Journal of Clinical Investigation
`Vol. 45, N0. 12, 1966
`
`17-Hydroxylation Deficiency in Man *
`
`EDWARD G. BIGLIERI, MARY ANNE HERRON, AND NORMA BRUST
`
`(From the Medical Services, San Francisco General Horpital, and the Department of
`Medicine, University of California School of Medicine, San Francisco, Calif.)
`
`The biosynthesis of steroid hormones 1 requires
`a number of hydroxylating enzymes. Deficiency
`of these enzymes is demonstrated by increased or
`decreased amounts of certain steroid metabolites in
`blood and urine and is best exemplified by the
`deficiency of 113-hydroxylase (1) and 21—hy—
`droxylase (2) in patients with congenital adrenal
`hyperplasia.
`17a-Hydroxylase activity is present
`in these disorders because of
`the increases in
`
`secretion of androgens and excretion of preg-
`nanetriol.
`In addition, a lack of 3—hydroxysteroid
`dehydrogenase has also been described (3).
`17-
`Hydroxylation is essential not only to the bio-
`synthesis of cortisol but also to the formation of
`
`* Submitted for publication July 19, 1966; accepted
`September 7, 1966.
`Supported by U. S. Public Health Service grant AM-
`06415 from the National Institute of Arthritis and Meta-
`bolic Diseases, National Institutes of Health. The stud-
`ies were carried out
`in the General Clinical Research
`Center, FR-83, San Francisco General Hospital,
`sup-
`ported by the Division of Research Grants and Facilities,
`National Institutes of Health.
`Presented in part at the Fifty-eighth Annual Meeting of
`the American Society for Clinical Investigation, Atlantic
`City, N. _I., May 1, 1966.
`Address requests for reprints to the Editoral OH-ice,
`Medical Services, San Francisco General Hospital, San
`Francisco, Calif.
`1The following trivial names are used in this paper:
`androstenedione = 4-androsten-3,17-dione; compound S =_
`11-deoxycortisol = 4-pregnen-17a,21—diol-3,20-dione;
`de-
`hydroepiandrosterone = 5-androsten-3;?-ol-17-one ; dexa-
`methasone = 1,4-pregnandien-9-fluoro-16oz-methyl-11fl,17a,
`21-triol-3,20-dione; etiocholanolone = 5fl-androstan-3a-
`ol-17-one; pregnanediol = Sfi-pregnan-3a,20a-diol; preg-
`nanetriol = 5ft-pregnan-3a,11oz,20a-triol;
`stilbestrol = di-
`ethylstilbestrol = a,o/diethyl-4,4’stilbenediol;
`tetrahydro-
`aldosterone = TH aldosterone = 5}?-pregnan-3a,1l,3,2l-tri-
`hydroxy-20 keto-18-ol ;
`tetrahydrocortisol = TI-IF =
`513-pregnan—3az,1Ifl,l7a,21-tetrol-20-one ;
`tetrahydrocorti-
`sone = THE = 5,8-pregnan-3a,17a,2l-triol-11,20—dione ;
`tet-
`rahydrodeoxycorticosterone = THDOC = 5/3-pregnan-3a,
`21-diol-20-one; tetrahydro-11-deoxycortisol = 5B-pregnan-
`3a,l7a,21-triol-20-one;
`and tetrahydro-18-OH-dehydro-
`corticosterone = TH-18-OH compound A = 5fl-pregnan-
`3a,18,21-trihydroxy-11,20-dione.
`
`gonadal hormones (4, 5), the androgenic steroids,
`androstenedione, testosterone, and, eventually, es-
`trogens (6). Deficiency of this enzyme system
`should be manifested clinically in both adrenal
`and gonadal abnormalities if the enzyme is similar
`in both glands. Furthermore, it is reasonable to
`assume that
`lack of 17-hydroxylated steroids
`would allow uninhibited release of adrenocortico-
`
`In such a circumstance in which cortisol
`tropin.
`secretion is absent, prolonged survival would not
`be anticipated because mineralocorticoids would
`be the only adrenal steroids produced. We will
`describe and discuss a patient with deficiency in
`17a-hydroxylase activity with possibly a second
`defect in production of aldosterone.
`
`Methods
`
`Care report
`
`The female patient M. H. was thegproduct of a full-term
`normal pregnancy and weighed 7 pounds at birth. Several
`severe episodes of bronchitis occurred before 5 years of
`age. The patient was hospitalized for an influenza-like
`syndrome when she was 5 years old;
`the mother
`(29
`years) and one sibling (11 months) died from “influenza”
`at this time. From 5 to 10 years of age the patient was
`absent from school about one-third of the time because
`of infections of
`the upper respiratory tract.
`She was
`hospitalized at age 9 for severe upper respiratory infec-
`tion, high fever, and unconsciousness.
