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1963 Turnover rates: disappearance and conjugation of free etiocholanolone Peter Berkeley Gregory Yale University
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Turnover Rates: Disappearance and Conjugation of Free Etiocholanolone
Peter B. Gregory Yale School of Medicine 1963
Dedicated to
George L, Cohn, Philip K» Bondy arid Morris Dillard
for their invaluable assistance Digitized by the Internet Archive in 2017 with funding from The National Endowment for the Humanities and the Arcadia Fund
https://archive.org/details/turnoverratesdisOOgreg 1
It is recognized that certain endogenous steroids xv^ith a 5-3eta configuration produce a febrile response in man, (1,2,3)
Several of these compounds may have well known physiologic functions 5 the temperature rise in the luteal phase of the menstrual period, with the concomitant increase of progesterone and its pyrogenic metabolites, pregnanolone and pregnanediol,
Other 5-Beta steroids such as 11-keto-pregnanolone, pregnane- dione, 21-hydro xy-pregnanedione, 11-Beta-hydroxy-etiocholanolone, etiocholanedione, and etiocholanolone have no known responsi¬ bility for temperature regulation in normal individuals, (3)
In 1957, Kappas and his collaborators (4,5) administered etiocholanolone intramuscularly to normal volunteers. The re¬ sponse to 100 mgm, was severe. Shaking chills occurred after a four to six hour latent period. Subsequent findings during the next five hours included x^reakness , muscular aches and pains, anorexia, sx^eating, severe headache, and temperature elevation to 103° F. The systemic reaction began to diminish at the eleventh hour and ceased by the eighteenth hour. Leukocytosis with a shift to the left, occasional acute thrombocytosis, and an eosinophilic reaction after two xveeks1 administration were also noted. Tolerance to administered etiocholanolone was not found; continued administration gave continued fever. Pre- treatment xvith cortisone and aspirin significantly reduced the systemic reaction. Conjugation of etiocholanolone completely removed its pyrogenic effect. (3) *
c
1 2
In 1953* Bondy, Cohn e_t a_1_ - (6) reported two patients with recurrent fever, abdominal pain, arthralgias and leukocytosis, who consistently excreted large amounts of etiecholanolone in
their urine during a febrile episode. It was also noted that during a febrile attack, unconjugated etiocholanolone was de¬ monstrable in their plasma.
Further studies by Bondy, Cohn, Castiglione (?) indicated
that normal individuals have a mean etiocholanolone concentra¬
tion of 0.46 jug/lOO ml, plasma, with a maximum level of 1,22 ng.
Fifteen patients with fever of known etiology, including three with Familial Mediterranean Fever, had etiocholanolone levels within this normal range. (8) Five "Etiocholanolone Fever" patients had plasma levels of 2.0-7.3 (average of 5.3) jig/100 ml. during the rising phase of their temperature peak. (8)
Attacks were not precipitated in patients Ttfith Etiocholan¬ olone Fever either with ACTH or steroid precursors of etiocho¬ lanolone, such as testosterone and 11-deoxy-cortisol. Both
ACTH and testosterone did, however, increase plasma levels of free dehydroepiandrosterone ( 0,1- 0,92) and free androsterone
( 0.1- S.5-9.2), without significant change in the etiocholan¬ olone level. Twenty-five mgra. of 11-keto-pregnanolone provoked a typical attack of fever in these patients. During this iatro¬ genic attack, large amounts of unconjugated etiocholanolone and dehydroepiandrosterone were found in their plasma.
The data suggested two steroid metabolic abnormalities might be responsible for Etiocholanolone Fever. The increased
1
plasma levels of free dehydroepiandrosterone and androsterone upon ACTH stimulation suggested a limitation of conjugating capacity for androgens. The high levels of etiocholanolone rather than androsterone during an attack required further ex¬ planation, The authors postulated a 17-ketosteroid metabolic pathway shift whereby etiocholanolone is produced from dehydro¬ epiandros terone at the expense of androsterone.
This study was designed to establish the disappearance and conjugation rates of etiocholanolone in normal individuals,
In add.ition9 these rates were studied under the influence of
ACTH (40 units) and 11-keto-pregnanolone in one subject (B.G.).
