ARL-TDR-63-17

0 CHARACTERIZATION OF HORMONAL STEROIDSOF THE CHIMPANZEE

Enrico Forchielli, Ph.D.Fred Elmadjian, Ph. D.

LU

C() TECHNICAL DOCUMENTARY REPORT NO. ARL-TDR-63-17

May 1963

_6571st Aeromedical Research Laboratory'Aeroipace Medical Division

Air Force Systems CommandSjHollotman Air Force Base, New Mexico

Project 6892, Task 68921

(Prepared under Contract No. AF 29(600)-2439 by'the Worcester Foundation for Experimental Biology,Shrewsbury, Massachusetts.)

O T

NOOT

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Do not return this copy. Retain or destroy.

FOREWORD

The authors ac1'owledge the invaluable participation of

Captain Erwin R. Archibald, Captain William E. Ward, and

Captain Thomas L. Gleason, M, who were project officers

during the course of these studies.

Appreciation is also expressed to Captain (Dr.) Jerry

Fineg, who as Chief of Vivarium, directed blood sampling and

urine collections, and Major (Dr.) Robert H. Edwards for his.

professional advice.

Recognition of the technical assistance of the following

personnel is also in order: MSgt Edward C. Dittmer, AtC

Charles H.S. Watkins, and AIC George V. Pegram.

The authors are deeply indebted to colleagues at the

Worcester Foundation for Experimental Biology, particularly

Dr. Constantin Gherondache, Mr. Edwin T. Lamson, and

Mr. Norman Gibree for the technical aspects relating to the

determinations reported in this document.

Finally, appreciation is expressed for the valuable adviceand support of Dr. Gregory Pincus and Dr. Ralph I. Dorfman.

ABSTRACT

This report attempts to establish (a) the qualitative and

quantitative nature of steroid hormones of the chimpanzee, and(b) to report changes observed in adrenal cortical functions

during simulated and actual space flights. Steroid determinations

made include: Blood: plasma 17-hydroxycorticosteroids (17-OHCS)

and cholesterol; Urine: 17-OHCS, 17-ketosteroids (17-KS), estrone,

e stradiol, estriol, pregnanediol, pregnanetriol, and aldosterone.

Quantitative chromatography of 17-KS was also accomplished.

Analyses were made on both the mature and immature chimpanzee.

PUBLICATION REVIEW

This technical documentary report has been reviewed and

is approved.

HAMILTON H. BLACKSHEARLt Colonel, USAF, MCCommander,6571st Aeromedical Research Laboratory

iii

CARE AND HANDLING OF THE SUBJECTS

The animals used in this study were handled in accordance

with the "Principles of Laboratory Animal Care" established by

the National Society for Medical Research.

iv

TABLE OF CONTENTSPage

I. INTRODUCTION AND PURPOSE ........... 1

II. EXPERIMENTAL DESIGN ...... ......... 1

III. CHARACTERIZATION OF STEROIDS . . . .... 2

A. 17-Hydroxycorticosteroids (17-OHCS) and17-Ketosteroids (17-KS) . . . . . .... . . . . 2

B. Chromatography of 17-KS..... ...... . 3

C. Aldosterone Excretion.... ...... 3

D. Plasma 17-Hydroxycorticosteroids ..... . . 3

E. Pregnanediol and Pregnanetriol Excretion. . . . 7

F. Estrogens . . . . . ... . . .* 0 0 0 * . . 7

IV. ADRENAL CORTICAL CHANGES DURINGSIMULATED AND ACTUAL SPACE FLIGHTS . . . . 10

A. 17-Hydroxycorticosteroids (17-OHCS) and17-Ketosteroids (17-KS) . . . * * * 0 . . . 10

B. Plasma Cholesterol and 17-Hydroxycorticosteroids . . . .. . . . . . . . . 13

V. CONCLUSIONS . .. . . . . . . . . . . . . . . . . . 14

VI RECOMMENDATIONS .i. . .14

REFERENCES .................. . . 15

LIST OF ILLUSTRATIONS

Table

I. 17-Hydroxycorticosteroids (17-OHCS) and17-Ketosteroids (17-KS) Excretion ofImmature Chimpanee on Control Day ........ 2