`Intravenous glu-
`cose therapy promptly restored consciousness, and hypo-
`glycemia was diagnosed but not documented. A similar
`episode and response to glucose therapy occurred a year
`later. A tonsillectomy was performed; continued post-
`operative nausea and vomiting necessitated hospitalization
`for 1 week.
`By 16 years of age the patient was 5 feet 3 inches tall
`and had not menstruated. Disabling infections of
`the
`upper
`respiratory tract were frequent
`throughout her
`adult life. At age 17,
`the basal metabolic rate and the
`glucose tolerance test were within normal
`limits, and a
`roentgenogram of the skull showed no abnormalities. The
`patient had grown 4 inches, but menses had not com-
`menced nor had secondary sex characteristics developed.
`An attempt
`to effect menses and development of sec-
`ondary sex characteristics with hormones was made; the
`breasts developed slightly but menstruation did not occur.
`
`1 946
`
`Amerigen Exhibit 1186
`Amerigen Exhibit 1186
`Amerigen v. Janssen IPR2016-00286
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`
`

`
`17-HYDROXYLATION DEFICIENCY IN MAN
`
`1947
`
`A physical examination at school revealed hypertension;
`blood pressure ranged between 150/110 and 180/130 mm
`Hg and remained elevated until age 24. Although the
`blood pressure was measured frequently between ages
`17 and 24, treatment for hypertension was not initiated.
`At 24 years, Premarin was administered for 3 months
`after which some vaginal spotting occurred. Small doses
`of Raudixin were administered to reduce hypertension,
`but the blood pressure continued to range between 140/
`100 and 180/110 mm Hg; medication was discontinued
`after a brief time because of unpleasant side effects. At
`26 years of age, the patient was 5 feet 9% inches tall. At
`27,
`the blood pressure was 180/120 mm Hg,
`tests for
`pheochromocytoma were negative, and an intravenous
`pyelogram showed no abnormalities. At 30, the systolic
`blood pressure was greater than 200 mm Hg for the
`first
`time. At 34, several episodes of marked muscle
`weakness prompted re—evaluation of
`the hypertension;
`low level serum K was discovered electrocardiographi-
`cally. A Trousseau sign was elicited frequently during
`measurement of blood pressure; numbness and tingling
`in the extremities had occurred for many years.
`In
`addition, episodes of partial hair loss had occurred since
`the patient was 24 years old.
`Physical examination. At the time of admission to the
`Clinical Study Center the patient was 35 years old. The
`blood pressure was 220/140 in the, supine position and
`194/120 mm Hg on standing. Pulse was 70 beats per
`minute and regular. Body weight was 75.0 kg. A Trous-
`seau sign was elicited. Funduscopic examination revealed
`arteriolar narrowing and arteriovenous nicking but no
`hemorrhage or exudate. The heart was not enlarged and
`no murmurs were heard. Examination of the lungs and
`abdomen revealed no abnormalities. No abdominal bruits
`were detected. The skin was extremely smooth with
`fine wrinkles at the corners of the mouth and eyes and
`some fawn-colored freckles on the malar surfaces. The
`ears were rigid. Axillary and pubic hair were not pres-
`ent, and the breasts were prepubertal in size. Gynecologic
`examination revealed the following: the clitoris and labia
`were small, the vagina was pink and nonestrogenized, the
`cervix was 1 cm in diameter, a tubular structure (2 X 2
`cm) was felt in the area of the uterus, no ovaries were
`felt, and a vaginal smear showed no estrogen effect.
`Initial laboratory data. The electrocardiogram showed
`flattening of the T-waves, U-waves compatible with hy-
`pokalemia, and left ventricular hypertrophy. The hemato-
`crit was 40 per 100 ml, and the hemoglobin, 13.5 g per
`100 ml. Eosinophils were 2 to 6% of the leukocyte count.
`Blood urea nitrogen was 12, serum creatinine, 0.8, and
`serum cholesterol, 233 mg per 100 ml, and protein-bound
`iodine was 6.7 ,ug per 100 ml. The following serum
`electrolyte measurements are based on five determina-
`tions: Na, 141 to 147; K, 2.7 to 3.2; CO2, 29 to 32; and
`Cl, 95 to 102 mEq per L. Exchangeable K determined
`by “K dilution and by the whole body counter was 24
`mEq per kg. Serum pH was 7.52. Results of urinalysis
`included a specific gravity of 1.010 and no evidence of
`proteinuria, glucose, or casts. The genotype was 46/XX.