A pilot study to determine the i_n vitro distribution of etio¬ cholanolone between red blood cells and plasma was also carried out.
METHODS AND MATERIALS
A: Disappearance of Radioactive Etiocholanolone
Subjects*, Five males and one female, ranging in age from nineteen to twenty-four, were used as subjects. No history of previous liver or endocrine disease was obtained. All were in good health at the time of experimentation. All experiments were carried out in the morning before breakfast.
One female (B.G.), aged twenty-eight, was used for the
ACTH and 11-ketopregnanolone studies mentioned above. She suffered from a recurrent fever of 101-102° F,, which occurred eight to nine days before the menstrual period and disappeared \
n . : • ' 4
with the onset of menstruation. No etiology was found. Liver function tests, hormone analyses and urinalysis ivere normal.
Experimental Procedure: An 18-gauge needle with a syringe of heparin, connected to a three-way stopcock was placed in an antecubital vein of the volunteer. Ten ml. of a pyrogen-free isotonic saline solution containing 8,6 micro-curies of tritium labelled etiocholanolone and 5 ml. of T-X824 (Evan's Blue) was injected intravenously.
Twenty ml. samples of blood were drawn into a heparinized syringe at zero time and 3, 6, 9, 12, 15* 20, 25, 30, 45, 60, and 90 minutes, after the administration of the isotope. The average time needed to remove the blood samples was thirty to fort}'- seconds.
At the conclusion of the experiment, the heparinized blood samples were placed in 60 ml, centrifuge tubes and centrifuged at approximately 2500 rpm for thirty minutes, (International
Centrifuge Model SBV) The volume of plasma removed after the above separation was measured to the 0,2 cc. by volumetric pipette. One ml. of plasma \ Extraction: The plasma was extracted seven times with twice volume reagent grade chloroform after the addition of 0.5 ml. of 1 N NaOH. The seven chloroform extracts were then pooled and air dried in Erlenmeyer flasks. The residue was ( I transferred quantitatively with four chloroform washes to 20 ml. counting vials. The sample extracts were counted in a Tri-Carb Scintillation Model 31k EX, for at least three ten-minute counts. This counter has an efficiency for tri¬ tium of 16-17$, %*rith a background of 180 DPM. A known amount of radioactive etiocholanolone was then added to each sample extract in order to correct for quenching. The values were recorded as Disintegrations per Minute per Mililiter (DPM/ml.) of plasma. The radioactive extracts of one subject (D.L.) were combined after the above procedures and air dried. The re¬ maining phosphor salt and the radioactive material was extracted with 20$ ethanol,* The extract was air dried and the residue transferred with Chloroform to Whatman No. 1 filter paper,for chromatography in the 1:1 Heptane: 96$ Methanol system of Bush and Willoughby, (9) Appropriate C-19 standards were applied concomitantly. The area of radioactivity was located with a strip scanner (Nuclear Chicago Model 1036). This radioactive area was eluted with absolute alcohol at 37° C. and the sample rechromatographed sequentially in the above system and in the heptane:propylene glycol system of Rubin. (10) The only radio¬ activity found corresponded to the standard etiocholanolone (from Mann Research Laboratories, New York, New York--Lot# C1263). * Previous experimentation with this method of extraction gave 85-90$ re covery of radioactive etiocholanolone with only one 20$ ethanol wash. • ■ r • 6 B: Explanation and Evaluation of Experimental Procedures Used- Above in the Determination of the Rate of Disappearance of Free Radioactive Etlocholanolone, In an initial experiment on S.B. (Graph No, 1), 4,3 micro -curies of radioactive etiocholanolone were injected and blood samples were drawn at zero time and at 15» 30, 60, 90, 120, 180, 2,40 and 1440 minutes. Detectable levels of radioactive etiocholanolone (chloroform extractable) were no longer present in the plasma