II. 17-Ketosteroids - Chimpanzee No. 3 (VICKIE) . . . . 4

M. Aldosterone Excretion in Immature FemaleChimpansee ....... . ..... . ....... 5

V

Table Page

MV Pregnanediol and Pregnanetroil Excretion ofImmiature Chimpanzee ... .. .. 5

V. Pregnanediol and Pregnanetriol on MatureFemale Chimpanzee *. . . a a 6a *0,*. 8

VI. Excretion of Estrogens in Immature MaleChimpanzee (No. 42) . . .. . .. . a...... 0 9

VII. Estrogen Excretion of Adult Female Chim-panzee "I~yCKeI" 6 . . . . . 10

VIII. Urinary 17-Ketouteroids . . 0 11

IX. Urinary 17-Hydroxycorticosteroids . . . . . . .a*12

X. Plasma Cholesterol.* * ** *aasa 13

vi

CHARACTERIZATION OF HORMONAL STEROIDSOF THE CHIMPANZEE

I. INTRODUCTION AND PURPOSE

The choice of the chimpanzee as a species of primate forstudying biological aspects of space flight was prompted by itscapabilities in psychomotor testing and its relatively smallsize. The animals studied were those participating in the Pro-ject Mercury program and were located at the Air Force MissileDevelopment Center, Holloman Air Force Base, New Mexico.Characterization of steroid hormones of the chimpanzee wasdesirable for purposes of evaluating the hormonal adjustment inspace flight. The resultb obtained would be of value to predictthe possible changes that might occur in human space flights.This is a preliminary report attempting to establish (a) qualita-tive and quantitative nature of steroid hormones of this species,and (b), to report changes observed in adrenal functions duringsimulated and actual space flights. Steroid determinations madeinclude: Blood: plasma 17-hydroxycorticosteroids (17-OHCS)(Ref. 1) and cholesterol (Ref. 2); Urine (17-OHCS) (Ref. 3), 17-ketosteroids (17-KS)(Ref. 4); Estrone, estradiol, estriol (Ref. 5),pregnanediol, pregnanetriol (Ref. 6), and aldosterone (Ref. 7).Quantitative chromatography of 17-KS was also accomplished(Ref. 8). Analyses were made on both the mature and immaturechimpanzee.

II. EXPERIMENTAL DESIGN

The immature chimpanzee was -,bjected to stresses involving(a) temperature humidity test (Th I): sitting 20 hours at tempera-tures of 70 and 80"F ("the thermally neutral"); 85, 90, 95, and100"F ("thermally stressful") with 50 percent humidity, withoutfood and water; (b) acceleration and deceleration tests (AD) simu-lating launch and re-entry conditions, and (c), actual suborbitalflight of chimpanzee No. 65 (HAM) (MR-2)*.

Each experiment was carried out over a 5-day period, withblood sample No. I collected on the morning of the first day(control), sample No. Z collected immediately after terminationof the stress, which took place on the second day, and sampleNo. 3 collected 48 hours after termination of the stress test.Urine collections were scheduled so that there were two collec-tions for each day: the first sample from 0730 to 1600 (day) andthe second sample from 1600 to 0730 (night) the following day,and so on for a total of ten samples.

*Mercury Redstone 2

IMl. CHARACTERIZATION OF STEROIDS

A. 17-Rydroxycorticosteroids (17-OHCS) and17-Ketosteroids (17-KS)

Table I presents the data of the control day which wasdivided into day (collection 0730-1600) and night (1600-0730)samples. It will be noted that the immature chimpanzee indicatesthe possibility of a diurnal variation in both 17-OHCS and 17-KSexcretion. The mean daily excretion of 17-OHCS of immaturechimpanzees is 2.8 mg. while that of the adult chimpanzee is3.8 mg. The 17-KS excretion of 4.0 mg. per day for the immaturechimpanzee is somewhat higher than the 17-OHCS excretion.

TABLE I

17-HYDROXYCORTICOSTEROIDS (17-OHCS) and17-KETOSTEROIDS (17-KS) EXCRETION OF

IMMATURE CHIMPANZEE ON CONTROL DAY*

DAY NIGHT(0730- 1600) (1600-0730)

17-OHCS .16 * .028 .10 : .017

(20) (23)

17-KS .23 *.030 .13 : .013

(9) (12)

*mg/Z0 mg. creatinine

There are two factors which should be mentioned inevaluating the above data. Chimpanzee urine, when processedfor 17-OHCS determinations, frequently gave a high pink blankvalue. Approximately 20 percent of the urine samples analyzedgave such high blanks that no value could be obtained. It ishighly possible that the tendency for high blanks reduced thetiter in a large proportion of determinations obtained. As forthe 17-KS, it was discovered that only 33 to 63 percent of thetotal Zimmerman titer was recovered quantitatively afterchromatography procedure.

