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Effect of a hepatocatalase preparation (caperase) on adrenal function in the guinea pig
Effect of a Hepatocatalase Preparation (Caperase) on Adrenal Function in the Guinea Pig By A.
ORIOL-BOSCHAND K. B. EXK-NES
The ability of a bovine hepatocatalase preparation to interfere with the in vitro function of the guinea pig adrenal has been studied. Administration of native or heat-denaturated hepatocatalase, but not of horse-radish peroxidase, resulted in a decreased production of PorterSilber chromogens by adrenal slices. Furthermore, such adrenals were also less responsive to ACTH stimulation.
Adrenal slices or adrenal homogenates from animals treated with hepatocatalase metabolized progesterone-4-Cl4 to C14-adrenal steroids at the same rate as adrenals from normal animals. Hepatocatalase interfered, however, with the capacity of the guinea pig adrenal to incorporate acetate-l-Cl4 into adrenal sterols and steroids.
H
has recently gained interest as an antiatherosclerotic drug due to its effect on blood cholesterol.l*a When injected into guinea pigs it will cause a decrease in the excretion of urinary corticosteroids.3 Since several factors could be involved in such phenomena we were interested in studying the relationship between hepatocatalase administration and adrenal function in vitro. It was thought that if hepatocatalase would diminish the production of 17-hydroxycorticosteroids or Porter-Silk chromogens in the guinea pig this could be due either to an effect on the peripheral metabolism of adrenal steroids, to a change in the metabolism of a known steroid precursor like progesterone to adrenal steroids, or to an interference with adrenal conversion of acetate to sterols and steroids. The investigation was deemed of some importance since adrenal function and development of pathological blood pressure may be causally related. EPATOCATALASE
MATERIALSAND METHODS Hartley strain male guinea pigs, body weight ranging
from 400-800 Gm. at the be-
ginning of the experiments, were injected with: (a) bovine hepatocatalase solved in 0.9 per cent sodium chloride; (b) 0.9 per cent sodium chloride, 100 Gm. body weight.
This group served
as control;
(c)
hepatocatalase
(HC)” dis0.1 ml. per
denaturated
heating for 1 minute at 100” C. and dissolved in 0.9 per cent sodium chloride; radish peroxidase dissolved in 0.9 per cent sodium chloride,
(d)
by
horse-
All injections were performed subcutaneously. The total amount of HC administered ranged from 5,000-100,000 Pevya unitst/Kg. body weight. Upon termination of injections the animals were killed by decapitation, the adrenals removed and freed of adhesive tissue, weighed immediately and quartered. Each gland was incubated separately in 10 ml. From the Department of Biological Chemistry, University of Utah CoUege of Medicine, Salt L&e CiQ, Utah. Received fur publication Dec. 4,1963. ‘We wish to thank Dr. Puig Muset, Barcelona, Spain, for the generous supply of hepatocatalase ( Caperase), tA Pevya unit is the amount of enzyme capable of decomposing 10 mg. of hydrogen peroxide in 1 minute (P.U.). 319 METABOLISM
VOL. 13, No. 4 (APRIL),
1964
320
OBIOL-BOSCH
of glucose-phosphate-Ringer buffer, pH 7.4, and the flasks oxygenated O,, 5 per cent CO, for 3 minutes.4 Incubations were performed at 37” C. in a shaking incubator (Research 1 hour at 100 strokes per minute. The incubation medium was then and incubation flasks washed with 1 ml. buffer which was added to mixture extracted 3 times, using 2 volumes of methylene dichloride ments where the effects of adrenocorticotropic hormone (ACTH)
AND
in 95
ELK-NES
per
cent
Specialties)
for
removed, the glands the medium, and this each time. In experion the in vitro pro-
duction of adrenal steroids were studied, the buffer was removed after the first hour of incubation and 10 ml. fresh buffer containing ACTH (8 units per 100 mg. tissue) added. Oxygenation was again done for 3 minutes and the flasks were then incubated for 2 hours. At the end of this time the medium was removed and extracted with methylene dichloride as described. Porter-Silber chromogens in the extracted medium were determined by the method of Eik-Ness and all results expressed as pg. cortisol/Gm. adrenal tissue/hour. The same progesterone.
buffer and incubation technic was used for the studies with radioactive Progesterone-4-C” with a specific activity of 0.1 pc.lpg. was used in the concentration of 0.1 PC. per 100 mg. adrenal tissue. The purity of the substrate was checked by paper chromatography running it first in methylcyclohexane,‘propylene glycols and then in hexane/formamide.’ Second chromatography was done after acetylation7 of the progesterone-like material in the methylcyclohexane ‘propylene glycol system. Only radioactive material behaving like authentic progesterone in 2 systems of chromatography
was used for incubation studies. When adrenal homogenates were Broeck glass homogenizer centrifuged for 10 minutes
employed,
the
glands
were
in glucose-phosphate-Ringer buffer. at 1000 x g to separate the cell debris.
homogenized
in a Ten-
The homogenate was Supernatant equivalent
to 200 mg. of tissue was incubated in each flask. In some flasks the medium also contained 4mM TPN, 3mM glucose-g-phosphate and a few crystals of glucose-6-phosphatedehydrogenase. After 2 hours incubation the mixture was extracted 3 times with 2 volumes of ethyl acetate each time, washed with l/10 volume 0.1 N sodium hydroxide and the washed ethyl acetate evaporated to dryness in a stream of nitrogen. The different progesterone metabolites contained in this residue were isolated by paper chromatography in hexane:benzene (1:l) i according to the following scheme: It was first chromatographed formamides until the solvent reached the front of the chromatogram. Three areas were then eluted from the dried paper strip: (a) a front portion which could contain progesterone and Ad-androstenedione; (b) a middle zone behaving chromatographically like either authentic li’a-hydroxyprogesterone, testosterone or deoxycorticosterone; and (c) the origin which could contain more polar steroids like the corticostcroids. Each time unknown radioactive metabolites were chromatographed, known and authentic steroids were chromatographed on parallel strips. The strips were scanned in a Haines type ultraviolet scanner for the detection of A’-3-ketosteroids. Radioactivity on the pnper strips was located by a Vanguard automatic Cl4 and Hs strip counter. All evaporations between chromatography were done in nitrogen. Zone A: Progesterone was separated from Ad-androstenedione by chromatography in hexane/formamide, the individual compounds being further purified by chromatography and final chromatography in hexane,’ in methylcyclohexane/propylene glycol, acetylation,’ formamide. Zone B: 17a-Hydroxyprogesterone was separated from testosterone after acetylation and chromatography of the acetylated product in hexane-henzine ( 1: 1) /formamide. Zone C: After rechromatographing this fraction in chloroform,/formamide 3 radioactive zones D, E and F were obtained. Zone D could contain ll/%hydroxyandrostenedione, corticosterone and Il-deoxycortisol. 11/3-Hydroxyanclrostenedione was separated from the other steroids by chromatography in benzene./formamide. Separation of corticosterone and II-deoxycortisol was accomplished in tohlene/propylene glycol,* keeping the chromatogram in the tank for lo-12 hours after the solvent had reac,hed the front of the chromatogMllS.