after 60 minutes. No significant radioactivity was found in the red blood cells after precipitation with ab¬ solute alcohol and extraction with chloroform. These data neces¬ sitated the following changes in procedures 1, Carry out future experiments to only 90 minutes, 2, Extract only the plasma for radioactivity, 3, Inject 8,6 micro-curies of radioactive etio¬ cholanolone rather than 4.1 micro-curies. The values for radioactive etiocholanolone (in S.B.) at the zero and 15 minute mark indicated the need to study etio¬ cholanolone clearance in more detail during the first few minutes after injection. Samples were taken at zero time and 3, 6, 9» 12, and 15 minutes thereafter. In order to correct any error introduced by incomplete plasma isotope mixing in samples taken so soon after injection, T-X824 (Evan’s Blue) was injected xcith the radioactive etiocholanolone, A correction factor was determined by comparing the optical density of the early sample with the optical density of the samples obtained after 15 minutes when complete mixing had taken place. The t c z factor was seldom greater than the error inherent in the optical density determination, except in the zero minute sample. Corrections in this sample ranged up to three times the obtained value, ivith the usual correction being approximately twice. Corrections in later samples were alxvays under 10$ of the original figure, NaOH was added to each sample initially since it de¬ creases the transfer of "quenching material" into the chloro¬ form, This observation was documented by the following experiment; 27,600 DPM of radioactive etiocholanolone were added to two equal samples of plasma, NaOH xvas added to one of the samples. Both samples were then extracted seven times with chloroform. The extracts were pooled, air-dried, and counted in the scintillation counter. The recovery of radioactivity was 5Qfo greater in the NaOH- treated sample. The decision to extract plasma seven times with chloro¬ form ivas determined by the following experiment: Two samples of plasma with known DPM of radioactive etiocholanolone were extracted ten times with twice volume chloroform. Each extract was counted separately in the scintillation counter. As per Graph No. 2, it is evident that significant radio¬ activity ( above background) remains through the seventh extraction. 88$ of the radioactivity was removed from one sample in three extractions. Another 7$ was recovered in the next four Seven chloroform extractions were performed c to insure that all the free steroid was removed and that the remaining radioactivity was conjugate only. Graphs Nos, 1 and 2 follow: 10000 1 Normal NaOH and Chloroform 12960 DPM Radio active bti ocholanolo ne added to two dI asma sampl es : 9000 1. 7.5 ml. P tored plasma —) 2. 10.0 ml. fresh plasma (X —) $ recovery without c orrec tion for 8000 quencning: (after 7 cnioroi ortn extractions/ 1. Stored plasma 95fo ^ . Fresh plasma 8 5# Q 7000 itaciioacriviry xn ser iai extractions Extract: Freslfi(DPM) Stored (DPM) 1 4686 7398 cO 2604 2o44O O )« l» 3 1518 1206 4 1110 468 6ooo ;> 780 6 264 90 7 9 6 42 Q 24 j- — 9 24 18 10 24 18 5000 X DPM 400 0 a —Tv \\ 300 o ——o (> 1 ( n' - 200 0 X ~ — L [- J i < 100 o N \ K X S. ! \D Serial Extractions l1 -10 ) 1 2 3 4 5 V " 1 C: Conjugation of Radioactive Etiocholanolone. The plasma residue after chloroform extraction for free steroid was then extracted three times with absolute alcohol and twice with n“butanol to recover the conjugated 1 (xrater soluble) etiocholanolone. The pooled n-butanol-alcohol extracts were transferred to test tubes and air dried,, Exactly 10 ml, of n-butanol was added to the residue, mixed thoroughly and allowed to stand at room temperature for at least twenty-four hours. One ml, of the n-butanol solution was pipetted into a 20 ml, counting vial and counted in a liquid phosphor scintillation counter as described for the free steroid. Correction for "quenching" was not performed because pure radioactive conjugate was