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Clinical Research Center Studies |
Division of Endocrinology and Metabolism (S.S.E., P.W.L.) and Kennedy Krieger Institute (S.N.), Johns Hopkins Medical Institutions, Baltimore, Maryland 21287; and the Section of Endocrinology and Metabolism, Emory Clinic (L.S.B.), Atlanta, Georgia 30322
Address all correspondence and requests for reprints to: Dr. Paul W. Ladenson, Division of Endocrinology and Metabolism, Department of Medicine, Johns Hopkins Hospital, 600 North Wolfe Street, Blalock 904, Baltimore, Maryland 21287-4904.
Abstract
Adrenoleukodystrophy (ALD) is an X-linked recessive disorder that destroys the white matter of the brain and is associated with adrenal insufficiency. The prevalence of adrenal dysfunction in 71 women carriers of the X-linked ALD gene was studied. These subjects were identified initially on the basis of being obligate carriers of the X-linked trait by pedigree analysis and were confirmed by plasma very long chain fatty acid levels consistent with a heterozygote status. One subject had well documented overt adrenal insufficiency, diagnosed and treated since age 9 yr. Among the remaining women, the mean serum 0800 h and 1 h post-ACTH cortisol concentrations [16 ± 7 (±SD) and 34 ± 8 µg/dL, respectively] were normal. All subjects had normal ACTH-stimulated serum cortisol levels, i.e. more than 20 µg/dL. However, 4 subjects (6%) had subnormal ACTH-stimulated aldosterone concentrations (mean, 9 ± 6 vs. 42 ± 16 ng/dL for other subjects; P = 0.001, by Mann Whitney rank sum test). Three of these women (75%) were taking nonsteroidal antiinflammatory agents (NSAIDs), whereas only 4 of 67 (6%) subjects with normal aldosterone responsiveness were NSAIDs users (P < 0.01, by Fisher’s exact test). Thus, NSAIDs use was associated with increased risk of hypoaldosteronism (odds ratio, 50.2; 95% confidence interval, 3.3–266; P < 0.002). Three of these four women had symptoms consistent with mineralocorticoid deficiency. Serum sodium and potassium concentrations were normal in all subjects. Basal and metyrapone-stimulated plasma ACTH concentrations were also normal in adequately tested subjects with and without mineralocorticoid insufficiency.
Five of eight subjects (63%) who underwent testing with synthetic ovine CRH (oCRH) had abnormalities. Three did not meet the criteria for adequate cortisol stimulation (i.e. >20 µg/dL) and had peak ACTH levels greater than 30 pg/mL. Two other subjects had exaggerated ACTH responses with normal cortisol levels.
There were no significant differences in the mean or median levels of very long chain fatty acid, C26:0, C24/22 ratios, or C26/22 ratios among the entire subject group, the subgroup with blunted aldosterone responses to ACTH, and the subgroup with blunted responses to oCRH (P > 0.05, by ANOVA and Kruskall-Wallis test for C26, C24/22 ratio, and C26/22 ratio).
We conclude that 1) adrenal cortical insufficiency rarely develops in ALD heterozygotes; 2) isolated mineralocorticoid insufficiency can occur in ALD heterozygotes, as has been previously reported to occur with autoimmune and acquired immunodeficiency syndrome-related adrenal dysfunction; 3) ALD heterozygosity may predispose these individuals to NSAID-related hypoaldosteronism; and 4) a subclinical decrease in glucocorticoid reserve, as measured by oCRH testing, may be present in a majority of these women. Aldosterone levels should be included in the ACTH stimulation testing when seeking evidence of adrenal insufficiency in affected women. NSAIDs should be considered a risk factor for the development of hypoaldosteronism in women heterozygous for ALD.