HEPATOCATALASE
Zone
E:
EFFECT
ON ADRENAL
could contain cortisone.
321
FUNCTION
It was chromatographed
in toluene/propylene-glycol
for 20 hours and thereafter it was acetylated and chromatographed in benzene/formamide. Zone F: should contain cortisol. It was chromatographed in chloroform/formamide for a total of 8 hours. The cortisol zone after being acetylated was rechromatographed in the same system. Studies on acetate-l-C14 incorporation into adrenal sterols and steroids were performed as follows: adrenal tissue from normal guinea pigs was weighed and placed in 4 incubation flasks containing 10 ml. of the buffer. The incubation was performed as described by Billiara for a total of 8 hours. Buffer was changed every second hour and 50 PC. acetatelCl4 was added each time to make a total addition of 200 PC. acetatelCr4. ACTH (8 units/100
mg. tissue)
was added
to each
flask before
the last 2 hour incubation
period.
While 2 of the flasks served as control, HC (4 x 10,000 P.U. ) was added to the other flasks each time fresh acetate-l-C14 was added. The steroids present in the final incubation medium were isolated and quantitated as described. After the incubation the adrenal tissue was homogenized in acetone-ethanol-ether (4:4:1) and the protein separated by filtration. The sterols present in the organic solvent were precipitated with tomatine fore and after saponification with ethanolic KOHla. The amount of sterols present determined by the Liebermann-Burchard reaction and the radioactivity incorporated
bewas into
the tomatine precipitable fractions was estimated as described by Kabasa et al.10 All isolated radioactive compounds were counted by liquid scintillation after being dissolved in 10 ml. scintillation fluid (4 Gm. 2,5-diphenyloxazole and 50 mg. 1.4-bis-2 (5-phenyloxazolyl)-benzene dissolved in 1000 ml. toluene). The counting time was set for an accuracy below the 5 per cent error. Statistical evaluations were done by the “p” test of Student. &WJLTS
The amount of Porter-Silber chromogens produced by adrenal slices in vitro are shown in table 1. Each figure represents the mean value from 8-12 incubation flasks. Experiments 2-6 show a statistically significant difference at the 5 per cent level between the 0.9 per cent sodium chloride injected and HC injected animals. Animals receiving small doses of HC behaved like control animals, and no difference in the production of Porter-Silber chromogens was furthermore established between animals receiving native HC and those given HC denaturated by heat (experiments 7 and 8). Finally, an 8day administration of horse-radish peroxidase dissolved in 0.9 per cent sodium chloride gave the same production of Porter-Silber chromogens as seen in animals given 0.9 per cent sodium chloride alone during the same period. A considerable variation in the control production of Porter-Silber chromogens was, however, observed ranging from a mean of 7.7 pg./Gm. adrenal weight/hour to 30.8 pg./Gm. adrenal weight/hour. The reason for this fluctuation is still unknown. In table 2 is recorded the Porter-Silber chromogen production following the addition of ACTH to an incubation medium containing adrenal slices. The production of Porter-Silber chromogens by adrenal slices of HC treated animals was lower than by those of the control animals, although only 2 of the groups fall into the 5 per cent significance category. No difference was noticed between animals receiving heat denaturated or the native HC preparation. Eight steroids were isolated and identified when progesterone-4-W” was
322
OBIOL-BOSCH
AND
EIIC-NES
used as substrate for adrenal slices and homogenates (table 3). Unknown compounds were also isolated and quantitated. The results are expressed as percentage of the total recovered radioactivity. No significant difference was found in the metabolism of progesterone to the various progesterone metabolites by slices and homogenates of adrenals from normal animals and from animals treated with HC (total dose 50,000 P.U./Kg. ) . Slices gave better yields of radioactivity in isolated cortisol, cortisone and corticosterone than those obtained with homogenates. Homogenates, however, in the presence of added cofactors showed the greatest capacity to metabolize progesterone as seen by the low amount of this compound left over at the end of the incubation period (table 3). Such homogenates, seemingly, metabolized progesterone mainly to compounds which could not be properly identified by th e systems of paper chromatography used. Homogenates without cofactors had less capacity for progesterone metabolism but 17a-hydroxyprogesterone and deoxycorticosterone, two direct metabolites of this substrate, were found in considerable amounts. Slices gave the greatest yield of II-deoxycortisol with deoxycorticosterone and 17a-hydroxyprogesterone being the next metabolites in order of magnitude. Very little radioactivity was found in cortisol and corticosterone regardless of incubation conditions. The ll-deoxy-compounds accounted for 40-50 per cent of the total converted radioactivity while only 10-E per cent was present in the 11-oxygenated steroids. The same trend was seen using homogenates. The incorporation of acetate-l-Cl4 b y adrenal slices into free and conjugated sterols and some steroids is presented in table 4. The presence of HC in the incubation medium inhibited the incorporation of radioactivity into all sterols while it influenced only moderately the total concentration in each fraction. Therefore, the specific activity of all the compounds which could be quantitated fell in the presence of HC. DISCUSSION
It has been shownll that corticosteroid production by the adrenal gland in vitro is an adequate index of the functional state of the gland at the moment of decapitation of the animal. Accepting this criterion one may imply that the administration of a hepatocatalase preparation lowered adrenal function in the guina pig. This effect seems dependent on the dose of HC used, since the low doses failed to inhibit corticosteroid production in vitro. Furthermore, high doses did not give additional inhibition over what 50,000 P.U./Kg. could achieve. The inhibitory effect of the HC preparation on adrenal function is slow as reported previously’” and as shown in experiments 3, 4 and 5 (table 1). Finally, the same high dose of HC produced greater inhibition when its time of administration was spread out. Since the heat denaturated HC was as active as the native preparation in decreasing PorterSilber chromogen production, it appears that the active factor is thermostable. In a recent report Caravaca and co-workers r3 described a factor capable of inhibiting cholesterol biosynthesis in the cell-free homogenate of the rat
HEPATOCATALASE
EFFECT
ON ADRENAL
323
FUNCTION
Table L-The In Vitro Production of Porter-Silber Chromogens by Adrenal Tissue of Guinea Pigs Subjected to Different Treatments before the Adrenals were Removed for Incubation Treatment
-Experiment NO.
