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Lack of effect of chronic administration of p-aminobenzoic acid on the secretion of 18-hydroxydeoxycorticosterone and corticosterone by the rat adrenal
551
LACK OF EFFECT OF CHRONIC ~ T R A T I O N ON ~ E
OF p-AMINOHENZOIC ACID
SECRETION OF 18-HYDROXYI~XYCORTICOS~ONE AND CORTICOSTERONE BY THE RAT ADRENAL.
g
Julio M. Cortes and Fernand G. P~ron With the technical assistance of Miss Charlotte Lilly.
The Worcester Foundation for Experimental Biology Shrewsbury, Massachusetts
Received April
2,1964
ABSTRACT
Male rats were fed p-sminobenzoic acid (PABA) in their diet for 12 weeks. Although body weight and absolute adrenal weights were decreased and thyroid weight increased, there was no effect of PABA administration on the secretion of 20,21-dihydroxy-18,20-epoxy-4-pregnen-3-one (18-OHDOC ) and ll~, 21-dihydroxy-4-pregnene-3,20-dione (corticosterone) by the rat adrenal gland. The quantitation of corticosterone and 18-OH-DOC was carried out by two methods which measure these steroids specifically rather than by the two component technique of analysis used by McCarthy et al.4 It was concluded from the results of these experiments that PABA had no direct effect on the rat adrenal gland per se, and that the abnormal corticosterone and P-S chromogen levels found by McCarthy et al. ~ in peripheral rat blood after PABA administration was not due to any apparent abnormal adrenal secretion of corticosterone or 18-OH-DOC. One of the main secretory products of the rat adrenal gland is 20, 21-dihydroxy-18, 20-epoxy-4-pregnene-3-one (18-OH-DOC) and its production, like that of ll~,21-dihydroxy-4-pregnen-3,20-dione (corticosterone) is at least in part under ACT~ control1.
It appears to account ex-
clusively for the Porter-Silber (P-S) chromogens isolated in rat adrenal venous blood1.
In spite of evidence2'3 showing it to have little mineral-
ocorticoid activity, the relatively large amounts which are secreted in normal stressed or hypophysectomized rats injected with ACTH causes one to
552
S T E R O I D S
3:5
suspect a physiological role for this substance. McCarthy et al. 4, reported that the goitrogenic effect produced by the prolonged administration of p-aminobenzoic acid (PABA) to male rats was accompanied by an absolute and relative (rag.per lO0 gm b.w. ) reduction in adrenal weight, an increase in peripheral plasma P-S chromogens and a drastic decrease in peripheral plasma corticosterone levels.
These authors
suggested that the mechanism whereby PABA manifested its effects was by a direct action on the adrenal gland and thus presumably affecting the circulating levels of adrenocorticosteroids.
From the foregoing report it
would be expected that the chronic administration of PABA to rats would lead to substantial changes in the level of adrenal venous blood 18-OH-DOC. It was also reasoned that these changes might lead to the detection of some manifestation of 18-OH-DOC hypersecretion and thus give a clue to the possible physiological role of this substance in the rat. Under the conditions of the experiments, the results obtained preclude at present a direct effect of PABA at the adrenal level.