ADRENOLEUKODYSTROPHY (ALD) is an X-linked recessive disorder that destroys the white matter of the brain and is associated with adrenal insufficiency (1, 2). The disease most commonly affects boys during early childhood and presents initially with either adrenal insufficiency or neurological manifestations, although most patients eventually develop both. Neurological involvement relentlessly progresses with white matter demyelination from caudal to rostral parts of the brain, eventually leading to complete disability, a vegetative state, and death. A less severe, related disorder, called adrenomyeloneuropathy (AMN) (3, 4), has its neurological onset in adolescent or adult males, may be clinically confused with multiple sclerosis (5), and is also associated with adrenal dysfunction (2, 6, 7). It has been previously reported (2) that 53% of heterozygotes have neurological signs and symptoms, varying in severity from mild hyperreflexia and vibratory sense impairment with little or no functional disability, to paraparesis requiring a wheelchair. Adrenal insufficiency has also been reported to occur in heterozygotes (2) as have pathological changes in their adrenal cortexes (8), but no systematic study has documented the prevalence of adrenal dysfunction in ALD heterozygotes.
The adrenal dysfunction in both ALD and AMN may involve any aspect of adrenal cortical function. Its severity can range from subclinical laboratory abnormalities to full-blown glucocorticoid- and mineralocorticoid-dependent disease (2, 9). Thus, patients can have only an elevated basal ACTH concentration (6), impaired cortisol and/or aldosterone responsiveness to ACTH stimulation without clinically evident disease, or clinical adrenal insufficiency.
Subclinical adrenal insufficiency has been described previously in other disorders affecting the adrenal cortex, including patients with autoimmune adrenal disease (10, 11), human immunodeficiency virus infection (12), and AMN (6). Consequently, we studied 71 heterozygote women carriers of ALD to define their adrenal status. Our hypothesis was that heterozygote carriers of ALD would demonstrate subtle abnormalities in adrenal function.
Subjects and Methods
Subject selection
Seventy-one women who were heterozygous for ALD were admitted to
the Johns Hopkins Hospital General Clinical Research Center for
hormonal testing (Table 1
). These subjects had
been previously screened and determined to be obligate heterozygotes by
pedigree analysis. Very long chain fatty acid (VLCFA) levels were
assayed as described previously (13) for C24:0, C26:0, and C22:0 by
gas-liquid chromatography. Seventy subjects demonstrated plasma levels
of VLCFA consistent with the heterozygote status; one subject had
normal plasma levels of VLCFA, but increased fibroblast C26:0
levels.
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A clinical evaluation, complete blood count, electrolyte panel, and serum liver and renal function parameters were performed in all subjects on admission. The women had 24-h urine collections for cortisol, aldosterone, and sodium determinations 1 day before stimulation testing. A standard 1-h ACTH stimulation test was performed on day 3. Specifically, 0800 h (basal) cortisol and supine aldosterone levels were determined; synthetic ACTH-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) (250 µg; Cortrosyn, Organon, West Orange, NJ) was administered as an iv bolus, and plasma for cortisol and aldosterone determinations was collected at 30 and 60 min.
A standard metyrapone test (14, 15, 16, 17) was begun on the evening of day 3. Specifically, metyrapone (Ciba-Geigy, Edison, NJ; 30 mg/kg) was administered at 2300 h, and plasma for ACTH, cortisol, and 11-deoxycortisol (11-DOC) determinations was obtained at 0800 h on the day of and the day after metyrapone administration. No subjects needed emergent glucocorticoid therapy during the metyrapone test.
On the morning of day 5, eight subjects underwent stimulation testing with ovine CRH (oCRH; Ferring Laboratories, Suffern, NY) (18, 19). Plasma for baseline ACTH and cortisol measurements was collected at approximately 0800 h. One microgram per kg oCRH was administered iv immediately following the baseline plasma collection. Plasma for ACTH and cortisol determinations was collected before and 15, 30, and 60 min after the injection of oCRH.