Total
Duration of Administration (days)
Dose Given
8
Inhibition % of Controls
None 7.9 + 0.89 7.7 + 0.63
0.9% NaCl Hepatocatalase 30,000 PU/Kg.
22
0.9% NaCl Hepatocatalase 50,000 PU/Kg.
6
0.9% NaCl Hepatocatalase 50,000 PU/Kg.
7
0.9% NaCl Hepatocatalase 50,000 PU/Kg.
8
5.8 t
.05
24
.02
28
.Ol
39
.05
50
.Ool
44
0.47
25.3 r+ 2.30 18.3 Xk 0.95 12.4 + 1.33 7.4 t
0.65
16.3 +- 2.80 8.1 k 0.98
0.9% NaCl Hepatocatalase 100,000 PU/Kg.
30.8 I!Z 2.56 12 17.1 zk 1.36
Heat Denat. Hepatocatalase 30,000 PU/Kg. Hepatocatalase 30,000 PU/Kg. Heat Denat. Hepatocatalase 50,000 PU/Kg. Hepatocatalase 50,000 PU/Kg. 0.9% NaCl Horse-Radish 250 mg./Kg.
Pt
7.9 + 0.03
0.9% NaCl Hepatocatalase 5,000 PU/Kg.
7
Porter-Silber Chromogens pg./Gm. tissue/hr. mean k s.e.*
6.4 + 0.53 11
22 5.7 + 0.03
8.2 k 0.90 9
8 7.5 -+ 1.08 9.9 I!z 0.64
Peroxidase
‘Standard error of the mean. tprobability of the difference
11
8 8.9 f
being
0.95
significant.
liver. This factor was present in the same HC preparation as used by us, and was aIso diaIyzable.13 Azarnoff l* has reported an inhibitory effect on mevalonic acid incorporation into cholesterol by liver homogenates from rats treated either with this preparation of HC or by an HC preparation obtained from hog liver. This author also observed that ashed HC still was inhibitory. If a chelating agent was added to ashed HC, a less marked inhibition was obtained. Thus it is likely that all the published inhibition of HC on the liver13J4 and its reported inhibition on the adrenal are due to the same
324
ORIOL-BOSCH
AND
ELK-NES
Table 2.-The Effect of ACTH Addition (8 units/100 mg. tissue) on the In Vitro Production of Porter-Silber Chromogens by Adrenals of Control and Hepatocatakzse Treated Guinea Pigs Treatment Experiment NO.
Total
Dose Given
Duration of Administration (days)
Porter-Silber Chromogens* !.a./Gm. tissue/hr. mean k s.e.t
0.9% NaCl 10
11
12
13
8.5r
Hepatocatalase 50,000
Inhibition % of Controls
Pt:
6
PU/Kg.
.l
39
.Ol
36
.05
44
5.2 + 1.12
0.9% NaCl Hepatocatalase 50,000 PU/Kg.
7
13.1 -e 1.50
0.9% NaCl Hepatocatalase 50,000 PU/Kg.
8
8.5 f
0.90
10.6 + 1.90 5.8 Y!Z0.60
Heat treated Hepatocatalase 50,000 PU/Kg. Hepatocatalase 50,000 PU/Kg.
8.2 t
0.60
8 9.7 i- 1.30
*The left and right adrenal from the same animal were incubated separately in the presence or absence of ACTH. The Porter-Silber data are recorded as the mean difference between ACTH stimulated and nonstimulated glands. +Standard error of the mean. #Probability of the difference being significant.
Table 3.-Metabolism of Progesterone-l-C14 by Adrenal Slices and Homogenates of Guinea Pigs Treated with 0.9% NaCl (Controls) or with 50,000 PU/Kg. Hepatocatalase over 8 Days. Data are Expressed as Mean Percentage (Duplicate Fksks) of Recovered Radioactivity Incubation
Slices
Compounds
Isolated
Progesterone 17a-OH-Progesterone 11-Deoxycorticosterone 11-Deoxycortisol Ah-Androstenedione ll&OH-Androstenedione c0rtis01 Cortisone Corticosterone Unknown radioactivity *4 mM TPN, 3 mM present. NI = Not identified.
Conditions
Homogenate
Control
Hepatocatalaae
29.6 9.8 11.4 21.2 3.3 0.9 4.1 4.2 2.3 21.7
11.3 6.1 16.8 21.1 4.3 1.0 4.0 4.4 3.1 27.6
glucose-6-phosphate
Control
27.5 14.7 5.8 NI 1.7 NI 1.8 NI 0.3 48.1 and
zz
31.6 16.6 7.1 NI 2.1 NI 1.1 NI 0.3 41.0
~___ glucose&-phosphate
Homogenate cofactors*
Control
3.4 2.2 1.5 1.3 NI NI NI NI 1.0 96.6
+
HepZXt0catalMe
4.5 2.2 0.7 1.1 NI NI NI NI 0.3 91.2
dehydrogenase
HEPATOCATALASE
EFFECT
ON
ADRENAL
by Adrenal
into Sterol and Steroid Slices. Hepatocatulase (40,000 PU/paSk) was Added to the Flasks Indicated ~Control
Isolated
Radioactivity cpm/Gm. adrenal tissue _~~_~
Free
Conjugated
11-Deoxyeorticceterone
precipitable
Radioactivity cpm/Gm. adrenal tissue
Specifle Activity cpm/mpM 169 x 105t
1,616
1.686,300
391 x 106f
1.321
644,300
1,410.000
460 x 10”
1,377
633,600
173 x 105
1,461
921,400
244 x I@t
1,632
636,060
136 x 1Vt
1,339
1,073,300
299 x l@
2,007
633,000
133 x 10’
3,670
-
2,670
-
Not
16,120
-
determined
13.620
-
Cortiaol
Flasks
Amount in fig./100 mg. tissue
1.232
Not
_activity
Hepatocatalaae Specitlc A&vita cpm/mpM
determined C0rt.ic00terone
tSpeciAc
Flasks
Amount in P”.(/$c$w.