1 ~ ~
AND I ~ I O I ~
Male Sprague-Dawley rats weighing 90 to llO gin. were fed ad libidum for a period of 12 weeks with a mixture of ground rat laboratory chow to which PABA was added (4% by weight). The rats were anesthesized with ether and cannulation of the left adrenal vein by the method of Lipscomb and Nelson5 was performed. Blood was withdrawn in a heparinized syringe for exactly l0 minutes after which the animals were killed by exanguination. The adrenals and thyroid were removed, cleaned of adherent tissues and weighed. Each adrenal venous sample was processed as previously described and 18-OH-DOC and cort$costerone were determined by modifications~, U of both the P-S reaction" and the fluorometric method of Silber et al. ~. In their studies, McCarthy et al. 4 made the simultaneous determination of corticosterone and 18-OH-DOC by measuring the absorbaucies obtained at 355 m~ and 410 m~ respectively, with the ethanolic P-S reagent (64 ml. of concentrated sulphuric acid, 36 ml. of water, 50 ml. of absolute ethanol and lO0 rag. of phenylhydrazine hydrochloride ). In the present report the determination of 18-OH-DOC was quantitated by taking readings
May 1964
S T E R 0 I D S
553
in the aqueous P-S reagent (64 ml. of concentrated sulphuric acid, 36 ml. of water and lOO rag. of phenylhydrazine hydrochloride) at 405 m~ 1 for reasons given below. A study of the behavior of mixtures of ll~,17G,21-trihydroxypregnene-3,20-dione (cortisone, see ref. 13) and corticosterone in amounts equal to those observed in rat plasma for 18-OH-DOC and corticosterone I was carried out in order to elucidate any possible differences of the spectra with the two P-S reagents referred to above. Two duplicate sets of samples of 2, 4 and 6 ~g. of corticosterone, 2 ~g. of cortisone and mixtures of 2 ~g. of cortisone with 2, 4 and 6 ~g. of corticosterone were used. Four ml. of methylene dichloride were added to the dry steroid samples. One set of samples was then treated with the aqueous P-S reagent and the other with the ethanolic P-S reagent. ~ e spectra obtained with the two reagents are shown in figures IA and IB. The maximum absorption peak for corticosterone treated with the ethanolic P-S reagent was found to be at 350 m~. At this wavelength, absorption was proportional to the concentration of corticosterone. The addition of corticosterone to cortisone gave rise to alterations of the absorption curves of cortisone although the absorption at 410 m~ and 350 m~ were found to be the sum of the respective absorbancies of cortisone and corticosterone at both wavelengths (Figure IB). On the other hand, with the aqueous P-S reagent. 2-6 ~g. of corticosterone showed no light absorption at the wavelengths studied (Figure IA). In this instance, the absorbancy of cortisone at 410 m~t although less intense than that produced by the ethanolic P-S was unaffected by 2-6 ~g. additions of corticosterone (Figure IA). As a result of these findings we had a choice of two P-S reagents for quantitating 18-OH-DOC. The aqueous P-S reagent was used in the present study because the above results indicated that 18-OH-DOC could be measured specifically without inter{erence from possible fluctuations in levels of corcicosterone or other Z~ o-heto steroids ~hich mioht be brought about by PABA administration.
RESULTS A$[D DISCUSSION ~%e results in Table I show that rats receiving PABA during 12 weeks gained less weight than the controls.
Their thyroids increased in lI
~eight, both absolute and relative to body weight, as previously reported ~. The weight of the adrenal glands was significantly less in the PABA group than the controls but there was no difference in the relative adrenal weight to body weicht in the group.
These results indicate that the de-
crease in adrenal weight is probably not due to an effect of PABA on this organ but is related to factor(s) leading to the observed failure in body weight gain.
~lis also agrees with findings obtained with different
554
S T E R O I D S
3:5
.24 .20 .16
.08 .04 0
_ 340
i 380
I
I 420
Wavelength
'
340
I
I
!
380
|
420
Wavelength
Figure IA~ left. Spectra obtained with 2 i~g. of cortisone (curve 4), and 2 ~g. of cortisone plus 2, 4, 6 i~g. of corticosterone respectively (curves 5, 6, 7) in the aqueous P-S reagent.
Figure IB 2 right. Spectra obtained with 2, 4, 6 ~g. of corticosterone (curves l, 2, 3]respectively, 2 ~g. of cortisone (curve 4) and 2 ~g. of cortisone to which were added 2, 4, 6 ~g. of corticosterone (curves 5, 6, 7) respectively in the ethanolic P-S reagent.
goitrogens where a decrease in adrenal weight is related to a failure in c~ i0, ii body weisht cain J' ~e
mechanism proposed by McCarthy, et al. 4 to e~plain the effect
of PAI~ on the peripheral icvels of P-S chromogens (iS-OH-DOC in this paper) and corticosterone was considered attractive.
Although their
results did not permit overlooki~ the possibility that adrenal atrophy may have resulted as a consequence of thyroid inhibition~ these authors concluded that a direct action of PABA on the adrenal gland would best account for the increased peripheral plasma levels of P-S chromogens and a decrease in plasma corticosterone.