not available. In one experiment{G,R,), the residue remaining after five extractions was transferred to counting vials and counted. No significant radioactivity icas observed. The extraction procedure was considered to effectively remove all conjugated radioactive steroid. D: Preliminary Identification of the Conjugate Fraction. The 9 ml, of conjugate fraction remaining after the 1 ml, removal for radioactivity determinations were utilized as follows: The conjugate fractions of C.K. and B.H. were combined in order( 0 Min/C.K. + 0 Min/B.H.; 3 Min/C.K, + 3 Min/B.H,,etc,), and placed in separate flube tubes.and air dried. The sides of the tube were washed with ethanol(5ml,) several times until the material was concentrated in the 10 narrow part of the tube. Each sample ivras transferred with absolute alcohol to Whatman No. 1 filter paper and chromato¬ graphed in the Ethyl Acetate:Hexane:Acetic Acid:Water, 120:80:60:1 if-Q system of Schneider and Lewbart(ll) with androsterone glucuronide and sulfate standards. (Etiocho- lanolone conjugate was not available.) The radioactive strips were then cut in serial one inch sections. Each section was placed into a separate counting vial and counted as described. The results were graphed as DPM/one inch of chromatograph s trip, The conjugate sample spotted on the filter paper was large and bulky. An attempt was made to purify the last six samples(20 Min-90 Min) by eluting the entire strip after the first chromatography run. The eluate was dried, re¬ spotted and chromatography was performed once more. Very little gross purification was achieved. E: Effect of ACTH and 11-keto-pregnanolone on the disappear¬ ance rates of radioactive etiocholanolone in one subjectXB.G.) Three etiocholanolone disappearance rate experiments were carried out on B.G. In this series, T-1824 was not used and quenching corrections were considered unnecessary since yello\\r color contamination was not visible in the extraction solutions. Experiment No. Is a, 8.6 micro-curies of radioactive etiocholanolone were injected intravenously. b. Serial blood samples were removed for 80 minutes after the injection. 11 Experiment No, 2; a. 4 l/2 hour infusion of 40 units ACTH, which was started k 3/h hours prior to intravenous administration of 8.6 micro-curies of radioactive etiocholanolone. b. Serial blood samples were removed for 80 minutes after the injection. Experiment No. 3' Part 1 a. 25.0 mgra. of 11-keto-pregnanolone were given intramuscularly 5 hours and 21 minutes prior to the first injection of 8.6 micro-curies of radioactive e tiocholanolone, b. Serial blood samples were removed for 60 minutes after the injection. c. Temperature was 100' F. 7 l/2 hours after the 11-keto-pregnanolone injection. d. One blood sample was drawn 189 minutes after the first radioactive etiocholanolone injection. Part 2 a. A second injection of 8.6 micro-curies of radio¬ active etiocholanolone was given 8 hours and. 52 minutes after the 11-keto-pregnanolone injection. b. Serial blood samples were drawn for 80 minutes after the second radioactive etiocholanolone injection. o c. Her temperature was 101 F. 10 hours after the 11-keto»pregnanolone injection. d. The experiment was terminated five minutes later with 200 mgm. of Solu-Cortef intravenously„ F: In Vitro Distribution of Radioactive Etiocholanolone Between Red Blood Cells and Plasma--Pilot Study 13) Twenty-one mililiters of blood, were drawn from a normal volunteer and placed in a 60 ml. centrifuge tube, which contained 90,5^0 PPM of radioactive etiocholanolone. The blood was mixed thoroughly to insure solution of the steroid. The hematocrit of this sample was determined by the micro¬ capillary tube method. The actual volume of red blood cells and plasma in the sample was calculated. Centrifugation for 20 minutes was carried out and a measured amount of plasma 12 was removed by pipette. The red cells and plasma were separately precipitated with absolute alcohol and extracted with chloroform. The