TPS
lTomatine
32.5
Vitro Zncorporation of Acetate-104
Table 4.-Zn
Material
FUNCTION
Not determined Not determined
1,330
-
1,336
-
3,330
-
6.920
-
0.76
6,760
23 x 10s
0.6’7
1.330
3.7 x 10J
1.46
3.400
21 x 105
0.64
1.335
10.2 x 105
sterols.
calculated
on basis
of
M.W.
of
336.
factor(s), an inorganic substance the nature of which is still unknown. To gain insight into the mechanism of adrenal inhibition by HC we considered several possibilities. Lowered production of corticosteroids could be due to an inability of the adrenal tissue to biosynthesize steroids from steroid precursors. Homogenate should provide a model system where the problem of cellular permeability would be nonexistent, while slices, representing a higher degree of organization, might imitate the in vivo situation. Furthermore, addition of a TPNH generating system to the homogenate should rule out the lack of available cofactors as the cause of decreased corticosteroid production. Since control animals and HC injected animals metabolized progesterone in a similar fashion, the mechanism of action of HC must be before progesterone is biosynthesized in the adrenal cortex. HC apparently decreased the utilization of acetate-l-Cl4 for sterol and steroid production by the adrenal gland. This inhibitory effect of HC showed an interesting peculiarity since a dissociation was observed between the radioactivity incorporated into the sterols and steroids and the quantitative amount of material (“cold material”) existing in these fractions. The decrease in utilization of the radioactive two carbon unit as precursor was more marked than the decrease in utilization of endogenous unlabeled precursors, resulting in a fall of specific activity. Although cholesterol biosynthesis has not been studied in as much detail in the adrenal tissue as in the liver, it probably follows similar pathways in both organs. If so, the effects of HC on the adrenal gland must occur somewhere before cholesterol is formed. Aza.rn~ff~~ has observed that in the liver HC acts on the conversion of mevalonic acid to isopentenyl pyrophosphate. The metabolic pattern obtained from incubations of guinea pig adrenals with progesterone showed some unexpected findings. From the reports in
326
ORIOL-BOSCH
AND
EIK-NES
the literature16J6 it is known that cortisol or hydroxylated derivatives of cortisol are the main corticosteroids produced by the guinea pig adrenal. We found 11-deoxycortisol and corticosterone as the compounds containing most of the radioactivity following incubation with either progesterone-4-C’ or acetate-1-C14. In the case of progesterone incubations, the ll&hydroxylase apparently failed to utilize the available 11-deoxy-compounds. No explanation is at hand to clarify this peculiar metabolic pattern seen also in prolonged incubations. 9 The production of labeled corticosterone from ac8tate-l-Cl4 or from progesterone-4-Cl4 is indeed in contrast to the reported inability of whole homogenates or minces of guinea pig adrenals to produce this compound from progesterone-4-C 14.i7 Only the isolated mitochondria of guinea pig adrenals produced labeled corticosterone when incubated with labeled progesterone. I7 Further knowledge of the localization of biochemical pools of adrenal precursors and their particular organization is needed to understand these problems. REJSXENCES 1. Laporte,
J., Puig Muset,
P., and Valde-
casas, F. G.: The action catalase upon cholesterol metabolites.
B&hem.
of hepatoand other
Pharmacol.
8:
161, 1961. -, and -: Action of hepatocatalase on experimental hypercholesterolemia. Biochem Pharmacol. 1 I :670, 1962. 3. Puig Muset, P., and Oriol-Bosch, A.: Estudios sobre la elimination de 17OH-corticosteroides urinarios en el 2. -,
cobayo: Accion de las enzimas lipoxidasa y catalasa. Rev. Iber. Endocrinol. 7:645, 1969. 4. Umbreit, W. W., Burris, R. H., and Stauffer, J. F.: In: Manometric TechMinneapolis, Burgess Pubniques. lishing Co., 1957, p. 1949. 5. Eik-Nes, K. B.: Determination of 17,21dihydroxy-20-ketosteroids in blood plasma.
J.
Clin.
Endocrinol.
17:502,
1957. 6. Savard, K.: Paper partition chromatography of C,, and q, ketosteroids.J. Biol. Chem. 202:457, 1953. Micromethods for the 7. Zaffaroni, A.: analysis of adrenocortical steroids. Rec. Prog. Honn. Res. 8:51, 1953. 8. -, Burton, R. B., and Keutmann, E. H.: Adrenal cortical hormones: analysis by paper partition chromatography and occurrence in the urine of normal persons. Science 111:6, 1950.
9. Billiar, of
R. B.: Utah,
Ph.D.
thesis,
University
1963.
10. Kabara, J. J., McLaughlin, J. T., and Riegel, C. A.: Quantitative microdetermination of cholesterol using tomatine as precipitating agent. Anal. Chem. 33:305, 1961. 11. van der Vies, J.: Corticoid production in oitfo as a test of adrenocortical function in rats. Acta Endocrinol. 33: 59, 1960. 12. Demand, H. A., Quevedo, M., and Oriol-Bosch, A.: Ueber den Einfluss der Katalase auf die oid-Ausscheidung
17-OH-Corticosterbei Meerschwein-
then. In Nowakowski, H.: Gewebs und Neurohormone. Berlin-Goettingen-Heidelberg, Springer Verlag, 1962, p. 336. 13. Caravaca, J., May, M. D., and Dimond, E. G.: Inhibition of squalene and cholesterol biosynthesis by hepatocatalase (Caperase). Biochem. Biophys. Res. Comm. 10:189, 1963. 14. Azarnoff, D. L., and Currah, G. L.: The effect of Caperase (bovine hepatocatalase) on cholesterol biosynthesis. Fed. Proc. 22269, 1963. 15. Heard, R. D. H., Bligh, E. G., Cann, M. C., Jellinck, P. H., D’DonneB, V. J., Rao, B. J., and Webt, J. L.: Biogenesis of the sterols and steroid hormones. Rec. Progr. Harm. Res.
HEPATOCATALASE
EFFECT
ON ADRENAL
FUNCTION
12:45, 1950. 16. Burstein, S., and Dorfman, R. I.: Hydrocortisone in normal guinea pig urine isolation and quantitative determination. J. Biol. Chem. 206:607, 1954.
327
17. Hoffman, F. G.: The suppression of corticosterone formation in vitro by the interaction of adrenal mitochondria and microsomes. Biochim. Biophys. Acta 65:13, 1962.
Albert0 Oriol-Bosch, M.D., U.S.P.H.S. Postdoctoral Fellow, Department of Biological Chemistry, University of Utah, Salt Lake City, Utah. Kristen B. Eik-Nes, M.D., Associate Professor, Department of Biological Chew&y, University of Utah, Salt Lake City, Utah.