The results of Table I do not
support this conclusion since steroid secretory capacity of the adrenal glana is the same in the control as in the PAl~-treated animals.
The re-
M a y 1964
S T E R O I D S
555
TABLE I ~ e Effect of PABA Administration on the Rat Adrenal Gland Weight, Thyroid ~eight, Body Weight and the Secretion of C~rticosterone and 18-OH-DOC by the Rat Adrenal Gland. Control Number of rats
PABA
ii
Rat weight, gm.
381
p
i!
+ 39
~
+ 28
~ .02
Adrenal weight, mg.
44.8 +
1.6
38.4 + i.$
< .001
Adrenal weight, relative
ii.9 +
2.4
ii.6 + 1.9
> .7
~nyroid ~eight, mg.
23.6 +
1.2
26.7 + I. 9
~ .02
6.2 +
i.0
8.0 + 1.3
~ .0!
Thyroid weight, relative Corticosterone, ~g.
2.69 +
0.66
2.73 + 0.78 > .9
18-OII-DOC, L~'g.
1.0
0.4
1.0
_~a_
+
~__.
+ 0.3 -.
.
.
> .7
.
Rats are ~res~unablymaximally stressed at the time when blood sampling was performed I. Relative weight, mg. per i00 gm. of body weight. Corticosterone and 18-OH-DOC secretion rate, ~g./adrenal/lO minute/lO0 ~ . body weight.
suits obtained by McCarthy et al.
4
suggest, therefore, that perhaps PABA
affects the levels of 18-OH-DOC and corticosterone circulating in the blood by a mechanism(s) which may involve the preferential breakdown or metabolism of corticosterone while allowing 18-OH-DOC to accumulate. One may question, however, the validity of using the two component 12 technique for analysing 18-OH-DOC and corticosterone ministered to rats 1.
4
when P A B A w a s ad-
The lack of specificity of the method is indicated
by the fact that the amount of "17-hydroxycorticosteroids" (P-S chromogens) detected by chromatography did not account for the concentrations of
4
cortisol-like material detected by the colorimetric techniques .
On the
other hand, using the aqueous P-S reagent, the values obtained by chromatography were close to those obtained by colorimetry I indicatin~ a sufficient-
556
S T E R O I D S
3:5
ly high degree of specificity to enable accurate determlinations DOC to be carried out in the present study.
The reason ~ y
of I$-OH-
a two compon-
ent technique would yield zero values for corticosterone in PABA-treated animals 4 remains unknown.
It is worthy of mention, however~ that the ~ o
component technique will not yield ~osolute corticosterone values.
The
absorbance which is obtained at 355 m~ with the ethanolic P-S reagent is due to the presence of a ~4 3-keto function of the steroid 7.
As can be
seen in a two component system (Fig~ire IB)~ the absorption values obtained at 350 m~. are the sum of the respective absorbancies of cortisone and corticosterone, both steroids possessing the &4 3-keto function. Because of this, corticosterone values obtained in plasma samples analysed by this method will be in error by a factor equal to a sum of the absorption values derived from other &4 3-keto steroids such as testosterone and other adrenocorticoids.
However, this may not prove to be a ser-
ious objection in using the two component technique because of the very I
low circulating levels of ~; 3-keto steroids besides 18-0H-DeC and corticosterone I.
Nevertheless,
in future experiments of this type it might be
well to carry out corticosterone determinations by the aforementioned techniques as well as by fluorometry which measures corticosterone specifically in the rat I. ~he possibility that PABA itself could interfere with the corticosterone readings taken at 355 m~ in the ethanolic P-S and result in zero readings was eliminated.
Addition of lOO~g, of PABA to 6 ~g. and 2 ~g.
of corticosterone and cortisone respectively did not affect the 355 or 410 m~.~ readings. of P ~
~lere is still the possibility that metabolic products
rather than PABA itself could interfere with the 355 n~ readings.
The reason for the very low peripheral cortieosterone levels in PABA treated animals therefore should be the object of further scrutiny.