radioactive free etiocholanolone was determined as outlined in Section A. Calculations included correction for radioactivity contained in plasma not removed from the red cells by centrifugation. RESULTS A: Disappearance and Conjugation of Radioactive Etiocholanolone. The disappearance of free radioactive etiocholanolone and its subsequent conjugation is shown for the first six experimental subjects in the following graphs, Nos. 3-S. Whereas a "hyperbolic" curve most closely fits the experimental values, exponential decay curves can also be applied. Three separate decay curves with three half lives are determined; Initially there is a very rapid fall-off of radioactivity with an approximate half-life of 1-2 minutes. This is folloxv'ed by a slower decay with an average half life of 15 minutes (range 12-17). This in turn is followed by still sloiirer decay, with an average half life of 65 minutes (range 28-102), The conjugate fraction rises rapidly with a peak reached in 8-20 minutes (average 15). The samples of S.B. were extracted for conjugate radio¬ activity more than six months after the experiment was performed. The values obtained in this s ubject do not corres pond with the other five, suggesting ir regular hydrolysis d uring the long storage period. The dec reased levels of con jugate in P.G. also reflect a prolonged time lag before ext raction. Graphs Nos. '}~8 folloxv: FREE AND CONJUGATED RADIOACTIVE C.K. 6/19/62 t ET10CH0LANOLONE IN PLASMA Minutes after Etioc’ tiocholanolone Etiocholano Exponen / a \ O 0 emi-Logarithmic jcles x 10 to the inch FREE AND CONJUGATED RADIOACTIVE ion ■mi-Loftarithmic :lcs x 10 to the inch FREE AND CONJUGATED RADIOACTIVE B.H. 6/6/62 & ETIOCHOLANCLONE IN PLASMA~ .‘mi-Lo£arithmic kies x 10 to the inch FREE AND CONJUGATED RADIOACTIVE G.R. 6/26/62 & Hyperbolic approximatio of conjuga mi-Logarithmic les x 10 to the inch FREE AND CONJUGATED RADIOACTIVE P.G. 3/26/62 STIOCHOLANOLONE IN PLASMA Minutes after Etiocholanolone E t i o ch.o 1 an o 1 o n e etiocholanol one Exponential Decay Curve § Exponential Decajr Curve f Exponential Decay Curve ocholanol Curve MINUTES A mi-Lo£arithmic les x 10 to the inch 14 B: Preliminary Identification of the Conjugate Fraction. Average recovery of the conjugate material from the chromatograph strips was 3^%* with a range of 16-80%. The six samples that were run twice in the same system gave an average recovery of 47%. The six samples run only once gave a recovery of 21%. Rechromatographing the samples in the same system did remove contamination, although it did not grossly appear to do so. After a 12-hour run in the Schneider-Lewbart acid system, the androgen sulfates as a class travel l/4-l inch from the origin. The androgen glucuronide travels approximately 6-8 inches from the origin. All the recovered radioactivity from the chromatographed etiocholanolone experimental samples peaked in the area corresponding to the glucuronide standard. This is shown in Graph No. 9 of the 25 minute sample and in the ac- cotnpanjang table (§l) for all the conjugate samples. The 20 minute sample was grossly contaminated before chromatography (see Graph No. 5-»the 20 minute sample of B.H.). Graph No, 9 follows: Table No. 1 follows: , Graph jf= 9 CO !N- i—I 'O H Sjo 0 OOOOHCN'AO^J-OOCMOOOOOO Q_,| C^N H PAN^O H V> L nl CM CM pH CM vc\ -3- pH £ tp CP •H 0) s m «=$ Pi P O -rH ON Pi -P a p p c5 W 3 ^ 0 O - Ph £ w Pi PI S P £ rH o P 0 K tyj c O O H 01 C°l4 'A'O NCO HN >OCO N ffi O -H I HrliHHHHrlH O -P o + Andros terone Glucuronid e ffi X w CP CO w £-h D H > H p-\ CP I >H H W cvi H Androsterone sulfate 01 O ' O ' O 0 ■ 0 o 1 0 O O s O 0 0 o VO CM CO cp -3' 0 MO CM CM CM rH Q tH rH Table No s •H H P in C © Chromatograph strip VO A •H VO JC3 Cl CM CA CM rH CM rH iA O o o »A cm VM O 0M| a o in p u in c a1 o 1 VV OHNCMAfVMVOivCO CM Cl N CMrHHr-irir-lrfl CMH' rH s pu p i I * ! 