`Steroid values were as follows: urinary excretion of
`
`Porter-Silber chromogens (5-ml urine sample) and tet-
`rahydro-11-deoxycortisol, and secretion of cortisol were
`0 mg per 24 hours; secretion of aldosterone was 10 Mg
`per 24 hours; and acid-hydrolyzable conjugate of aldoster-
`one was less than 1 pg per 24 hours. A ketosteroid value
`of 5 mg per 24 hours was obtained but was later con-
`sidered a result of interference from increased amounts
`of
`the metabolites of corticosterone because individual
`ketosteroids were very low.
`All studies were performed in the Clinical Study Center
`at San Francisco General Hospital periodically over a
`2-year period. The patient was put on a constant meta-
`bolic diet, which contained 10 mEq Na and 74 mEq K per
`day by diet analysis.
`In some studies 6 g NaCl was added
`to increase Na intake to 122 mEq per day.
`All basal steriod measurements were performed while
`the patient was on 122 mEq Na intake. The administra-
`tion of corticotropin intramuscularly, 40 U every 12
`hours for 5 days, and of angiotensin was also performed
`on this sodium intake. Pituitary suppression was achieved
`by administration of dexamethasone and cortisol. Levels
`of Na and K were measured in the urine daily and in
`the serum frequently by internal standard flame pho-
`tometry. Serum chloride was measured by the Cotlove
`titrimeter technique (7) and serum CO2 by titrimetric
`and Van Slyke analysis (8). The Na, K, and N con-
`tents were measured after acid digestion of samples of
`diet and stool.
`
`Steroid measurements
`
`Plasma. Cortisol and corticosterone were measured by
`a double isotope dilution derivative technique (9). The
`half-life of corticosterone was determined after the intra-
`venous administration of corticosterone-4-“C (10). Al-
`dosterone was measured by the constant
`infusion (d-
`aldosterone-1-2-’H)
`technique of Tait, Tait, Little, and
`Laumas (11). Progesterone was measured by the double
`isotope derivative technique
`(12).? Testosterone
`(13)
`and androstenedione (14) were determined by the double
`isotope derivative technique.3
`~
`Secretion. The methods used to measure secretory
`rates of cortisol, corticosterone, and aldosterone have
`been reported previously (15). Cortisol and corticos-
`terone were determined by the double isotope dilution
`technique and aldosterone by the double isotope deriva-
`tive technique.
`The secretory rate of deoxycorticosterone (DOC) was
`determined as follows: A 3-,uc dose of DOC-1-2-“H was
`administered intravenously, and urine was collected 24
`and 48 hours thereafter. During the first 24 hours, 75%
`of the injected dose was excreted. At most, only an
`additional 5% was excreted in the subsequent 96 hours.
`Urine samples were first washed with 2 vol of ethyl ace-
`
`2 Measurement done by Mr. Richard Underwood, Sears
`Surgical Laboratory, Boston City Hospital, Boston,
`Mass.
`3 Determination performed by Dr. Charles Lloyd,
`Worcester Foundation for Experimental Biology, Shrews-
`bury, Mass.
`
`

`
`1948
`
`E. G. BIGLIERI, M. A. HERRON, AND N. BRUST
`
`TABLE I
`
`Protein hormone measurements
`
`Chromatographic system for isolation of THDOC*
`
`System
`
`Solvent ratios
`
`I
`
`V
`
`Cyclohexane 100: benzene 40: methanol
`100:water 20
`II Decalin 100:nitromethane 50:methanol 50
`III
`Cyclohexane 100:nitromethane 50:methanol
`50
`IV Methylcyclohexane l00:methanol 100:water
`50 (reverse phase)
`Iso-octane system of Pasqualini and Jayle
`(16)
`VI Decalin 100:nitromethane 50:methanol 50
`VII
`Cyclohexane 100:nitromethane 50:methanol
`50
`Same as IV
`
`VIII
`
`* TH DOC = tetrahydrodeoxycorticosterone.
`
`tate, and the pH was adjusted to 4.5 and hydrolyzed with
`1% of the volume of the sample with Ketodase. Samples
`were incubated for 24 hours. The steroid was extracted
`with CCI4, washed, and chromatographed in system I
`(Table I). The THDOC was eluted and chromato-
`graphed in systems II and III, then eluted, dried, and
`acetylated with acetic anhydride-“C for 24 hours. The
`THDOC diacetate was then chromatographed in systems
`IV through VIII. The THDOC diacetate of the final
`chromatogram was eluted, dried, dissolved in toluene with
`2,5-diphenyloxazole and 1,4-di-2-phenyloxazole, and “H
`and “C were counted simultaneously in a liquid scintilla-
`tion spectrometer.