May 1964
S T E R 0 I D S
557
ACEI{OWLEDGEMENT
This work was supported in part by Grant A-4899 from the National Institute of IIealth and by a Public }lealth Service research career development award No. AM-K3-19, 190 awarded to one of us (F. P.).
REFERENCES
i.
Cort6s, J. M., Peron, F. G. and Dorfman, R. I. 7 i~ ~ (1963 ).
2.
Birmingham, Mo and Ward, P. J.
3.
P~ron, F. G. Unpublished Observation.
4.
ENDOCRINOLOGY 73,
J. BIOL. CHEM. 236, 1661 (1961).
McCarthy, J. L., Corley, R. C. and Zarrow, M . V . 197, 693 (1959).
5.
Lipscombj H. S. and Nelson, D. H.
6.
Moncloa, F., P6ron, F. G. and Dorfman, R. I.
AM. J. PHYSIOL.
ENDOCRINOLOGY 7_~1, 13 (1962). K%VDOCRINOLOGY 6_~5, 717
(19~9). 7.
Silber, R. H. and C. C. Porter, J. BIOL. CHilli. 210, 923 (1954).
8.
Silber, R. H., Busch, R. D. and Oslapas, R.
CLIN. CHEM. ~, 278
(1958). 9.
Leblond, C. P. and Hoff, H. E. ENDOCRINOLOGY 35, 229 (1944).
lO.
Gabrilove, J. L. and Soffer, L. J. ENDOCRINOLOGY 47, 461 (1950).
!I.
Wong, T.~ IIogness, J. R. and Williams, R. H.
PROC. SOC. EXPTL. BIOL.
~mo. 8_2, 598 (1953). 12.
Vickerstaff, T. THE PHYSICAL C~H]~MISTRY OF DYEING (2nd ed.) London Interscience, 1954.
13.
Cortisone was used instead of 18-0H-DOC because of the unavailabity of the latter (see ref. 1). Since cortisone and 18-OH-DOC have approximately the same absorption maxima and the same chromogenic spectra over the range 330-450 m!~ the use of cortisone instead of 18-OH-DOC is presumed valid for the above work 1.
LACK OF EFFECT OF CHRONIC ~ T R A T I O N ON ~ E
OF p-AMINOHENZOIC ACID
SECRETION OF 18-HYDROXYI~XYCORTICOS~ONE AND CORTICOSTERONE BY THE RAT ADRENAL.
g
Julio M. Cortes and Fernand G. P~ron With the technical assistance of Miss Charlotte Lilly.
The Worcester Foundation for Experimental Biology Shrewsbury, Massachusetts
Received April
2,1964
ABSTRACT
Male rats were fed p-sminobenzoic acid (PABA) in their diet for 12 weeks. Although body weight and absolute adrenal weights were decreased and thyroid weight increased, there was no effect of PABA administration on the secretion of 20,21-dihydroxy-18,20-epoxy-4-pregnen-3-one (18-OHDOC ) and ll~, 21-dihydroxy-4-pregnene-3,20-dione (corticosterone) by the rat adrenal gland. The quantitation of corticosterone and 18-OH-DOC was carried out by two methods which measure these steroids specifically rather than by the two component technique of analysis used by McCarthy et al.4 It was concluded from the results of these experiments that PABA had no direct effect on the rat adrenal gland per se, and that the abnormal corticosterone and P-S chromogen levels found by McCarthy et al. ~ in peripheral rat blood after PABA administration was not due to any apparent abnormal adrenal secretion of corticosterone or 18-OH-DOC. One of the main secretory products of the rat adrenal gland is 20, 21-dihydroxy-18, 20-epoxy-4-pregnene-3-one (18-OH-DOC) and its production, like that of ll~,21-dihydroxy-4-pregnen-3,20-dione (corticosterone) is at least in part under ACT~ control1.
It appears to account ex-
clusively for the Porter-Silber (P-S) chromogens isolated in rat adrenal venous blood1.
