THE URINARY EXCRETION OF 17-KETOSTEROIDS BY MEN 
UNDER FIELD CONDITIONS IN EXTREME COID* 
by 
L. E, Osgood, Capto, MoCo end W, J. Zimmerman, Capto, MoC0 
with the technical assistance of 
E. W. King and 0. H. Diffley 
from 
Medical Department Field Research Laboratory 
Fort Knox, Kentucky- 
27 October 19h9 
♦Sub-project under Study of Physiological Effects of Coldo Approved 
2U September 19U?« MDFRL Project No* 6-6u-12-02-(ll)* Project Noc. 6-61i-12-02 
Sub-project MDFRL 02-(ll) 
MEDEA 
27 October 19^9 
ABSTRACT 
THE URINARY EXCRETION OF 17-KETOSTEHOIDS BY HEN 
UNDER FIELD CONDITIONS IN EXTREME COLD 
OBJECT 
To determine the excretion of 17-ketosteroids on a group of seven 
subjects participating in two bivouac exercises, first under conditions 
of a temperate climate, and later under arctic conditions, which were 
so designed as to be similar except for the factor of extreme cold0 
RESULTS 
When the excretion of 17-ketosteroids by seven healthy soldiers 
under field conditions in a temperate climate was compared with their 
excretion of these substances under field conditions in extreme cold*, 
no significant changes were noted0 
CONCLUSIONS 
The determination of the excretion of 17-ketosteroids in the urine 
did not indicate a stress due to cold under the circumstances described 
in this reportc 
RECOMMENDATIONS 
Since one cannot suppose that urinary excretion of these steroids 
reflects conditions in the circulating blood, studies should bo directed 
toward finding a convenient and reliable method for the determination 
of 17-ketosteroids or their precursors in blood0 
There are numerous factors which affect the level of urinary excre- 
tion other than the actual amount of active principle secreted into the 
bloodo Attemnts should be made to determine what factors modify the 
pathway of metabolism of the active compounds and what affect variation 
in kidney and liver function has in determining the level of ©xcretiono 
Submitted by* 
L. G. Osgood, Capt*, MoCo 
Wo Jo Zimmerman, Capto, M,C0 
E. W. King, Biochemist 
G. Ho Diffley, Biochemist 
Approved; 
RAY (fJL ykOOS {/ c/ 
Director of Research 
7 
Approved 
Lt. coio, i4»c'/* 
Commanding THE URINARY EXCRETION OF 17-KETOSTEROIDS BY MSN 
UNDER FIEID CONDITIONS IN EXTREME CCID 
lo INTRODUCTION 
Many observations have been recorded showing a relationship of the 
adrenal cortex to the process of adaptation to stress (1, 2, 3> 14)0 
The determination of the excretion, of 17-ketostoroids has been employed 
as a means of studying certain aspects of adrenal cortical functionso 
A study of the excretion of the 17-ketostoroids was, therefore, in- 
cluded in the observations carried out on a group of seven subjects 
participating in a bivouac exercise under arctic conditions at Ft® 
Churchill, Canada® It was anticipated that changes in the excretion of 
the 17-ketosteroids would provide an objective measure of the response 
to the stress (cold and exercise) to which the men were subjected® As 
a basis for comparison with the Ft® Churchill conditions, the excretion 
of these steroids was determined (previous to the Ft® Churchill bivouac) 
for the same group of subjects during a bivouac at Ft* Knox, Kentucky 
under conditions which were so designed as to be similar in every way 
except for the factor of extreme cold® 
I!® EXPERIMENTAL 
A® Ft. Knox and Ft. Churchill Bivouacs 
Complete details of the two bivouacs and the major investiga- 
tion will be described in another report (5)j for the present purpose 
the conditions of the study may be summarized briefly® The subjects 
were seven healthy male enlisted volunteers® After a month of physical 
conditioning (daily hikes), the lU-day control bivouac was carried out 
at Ft® Knox during the month of November, lRh7o Throughout this test 
period, the men marched 13-15 miles a day except that no marches were 
scheduled on the two Sundays that were included in the bivouac period® 
The diet consisted of the Army **C-2M ration, and the clothing worn dur- 
ing this period was the Army fatigue uniform® 
The subjects -were taken to Ft. Churchill, Canada early in 
January, 19ii8o After a oeriod of conditioning which included the 2-week 
Canadian arctic indoctrination course, a second 13-day bivouac exercise 
was carried out during February, 19U8o The men hiked (snow-shoes) h to 
6 hours a day, and the average exposure to the outdoor weather was 6d 
hours per day for the entire periodo Their food was again the ltC—2H 
rations Regulation arctic uniforms were worn outdoorSs 
