S. Post-Bed Rest 5.57 3.04 162 TABLE III I CONTINUED STATISTICAL COMPARISON OF URINARY 17 -KETOSTEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS --OF THE STUDY PART.!_. ALL SUBJECTS Standard lit II Population Compared Means Probability Deviation Value Pre-Bed Rest 5. 34 3.16 7.8299 P< 0.001 Bed Rest One 8.48 3.53 Pre-Bed Rest 5. 34 3.16 2.3229 P< 0. 05 Interim Ambulatory 4. 37 2.40 Pre-Bed Rest 5. 34 3 .16 1. 6882 P< 0.10 Bed Rest Two 5.99 3.26 Pre-Bed Rest 5. 34 3.16 0.4742 N.S. Post-Bed Rest 5 .13 2.80 Bed Rest One 8.48 3.53 P< 0. 001 Interim Ambulatory 4.37 2.40 9.1211 Bed Rest One 8.48 3.53 6.2775 P< 0.001 Bed Rest Two 5.99 3.26 Bed Rest One 8.48 3.53 Post-Bed Rest 5. 13 2.80 7.0750 P Interim Ambulatory 4.37 2.40 Bed Rest Two 5.99 3.26 3.8292 P< 0.001 Interim Ambula tonr 4.37 2.40 Post-Bed Rest 5 .13 2.80 1 . 777 3 P<0.10 Bed Rest Two 5.99 3.26 Post-Bed Rest 5 .13 2.80 1.9251 P< 0.10 163 TABLE IV STATISTICAL COMPARISON OF URINARY 17-KETOSTEROIDS BETWEEN PAIRS OF THE DIFFERENT PERIODS BY GROUPS OF SUBJECTS EXERCISING REGULARLY AND "AT WILL" II t II Populations Compared Means Standard Probability Deviation Value BED REST ONE - NO EXERCISE Subjects who Exerc is eel Regularly during Bed Rest Two 7.62 2.47 3.0322 P< 0. 01 Subjects who Exercised "At Will" during Bed Rest Two 9.33 c! • 1 G BED REST TWO Subjects who Excrc is eel Regularly during Bed Rest Two 5.82 3. 09 0.6225 N .S. Subjects who Exercised "At Will" during Bed Rest Two 6. 16 3.41 164 TABLE V STATISTICAL ANALYSIS OF URINARY 17-KETOSTEROIDS BETWEEN GROUPS OF SUBJECTS EXERCISING REGUIARLY AND "AT WILL" DURING BED REST ONE AND BED REST TWO S ta nda rd lit II Populations Compared Means Probability Deviation Value SUBJECTS EXERCISING "AT WILL" (M I EE I GG) Bed Resl One 9.33 4 .16 A.9231 P<0.001 Bed Rest Two 6 .16 3.42 ·-- SUBJECTS EXERCISING REGULARLY (BB I rr I HH) Bed Rest One 7.G2 2 .4 7 3.8657 P<0.001 Bed Rest Two 5.82 3. 09 165 TABLE VI STATISTICAL COMPARISON OF URINARY 17-KETOSTEROID EXCRETION AT DIFFERENT TIMES OF THE DAY J?ART !::_. SUBJECTS EXERCISING REGUlARLY (BB I FF I AND HH) Standard "t" Populations Compared Means Probability Deviation Value Subject BB 12 Noon - 8 PM 0.358 0.194 1.1212 N.S. S PM -SAM 0.299 0.152 12 Noon - 8 PM 0.35S 0.194 2.7909 P< 0.01 SAM - 12 Noon 0.55S 0.290 S PM -SAM 0.299 0 .152 3.6675 P<0.001 SAM -12 Noon 0.55S 0.290 Subject FF 12 Noon - 8 PM 0.281 0.14 9 8 PM - 8 AM 0.269 0.159 0.2667 N .S. 12 Noon - 8 PM 0.281 0.149 SAM- 12 Noon 0.3S4 0.187 2. 04 55 P< 0. 05 S PM -SAM 0.269 0.159 8 AM - 12 Noon 0.384 0.187 2.2240 P< 0. 05 Subject HH 12 Noon - 8 PM 0.246 0.169 0.2157 8 PM - 8 AM 0.256 0.136 N .S. 12 Noon - 8 PM 0.246 0.169 3. 