In spite of evidence2'3 showing it to have little mineral-
ocorticoid activity, the relatively large amounts which are secreted in normal stressed or hypophysectomized rats injected with ACTH causes one to
552
S T E R O I D S
3:5
suspect a physiological role for this substance. McCarthy et al. 4, reported that the goitrogenic effect produced by the prolonged administration of p-aminobenzoic acid (PABA) to male rats was accompanied by an absolute and relative (rag.per lO0 gm b.w. ) reduction in adrenal weight, an increase in peripheral plasma P-S chromogens and a drastic decrease in peripheral plasma corticosterone levels.
These authors
suggested that the mechanism whereby PABA manifested its effects was by a direct action on the adrenal gland and thus presumably affecting the circulating levels of adrenocorticosteroids.
From the foregoing report it
would be expected that the chronic administration of PABA to rats would lead to substantial changes in the level of adrenal venous blood 18-OH-DOC. It was also reasoned that these changes might lead to the detection of some manifestation of 18-OH-DOC hypersecretion and thus give a clue to the possible physiological role of this substance in the rat. Under the conditions of the experiments, the results obtained preclude at present a direct effect of PABA at the adrenal level.
1 ~ ~
AND I ~ I O I ~
Male Sprague-Dawley rats weighing 90 to llO gin. were fed ad libidum for a period of 12 weeks with a mixture of ground rat laboratory chow to which PABA was added (4% by weight). The rats were anesthesized with ether and cannulation of the left adrenal vein by the method of Lipscomb and Nelson5 was performed. Blood was withdrawn in a heparinized syringe for exactly l0 minutes after which the animals were killed by exanguination. The adrenals and thyroid were removed, cleaned of adherent tissues and weighed. Each adrenal venous sample was processed as previously described and 18-OH-DOC and cort$costerone were determined by modifications~, U of both the P-S reaction" and the fluorometric method of Silber et al. ~. In their studies, McCarthy et al. 4 made the simultaneous determination of corticosterone and 18-OH-DOC by measuring the absorbaucies obtained at 355 m~ and 410 m~ respectively, with the ethanolic P-S reagent (64 ml. of concentrated sulphuric acid, 36 ml. of water, 50 ml. of absolute ethanol and lO0 rag. of phenylhydrazine hydrochloride ). In the present report the determination of 18-OH-DOC was quantitated by taking readings
May 1964
S T E R 0 I D S
553
in the aqueous P-S reagent (64 ml. of concentrated sulphuric acid, 36 ml. of water and lOO rag. of phenylhydrazine hydrochloride) at 405 m~ 1 for reasons given below. A study of the behavior of mixtures of ll~,17G,21-trihydroxypregnene-3,20-dione (cortisone, see ref. 13) and corticosterone in amounts equal to those observed in rat plasma for 18-OH-DOC and corticosterone I was carried out in order to elucidate any possible differences of the spectra with the two P-S reagents referred to above. Two duplicate sets of samples of 2, 4 and 6 ~g. of corticosterone, 2 ~g. of cortisone and mixtures of 2 ~g. of cortisone with 2, 4 and 6 ~g. of corticosterone were used. Four ml. of methylene dichloride were added to the dry steroid samples. One set of samples was then treated with the aqueous P-S reagent and the other with the ethanolic P-S reagent. ~ e spectra obtained with the two reagents are shown in figures IA and IB. The maximum absorption peak for corticosterone treated with the ethanolic P-S reagent was found to be at 350 m~. At this wavelength, absorption was proportional to the concentration of corticosterone. The addition of corticosterone to cortisone gave rise to alterations of the absorption curves of cortisone although the absorption at 410 m~ and 350 m~ were found to be the sum of the respective absorbancies of cortisone and corticosterone at both wavelengths (Figure IB). On the other hand, with the aqueous P-S reagent. 2-6 ~g. of corticosterone showed no light absorption at the wavelengths studied (Figure IA). In this instance, the absorbancy of cortisone at 410 m~t although less intense than that produced by the ethanolic P-S was unaffected by 2-6 ~g. additions of corticosterone (Figure IA). As a result of these findings we had a choice of two P-S reagents for quantitating 18-OH-DOC. The aqueous P-S reagent was used in the present study because the above results indicated that 18-OH-DOC could be measured specifically without inter{erence from possible fluctuations in levels of corcicosterone or other Z~ o-heto steroids ~hich mioht be brought about by PABA administration.