`tetrahydrocortisol and
`Urinary excretion. Levels of
`tetrahydrocortisone were determined by measurement of
`Porter-Silber chromogens (17) and those of tetrahydro-
`ll-deoxycortisol, by the technique of Henke, Doe, and
`Jacobson (18) . Tetrahydro-18-hydroxy-1l-dehydrocorti-
`costerone was measured (19)! Dehydroepiandrosterone,
`etiocholanolone, androsterone, pregnanetriol, and preg-
`nanediol were measured by gas liquid chromatography.-'*
`Total biologically active estrogens were determined by
`the method of Maddock and Nelson (20).° Acid-hydro-
`lyzable conjugate of aldosterone and TH aldosterone
`were measured by the double isotope dilution deriva-
`tive technique (21). Tetrahydrodeoxycorticosterone ex-
`cretion was measured by a double isotope dilution deriva-
`tive technique. Tritium-labeled THDOC was prepared en-
`zymatically from DOC-1-2“-H (21). Approximately 6,000
`cpm of THDOC-1-23-H was added to the urine samples.
`Portions were extracted, hydrolyzed,
`acetylated,
`and
`chromatographed as described for DOC secretion.
`
`4 Dr. Stanley Ulick, Veterans Administration Hospital,
`Bronx, N. Y.
`5Dr. Roberto Rivera, Syntex Research Laboratory,
`Palo Alto, Calif.
`~
`“Dr. C. Alvin Paulsen, University of Washington
`School of Medicine, Seattle, Wash.
`
`Time
`
`hours
`
`8
`
`96
`28
`
`4
`
`6
`
`48
`12
`
`Blood. Plasma ACTH was measured by the method
`of Lipscomb and Nelson (22)." Renin was determined
`by a modification of the method of Boucher, Veyrat, De
`Champlain, and Genest
`(23).8 Serum growth hormone
`was determined by an adaptation of radioimmunoassay
`technique (24).9
`Urine. The urine was assayed for follicle-stimulating
`hormone with the Steelman-Pohley technique (25).
`
`Miscellaneous measurements
`
`Angiotensin sensitivity. The amount of angiotensin II
`amide required to raise diastolic and systolic blood pres-
`sures 20 mm Hg was determined by a technique similar
`to that of Kaplan and Silah (26).
`Insulin sensitivity.
`Insulin sensitivity was assessed
`after intravenous administration of .05 kg crystalline in-
`sulin 16 hours after fasting by measuring blood sugar
`(27) and levels of
`free fatty acids and glycerol
`in
`plasma.“
`
`Results
`
`C-21 steroids derived from progesterone. The
`concentration of progesterone in plasma was 0.21
`pg per 100 ml (normal is 0.11 to 1.04), and that
`of pregnanediol in urine ranged from 2 to 11 mg
`per 24 hours (normal is 2 to 5) in three consecu-
`tive 24-hour collections. Urinary THDOC was
`500 pg per 24 hours (normal is 7 to 25), and se-
`cretion of DOC was 4.0 mg per 24 hours (normal
`is 0.050 to 0.160). The concentration of corti-
`costerone in plasma ranged from 21 to 31 pg per
`100 ml
`(normal is < 1) and showed a diurnal
`rhythm (25 at 8 a.m. and 9.1 pg per 100 ml plasma
`at 6 p.m.). The secretory rate of corticosterone
`was 112 to 124 mg per 24 hours (normal is 0.9 to
`4.4). The TH-18—OH compound A in urine was
`675 pg per 24 hours (normal is < 5 X TH aldos-
`terone). No aldosterone was detected in plasma
`(normal is < 0.01 pg per 100 ml plasma)-. The
`secretory rate of aldosterone ranged from 10 to 18
`pg per 24 hours on four occasions (normal is 60
`to 168). Urinary levels of acid-hydrolyzable con-
`
`7Dr. E. M. Gold, Veterans Administration Hospital,
`Los Angeles, Calif.
`8Determination done in the laboratories of Dr. J. W.
`Conn, University of Michigan Medical Center, Ann
`Arbor, Mich.
`9 Dr. G. Grodsky, Metabolic Unit, University of Cali-
`fornia Medical Center, San Francisco, Calif.
`10 Free fatty acids and glycerol were measured by Dr.
`Richard Havel, University of California Medical Center,
`San Francisco, Calif.
`
`

`
`17-HYDROXYLATION DEFICIENCY IN MAN
`
`1949
`
`FIG. 2. EFFECT or PITUITARY SUPPRESSION DURING 122
`MEQ NA INTAKE. Note that dexamethasone produced
`Na diuresis, K retention with correction of hypokale-
`mia, and weight loss.
`
`-he control
`
`rates of secretion of
`
`corticosterone and aldosterone were 112 mg and
`10 [Lg per 24 hours, respectively.