2 7 02 P< 0. 01 8 AM - 12 Noon 0 ..:168 0.275 8 PM - 8 AM 0.256 0.136 8 AM - 12 Noon 0.46S 0.275 3.3352 P< 0.001 166 TABLE YJ, CONTINUED STATISTICAL COMPARISON OF URINARY 17 -KETOSTEROID EXCRETION AT DIFFERENT TIMES OF THE DAY PART~· SUBJECTS EXERCISING "AT WILL II (M I EE I AND GG) Standard lit II Populations Compared Means Probability Deviation Value Subject AA 12 Noon - 8 PM 0.288 0.18 3 8 PM - 8 AM 0. 2 59 0.15 0 0.5923 N .S. 12 Noon - 8 PM 0.288 0. 18 3 2 . 94 04 p < 0. 01 8 AM - 12 Noon 0. 5 06 0.300 8 PM - 8 AM 0. 2 59 0. 15 0 3.5491 P< 0. 001 8 AM - 12 Noon 0. 506 0.300 Subject EE 12 Noon - 8 PM 0.429 0.279 8 PM - 8 AM 0. 299 0.222 1.6991 P< 0 .l 0 12 Noon - 8 PM 0.429 0.279 8 AM - 12 Noon 1. 2 02 0. 745 4.6226 P<0.001 8 PM - 8 AM 0. 2 99 0.222 5.3224 P<0.001 8 AM - 12 Noon 1. 2 02 0.745 Subject GG 12 Noon - 8 PM 0. 3 01 0.213 0.848 N.S. 8 PM - 8 AM 0.353 0.187 12 Noon - 8 PM 0. 3 01 0. 213 3.2822 p < 0. 01 8 AM - 12 Noon 0. 540 0.273 8 PM - 8 AM 0.353 0.187 2.6392 p < 0. 01 12 Noon - 8 PM 0.540 0. 2 7 3 167 TABLE VI I CONTINUED STATISTICAL COMPARISON OF URINARY 17-KETOSTEROID EXCRETION AT DIFFERENT TIMES OF THE DAY PART g. ALL SUBJECTS Stu nda rd lit II Populo. tions Compared Means Probability Deviation Value 12 Noon - 8 PM 0.317 0. 060 0.8316 N.S. 8 PM - 8 AM 0.289 0.033 12 Noon - 8 PM 0.317 0.060 8 AM - 12 Noon 0.610 0.271 2.1522 P< 0.10 8PM - 8 AM 0.289 0.033 8 AM - 12 Nncl!1 0.610 0.271 2.3974 p < 0. 05 168 TABLE VII CORRELATION COEFFICIENT DERIVED BETWEEN 17-KETOSTEROID EXCRETION AND SEVEN INDEPENDENT VARIABLES (ALI:_ SUBJECTS) PART A. PRE-BED REST Corre lu. tion Level Vu.riab les Coefficient of r Siqnifica nee 17- Kc tos Leroids Creu tinine 0 .1944 P< 0.10 17- Kc Los Lcroids Hydroxyproline 0.3071 P< 0. 01 17-Kc Los Leroids 0.0521 N.S. ToLu l Calcium Excretion 17- Kc Los Lero ids 0.0264 N.S. Urinary Calcium 17-KeLosLeruicls 0.0922 N.S. To LC\ l NiiTOCJCl1 l 7- Kc tos Lcro ids 0.1687 P<0.10 17-Hydroxycorlicos Lcroic!s 1 7- Kc tos teroicls 0.1471 N.S. Crca tine 169 TABLE VII I CONTINUED CORRELATION COEFFICIENT DERIVED BETWEEN 17-KETOSTEROID EXCRETION AND SEVEN INDEPENDENT VARIABLES (ALL SUBJECTS) PART B. BED REST ONE Correlation Level Variables Coefficient of Y' Significance 17-Ketos teroids 0.0039 N .S. Creatinine 17-Ketosteroids 0 .1 04 9 N .S. Hydroxyproline 17-Ketos teroids 0. 059 0 N.S. Total Calcium Excretion 17-Ketos teroids 0. 