RESULTS A$[D DISCUSSION ~%e results in Table I show that rats receiving PABA during 12 weeks gained less weight than the controls.
Their thyroids increased in lI
~eight, both absolute and relative to body weight, as previously reported ~. The weight of the adrenal glands was significantly less in the PABA group than the controls but there was no difference in the relative adrenal weight to body weicht in the group.
These results indicate that the de-
crease in adrenal weight is probably not due to an effect of PABA on this organ but is related to factor(s) leading to the observed failure in body weight gain.
~lis also agrees with findings obtained with different
554
S T E R O I D S
3:5
.24 .20 .16
.08 .04 0
_ 340
i 380
I
I 420
Wavelength
'
340
I
I
!
380
|
420
Wavelength
Figure IA~ left. Spectra obtained with 2 i~g. of cortisone (curve 4), and 2 ~g. of cortisone plus 2, 4, 6 i~g. of corticosterone respectively (curves 5, 6, 7) in the aqueous P-S reagent.
Figure IB 2 right. Spectra obtained with 2, 4, 6 ~g. of corticosterone (curves l, 2, 3]respectively, 2 ~g. of cortisone (curve 4) and 2 ~g. of cortisone to which were added 2, 4, 6 ~g. of corticosterone (curves 5, 6, 7) respectively in the ethanolic P-S reagent.
goitrogens where a decrease in adrenal weight is related to a failure in c~ i0, ii body weisht cain J' ~e
mechanism proposed by McCarthy, et al. 4 to e~plain the effect
of PAI~ on the peripheral icvels of P-S chromogens (iS-OH-DOC in this paper) and corticosterone was considered attractive.
Although their
results did not permit overlooki~ the possibility that adrenal atrophy may have resulted as a consequence of thyroid inhibition~ these authors concluded that a direct action of PABA on the adrenal gland would best account for the increased peripheral plasma levels of P-S chromogens and a decrease in plasma corticosterone.
The results of Table I do not
support this conclusion since steroid secretory capacity of the adrenal glana is the same in the control as in the PAl~-treated animals.
The re-
M a y 1964
S T E R O I D S
555
TABLE I ~ e Effect of PABA Administration on the Rat Adrenal Gland Weight, Thyroid ~eight, Body Weight and the Secretion of C~rticosterone and 18-OH-DOC by the Rat Adrenal Gland. Control Number of rats
PABA
ii
Rat weight, gm.
381
p
i!
+ 39
~
+ 28
~ .02
Adrenal weight, mg.
44.8 +
1.6
38.4 + i.$
< .001
Adrenal weight, relative
ii.9 +
2.4
ii.6 + 1.9
> .7
~nyroid ~eight, mg.
23.6 +
1.2
26.7 + I. 9
~ .02
6.2 +
i.0
8.0 + 1.3
~ .0!
Thyroid weight, relative Corticosterone, ~g.
2.69 +
0.66
2.73 + 0.78 > .9
18-OII-DOC, L~'g.
1.0
0.4
1.0
_~a_
+
~__.
+ 0.3 -.
.
.
> .7
.
Rats are ~res~unablymaximally stressed at the time when blood sampling was performed I. Relative weight, mg. per i00 gm. of body weight. Corticosterone and 18-OH-DOC secretion rate, ~g./adrenal/lO minute/lO0 ~ . body weight.
suits obtained by McCarthy et al.
4
suggest, therefore, that perhaps PABA
affects the levels of 18-OH-DOC and corticosterone circulating in the blood by a mechanism(s) which may involve the preferential breakdown or metabolism of corticosterone while allowing 18-OH-DOC to accumulate. One may question, however, the validity of using the two component 12 technique for analysing 18-OH-DOC and corticosterone ministered to rats 1.
4
when P A B A w a s ad-
The lack of specificity of the method is indicated
by the fact that the amount of "17-hydroxycorticosteroids" (P-S chromogens) detected by chromatography did not account for the concentrations of
4
cortisol-like material detected by the colorimetric techniques .