The two bivouac exercises were carried out under as nearly 
identical conditions as possible (personnel, food, activity, procedure) 
except for the difference of environmental temperature and the clothing 
worn® Thus two realistic field situations were attained which could be 
compared directly with respect to physiologic measures under temperate 
and arctic temperatures, respectively®. The temperature and wind condi- 
tions during the two exercises are summarized in Table 1® TABLE 1 
SUMMARY OF CONDITIONS OF TEMPERATURE AND WIND 
DURING THE WO BIVOUAC EXERCISES 
Bivouac 
Mean Terno* 
(deg. F) 
Temp. Range Mean Wind Vel* Wind Velo Mean 
(deg. F) (mcDohc) Range (mop0hB) Windchill 
Ft. Knox 
Ul.3 
?7=5 to 6?.8 i4c8 0 to 37 590 
Ft. Churchill -19=7 
-37 =0 to l?cO 16o5 0 to Iih 1790 
Bo Methods 
lo Collection and preservation of urine« 
Complete urine collections were made during both bivouac 
periods o "Waking** urines were collected from 0600 through the last void- 
ing before retiring; "sleeping** urines were collected through the first 
morning urine at 06000 An exact record of the time elapsed during each 
collection res made in order that the hourly output for each neriod might 
be calculated0 
The urine was voided directly into tinned metal cans and 
brought to the laboratory within 2h hours after the beginning of any 
collectiono At Ft. Churchill, the samples became frozen in the collecting 
cans within a few minutes after voiding and were allowed to thaw at room 
temperature after arriving at the laboratory» 
When the urines were received at the laboratory, the volumes 
were measured and a sample (usually ?50 ml.) was set aside for the steroid 
determinations. Since some of the specimens were alkaline in reaction 
during the bivouac at Ft. Knox, the pH of such samples set aside for 
steroid analyses was adjusted to 5 with hydrochloric acid (nitrazine 
paper). During the Ft. Churchill bivouac, all specimens were acid in 
reaction and consequently the pH was not adjusted. The samples were 
then frozen rapidly and were maintained in the frozen state until the 
time of analysis. 
2o The 17-ketosteroid analytical procedure. 
Hydrolysis and extraction of the urines for separation of the 
ketonic neutral fraction were carried out according to the procedure out- 
lined by Pincus (6). This method includes the micro-Girard separation 
of ketonic from nonketonic substances using Girard's reagent T. The 
colorimetric determination of the 17-ketosteroid content of the ketonic 
fraction was carried out according to the method of Callow, Callow and 
Emmons (7)<> This is the Zimmerman reaction employing an alcoholic potas- 
sium hydroxide solution„ All results are reported in terms of dehydrclso- 
androsterone which was used as a standard. The ether used for extraction 
was checked daily for substances oxidizing potassium iodide and none was 
employed which showed a positive test. The accuracy and reproducibility of the method ss carried out 
in this laboratory are indicated by the following observationst 
Twenty pairs of duplicate determinations were carried out at 
the beginning of the study; after that only a single analysis was done 
on each sample0 Of these twenty pairs, thirteen showed a difference 
(between duplicates) of 0-5$ of the mean for the two values, and eighteen 
agreed within 10$. 
At intervals during the course of the analytical work the whole 
procedure was checked by processing 100 or 200 micrograms of dehydroiso- 
androsterone added to 100 mlo of distilled water. Hie average error in 
recovery for nine such tests was 13$. However, individual errors in 
recovery ranged from -21$ to +15$ = 
Co Results 
The mean 2U-hour excretion of 17-ketosteroids fof each subject 
during the Ft. Knox and Ft, Churchill bivouacs is listed in Table 20 
Inspection of these data will show that there is no significant differ- 
ence between the means for the two bivouacs in any one of the seven 
subjectso Neither was there any definite trend in the level of 17- 
ketosteroid excretion during either bivouac exercise for the group as a 
whole. The values for the 17-ketosteroid excretion for the most part 
fell within the normal ranges reported by others (8)0 
TABLE 2 
MEAN DAILY 17-KET0STER0ID EXCRETIONJ 
RATIO OF MEAN HOURLY WAKING TO SLEEPING OUTPUT 
Sub- 
ject 
Bivouac 
Number of 
Determine 
Mean 17 KcSc 
(mgo/2ii hrs0) 
Mean 
Deviation 
Waking/Sleeping 
Output 
1 
Fto Knox 
Ih 
9o5 
4- loU 
1.3 
Ft. Churchill 
13 
6o? 