082 0 N.S. Urinary Calcium 17-Ketos teroids 0 .1 04 3 N.S. Total Nitrogen 17-Ketos teroids 0.1430 N .S. 17-Hydroxycorticos teroids 1 7-Ketos teroids 0.1471 N.S. Creatine 170 TABLE VIII CORRELATION COEFFICIENT DERIVED BETWEEN 17-HYDROXYCORTICOSTEROID EXCRETION AND SEVEN INDEPENDENT VARIABLES (ALL SUBJECTS) ---PART A. PRE -BED REST Correlation Level Variables Coefficient of Siqnificance 17-Hydroxycorticosteroids 0.1150 N .S. Creatinine 17-Hydroxycorticos teroids 0 .1100 N .S. Hydroxyproline 17-Hydroxycorticosteroids 0.1272 N.S. Total Calcium Excretion 17-Hydroxycorticosteroids 0.1078 N .S. Urinary Ca lei urn 1 7-Hydroxycorticosteroids 0 .ll 07 N .S. Total Nitrogen 17 -Hydroxycorticosteroids 0.1687 P< 0.10 17-Ketosteroids 17-Hydroxycorticos teroids 0.0652 N .S. Creatine 171 TABLE vII I I CONTINUED CORRELATION COEFFICIENT DERIVED BETWEEN 17-HYDROXYCORTICOSTEROID EXCRETION AND SEVEN INDEPENDENT VARIABLES (ALL SUBJECTS) ---PART B. ------BED REST ONE Correlation Level Variables Coefficient of Siqnificance 17-Hydroxycorticosteroids Creatinine 0.2909 p < 0. 01 -- 17-Hydroxycorticosteroids Hydroxyproline 0.0292 N.S. 17-Hydroxycorticos teroids Total Calcium Excretion 0.2681 p < 0. 01 17-Hydroxycorticos teroids Urinary Calcium 0 . 12 08 N .S. 1 7-Hydroxycorticos teroids Total Nitrogen 0.1492 N .S. l 7-Hydroxycorticos teroids 17-Ketosteroids 0.1430 N .S. 1 7-Hydroxyc orticos teroids Creatine 0.2237 P < 0. OS TABLES FOR PART II OF THE STUDY Including Excretion of 17 -Hydroxycorticosteroids During Pre -Bed Rest Period by Eight Subjects Participating in a 56-Day Bed Rest Study Con- ducted in 1969 {Subjects 1A, 2A, 3A, 4A, 6A, 7A, SA, and 9A) 172 173 TABLE IX URINARY 17-HYDROXYCORTICOSTEROID EXCRETION DURING PRE-BED REST (Milligrams per 24 hours) PART !2_. SUBJECTS 1A, ZA, 3A, AND 4A Day Subject 1A Subject 2A I Subject 3A Subject 4A 1 0.929 4.507 3.700 1. 764 2 4 .1 00 0.790 3.250 4. 04 0 3 5.700 6.650 3.996 0.784 4 5. 017 3.092 1 . 310 1. 770 5 3.410 3.990 15.300 4.310 6 4.450 G. 034 4. 720 10.680 7 4.824 2.330 4. 88 0 5.460 8 1. 62 0 0.000 7.697 7. 080 9 8.660 4.260 4.510 0.620 10 2 .16 0 5.550 2 .1 01 0.315 ll 3.296 6.553 2 .117 3.423 12 1.802 4.582 8.300 3.390 13 15.712 7.990 4.670 6.700 14 5.790 3.324 4. 067 6. 040 15 8.390 4.958 3,095 6 .608 16 7.300 2. 671 4.240 4 .4 08 MEAN 5.198 4. 206 4.872 4.212 174 TABLE IX I CONTINUED URINARY 17-HYDROXYCORTICOSTEROID EXCRETION DURING PRE -BED REST (Milligrams per 24 hours) PART~· SUBJECTS 6A, 7A, SA, AND 9A Day Subject 6A Subject 7A Subject SA Subject 9A 1 3. 56 0 2.710 1 . 95 0 9.970 2 4.497 2.930 3.629 2. 097 3 6. 64 0 4. 96 0 2.600 0.899 4 7.870 1 .4 70 10. 08 0 2. 000 5 4.200 0.056 9.050 15.096 6 4. 56 0 l . 2 9 0 1 s. 84 0 8.430 7 0.884 0.145 ·1 .1 04 3.530 8 2. 34 0 7. 080 2.450 3.200 9 6.950 0.479 0.16 0 4.220 l 0 4.640 0. 605 1. 36 0 2. 100 ll 5. 964 3.257 9. 