Effect of ACTH on Distribution of Ascorbic Acid in Rat Adrenal Gland as Determined by Direct Microchemical Analysis
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Original Repository: Alan Mason Chesney Medical Archives. Daniel Nathans Collection
Reproduced with permission of Joanne Nathans.
Medical Subject Headings (MeSH):
Choosing Research, 1955-1962
Since the discovery that the ascorbic acid content of the adrenal gland changes with adrenal stimulation, this fact has been
widely used both to assess stress reactions in experimental animals and as a means of assaying ACTH. (1) By measuring total
adrenal ascorbic acid it has been found that the amount of this substance falls rapidly after acute stress or ACTH administration,
reaching a minimum in one to three hours (falling to about 40 percent of original value in rats). (1)
The distribution of ascorbic acid in the rat adrenal has been extensively investigated by Greep and Deane (2) and by Deane
and Morse (3). They used a histochemical technique dependent on reduction of silver ion by ascorbic acid. Their results show
silver-reducing material in all zones of the gland, with a fall following a single dose of ACTH limited to the zona fasciculata
and reticularis. After prolonged stress (starvation) the silver-reducing material seemed normal in amount.
The limits of this histochemical technique are 1) that it is qualitative rather than quantitative, and 2) that one cannot
be sure that the position of the ascorbic acid or the finally visualized material has not changed during the procedure. The
present study was undertaken to determine quantitatively by direct microchemical analysis of adrenal slices the distribution
of ascorbic acid in the various zones of the rat adrenal gland and to determine changes after ACTH administration.
The general method employed was the Linderstrom-Lang technique as modified by Lowry (4). The animals were rapidly killed by
decapitation, the adrenals promptly removed and dropped into liquid nitrogen. These were later mounted at a temperature of
-20 degrees Centigrade, and 20 mu section of the frozen gland were cut. These were dehydrated at low temperatures and finally
the desired zone of the gland, which could be readily identified in frozen sections was dissected out, transferred to a test
tube, and chemical analysis for ascorbic acid, cholesterol* and protein carried out.
Ascorbic acid was determined by the dinitrophenylhydrazine method as adapted for small samples by Lowry, Lopez, and Bessey
(5). Protein content of each section was used as a reference and was determined by the method of Lowry, Rosebrough, Farr,
and Randall (6).
Adult female rats weighing 215 to 270 gm. were used. One adrenal from each rat was analysed. There were 3 control animals;
2 others were sacrificed 30 minutes after a single intravenous dose of ACTH (1 mg.), 2 90 minutes after the same dose and
1 animal sacrificed 36 hrs. after the last of eight 1 mg. doses of ACTH given over a 3-day period.
The results are presented in detail in tables I through IV and plotted in the accompanying figure. The figure was constructed
by plotting the average values obtained in all the animals of a given group, the vertical lines representing the standard
error of the mean (S.E.) for each value.** As shown, the normal rat adrenal has the highest concentration of ascorbic acid
in the zona fasciculata
*The cholesterol studies were done by Mr. Wayne Albers of the Dept. of Pharmacology and are not reported here.
**S.E. was calculated by the following formula: S.E. Sd2/n(n-1)
and only slightly less in the inner zona reticularis. Following acute stimulation with ACTH the ascorbic acid concentration
falls rather uniformly in all zones of the cortex except the glomerulosa, where after 90 minutes there is a slight fall of
questionable significance. This constancy of glomerulosa ascorbic acid re-emphasizes the probable independe of the rat zona
glomerulosa from the pituitary.
After prolonged stimulation with ACTH the reticularis was almost unrecognizable as such in frozen section due to marked widening
of the fasciculata. Sections taken from the inner fasciculata zone down to the medulla are plotted as F-R. It is seen that
with chronic stimulation ascorbic acid concentration remains at normal levels in the fascibulata and possibly increases in
the zone between the fasciculata and the medulla. (Note that only one animal was used for chronic stimulation.)
The adrenal gland with its rather sharply demarcated zones has been found to be readily susceptible to Lowry's technique
of direct chemical analysis of dissected tissue slices. The present study of the distribution of ascorbic acid in the rat
adrenal has in general confirmed earlier work using histochemical techniques and has quantitated the results. The significance
of the adrenal ascorbic acid changes with stress or ACTH have yet to be determined. There is no clear evidence of a specific
role in steroid metabolism (7).
Using the method described above, Albers of the Dept. of Pharmacology has extended the study of the "microchemical anatomy"
of the adrenal by "mapping out" cholesterol and various enzymes in the different zones of the gland.
1) Direct microchemical analysis of dissected slices of rat adrenal for ascorbic acid and protein is reported.
2) The distribution of ascorbic acid in the various zones of the adrenal is plotted and the results show the highest concentrations
in the zona fascibulata and inner zona reticularis.
3) Following acute ACTH stimulation, the ascorbic acid concentration falls rather uniformly throughout the cortex with the
exception of the glomerulosa where it remains approximately constant or falls slightly. With chronic stimulation by ACTH the
fascibulata broadens and maintains its ascorbic acid content while the area adjacent to the medulla shows increased concentration
of this substance.
This work was done with Dr. O.H. Lowry and Mr. Wayne Albers of the Department of Pharmacology, Washington University School