ORIOL-BOSCHAND K. B. EXK-NES
The ability of a bovine hepatocatalase preparation to interfere with the in vitro function of the guinea pig adrenal has been studied. Administration of native or heat-denaturated hepatocatalase, but not of horse-radish peroxidase, resulted in a decreased production of PorterSilber chromogens by adrenal slices. Furthermore, such adrenals were also less responsive to ACTH stimulation.
Adrenal slices or adrenal homogenates from animals treated with hepatocatalase metabolized progesterone-4-Cl4 to C14-adrenal steroids at the same rate as adrenals from normal animals. Hepatocatalase interfered, however, with the capacity of the guinea pig adrenal to incorporate acetate-l-Cl4 into adrenal sterols and steroids.
H
has recently gained interest as an antiatherosclerotic drug due to its effect on blood cholesterol.l*a When injected into guinea pigs it will cause a decrease in the excretion of urinary corticosteroids.3 Since several factors could be involved in such phenomena we were interested in studying the relationship between hepatocatalase administration and adrenal function in vitro. It was thought that if hepatocatalase would diminish the production of 17-hydroxycorticosteroids or Porter-Silk chromogens in the guinea pig this could be due either to an effect on the peripheral metabolism of adrenal steroids, to a change in the metabolism of a known steroid precursor like progesterone to adrenal steroids, or to an interference with adrenal conversion of acetate to sterols and steroids. The investigation was deemed of some importance since adrenal function and development of pathological blood pressure may be causally related. EPATOCATALASE
MATERIALSAND METHODS Hartley strain male guinea pigs, body weight ranging
from 400-800 Gm. at the be-
ginning of the experiments, were injected with: (a) bovine hepatocatalase solved in 0.9 per cent sodium chloride; (b) 0.9 per cent sodium chloride, 100 Gm. body weight.
This group served
as control;
(c)
hepatocatalase
(HC)” dis0.1 ml. per
denaturated
heating for 1 minute at 100” C. and dissolved in 0.9 per cent sodium chloride; radish peroxidase dissolved in 0.9 per cent sodium chloride,
(d)
by
horse-
All injections were performed subcutaneously. The total amount of HC administered ranged from 5,000-100,000 Pevya unitst/Kg. body weight. Upon termination of injections the animals were killed by decapitation, the adrenals removed and freed of adhesive tissue, weighed immediately and quartered. Each gland was incubated separately in 10 ml. From the Department of Biological Chemistry, University of Utah CoUege of Medicine, Salt L&e CiQ, Utah. Received fur publication Dec. 4,1963. ‘We wish to thank Dr. Puig Muset, Barcelona, Spain, for the generous supply of hepatocatalase ( Caperase), tA Pevya unit is the amount of enzyme capable of decomposing 10 mg. of hydrogen peroxide in 1 minute (P.U.). 319 METABOLISM
VOL. 13, No. 4 (APRIL),
1964
320
OBIOL-BOSCH
of glucose-phosphate-Ringer buffer, pH 7.4, and the flasks oxygenated O,, 5 per cent CO, for 3 minutes.4 Incubations were performed at 37” C. in a shaking incubator (Research 1 hour at 100 strokes per minute. The incubation medium was then and incubation flasks washed with 1 ml. buffer which was added to mixture extracted 3 times, using 2 volumes of methylene dichloride ments where the effects of adrenocorticotropic hormone (ACTH)
AND
in 95
ELK-NES
per
cent
Specialties)
for
removed, the glands the medium, and this each time. In experion the in vitro pro-
duction of adrenal steroids were studied, the buffer was removed after the first hour of incubation and 10 ml. fresh buffer containing ACTH (8 units per 100 mg. tissue) added. Oxygenation was again done for 3 minutes and the flasks were then incubated for 2 hours. At the end of this time the medium was removed and extracted with methylene dichloride as described. Porter-Silber chromogens in the extracted medium were determined by the method of Eik-Ness and all results expressed as pg. cortisol/Gm. adrenal tissue/hour. The same progesterone.
buffer and incubation technic was used for the studies with radioactive Progesterone-4-C” with a specific activity of 0.1 pc.lpg. was used in the concentration of 0.1 PC. per 100 mg. adrenal tissue. The purity of the substrate was checked by paper chromatography running it first in methylcyclohexane,‘propylene glycols and then in hexane/formamide.’ Second chromatography was done after acetylation7 of the progesterone-like material in the methylcyclohexane ‘propylene glycol system. Only radioactive material behaving like authentic progesterone in 2 systems of chromatography
was used for incubation studies. When adrenal homogenates were Broeck glass homogenizer centrifuged for 10 minutes
employed,
the
glands
were
in glucose-phosphate-Ringer buffer. at 1000 x g to separate the cell debris.
homogenized
in a Ten-
The homogenate was Supernatant equivalent
to 200 mg. of tissue was incubated in each flask. In some flasks the medium also contained 4mM TPN, 3mM glucose-g-phosphate and a few crystals of glucose-6-phosphatedehydrogenase. After 2 hours incubation the mixture was extracted 3 times with 2 volumes of ethyl acetate each time, washed with l/10 volume 0.1 N sodium hydroxide and the washed ethyl acetate evaporated to dryness in a stream of nitrogen. The different progesterone metabolites contained in this residue were isolated by paper chromatography in hexane:benzene (1:l) i according to the following scheme: It was first chromatographed formamides until the solvent reached the front of the chromatogram. Three areas were then eluted from the dried paper strip: (a) a front portion which could contain progesterone and Ad-androstenedione; (b) a middle zone behaving chromatographically like either authentic li’a-hydroxyprogesterone, testosterone or deoxycorticosterone; and (c) the origin which could contain more polar steroids like the corticostcroids. Each time unknown radioactive metabolites were chromatographed, known and authentic steroids were chromatographed on parallel strips. The strips were scanned in a Haines type ultraviolet scanner for the detection of A’-3-ketosteroids. Radioactivity on the pnper strips was located by a Vanguard automatic Cl4 and Hs strip counter. All evaporations between chromatography were done in nitrogen. Zone A: Progesterone was separated from Ad-androstenedione by chromatography in hexane/formamide, the individual compounds being further purified by chromatography and final chromatography in hexane,’ in methylcyclohexane/propylene glycol, acetylation,’ formamide. Zone B: 17a-Hydroxyprogesterone was separated from testosterone after acetylation and chromatography of the acetylated product in hexane-henzine ( 1: 1) /formamide. Zone C: After rechromatographing this fraction in chloroform,/formamide 3 radioactive zones D, E and F were obtained. Zone D could contain ll/%hydroxyandrostenedione, corticosterone and Il-deoxycortisol. 11/3-Hydroxyanclrostenedione was separated from the other steroids by chromatography in benzene./formamide. Separation of corticosterone and II-deoxycortisol was accomplished in tohlene/propylene glycol,* keeping the chromatogram in the tank for lo-12 hours after the solvent had reac,hed the front of the chromatogMllS.