2

B. Chronatography of 17-KS

Preliminary results of chromatography analysis of theurine of immature chimpanzees indicated the presence of 118-hydroxyetiocholanolone, 110-hydroxyandrosterone, dehydroepi-androsterone and traces of etiocholanolone and androsterone.Quantitative estimations were not feasible because of the smallamounts. The quantitative results of three daily collectionsobtained from adult chimpanzee No. 3 (VICKIE) are presentedin Table II. During the course of the collection there was somequestion of whether chimpanzee No. 3 (VICKIE) was pregnant;however, on 3 April 1961, menstruation was recorded for thischimpanzee.

All samples were hydrolyzed enzymatically with 0-glucuronidase and chemically using the solvolysis method (Ref. 9).Individual 17-KS were analyzed after paper chromatographicseparation. It should be noted that only 33 to 63 percent of thetotal 17-KS was recoverable quantitatively. Normal human urineso processed in our laboratories indicate 80 to 90 percentrecoverable material. It appears from these data that signifi-cant amounts of non-specific chromogens are measurable inthe total 17-KS urine of thin adult female chimpanzee. Theseresults permit us to consider that the 17-KS excretion of themature female chimpanzee parallels qualitatively and quantita-tively the data observed for the human (Ref. 10, 11, 12, 13, 14).

C. Aldosterone Excretion

Twenty-four hour urine samples are necessary foraldosterone determination. The values for the immature chim-panzee, ranging from 0.00 to 2.2 mcg. per day are presented inTable MrI. We have had results indicating that the immaturechimpanzee is capable of excreting as high as 27.7 mcg. perday under the stress of 90OF and the deprivation of both foodand water.

D. Plasma 17-Hydroxycorticosteroids

All the data available for plasma 17-OHCS on the imma-ture chimpanzee are depicted in the figure on page 6. It will benoted that the values for blood samples No. 1 (pre-stress) andNo. 3 (post-stress) show similar distributions indicating thatthe "basal',17-OHCS ranges from 10 to 20 mcg. percent. Normalvalues for human subjects as well as for the Rhesus monkey arewithin this range.

3

TABLE II

17-KETOSTEROIDS - CHIMPANZEE NO. 3 (VICKIE)

mcg/l. 0 g. creatinine

Total 17-KS 4430 8230 3600

Chromatography of 17-KS

11-OH-E 222 355 240

11-OH-A 139 139 100

11-K-E 612 854 948

11-K-A 185 299 73

DHEA 408 512 483

Etio 231 270 204

Andro 174 244 225

Pre-andro 78 40 48

Polar Res. 26 46 256

Abbreviations:

11-OH-E: 1 1-Hydroxyetiocholanolone11-OH-A: 116 -Hydroxyandrosterone1 1-K-E: 11 -Ketoetiocholanolone11-K-A: 11 -KetoandrosteroneDHEA: DehydroepiandrosteroneEtio: EtiocholanoloneAndro: Androsterone

Pre-andro: Any material on chromatogram lesspolar than androsterone.

Polar Res: Chromogenic material remaining at theorigin of paper chromatogramns.

4

TABLE III

ALDOSTERONE EXCRETION INIMMATURE FEMALE CHIMPANZEE

Chimpanzee Total AldosteroneNo. Creatinine mcg/0. 5 g Creat.**

35 372 0.60

41* 421 23.70

46 500 1.00

46 423 1.20

46 465 2.20

49 645 0.0049* 813 27.70

50 692 1.50

* After Stress*, Equivalent to daily excretion

TABLE IV

PREGNANEDIOL AND PREGNANETRIOL EXCRETIONOF IMMATURE CHIMPANZEE*

N Mean

Pregnanediol 23 .23

(.04-.60)

Pregnanetriol 22 .40

(.18-.83)

5

0OI

2oJ*6

to'

S!2N 11VC-

* ** 06

E. Pregnanediol and Pregnanetriol Excretion

The pregnanediol and pregnanetriol values for theimmature chimpanzee are presented in Table IV. We wish topoint out the range of values depicted in the table, especiaflythe levels of low concentration.