`E[feet of pituitary suppression on Na, K, and
`aldosterone excretion, serum K, and body weight
`during 122 mEq Na intake (Figure 2). Adminis-
`tration of dexamethasone, 0.5 mg every 12 hours,
`for 6 days eifected prompt Na diuresis, decrease
`in urinary K, increase in serum K from 2.9 to 4.5
`mEq per L, and loss of weight. Cumulative Na
`excretion was 372 mEq and cumulative K reten-
`tion, based on measurements made in urine, was
`242 mEq per L. There was no change in aldos-
`terone excretion; values were < 1 ,u.g per 24 hours.
`Ejfect of restricted Na intake and pituitary sup-
`pression on electrolyte balance, steroids, and body
`weight (Figure 3). A 4-day control study after
`equilibration on 122 mEq Na intake was carried
`out.
`Potassium balance was slightly negative,
`and serum K levels were low. Corticosterone
`
`secretion was 124 mg per 24 hours, and plasma
`level was 20 pg per 100 ml. Aldosterone excre-
`tion was 1 pg per 24 hours, and secretion on day 1
`was 10 pg per 24 hours. On day 4, THDOC was
`500 ;;.g per 24 hours.
`At the end of the control period added NaCl
`was removed from the diet. Renal sodium con-
`servation on the sodium-limited diet was accom-
`
`plished in the first 2 days of the 9-day study (even
`though a mild gastroenteric disorder and fever _
`produced a brief negative Na and K balance on
`day 6. Potassium was retained, and serum K
`increased slightly. Aldosterone excretion remained
`at 1 pg per 24 hours, and after 9 days THDOC
`
`jugate of aldosterone were always < l [Lg per 24
`hours (normal is 5 to 20), and those of TH aldos-
`terone ranged from 0 to 5 ;;.g per 24 hours (normal
`is 15 to 60).
`C-21 steroids derived from 17a—OH-progester-
`one. None of the C-21 steroids derived from 17a-
`
`OH—progesterone were detected in blood and urine.
`The secretory rate of cortisol was 0 (normal is 10
`to 30 mg per 24 hours).
`C-19 steroids
`and estrogens derived from
`1 7a-OH—progesterone and 1 7a-OH—pregnanolone.
`Urinary dehydroepiandrosterone,
`androsterone,
`and etiocholanolone levels were 0.11 to 0.17 (nor-
`mal is 1.34), 0.l3 to 0.24 (normal is 3.12), and
`0.20 to 0.40 (normal is 1.0 to 3.0) mg per 24 hours, ‘
`respectively.
`Plasma testosterone was 0.014 pg
`per 100 ml (normal is 0.037), and androstenedione
`was 0.068 pg per 100 ml (normal is 0.140). The
`total amount of biologically active estrogens in
`urine was < 0.2 pg-Eq estradiol benzrate per 24
`hours (normal is 0.4 to 0.7).
`Protein hormone measurements. Plasma levels
`
`of ACTH were 2.0 at 8 a.m. and 1.6 mU per 100
`ml at 6 p.m. No plasma renin activity was de-
`tected after 4 days of 10 mEq Na intake and 4
`hours of standing. The amount of angiotensin II
`amide required to produce a 20 mm Hg blood pres-
`sure increase was only 2.7 m,u.g per kg per min-
`ute (normal is 5 to 11). The level of follicle-stim-
`ulating hormone was 138 international units (IU)
`per 24 hours
`(highest normal
`is 20).
`Serum
`growth hormone levels were normal at 0.5 mU per
`ml.
`
`Infusion of corticosterone-4-“C. The biological
`half-time of infused corticosterone-4—“‘C was 50
`
`minutes (normal is 60 minutes). The pool size
`was 8.2 mg (normal is .3).
`
`
`
`
`
`
`
`
`ALDOSTERONE
`ug/24HR
`
`
`
`
`O
`
`CORTICOTROPIN 40 UNITS IM. EVERY I2 HOURS FOR 5 DAYS
`
`FIG. 1. RESPONSE TO CONTINUED ADMINISTRATION or
`CORTICOTROPIN. Note little change in aldosterone and
`corticosterone secretion.
`
`

`
`3:3‘
`
`
`
`
`
`20
`no Zbhiicbsrcnom
`ug %
`0
`
`1950
`
`MM!-1
`:5 van 9
`
`E. G. BIGLIERI, M. A. HERRON, AND N. BRUST
`
`ocxmsrmson: .5 mg/6 an
`
`FECAL Imsq/24 an
`
`__
`
`
`
`
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`’/7
`%/I// 7
`gggkgfcnn we
`O
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`
`_________H
`
`POTASSIUM
`
`?.":E,’§’z‘3‘Ha
`
`I00
`
`§é:::EE:CTff:,’T‘ERoNE 5°
`0
`500
`
`THDOC
`EXCRETION
`
`ug/24 HR
`
`BODY
`waneur
`
`300
`
`‘Go
`
`78
`
`75
`
`74
`JUNE |9
`
`I
`
`2|
`
`23
`
`2 5
`
`2 7
`
`29
`
`I
`
`3
`
`7
`
`9
`
`il
`
`I3
`
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`
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`
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`
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`
`DAYS
`
`EFFECT 01-‘ RESTRICTED NA INTAKE AND PITUITARY SUPPRESSION ON ELECTROLYTE
`FIG. 3.