HEPATOCATALASE
Zone
E:
EFFECT
ON ADRENAL
could contain cortisone.
321
FUNCTION
It was chromatographed
in toluene/propylene-glycol
for 20 hours and thereafter it was acetylated and chromatographed in benzene/formamide. Zone F: should contain cortisol. It was chromatographed in chloroform/formamide for a total of 8 hours. The cortisol zone after being acetylated was rechromatographed in the same system. Studies on acetate-l-C14 incorporation into adrenal sterols and steroids were performed as follows: adrenal tissue from normal guinea pigs was weighed and placed in 4 incubation flasks containing 10 ml. of the buffer. The incubation was performed as described by Billiara for a total of 8 hours. Buffer was changed every second hour and 50 PC. acetatelCl4 was added each time to make a total addition of 200 PC. acetatelCr4. ACTH (8 units/100
mg. tissue)
was added
to each
flask before
the last 2 hour incubation
period.
While 2 of the flasks served as control, HC (4 x 10,000 P.U. ) was added to the other flasks each time fresh acetate-l-C14 was added. The steroids present in the final incubation medium were isolated and quantitated as described. After the incubation the adrenal tissue was homogenized in acetone-ethanol-ether (4:4:1) and the protein separated by filtration. The sterols present in the organic solvent were precipitated with tomatine fore and after saponification with ethanolic KOHla. The amount of sterols present determined by the Liebermann-Burchard reaction and the radioactivity incorporated
bewas into
the tomatine precipitable fractions was estimated as described by Kabasa et al.10 All isolated radioactive compounds were counted by liquid scintillation after being dissolved in 10 ml. scintillation fluid (4 Gm. 2,5-diphenyloxazole and 50 mg. 1.4-bis-2 (5-phenyloxazolyl)-benzene dissolved in 1000 ml. toluene). The counting time was set for an accuracy below the 5 per cent error. Statistical evaluations were done by the “p” test of Student. &WJLTS
The amount of Porter-Silber chromogens produced by adrenal slices in vitro are shown in table 1. Each figure represents the mean value from 8-12 incubation flasks. Experiments 2-6 show a statistically significant difference at the 5 per cent level between the 0.9 per cent sodium chloride injected and HC injected animals. Animals receiving small doses of HC behaved like control animals, and no difference in the production of Porter-Silber chromogens was furthermore established between animals receiving native HC and those given HC denaturated by heat (experiments 7 and 8). Finally, an 8day administration of horse-radish peroxidase dissolved in 0.9 per cent sodium chloride gave the same production of Porter-Silber chromogens as seen in animals given 0.9 per cent sodium chloride alone during the same period. A considerable variation in the control production of Porter-Silber chromogens was, however, observed ranging from a mean of 7.7 pg./Gm. adrenal weight/hour to 30.8 pg./Gm. adrenal weight/hour. The reason for this fluctuation is still unknown. In table 2 is recorded the Porter-Silber chromogen production following the addition of ACTH to an incubation medium containing adrenal slices. The production of Porter-Silber chromogens by adrenal slices of HC treated animals was lower than by those of the control animals, although only 2 of the groups fall into the 5 per cent significance category. No difference was noticed between animals receiving heat denaturated or the native HC preparation. Eight steroids were isolated and identified when progesterone-4-W” was
322
OBIOL-BOSCH
AND
EIIC-NES
used as substrate for adrenal slices and homogenates (table 3). Unknown compounds were also isolated and quantitated. The results are expressed as percentage of the total recovered radioactivity. No significant difference was found in the metabolism of progesterone to the various progesterone metabolites by slices and homogenates of adrenals from normal animals and from animals treated with HC (total dose 50,000 P.U./Kg. ) . Slices gave better yields of radioactivity in isolated cortisol, cortisone and corticosterone than those obtained with homogenates. Homogenates, however, in the presence of added cofactors showed the greatest capacity to metabolize progesterone as seen by the low amount of this compound left over at the end of the incubation period (table 3). Such homogenates, seemingly, metabolized progesterone mainly to compounds which could not be properly identified by th e systems of paper chromatography used. Homogenates without cofactors had less capacity for progesterone metabolism but 17a-hydroxyprogesterone and deoxycorticosterone, two direct metabolites of this substrate, were found in considerable amounts. Slices gave the greatest yield of II-deoxycortisol with deoxycorticosterone and 17a-hydroxyprogesterone being the next metabolites in order of magnitude. Very little radioactivity was found in cortisol and corticosterone regardless of incubation conditions. The ll-deoxy-compounds accounted for 40-50 per cent of the total converted radioactivity while only 10-E per cent was present in the 11-oxygenated steroids. The same trend was seen using homogenates. The incorporation of acetate-l-Cl4 b y adrenal slices into free and conjugated sterols and some steroids is presented in table 4. The presence of HC in the incubation medium inhibited the incorporation of radioactivity into all sterols while it influenced only moderately the total concentration in each fraction. Therefore, the specific activity of all the compounds which could be quantitated fell in the presence of HC. DISCUSSION
It has been shownll that corticosteroid production by the adrenal gland in vitro is an adequate index of the functional state of the gland at the moment of decapitation of the animal. Accepting this criterion one may imply that the administration of a hepatocatalase preparation lowered adrenal function in the guina pig. This effect seems dependent on the dose of HC used, since the low doses failed to inhibit corticosteroid production in vitro. Furthermore, high doses did not give additional inhibition over what 50,000 P.U./Kg. could achieve. The inhibitory effect of the HC preparation on adrenal function is slow as reported previously’” and as shown in experiments 3, 4 and 5 (table 1). Finally, the same high dose of HC produced greater inhibition when its time of administration was spread out. Since the heat denaturated HC was as active as the native preparation in decreasing PorterSilber chromogen production, it appears that the active factor is thermostable. In a recent report Caravaca and co-workers r3 described a factor capable of inhibiting cholesterol biosynthesis in the cell-free homogenate of the rat
HEPATOCATALASE
EFFECT
ON ADRENAL
323
FUNCTION
Table L-The In Vitro Production of Porter-Silber Chromogens by Adrenal Tissue of Guinea Pigs Subjected to Different Treatments before the Adrenals were Removed for Incubation Treatment
-Experiment NO.