On the
other hand, using the aqueous P-S reagent, the values obtained by chromatography were close to those obtained by colorimetry I indicatin~ a sufficient-
556
S T E R O I D S
3:5
ly high degree of specificity to enable accurate determlinations DOC to be carried out in the present study.
The reason ~ y
of I$-OH-
a two compon-
ent technique would yield zero values for corticosterone in PABA-treated animals 4 remains unknown.
It is worthy of mention, however~ that the ~ o
component technique will not yield ~osolute corticosterone values.
The
absorbance which is obtained at 355 m~ with the ethanolic P-S reagent is due to the presence of a ~4 3-keto function of the steroid 7.
As can be
seen in a two component system (Fig~ire IB)~ the absorption values obtained at 350 m~. are the sum of the respective absorbancies of cortisone and corticosterone, both steroids possessing the &4 3-keto function. Because of this, corticosterone values obtained in plasma samples analysed by this method will be in error by a factor equal to a sum of the absorption values derived from other &4 3-keto steroids such as testosterone and other adrenocorticoids.
However, this may not prove to be a ser-
ious objection in using the two component technique because of the very I
low circulating levels of ~; 3-keto steroids besides 18-0H-DeC and corticosterone I.
Nevertheless,
in future experiments of this type it might be
well to carry out corticosterone determinations by the aforementioned techniques as well as by fluorometry which measures corticosterone specifically in the rat I. ~he possibility that PABA itself could interfere with the corticosterone readings taken at 355 m~ in the ethanolic P-S and result in zero readings was eliminated.
Addition of lOO~g, of PABA to 6 ~g. and 2 ~g.
of corticosterone and cortisone respectively did not affect the 355 or 410 m~.~ readings. of P ~
~lere is still the possibility that metabolic products
rather than PABA itself could interfere with the 355 n~ readings.
The reason for the very low peripheral cortieosterone levels in PABA treated animals therefore should be the object of further scrutiny.
May 1964
S T E R 0 I D S
557
ACEI{OWLEDGEMENT
This work was supported in part by Grant A-4899 from the National Institute of IIealth and by a Public }lealth Service research career development award No. AM-K3-19, 190 awarded to one of us (F. P.).
REFERENCES
i.
Cort6s, J. M., Peron, F. G. and Dorfman, R. I. 7 i~ ~ (1963 ).
2.
Birmingham, Mo and Ward, P. J.
3.
P~ron, F. G. Unpublished Observation.
4.
ENDOCRINOLOGY 73,
J. BIOL. CHEM. 236, 1661 (1961).
McCarthy, J. L., Corley, R. C. and Zarrow, M . V . 197, 693 (1959).
5.
Lipscombj H. S. and Nelson, D. H.
6.
Moncloa, F., P6ron, F. G. and Dorfman, R. I.
AM. J. PHYSIOL.
ENDOCRINOLOGY 7_~1, 13 (1962). K%VDOCRINOLOGY 6_~5, 717
(19~9). 7.
Silber, R. H. and C. C. Porter, J. BIOL. CHilli. 210, 923 (1954).
8.
Silber, R. H., Busch, R. D. and Oslapas, R.
CLIN. CHEM. ~, 278
(1958). 9.
Leblond, C. P. and Hoff, H. E. ENDOCRINOLOGY 35, 229 (1944).
lO.
Gabrilove, J. L. and Soffer, L. J. ENDOCRINOLOGY 47, 461 (1950).
!I.
Wong, T.~ IIogness, J. R. and Williams, R. H.
PROC. SOC. EXPTL. BIOL.
~mo. 8_2, 598 (1953). 12.
Vickerstaff, T. THE PHYSICAL C~H]~MISTRY OF DYEING (2nd ed.) London Interscience, 1954.
13.
Cortisone was used instead of 18-0H-DOC because of the unavailabity of the latter (see ref. 1). Since cortisone and 18-OH-DOC have approximately the same absorption maxima and the same chromogenic spectra over the range 330-450 m!~ the use of cortisone instead of 18-OH-DOC is presumed valid for the above work 1.