`BALANCE AND STEROID MEASUREMENTS. THDOC = tetrahydrodeoxycorticosterone.
`
`was 285 pg per 24 hours. Blood pressure was
`unaffected during this period.
`Sodium restriction was continued, and dexa-
`methasone, 0.5 mg every 6 hours, was adminis-
`tered for 9 days. Negative Na balance occurred
`and K retention increased. Serum K gradually
`increased to 5.5 mEq per L. Secretion of corti-
`costerone decreased to 4.4 mg per 24 hours and
`plasma corticosterone to 6 pg per 100 ml. The
`secretory rate of DOC was 44 pg per 24 hours,
`and THDOC was 7.0 Aldosterone excretion was
`
`‘
`
`still reduced at 1 pg per 24 hours. Blood pressure
`fell and was 120/80 mm Hg on day 8 of the sup-
`pression period. The patient lost a total of 2.2 kg
`body weight.
`After dexamethasone was discontinued, NaCl
`Sodium balance was
`
`_ was added again to the diet.
`progressively positive, K balance was negative,
`and serum K gradually decreased from 5.5 to 4.0
`mEq per L. Corticosterone secretion increased to
`99 mg per 24 hours; plasma corticosterone, to 22
`
`pg per 100 ml; and THDOC excretion, to 180 pg
`per 24 hours.
`Effect of DOC and metyrapone on electrolytes
`and steroids (Figure 4). During the 2 days of
`treatment with DOC acetate (20 mg per 24 hours)
`Na retention was 114 mEq. There was little
`change in K balance, although serum K decreased
`from 3.5 to 3.2 mEq per L.
`During the 3 days of metyrapone administration,
`Na excretion fluctuated slightly, but a negative
`urinary K balance of 40 mEq was noted. Excre-
`tion of THDOC on the third day was 4,850 pg
`per 24 hours. The concentration of corticosterone
`in plasma decreased on the fourth day from the
`control level of 35.4 to 18.9 pg per 100 ml; in the
`subsequent 4 days after discontinuance of metyra—
`pone the concentration increased to 27.8 pg per
`100 ml.
`
`Nitrogen and carbohydrate metabolism. Al-
`though corticosterone secretion was approximately
`60 times the normal rate, N balance was slightly
`
`

`
`17-HYDROXYLATION DEFICIENCY IN MAN
`
`1951
`
`DEXAMHMASONE 0.5 mg/I2 Ml
`v-arr loot/24 an
`
`COll'|$Ol so ng/24 Ml
`SVIIIESYIOI
`I “/24 MI
`
`20
`
`O
`
`PLASMA B
`MD %
`
`I/’°\0~__°‘
`
`,
`
`--o--.°’_- .0--o---o.__°
`
`4
`
`8
`
`I6
`I2
`WEEKS
`
`20
`
`24
`
`28
`
`FIG. 5. COURSE or TREATMENT. 9¢xFF= 9rx-fluorohydro-
`cortisone; B = corticosterone.
`
`the level of serum K increased to 4.5 mEq per L
`and remained there. Blood pressure again re-
`turned to the normal range. Stilbestrol, 1 mg per
`day, was administered for 4 weeks; all measure-
`ments remained the same except
`the level of
`serum corticosterone, which was three times higher
`1 than that observed during administration of dexa—
`methasone by itself. When cortisol and stilbestrol
`were discontinued,
`the level of corticosterone in
`plasma promptly rose to 25 ,ug per 100 ml. During
`the course of treatment upper respiratory infec-
`tions and oral mucosal sores did not develop and
`stamina increased.
`
`Discussion
`
`A marked deficiency of adrenal 17a-hydroxylase
`activity is indicated by the absence of C-21 steroids
`derived from 17a-OH—progesterone and the mini-
`mal amounts of C-19 steroids derived from 17a-
`OH-progesterone and 17a—OH—pregnenolone. Fur-
`thermore,
`the negligible
`estrogen production,
`manifested clinically by the absence of secondary
`sex characteristics,
`the lack of estrinization, and
`the negligible amount of estrogens in urine, indi-
`cates additional evidence of diminished l7—hy-
`droxylation in the ovary.
`The only steroids
`detected were those not dependent on this enzy-
`matic process, namely, progesterone, DOC, corti-
`costerone (B), and TH—18-OH compound A.