Data were also obtained on the urinary excretion ofpregnanediol and pregnanetriol of three adult female chimpan-zees (Table V). Chimpanzee No. 132 (FITA) was sampled on26 June 1961. According to the records, this sample wasobtained during post-ovulatory phase of the menstrual cycle.Chimpanzee No. 135 (BETA) was sampled on 23 May 1961.This sample appears to have been obtained in mid-cycle.Chimpanzee No. 3 (VICKIE) was sampled on 28 March 1961and on 31 March 1961. Chimpanzee No. 3 (VICKIE) was sus-pected of being pregnant during the period of the sampling. Thefact that the animal did flow on 3 April 1961 does not rule outthis possibility. The pregnanediol data supports the view thatchimpanzee No. 3 (VICKIE) may have been pregnant; the data onthe estrogens to be presented in the next section also supportsthis view. The relatively low values for both pregnanediol andpregnanetriol for chimpanzee No. 135 (BETA) is consistent withvalues expected at mid-cycle. The value of 1.21 mg. obtainedindicates that chimpanzee No. 132 (FITA) was sampled duringthe luteal phase of the cycle.

F. Estrogens

The urine obtained from an immature male chimpanzeewas hydrolyzed successively by glucuronidase and the solvolysismethod for urinary steroid sulfates, was extracted and chroma-tographed for separation of estrane (EI), estradiol (E2 ) andestriol (E 3 ). Quantitative estimation of the separated estrogenswas made fluorometrically. The results (Ta'le VI) indicate thatthe urine of the immature male chimpanzee ontains El, E2 ,and E 3 principally as a glucuronide and sulfate. E1 appearsprinciplally as a sulfate, while E2 and E3 are mainly present inthe urine as glucuronides. We do not have data from pre-adolescent humans for comparative purposes. We are awarethat the values presented in Table VI are much higher than thosereported in the literature by other methods.

Estrogen analysis was performed on three samplesobtained from chimpanzee No. 3 (VICKIE) (see above) on thefollowing dates: 27 March, 28 March, and 31 March 1961. Thedata for total estrogens are presented in Table VI.

0

4)j

Ine

134

I n Go N

0 ~

04

I 440 Jz l

TABLE VI

EXCRETION OF ESTROGENSIN IMMATURE MALE CHIMPANZEE (NO. 42)

mcg/.5 GM. creatinine*

% of TotalB-Gluc.** S04*** Total (E l + E2 + E 3 )

10 May 1960

Estrone (E l ) .90 1.26 2.16 37.7

Estradiol (E2 ) .73 .48 1.21 22.2

Estriol (E 3 ) 1.42 .65 2.07 38.1

11 May 1960

Estrone .75 .86 1.61 41.1

Estradiol .66 .41 .97 24.7

Estriol .95 .39 1.34 34.2

*Equivalent to daily excretion

**$-Glucuronidase hydrolysis: includes free (trace amounts)plus glucuronides

***Sulfates in addition to values under**

It may be noted that the estrogen values, especially for estriol,are surprisingly high. Published data (Ref. 5) give the followingvalues for the three estrogens in early pregnancy urine: E1 ,3.5 - 13.5 mcg/L; E2 , 1.6 - 6.2 mcg/L; and E 3 , 60.6 - 194.4 mg/L.It is of interest that the data presented on chimpanzee No. 3(VICKIE) compares favorably with the published results. Thedata on pregnanediol as well as estrogens support the possibilitythat chimpanzee No. 3 (VICKIE) was pregnant at the time of thesampling, even though flow was recorded on 3 April 1961.

TABLE VII

ESTROGEN EXCRETION OFADULT FEMALE CHIMPANZEE "VICKIE"

Total Estrogens

mcg/1. 0 g. creatinine

27 March 1961 28 March 1961 31 March 1961

Estrone 0.185 0.285 3.60

Estradiol 3.90 8.25 7.7

Estriol 133.5 160.5 30.5

IV. ADRENAL CORTICAL CHANGES DURING SIMULATED ANDACTUAL SPACE FLIGHTS

A. 17-Hydroxycorticosteroids (17-OHCS) and17-Ketosteroids (17-KS)

In Table VIII are presented the 17-KS results, indicatingvarying degrees of increases in this index during the stress(samples No. 3 and 4). Of particular interest are samples No. 4of THT-17 (animal No. 42) and THT-18 (animal No. 35). Thediurnal variation of 17-KS may be observed in the data. InTable IX are depicted the 17-OHCS data. Here again, we wishto draw attention to the increased 17-OHCS excretion, especiallyin THT 11 and 17. The urine data available from chimpanzeeNo. 65 (HAM) (MR-Z) during the suborbital flight, indicate mod-erate to marked increases in 17-OHCS. The increases in both17-OHCS and 17-KS observed during the suborbital and orbitalflight were in the same order as those observed in the severetemperature humidity test stresses.