" 0.6 
1.U 
2 
Fto Knox 
12 
13 08 
1.9 
1.U 
Ft. Churchill 
9 
lit .3 
lo2 
lo2 
3 
Ft0 Knox 
13 
llio6 
1.7 
1.6 
Ft. Churchill 
13 
11.9 
2o5 
1.2 
U 
Fto Knox 
12 
11 oh 
1.8 
1.3 
Ft. Churchill 
10 
10 o 7 
1.6 
loli 
5 
Ft. Knox 
13 
7d 
1-3 
\ 1.3 
Ft. Churchill 
12 
7o6 
2 o2 
1.2 
6 
Fto Knox 
12 
9d 
1.2 
lol 
Ft. Churchill 
10 
8o0 
0c9 
lo2 
7 
Ft. Knox 
11 
8o£ 
1.3 
1.0 
Fto Churchill 
13 
8o3 
1.3 
1.6 
3 The diurnal variations in 17-ketosteroid output which has been 
studied by Pincus (9) was noted in these subjects as shown by the ratio 
of the mean hourly outputs during the waking and sleeping hours (Table 
2) c The ratio of waking to sleeping hourly output was greater then lo0 
in 792* of the individual values recorded0 However, there was no consis- 
tent change in the ratios for the as a whole during the Ft» Churchill 
bivouac as compared with the ratios at Ft. Knox0 
III* DISCUSSION 
No change was found in the excretion of 17-ketosteroids which might 
have been expected as the result of the cold stress to which these men 
were subjected during the Ft, Churchill bivouac» Neither the 2U-hour 
totals, nor the ratio of the excretion during the day (when the exposure 
was greatest) to that at night (when it was least) showed any consistent 
changeo The findings seem to indicate that the subjects may have been 
adapted to the stress (cold and exercise) during the bivouac period at 
Ft* Churchill prior to the first urine collections0 No 17-ketosteroid 
determinations were carried out during the period immediately after arri- 
val at Ft* Churchillo It is quite possible that a change in 17-ketosteroid 
excretion might have occurred during that time* However* the main object 
of this investigation was to compare the 17-ketosteroid excretion during 
two bivouac exercises9 first under conditions of a temperate climate 
and later under arctic conditions which were so designed as to be simi- 
lar except for the factor of extreme cold0 The results reported on the 
17-ketosteroid excretion are in accord with the findings of Stein £t al0> 
who found no significant change in 17-ketosteroid excgetion following 
reneated exposure of subjects to a temperature of -20 F. for £ hours in 
a cold chamber (lO)o. 
IV o CONCISIONS 
The 17-ketosteroid excretion of seven healthy male subjects was not 
significantly changed during a field exercise at Fto Churchill, Canada 
under conditions of extreme cold from the levels of excretion recorded 
for the same men during a similar exercise under temperate climatic condi- 
tions at Fto Knoxo A diurnal variation in 17-ketosteroids was noted in 
these subjects, the hourly excretion during the waking hours being consis- 
tently greater than that during sleepe The measurement of the excretion 
of 17-ketosteroids during the two bivouac periods seems to indicate that 
the subjects may have already been adapted to the stress (cold and exer- 
cise) during the period at Fto Churchill prior to the first urine collec- 
tions o 
Vo RECOMMENDATIONS 
Since one cannot suppose that urinary excretion of these steroids 
reflects conditions in the circulatory blood, studies should be directed 
toward finding a convenient and reliable method for the determination of 
17-ketosteroids or their precursors in blood» 
There are numerous factors which affect the level of urinary excre- 
tion other than the actual amount of active principle secreted into the bloodo Attempts should be made to determine what factors modify the 
pathway of metabolism of the active compounds and what affect variation 
in kidney and liver function has in determining the level of excretion» 
VI o BIBLIOGRAPHY 
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adrenal cortical hypertrophye Endocrinology 32: 373> 19b3° 
2o Selye, Ho The general adaptation syndrome and the diseases 
of adaptation© J, Clin© Endocrinol0 6: 117, 19b6© 
3 o Pincus, Go Studies of the role of the adrenal cortex in the 
stress of human subjects, in Recent progress in hormone researcho 
New York, Academic Press 123* 19b7 = 
bo Long, Co No Ho The relation of cholesterol and ascorbic acid 
to the secretion of the adrenal cortex in Recent progress in 
hormone researcho New York, Academic Press Is 99> 19b?o 
9° Molnar, G. W© et al© Studies of physiological problems under 
field conditions in extreme cold© To be published© 
60 Pincus, Go The analysis of human urines for steroid substances© 
J. Clin. Endocrinol. $s 291, 19b5© 
7® Callow, N. Ho, R. K. Callow and C. W. Emmons, Colorimetric 
determination of substances containing the grouping CHpCO in 
urine extracts as an indication of androgen content, Biochem0 
J, 32: 1312, 1938c 
8o Talbot, N. B, and A. M. Butlere Urinary 17-ketosteroid assays in 
clinical medicine© J© Clin. Endocrinol. 2t 72b, 19b20 
9* Pincus, G© A diurnal rhythm in the excretion of urinary keto- 
steroids by young men, J. Clin© Endocrinol© _3: 19b3o 
LOo Stein, J, H., R© A. Bader, J. W, Eliot and D. E, Bass© A study 
of hormonal alterations in men exposed to heat and cold stress© 
Environmental Protection Section Report No© IbO, Quartermaster 
Climatic Research Laboratory, Lawrence, Massachusetts, 27 December 
19b8©