6 3 0 3.816 12 5.261 2.334 5. 38 0 3. 06 0 13 8. 086 1. 56 0 6.790 5.890 14 9. 060 2.4SO 7.330 2.664 15 5. 02 5 5. 04 0 4 .1 07 10.770 16 9.64 0 3. 03 0 6. 314 9.770 MEAN 5.574 2.464 5.673 5.426 175 TABLE X URINARY EXCRETION OF 17-HYDROXYCORTICOSTEROIDS DURING PRE-BED REST AT DIFFERENT PERIODS OF THE DAY (Milligrams per hour) PART Jl. SUBJECT 1A Day 12Noon-8P.M. 8 P.M.-8 A.M. 8 A.M. -12 Noon 1 0.042 0.028 0.655 2 0. 006 0.099 0.027 3 0.009 0 .133 3.510 4 0.199 0.17 5 0.525 5 0.220 0.233 0.958 6 0.326 0. 08 7 0.913 Mcun 0.134 0.126 1.098 PARTB. SUBJECT2A Day 12 Noon-S P.M. 8 P.M. -8 i\. M. 8 A . M . -12 Noon 1 0. 309 0. 192 0.490 2 0. 2 68 0. 048 0.465 3 0.054 0.563 0. 2 00 4 0.078 0.058 0.450 5 o... J42 0. 04 0 0.325 6 0.038 0.089 0.325 Mean 0.198 0.16 5 0.376 176 TABLE ;s_ I CONTINUED URINARY EXCRETION OF 17-HYDROXYCORTICOSTEROIDS DURING PRE-BED REST AT DIFFERENT PERIODS OF THE DAY (Milligrams per hour) PART g. SUBJECT 3A Day 12 Noon-S P.M. 8 P.M.-8 A.M. 8 A.M.-12 Noon 1 0.150 0.065 0.034 2 0.195 0.12 5 1 • 310 3 0.085 0.166 0.500 -- 4 0.352 0.103 0. 004 5 0.125 0.076 0.295 6 0.063 0.142 0.510 Mean 0.162 0.113 0.442 PART Q. S UI3JECT 4A Day 12 Noon- 8 P.M. 8 P . M . - 8 A . tv'I • 8 A . M . - 12 Noon 1 0.212 0.077 0. 2 00 2 0.325 0. 066 0.000 3 0. 6 06 0.024 0.385 4 0.250 0.1.:16 0.572 5 0.312 0.324 0.054 6 0.214 0. 083 0.425 Mean 0.320 0 .12 0 0.273 177 TABLE ~I CONTINUED URINARY EXCRETION 0 F 17-HYDROXYCORTICOS TEROIDS DURING PRE-BED REST AT DIFFERENT PERIODS OF THE DAY (Milligrams per hour) PART~· SUBJECT 6A Day 12 Noon-8 P.M. 8 P.M.-8 A.M. 8 A. M . -12 Noon 1 0.138 0.333 0.216 2 0.089 0.104 0.825 -- 3 0.266 0.415 0.245 4 0.4 69 0.175 0.8 03 5 0.541 0.010 0.144 6 0.330 0.217 1.100 Mean 0 0 306 0 0 209 0.556 PART£. SUBJECT 7A Day 12 Noon-8 P.M. 8 P.M.-8 A.M. 8 A . M . -12 Noon 1 0.021 0.236 0.065 2 0.224 0 0 042 0.008 3 0.010 0. 040 0.250 4 0.14 0 0 0 029 0.254 5 0.352 0. OS 1 0.4 02 6 0.026 0.055 0.540 Mean 0.129 0.076 0.253 178 TABLE~' CONTINUED URINARY EXCRETION OF 17-HYDROXYCORTICOSTEROIDS DURING PRE -BED REST AT DIFFERENT PERIODS 0 F THE DAY (Milligrams per hour) PARTQ. SUBJECT8A Day 12 Noon- 8 P . M. 8P.M.-8A.M. 8 A . M • -12 Noon 1 0.375 0.476 0. 2 30 2 0 .161 0.298 0.128 3 0.200 0. 32 7 0.318 4 0. 3 02 0. 2811 0.375 5 0.016 0.295 0. 110 6 0.321 0.312 0.000 Mean 0.229 0.332 0.194 Day 12 Noon- 8 P.M. 8 P.M. -8 I\. M. 8 l\.M.-12 Noon 1 0.436 0. 02 3 0.012 2 0.138 0.054 0. 327 3 0.088 0.000 1 . 