10

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B. Plasma Cholesterol and 17-Hydroxycorticosteroids

The plasma cholesterol data are presented in Table X.Increases in plasma cholesterol are observed in both "thermallyneutral" and "thermally stressful" conditions. No correLationis evident betwe the increases in plasma cholesterol and theincreasing stress experienced by the chimpanzee in terms oftemperature. It is significant to observe that where the experi-ments were terminated before the full programmed 20-hourperiod, no consistent increases were observed in this index.We wish to emphasize that plasma cholesterol is not beingpresented as an index of adrenal cortical function. The data areof interest only in the finding that this measure showed increasesin both "thermally neutral" and "thermally stressful" conditionswhen the experiments were run through the full 20-hour period.

TABLE X

PLASMA CHOLESTEROL

ms. %

THT Animal No. Temp. F Pro- Stress Post-

4 46 80 271 297 281

5 35 80 196 233 196.

8 41 80 218 354 324

10 42 80 165 226 184

13 42 80 156 208 196

11 42 85 133 201 145

12 44 85 208 199 173

14 35 85 242 304 234

18 50 90 253 285 28018 35 90 242 296 248

19 41 95 219 344 292

23 41 100 269 340 381

23 50 100 280 279 ---

24 35 100 273 306 269

24 46 100 259 250 249

25 49 100 292 342 252

25 50 100 263 250 243

113

The plasma 17-OHCS data are presented in the figureon page 6. As mentioned previously, the "basal" 17-OHCS valueof an immature chimpanzee may be considered to be approxi-mately 16 mcg percent. Quantitative increases in sample No. 2(stress sample) of the 17-OHCS data were observed with increasesin temperature. The ceiling appears to be approximately 70 mcgpercent. It is noted that AD- 1 shows less of an increase thanAD-2. Furthermore, the result obtained on chimpanzee No. 65(HAM) (MR-2) immediately after the suborbital flight, was some-what less than the value obtained in the 20-hour stress periodunder 95OF conditions. Interpretation of the above data indifficult due to the varying degrees of time lapse from the pointof termination of the stress to the time of sampling.

V. CONCLUSIONS

Preliminary data characterizing steroids of the chimpanzeeindicate similarity both qualitatively and quantitatively withthose found in the human.

VI. RECOMMENDATIONS

These studies on steroidology of the chimpanzee should becontinued with the modern technics now available. This can beaccomplished by using the double isotope method (H3 and C14}which will give accurate secretion rates of steroid hormonessuch as that of the adrenal and gonads. Consideration should begiven to developing technics using principles of automation andtelemetry for evaluation of adrenal and gonad function during andafter space flights.

14

REFERENCES

1. Nelson, D. H. and L. T. Samuels. 3. Clin. Endocrinol.12, 519, 1952.

2. Kingsley, G. R. and Schaffert. 3. Biol. Chem. 180, 315, 1949.

3. Porter, C.C. and R.H. Silber. 3. Biol. Chem. 185, 201, 1950.

4. Pincus, G. J. Clin. Endocrin. 3, 195, 1943.

5. Finkelstein, M., R. 3ewelewicz, 0. Klein, V. Pfeiffer andS. Sodmoriah-Beck, (1960). In Prenatal Care, pp. 12-26,P. Noordhoff, Groningen, The Netherlands.

6. Stern, M. I. J. Endocrinol. 16, 180, 1957.

7. Mattox, V. R. and M.L. Lewbart. 3. Clin. Endocrinol. &Metab. 19, 115, 1960.

8. Rubin, B. L., R. I. Dorfman and G. Pincus. J. Biol. Chem.203, 626, 1953.

9. Burstein, S. and S. Lieberman. 3. Biol. Chem. 233,

331, 1958.

10. Masuda, M. and T. H. Holmes. Pediatrics 19, 424, 1957.

11. Rubin, B. L., R. I. Dorfman and G. Pincus. Recent Progr.Hormone Res. 9, 213, 1954.

12. Kappa*, A., O.H. Pearson, C.D. West and T. F. Gallagher.J. Clin. Endocrin. & Metab. 16, 517, 1956.

13. Kappas, A. and T. F. Gallagher. 3. Clin. Invest. 33, 1566,1955.

14. Dobriner, K. J. Clin. Invest. 32, 940, 1953.

25

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