`Secretory rates of these steroids were greater than
`normal, permitting survival of the patient. The
`minimal amount of aldosterone secreted in. the
`
`presence of increased secretion of B and excretion
`of TH-18-OH compound A may also indicate a
`second biosynthetic defect either in the conversion
`of
`l8—OH-B to aldosterone because of
`lack of
`
`positive, 0.2 to 2.0 g per 24 hours over a 30-day
`period. Levels of blood glucose were within nor-
`mal limits, and sensitivity to insulin was normal.
`Levels of free fatty acids and glycerol in plasma
`were considerably elevated at 1.51 and 0.252
`,u.moles per ml,
`respectively, but decreased nor-
`mally in response to insulin. Measurements of
`growth hormone in serum were within normal
`limits and increased to 3.5 mU per ml after admin-
`istration of insulin.
`
`(Figure 5). After the
`treatment
`Course of
`studies had been completed, the patient was treated
`during the first 12 weeks with dexamethasone, 0.5
`mg each 12 hours, orally. Blood pressure was
`maintained within the normal range, but the level
`of serum K began to rise and salt craving was
`noted by the patient.
`She increased the NaCl
`intake to 12 to 16 g per day. When the serum
`potassium level reached 6.7 mEq per L, 9a-fluoro-
`hydrocortisone, 100 pg per day, was given orally;
`serum K returned to normal levels, but a marked
`gain in weight occurred. Plasma corticosterone
`gradually decreased to a normal level (0.8 [Lg per
`100 ml). Dexamethasone and 9a-fluorohydrocor-
`tisone therapy were discontinued; the concentra-
`tion of plasma corticosterone rose to 20 pg per
`100 ml, and the level of serum K dropped to 3.8
`mEq per L.
`After 6 weeks cortisol, 30 mg per day, was
`given orally for 10 weeks. The concentration of
`plasma corticosterone fell to 3 pg per 100 ml, and
`
`DOC A(IVuM
`10 mglm/21 HE
`
`MEYVRAPONE
`A 5 gm/21 Ni
`INTAKE
`,,
`________ __ -11 ____ II fll
`
`URINARY
`Na
`mEq/24 HR
`
`‘O0
`
`50
`
`SERUM K
`
`URINARY K
`
`50‘
`
`BODY
`
`'/
`mEq/24 HR
`WE$HT
`
`JULY19|
`
`121'
`
`J23L
`
`‘Z51
`
`129‘
`
`131
`AUG 1
`
`i
`
`i
`
`3
`
`‘Z71
`DAYS
`
`FIG. 4. EFFECT or DEOXYCORTICOSTERONE (DOC) ACETATE
`AND METYRAPONE ON URINARY NA AND K.
`
`in the conversion of
`dehydrogenase activity or
`angular methyl group of B to an aldehyde.
`
`

`
`1952
`
`E. G. BIGLIERI, M. A. HERRON, AND N. BRUST
`
`The secretory rates of B and DOC were far
`greater
`than rates observed after
`infusion of
`ACTH (10). Plasma levels of B were continu-
`ally elevated and varied diurnally, and the pool
`size was much greater than normal. Metabolism
`of B was normal,
`indicated by the normal half-
`life. Corticosterone is not generally considered a
`mineralocorticoid,'but the amounts secreted in this
`patient readily explain the presence of hypokalemic
`alkalosis, hypertension, and eosinophilia. Conn,
`'Fajaris, and Louis demonstrated that
`similar
`amounts produce Na retention, K loss, and serum
`K reduction (28). Corticosterone does not affect
`the circulatory eosinophil count or the excretion
`of ketosteroids and cortisol metabolites.
`Short-
`
`term administration of B has no apparent effect
`on blood pressure, but continued and excessive
`secretion of this mineralocorticoid would be ex-
`
`' pected to cause hypertension eventually. The in-
`creased production of DOC also contributed to
`hypertension and hypokalemia. Mineralocorti-
`coids have little, if any, effect on release of ACTH,
`as previously suggested, by failure to alter the
`urinary metabolites of cortisol (28). However,
`because such large amounts of B are secreted
`there appears to be some control exerted on
`ACTH secretion in the absence of cortisol. Pig-
`mentation did not increase in this patient. Plasma
`ACTH was slightly above the normal ran e and
`fluctuated diurnall
`.
`
`
`
`
`
`he levels of ACTH observed,
`
`including the persistent diurnal pattern, are quite
`similar to those in patients with congenital adrenal
`hyperplasia (29).
`The levels of corticosterone secretion might be
`expected to affect carbohydrates and protein
`metabolites.
`However,
`growth was
`normal
`throughout
`life. Negative N balance was not
`detected during a 1-month study. Administration
`of B prevents hypoglycemia after fasting in pa-
`tients with Addison’s disease and decreases carbo-
`
`hydrate tolerance in normal subjects (28). The
`blood glucose levels after fasting, the oral glucose
`tolerance test, and the insulin sensitivity test were
`normal in this patient. Thus, some glucocorticoid
`activity could be ascribed to the large quantities
`
`of corticosterone secreted. The frequency and .