Total
Duration of Administration (days)
Dose Given
8
Inhibition % of Controls
None 7.9 + 0.89 7.7 + 0.63
0.9% NaCl Hepatocatalase 30,000 PU/Kg.
22
0.9% NaCl Hepatocatalase 50,000 PU/Kg.
6
0.9% NaCl Hepatocatalase 50,000 PU/Kg.
7
0.9% NaCl Hepatocatalase 50,000 PU/Kg.
8
5.8 t
.05
24
.02
28
.Ol
39
.05
50
.Ool
44
0.47
25.3 r+ 2.30 18.3 Xk 0.95 12.4 + 1.33 7.4 t
0.65
16.3 +- 2.80 8.1 k 0.98
0.9% NaCl Hepatocatalase 100,000 PU/Kg.
30.8 I!Z 2.56 12 17.1 zk 1.36
Heat Denat. Hepatocatalase 30,000 PU/Kg. Hepatocatalase 30,000 PU/Kg. Heat Denat. Hepatocatalase 50,000 PU/Kg. Hepatocatalase 50,000 PU/Kg. 0.9% NaCl Horse-Radish 250 mg./Kg.
Pt
7.9 + 0.03
0.9% NaCl Hepatocatalase 5,000 PU/Kg.
7
Porter-Silber Chromogens pg./Gm. tissue/hr. mean k s.e.*
6.4 + 0.53 11
22 5.7 + 0.03
8.2 k 0.90 9
8 7.5 -+ 1.08 9.9 I!z 0.64
Peroxidase
‘Standard error of the mean. tprobability of the difference
11
8 8.9 f
being
0.95
significant.
liver. This factor was present in the same HC preparation as used by us, and was aIso diaIyzable.13 Azarnoff l* has reported an inhibitory effect on mevalonic acid incorporation into cholesterol by liver homogenates from rats treated either with this preparation of HC or by an HC preparation obtained from hog liver. This author also observed that ashed HC still was inhibitory. If a chelating agent was added to ashed HC, a less marked inhibition was obtained. Thus it is likely that all the published inhibition of HC on the liver13J4 and its reported inhibition on the adrenal are due to the same
324
ORIOL-BOSCH
AND
ELK-NES
Table 2.-The Effect of ACTH Addition (8 units/100 mg. tissue) on the In Vitro Production of Porter-Silber Chromogens by Adrenals of Control and Hepatocatakzse Treated Guinea Pigs Treatment Experiment NO.
Total
Dose Given
Duration of Administration (days)
Porter-Silber Chromogens* !.a./Gm. tissue/hr. mean k s.e.t
0.9% NaCl 10
11
12
13
8.5r
Hepatocatalase 50,000
Inhibition % of Controls
Pt:
6
PU/Kg.
.l
39
.Ol
36
.05
44
5.2 + 1.12
0.9% NaCl Hepatocatalase 50,000 PU/Kg.
7
13.1 -e 1.50
0.9% NaCl Hepatocatalase 50,000 PU/Kg.
8
8.5 f
0.90
10.6 + 1.90 5.8 Y!Z0.60
Heat treated Hepatocatalase 50,000 PU/Kg. Hepatocatalase 50,000 PU/Kg.
8.2 t
0.60
8 9.7 i- 1.30
*The left and right adrenal from the same animal were incubated separately in the presence or absence of ACTH. The Porter-Silber data are recorded as the mean difference between ACTH stimulated and nonstimulated glands. +Standard error of the mean. #Probability of the difference being significant.
Table 3.-Metabolism of Progesterone-l-C14 by Adrenal Slices and Homogenates of Guinea Pigs Treated with 0.9% NaCl (Controls) or with 50,000 PU/Kg. Hepatocatalase over 8 Days. Data are Expressed as Mean Percentage (Duplicate Fksks) of Recovered Radioactivity Incubation
Slices
Compounds
Isolated
Progesterone 17a-OH-Progesterone 11-Deoxycorticosterone 11-Deoxycortisol Ah-Androstenedione ll&OH-Androstenedione c0rtis01 Cortisone Corticosterone Unknown radioactivity *4 mM TPN, 3 mM present. NI = Not identified.
Conditions
Homogenate
Control
Hepatocatalaae
29.6 9.8 11.4 21.2 3.3 0.9 4.1 4.2 2.3 21.7
11.3 6.1 16.8 21.1 4.3 1.0 4.0 4.4 3.1 27.6
glucose-6-phosphate
Control
27.5 14.7 5.8 NI 1.7 NI 1.8 NI 0.3 48.1 and
zz
31.6 16.6 7.1 NI 2.1 NI 1.1 NI 0.3 41.0
~___ glucose&-phosphate
Homogenate cofactors*
Control
3.4 2.2 1.5 1.3 NI NI NI NI 1.0 96.6
+
HepZXt0catalMe
4.5 2.2 0.7 1.1 NI NI NI NI 0.3 91.2
dehydrogenase
HEPATOCATALASE
EFFECT
ON
ADRENAL
by Adrenal
into Sterol and Steroid Slices. Hepatocatulase (40,000 PU/paSk) was Added to the Flasks Indicated ~Control
Isolated
Radioactivity cpm/Gm. adrenal tissue _~~_~
Free
Conjugated
11-Deoxyeorticceterone
precipitable
Radioactivity cpm/Gm. adrenal tissue
Specifle Activity cpm/mpM 169 x 105t
1,616
1.686,300
391 x 106f
1.321
644,300
1,410.000
460 x 10”
1,377
633,600
173 x 105
1,461
921,400
244 x I@t
1,632
636,060
136 x 1Vt
1,339
1,073,300
299 x l@
2,007
633,000
133 x 10’
3,670
-
2,670
-
Not
16,120
-
determined
13.620
-
Cortiaol
Flasks
Amount in fig./100 mg. tissue
1.232
Not
_activity
Hepatocatalaae Specitlc A&vita cpm/mpM
determined C0rt.ic00terone
tSpeciAc
Flasks
Amount in P”.(/$c$w.