2 9 7 4 0.200 0.013 0.226 5 0.812 0.238 0.355 6 0.260 0.491 0.275 Ivlean 0.322 0.136 0.415 179 TABLE XI STATISTICAL COMPARISON OF URINARY 17-HYDROXYCORTICOSTEROID EXCRETION AT DIFFERENT PERIODS OF THE DAY DURING PRE-BED REST PART A. SUBJECTS 1A, 2A, 3A Standard lit II Means Probability Populations Campa red I Deviation Value Subject 1A 12 Noon - 8 PM 0.134 0 .12 2 0.1156 N .S. 8 PM - 8 AM 0.126 0.066 12 Noon - 8 PM 0.134 0 .12 2 l . 7 !! s 3 N .S. 8/\M - 12 Noon 1.098 1 .121 8 PM - 8 AM 0 .12 6 0. 066 1.7668 N .S. 8 AM - 12 Noon 1.098 ] . 12 1 Subject 2A 12 Noon - 8 PM 0.198 0.151 0.2831 N .S. 8 PM - 8 AM 0.165 0.185 12 Noon - 8 PM 0.198 0.151 8 AM- 12 Noon 0.376 0. 102 1.9861 P< 0.10 8PM - 8 AM 0.165 0.185 8 AM - 12 Noon 0.376 0.102 2.0354 P< 0.10 Subject 3A 12 Noon - 8 PM 0 .162 0.095 8 PM - 8 AM 0.113 0.036 0. 98 06 N .S. 12 Noon - 8 Pf\/1 0 .162 0.095 8 1:'\M - 12 Noon 0.442 0.436 1.2826 N .S. 8PM - 8 AM 0.113 0.036 1 . 536 3 8 .2\M - 12 Noon 0.442 0.436 N .S. 180 TABLE ?:U I CONTINUED STATISTICAL COMPARISON OF URINARY 17-HYDROXYCORTICOSTEROID EXCRETION AT DIFFERENT PERIODS OF THE DAY DURING PRE-BED REST PART~· SUBJECTS 4A, 6A, 7A llo. II Standard L Probability Populations Compared I Means Deviation Value Subject 4A 0.135 12 Noon - 8 PM 0.320 2.4413 p < 0. 05 8 PM - 8 AM 0.12 0 0.098 12 Noon - 8 PM 0. 32 0 0.135 0.3916 N .S. 8 AM - 12 Noon 0. 2 73 0. 2 05 8 PM - 8 AM 0.12 0 0.098 1.36?30 N .S. BAM - 12 Noon 0. 2 73 0.205 Subject 6A 0. 306 0.16 3 12 Noon - 8 PM 0.9304 N.S. 8 PM - 8 AM 0. 2 09 0. 13 5 0. 3 06 0. l 6 3 12 Noon - 8 PM 1 . 2 6 88 N .S. 8 AM - 12 Noon 0.556 0.368 0. 2 09 0.135 8 PM - 8 AM 1. 8052 N.S. BAM - 12 Noon 0.556 0.368 Subject 7 A 0.12 6 12 Noon - 8 PM 0 .12 9 0.7494 N .S. 8 PM - 8 AM 0.076 0.072 0. 12 9 0.126 12 Noon - 8 PM 1.1441 N .S. 8 AM - 12 Noon 0.253 0.183 0.076 0.072 8 PM - 8 AM 1. 84 72 P< 0.10 8 AM - 12 Noon 0. 2 53 0. 183 1S1 TABLE X I I CONTINUED STATISTICAL COMPARISON OF URINARY 17-HYDROXYCORTICOSTEROID EXCRETION AT DIFFERENT PERIODS OF THE DAY DURING PRE-BED REST PART C. SUBJECTS SA, 9A, ALL SUBJECTS S ta ncla rd lit II Populations Compared Means Probability Deviution Value Subject SA 12 Noon - S PM 0. 229 0.12 0 1.5368 N .S. 8PM -SAM 0.332 0. OGG 12 Noon - S PM 0.229 0.12 0 0.4151 N .S. 8 l\M - 12 Noon 0.194 0.128 8PM - 8 AM 0.332 0. 066 1.%29 P< 0.10 8 l\M - 12 Noon 0.1911 0. 12 8 Subject 9A 12 Noon - 8 PM 0.322 0.245 1.2528 N .S. 8PM - n 1'\M 0.137 0 .1 78 12 Noon - 8 PM 0.322 0 . 2 :l s 0.3976 N.S. 8 i\M - 12 Noon 0.415 0.410 8PM - 8 l'd'vi 0.137 0.178 1.2745 8 1'\M - 12 Noon 0.415 0.410 N .S. All Subjects 12 Noon - 8 Prvr 0.225 0.169 2 . 02 4 p < 0. 05 8 Pi'vi - 8 Aivi 0.1 GO 0.138 s 12 Noon - 8 P !vi 0.22S 0.169 2.6595 p < 0. 01 8 Ai'vi - 12 Noon 0. 4 51 0.551 S P iVI - s Al'-/1 0.160 0 .13S 3.47SO S i\I'vi - 12 Noon 0. 4 51 0.551 P< 0.001