1 00 tv CA tH A tH Avovmc'tv,tvah i rHr"lr-l CO A A «a* P H w rHrlHHHrHHrHHHHHHHHHHH CM Cl01 CM ClC>>^Av-VOvMv'vAvMvAv rH CMCAA”VMVd>tvCO CM rH CM VM i »s •=t rH ClCAA-vAVOEvCOCMo rH CMCAAVMVCtv CM CM rH rH H AHHVMO00VO A AOCdOMtvCMrH NH O00A6OM CM MOPHtv£vjvo »A CMVOA CAA NHtvVMCACT\tv-VOVO‘-ACO rH 0s!CMVMOAMO{N- A tHCAtvCM A 00CMVOCAtv VDOJAOGaONWVDAO rH 'ArjH rH VOVM\.f\V\ pH A"00VOCMCAHfH A HO)CMoCOVOAAva «H AHrHiH O AVMMm00 pH A'COCMfH VM tvCAVO rH CAVA0D H tvVM CM IVA CM tH CA cfHtvVO rH OMVM©A00Cl rH CMCAH if CMdlH A CMtv N.VO rrltv CM rH pH r* CM A O A-CA CM »A CM00A i ^! cm : rH CA cis A p o p a O H W P CM ClC.lC? A* ACArH HHrHHHHHH O VMAClIVVO VM CMtHOMA o vm o VM VO CM VM rH CM VM o *A 12 C : Effect of ACTI-I arid 11-Keto-Pregnanolone on the Disappearance Rates of Radioactive Etiocholanolone in~One Subject (B,G.). The curves obtained from the three experiments outlined in Section E of Methods and Materials are reproduced on Graphs Nos. 10-12, The four experimental curves do not differ materi¬ ally in slope or in character. Again, initial rapid disappear¬ ance of radioactive etiocholanolone is noted. This is followed by two decay curves with half lives of 13 and Jk respectively. These are within the range of values found in the six normal volunteers. Di In Vitro Distribution of Radioactive Etiocholanolone Between Red Blood Cells and Plasma A pilot study gave a distribution of free etiocholanolone as fo1lows: 8°/o in association with red blood cells 92f0 in association with plasma. Graphs Nos. 10-12 follow; : EFFECT OF ACTH AND 11-KET0PREG-NANOLONE 7/5/62 & DISAPPEARANCE RATES OF RADIOACTIVE CLONE: 1005 Exponential Expor mi-Loftarithmic lcs x 10 to the inch EFFECT OF ACTH AND 11-KETOPREGNANOLONE B.G. 7/7/62$ ON THE DISAPPEARANCE RATES OF RADIOACTIVE BTIOCHOLAN PLONK: EXPERIMENT ^ 2 (POST ACTH) onential ?mi-Lo£arithmic :les x 10 to the inch EFFECT OF ACTII AND 11 -KBTOPREGNANOLONE ON THE DISAPPEARANCE RATES OF RADIOACTIVE IOCHOLANOLONE: EXPERIMENT 2 PARTS 11-KETOPREGNANOLONE) Five hours and twenty-one minutes Minutes a clone Etiocholano ight hour y-two minutes iocholano lone ) t t t 16 DISCUSSION Disappearance rates and half lives have been determined for a number of steroids. Cortisol has a half life of 80 minutes; corticosterone t l/2 = approximately 505 andro- sterone t l/2 = 20 minutes(14-18). Slaunwhite and Sandberg(l6) established disappearance rates for etiocholanolone in five female subjects with carcinoma of the breast. Straight line approximation from their 15, 30 and 60 minute samples gave a half life of 19 minutes. The disappearance rate of testosterone was also studied(16): two phases were found with half lives of 8 minutes and 80 minutes. In other studies (19-22) testosterone was observed to have half lives of 11 and 100 minutes; Estradiol and estrone had half lives of 20 and 70 minutes; 11-hydroxy-andros tenedione, t l/2 = 30 and 80 minutes; Dehydroisoandrosterone, t l/2 = 17 and 125 minutes. The conjugate samples in the steroids with two- phase decay curves( two half lives) consistently reached a peak within 15-30 minutes and declined slowly thereafter(19-21). Our studies on the disappearance rate of etiocholano¬ lone clearly suggest three separate phenomena. Initially, there is a very rapid fall-off with an approximate half life of 1-2 minutes. It has been stated(13,23) that distribution of steroid between plasma and red cells is instantaneous in vivo and in vitro. The percent of steroid associated with the red blood cells varies considerably; 6fo of blood 1L corticosterone is in the red cell as contrasted i^ith 31$ (24,25) of blood estradiol. The possibility arose, therefore, that this rapid fall-off in radioactive etiocholanolone might in part be due to rapid transfer into the red cells. The initial experiment on S.B, demonstrated that little if any radio¬ active etiocholanolone is present in the red cells in vivo after intravenous infusion. An in vitro pilot study demonstrated that only 8$ of the steroid dose was actually associated with the red cells, the rest remaining in the plasma. These two studies make it unlikely that the rapid