`severity of upper respiratory infections and the
`persistence of oral sores can be attributed to lack
`of cortisol and to minimal glucocorticoid activity
`of corticosterone. This is supported by the symp-
`tomatic improvement after dexamethasone and
`cortisol
`therapy. Levels of free fatty acids and
`glycerol in plasma were elevated in the presence
`of normal carbohydrate tolerance. The reason for
`this abnormality is not clear.
`It is unlikely that
`increased production of ACTH is responsible be-
`cause ACTH levels were not particularly elevated.
`Lack of cortisol, normal amounts of growth hor-
`mone, and greater than normal amounts of B may
`be factors involved in this observation. Because
`B and DOC have little or no effect on release of
`
`ACTH and because circulating cortisol is absent,
`minimal
`inhibition of ACTH secretion could be
`
`a major factor in this disorder. Evidence for
`ACTH as the cause of the increased secretion of
`
`B and DOC, the hypertension, and the hypoka-
`lemia was effectively demonstrated by the correc-
`tion of these abnormalities during slight pituitary
`suppression with dexamethasone and cortisol.
`Treatment with dexamethasone for 12 weeks ef-
`
`fected the same response, but it was‘ apparent that
`no mineralocorticoid activity was present, since
`serum K increased, salt craving occurred, and al-
`dosterone was absent
`in the urine. These re-
`
`sponses did not occur with comparable doses of
`cortisol for the same period of time, although blood
`pressure and plasma corticosterone levels were
`essentially the same. During the 4 weeks in which
`stilbestrol was administered in addition to cortisol
`
`all measurements remained the same except plasma
`corticosterone levels. The increase in B in plasma
`from 0.8 to 3 pg per 100 ml might be attributed
`to the effect of estrogens on steroid—binding
`globulin (30).
`A second biosynthetic defect may also be pres-
`ent. The diminished secretion and excretion of
`
`aldosterone remained essentially unchanged during
`the continued administration of ACTH and the
`
`, 9 days of Na restriction and after the marked
`natriuresis
`that occurred with dexamethasone
`
`therapy. The negligible aldosterone secretion and
`excretion in this patient are similar to those ob-
`served after unilateral adrenalectomy for an aldos-
`terone-producing tumor (31).
`In these patients
`
`

`
`17-HYDROXYLATION DEFICIENCY IN MAN
`
`1953
`
`the reduced aldosterone secretion is a consequence
`of prolonged suppression of renin secretion (32).
`The absence of plasma renin in this patient most
`likely results from excessive secretion of DOC
`and B, but whether or not this explains the lack
`of aldosterone secretion is not known at this time
`
`(33). Before a second biosynthetic defect can be
`implicated, measurement of aldosterone secretion
`during more prolonged suppression of ACTH pro-
`duction is necessary. Precedence for such an ab-
`normality can be found in patients with an aldos-
`terone biosynthetic defect, with Na loss, with
`extensively increased secretion of 18—OH-B, and
`with slight increases in B secretion. Treatment
`with DOC and a high Na diet reduce the secretory
`rates of
`l8—OH—B and corticosterone in these
`
`patients (34).
`Whether or not “escape” from salt-retaining
`hormones occurred in this patient
`is not clear.
`Hypervolemia ‘was not present. Excessive Na
`retention and K depletion were demonstrated by
`the diuresis of Na and retention of K during
`suppression of DOC and B and by the extremely
`low total body K. However, escape may not have
`occurred because administration of DOC acetate
`
`for 3 days produced further retention of Na and
`slight decrease in serum K.
`Metyrapone, an 11/3—OH inhibitor, reduced the
`concentration of corticosterone in plasma by 50%
`and increased DOC production but had little effect
`on urinary electrolytes. The amount of corti-
`costerone present during these two periods may
`have modified the Na response to exogenous and
`endogenous DOC:
`
`‘ 17-Hydroxylation of steroids occurs only in the
`adrenal glands and the gonads. The absence of
`secondary sex characteristics and the negligible
`excretion of estrogens strongly suggest that the
`demonstrated 17-hydroxylase deficiency in the
`adrenal glands also occurs in the ovaries.
`In
`addition, the level of follicle—stimulating hormone
`was pathologically increased in this estrogen-defi-
`cient patient. The presence of a similar enzymatic
`defect
`in both the ovary and the adrenal gland
`suggests that the enzyme may be determined by a
`single gene. We do not know whether a similar
`defect was present in the sibling who died at 11
`months of age or whether a partial defect was
`present in the mother.
`
`Summary
`
`A pa