TPS
lTomatine
32.5
Vitro Zncorporation of Acetate-104
Table 4.-Zn
Material
FUNCTION
Not determined Not determined
1,330
-
1,336
-
3,330
-
6.920
-
0.76
6,760
23 x 10s
0.6’7
1.330
3.7 x 10J
1.46
3.400
21 x 105
0.64
1.335
10.2 x 105
sterols.
calculated
on basis
of
M.W.
of
336.
factor(s), an inorganic substance the nature of which is still unknown. To gain insight into the mechanism of adrenal inhibition by HC we considered several possibilities. Lowered production of corticosteroids could be due to an inability of the adrenal tissue to biosynthesize steroids from steroid precursors. Homogenate should provide a model system where the problem of cellular permeability would be nonexistent, while slices, representing a higher degree of organization, might imitate the in vivo situation. Furthermore, addition of a TPNH generating system to the homogenate should rule out the lack of available cofactors as the cause of decreased corticosteroid production. Since control animals and HC injected animals metabolized progesterone in a similar fashion, the mechanism of action of HC must be before progesterone is biosynthesized in the adrenal cortex. HC apparently decreased the utilization of acetate-l-Cl4 for sterol and steroid production by the adrenal gland. This inhibitory effect of HC showed an interesting peculiarity since a dissociation was observed between the radioactivity incorporated into the sterols and steroids and the quantitative amount of material (“cold material”) existing in these fractions. The decrease in utilization of the radioactive two carbon unit as precursor was more marked than the decrease in utilization of endogenous unlabeled precursors, resulting in a fall of specific activity. Although cholesterol biosynthesis has not been studied in as much detail in the adrenal tissue as in the liver, it probably follows similar pathways in both organs. If so, the effects of HC on the adrenal gland must occur somewhere before cholesterol is formed. Aza.rn~ff~~ has observed that in the liver HC acts on the conversion of mevalonic acid to isopentenyl pyrophosphate. The metabolic pattern obtained from incubations of guinea pig adrenals with progesterone showed some unexpected findings. From the reports in
326
ORIOL-BOSCH
AND
EIK-NES
the literature16J6 it is known that cortisol or hydroxylated derivatives of cortisol are the main corticosteroids produced by the guinea pig adrenal. We found 11-deoxycortisol and corticosterone as the compounds containing most of the radioactivity following incubation with either progesterone-4-C’ or acetate-1-C14. In the case of progesterone incubations, the ll&hydroxylase apparently failed to utilize the available 11-deoxy-compounds. No explanation is at hand to clarify this peculiar metabolic pattern seen also in prolonged incubations. 9 The production of labeled corticosterone from ac8tate-l-Cl4 or from progesterone-4-Cl4 is indeed in contrast to the reported inability of whole homogenates or minces of guinea pig adrenals to produce this compound from progesterone-4-C 14.i7 Only the isolated mitochondria of guinea pig adrenals produced labeled corticosterone when incubated with labeled progesterone. I7 Further knowledge of the localization of biochemical pools of adrenal precursors and their particular organization is needed to understand these problems. REJSXENCES 1. Laporte,
J., Puig Muset,
P., and Valde-
casas, F. G.: The action catalase upon cholesterol metabolites.
B&hem.
of hepatoand other
Pharmacol.
8:
161, 1961. -, and -: Action of hepatocatalase on experimental hypercholesterolemia. Biochem Pharmacol. 1 I :670, 1962. 3. Puig Muset, P., and Oriol-Bosch, A.: Estudios sobre la elimination de 17OH-corticosteroides urinarios en el 2. -,
cobayo: Accion de las enzimas lipoxidasa y catalasa. Rev. Iber. Endocrinol. 7:645, 1969. 4. Umbreit, W. W., Burris, R. H., and Stauffer, J. F.: In: Manometric TechMinneapolis, Burgess Pubniques. lishing Co., 1957, p. 1949. 5. Eik-Nes, K. B.: Determination of 17,21dihydroxy-20-ketosteroids in blood plasma.
J.
Clin.
Endocrinol.
17:502,
1957. 6. Savard, K.: Paper partition chromatography of C,, and q, ketosteroids.J. Biol. Chem. 202:457, 1953. Micromethods for the 7. Zaffaroni, A.: analysis of adrenocortical steroids. Rec. Prog. Honn. Res. 8:51, 1953. 8. -, Burton, R. B., and Keutmann, E. H.: Adrenal cortical hormones: analysis by paper partition chromatography and occurrence in the urine of normal persons. Science 111:6, 1950.
9. Billiar, of
R. B.: Utah,
Ph.D.
thesis,
University
1963.
10. Kabara, J. J., McLaughlin, J. T., and Riegel, C. A.: Quantitative microdetermination of cholesterol using tomatine as precipitating agent. Anal. Chem. 33:305, 1961. 11. van der Vies, J.: Corticoid production in oitfo as a test of adrenocortical function in rats. Acta Endocrinol. 33: 59, 1960. 12. Demand, H. A., Quevedo, M., and Oriol-Bosch, A.: Ueber den Einfluss der Katalase auf die oid-Ausscheidung
17-OH-Corticosterbei Meerschwein-
then. In Nowakowski, H.: Gewebs und Neurohormone. Berlin-Goettingen-Heidelberg, Springer Verlag, 1962, p. 336. 13. Caravaca, J., May, M. D., and Dimond, E. G.: Inhibition of squalene and cholesterol biosynthesis by hepatocatalase (Caperase). Biochem. Biophys. Res. Comm. 10:189, 1963. 14. Azarnoff, D. L., and Currah, G. L.: The effect of Caperase (bovine hepatocatalase) on cholesterol biosynthesis. Fed. Proc. 22269, 1963. 15. Heard, R. D. H., Bligh, E. G., Cann, M. C., Jellinck, P. H., D’DonneB, V. J., Rao, B. J., and Webt, J. L.: Biogenesis of the sterols and steroid hormones. Rec. Progr. Harm. Res.
HEPATOCATALASE
EFFECT
ON ADRENAL
FUNCTION
12:45, 1950. 16. Burstein, S., and Dorfman, R. I.: Hydrocortisone in normal guinea pig urine isolation and quantitative determination. J. Biol. Chem. 206:607, 1954.
327
17. Hoffman, F. G.: The suppression of corticosterone formation in vitro by the interaction of adrenal mitochondria and microsomes. Biochim. Biophys. Acta 65:13, 1962.
Albert0 Oriol-Bosch, M.D., U.S.P.H.S. Postdoctoral Fellow, Department of Biological Chemistry, University of Utah, Salt Lake City, Utah. Kristen B. Eik-Nes, M.D., Associate Professor, Department of Biological Chew&y, University of Utah, Salt Lake City, Utah.