plasma fall-off is due to red blood cell uptake. Consequently, we can only echo Migeon(l4) who postulated a rapid extra- vascular distribution for cortisol, site unknown. The second decay curve has a half life of 15 minutes with a range of 12-17 minutes. Our T-1824 studies and the T-1824 literature(26) emphasize that complete plasma mixing has taken place by ten minutes in normal individuals. Our etiocholanolone decay curve(t l/2 = 15 minutes) persists through 30 minutes after the steroid injection. The curve cannot be a result of plasma mixing alone. As has been considered in the past(l4), it may result from the mixing of the radioactive steroid with the endogenous etiocholano¬ lone pool. Our last decay curve with a half life of 65 minutes corresponds to the half life for cortisol and with the second half lives of testosterone, estrone, estradiol, and 11-hydroxy-androstenedione(14-21). Etiocholanolone disappears from the plasma in three phases with distinctly different half lives. We must assume that the disappearance of free etiocholanolone represents its conjugation, as by far the most prominent metabolic pathway for etiocholanolone is conjugation(5). A possible explanation for the decay curves follows. Our preliminary identification of the conjugate fraction suggests that most of the conjugate is glucuronide. It is known that etiocholanolone glucuronide is rapidly cleared by the kidney(177 ml, of plasma/minute).(17) The formation of conjugate, then, is the effective metabolic end point for etiocholanolone. In so far as the decay curve(t l/2 = 65) represents the final disappearance rate of etiocholanolone, it seems reasonable to conclude that this curve(t l/2 = 65) represents turnover of the free steroid into the conjugated, which is then excreted. The faster decay curve(t l/2 - 15) must represent some step or steps preliminar)^ to glucuronide formation. Perhaps enzyme binding is the more rapid step represented. In this case, the binding would be to glucuronysl transferase which is present in microsomes(27,28). The (t l/2 - 15) decay curve, therefore, represents the disappearance of radioactive etiocholanolone resulting from the processes of '’binding" and "conjugation." With the saturation of the "enzyme" at the 20«=30 minute mark, the disappearance curve assumes the rate of the rate-limiting step(t l/2 - 65), which we consider to be "conjugation." 11 The curves for the conjugation of radioactive etio- cholanolone tend to confirm the above explanation if we consider the following: 1. The rapid rise of conjugated steroid emphasizes that etiocholanolone is quickly and predominantly conjugated. 2. The peak of the conjugated steroid at 8~20 minutes closely approximates the postulated "enzyme saturation" at the 20-30 minute mark. 3. The slow decay of conjugated etiocholanolone after the peak, despite the rapid clearance by the kidney, strongly suggests that conjugation is continuing during this period. If so, the slow decay eurv@(t i/2 - 65) must reflect the passage of free steroid out of the blood stream, through the saturated preliminary processes, binding, etc., through the unknown rate-limiting step, and finally out into the blood stream as conjugated etiocholanolone. We must reemphasize here that the decay curve(t 1./2 s 15) may not represent one of the steps in conjugation. It may Qnl)r reflect the mixing of radioactive steroid with the endogenous steroid pool. The conclusion is still that some step in the conjugating process is the rate-limiting one and that it is reflected in the decay curve(t 1/2 - 65). The disappearance and conjugation rates obtained experimentally for etiocholanolone correspond well with those observed in other laboratories for testosterone, estrone, estradiol, 11-hydroxy-androstenedione and •: 20 dehydroisoandrosterone(14-22), This suggests that these steroids have a common metabolic pathway in man. The experiments on B.G. with ACTH and Xl-keto- pregnanolone document the fact that these hormones have no recognizeable effect on the metabolism of radioactive etiocholanolone» Further experimentation should be designed to determine if etiocholanolone disappearance and conjugation rates are abnormal in Etiocholanolone Fever patients. As it is known that 11-keto-pregnanolone will provoke an attack in these patients, etiocholanolone disappearance studies should be carried out under the influence of this hormone. Further work on the conjugate fraction is necessary in order to determine whether sulfation also occurs. SUMMARY 1, Six normal subjects aged 19-24 were given 8.6 micro¬ curies of radioactive etiocholanolone intravenously, Serial blood samples were drawn through ninety minutes. The plasma was analyzed for free and conjugate radioactivity, 2. The disappearance curves for free etiocholanolone obtained experimentally demonstrated three separate half lives : a. t l/2 = 1-2 minutes b. t 1/2 » 15 minutes c. t l/2 - 65 minutes The conjugate fraction rose rapidly to peak at 15 minutes and then dropped sloxtfly over szozurt - p }i>£ minutes. Preliminary identification procedures suggest that the conjugate is predominantly glucuronide. 4, A possible explanation for the three separate half lives is offered: a. (t l/2 = 1-2 minutes); extravascular distribution. b. (t 1/2 = 15 minutes); ? early step in conjugation; ? binding to glucuronysl transferase, c. (t l/2 = 65 minutes); rate limting step in conjugation. 5, In one subject (B.G.), etiocholanolone disappearance rates were studied under the influence of ACTH and 11-keto- pregnanolone, No deviation from normal was observed, 6, An in vitro study revealed the distribution of radioactive etiocholanolone between red cells and plasma to be : a, 8$ associated with the red cells, b, 92% associated with plasma. 22 BIBLIOGRAPHY 1, Buxton, C.L,, Atkinson, W.B.: Hormonal factors involved in the regulation of basal body temperature during the menstrual cycle and pregnancy, J. Clin* Endocr,, 8, 544, 19^8. 2. Davis, M.E., Fugo, N.W. ; The cause of physiologic basal temperature changes in women, J. Clin. Endocr., 8, 550, 1948. 3. Kappas, A., Saybell, ¥» , Fukushima, D.K., Gallagher, T.F., Studies on pyrogenic steroids in man, Trans, Assoc. Amer. 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Sandberg, A,A. and Slaunwhite, W.R., Studies on phenolic steroids in human subjects: 2, The metabolic fate and hepato-biliary-enteric circulation of C-l4 estrone and C-l4 estradiol in women., J. Clin, Invest., 36,, 1266, 1957. 22. Horton, R,, Forsham, P.H., Metabolism of dehydroiso- androsterone, Clinical Research, 11, 114, 1963. 23. Migeon, C.J., Lescure, O.L., Antoniades, H.N., Further- in vitro studies with 16-C-lU estrone: Distributions between plasma protein fractions and red blood cells of man, The Johns Hopkins Bulletin, 106, 317, I960. 2k. Sandberg, A.A., Slaunwhite, W.R., The binding of steroids and steroid conjugates to human plasma proteins, Recent Prog. Hormone Res., 1_3, 209, 1957. 25. Goldzieher, J.W., Baker, R.E., Nyman, M.A., The dis¬ tribution of estrogens betii/een red cells and serum., Endocrinology, 65., 2, 310, 1959. 26. Warner Chilcott- Evan's Blue (T-l82if-)-- For determination of circulating blood volume. 27. Isselbacher, K.J., Axelrod, J., Enzymatic Formation of corticosteroid glucuronides, JACS, 77, 1070, 1955. 28. Isselbacher, K.J., Enzymatic mechanisms of hormone metabolism; 2. Mechanism of hormonal glucuronide formation., Rec. Prog. Hormone Res,, 1_2, 13^, 1956, i YALE MEDICAL LIBRARY Manuscript Theses Unpublished theses submitted for the Master's and Doctor's degrees and deposited in the Yale Medical Library are to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but passages must not be copied without permission of the authors, and without proper credit being given in subsequent written or published work. This thesis by Peter 8. y has been used by the following persons, whose signatures attest their acceptance of the above restrictions. NAME AND ADDRESS m £. Suo S-b-^jh a/,y. w. /ovfey