Laboratory determinations
Plasma ACTH concentrations were measured using a commercially available immunoradiometric assay kit (Corning-Nichols Institute, San Juan Capistrano, CA) with a reference range of 9–52 pg/mL. The intra- and interassay coefficients of variation were 3.1% and 7.0%, respectively. 11-DOC was measured by RIA on samples after extraction and chromatography (SmithKline Beecham, King of Prussia, PA). Intraassay coefficients of variation for 11-DOC were 8.9% for the lower range of the values (0.16–0.3 µg/dL) and 6.5% for the upper range of values (2–5 µg/dL). Interassay coefficients of variation for 11-DOC were 17.7% for the lower range and 11.3% for the upper range of values. The normal postmetyrapone 11-DOC range was 8–25 µg/dL (SmithKline Beecham). VLCFA (Kennedy Krieger Institute Laboratory, Baltimore, MD) (13) ranges for heterozygotes are: plasma C26:0, 0.82 ± 0.32 µg/mL (±SEM); C24/22, 1.20 ± 0.24; and C26/22, 0.04 ± 0.02.
Plasma cortisol concentrations were determined using a commercially available fluorescence polarization assay (Abbott-TDx, North Chicago, IL). The intra- and interassay coefficients of variation were both 6.6%. Twenty-four-hour urinary free cortisol (UFC) values (normal range, 46–131 µg/24 h, based on 20 healthy control subjects) were determined by fluorescence polarization (Abbott-TDx). The intra- and interassay coefficients of variation for this assay were 13% and 20%, respectively (Johns Hopkins Hospital Pediatric Endocrine laboratory, Baltimore, MD).
Plasma aldosterone and urinary aldosterone values were determined by RIA (Diagnostic Products Corp., Los Angeles, CA). Urine samples were hydrolyzed and extracted before the measurement of aldosterone. The intra- and inter-assay coefficients of variation for the plasma aldosterone measurements were 20.9% and 5.5%, respectively. The intra- and interassay coefficients of variation for the urinary aldosterone measurements were 12.6% and 8.5%, respectively. The reference range for basal plasma aldosterone based on a normal salt diet was 0–41.8 ng/dL for females. The post-ACTH plasma aldosterone reference range was 18–35 ng/dL.
Patients were given a normal diet during their stay at the General Clinical Research Center, and their 24-h urinary sodium levels were determined. As no subject had clinical evidence of sodium retention, the 24-h urinary sodium level was used as a reflection of salt intake. The 24-h urinary aldosterone reference range used was dependent on salt intake. At 10 mEq/day of sodium intake, the range was 17–44 µg/24 h; at 100–200 mEq sodium/day, the range was 2–16 µg/24 h; for greater than 200 mEq sodium/day, the range was 0–6 µg/24 h.
Statistical methods
All values are expressed as the mean ± SD.
When data were normally distributed, a two-sample unpaired t
test was used for comparisons. Because peak aldosterone levels were not
normally distributed, a nonparametric test (Mann-Whitney rank sum) (20)
was used to compare the groups with and without normal peak aldosterone
concentrations. The Fisher’s exact test (20) was used to compare the
proportion of NSAID users in the group with low aldosterone levels to
the group with a normal peak aldosterone value. A one-way ANOVA (20)
was used to compare biochemical data in Table 1C
among three groups:
the whole subject population, the low peak aldosterone group, and the
group with abnormal oCRH test results. The Kruskall-Wallis test (20)
was also applied. Significant multiple comparisons yielded P
values shown in Table 1C. Computer assistance for statistical analysis
was obtained using the PROPHET computer program (21) at the Outpatient
General Clinical Research Center of the Johns Hopkins Hospital.
Results
Clinical and routine biochemical parameters
One 14-yr-old subject (ST) had well documented clinical and biochemical adrenal insufficiency. She had been treated with hydrocortisone and fludrocortisone since age 9 yr, when she presented with intermittent abdominal pain, episodic dehydration, orthostatic hypotension, and hyperpigmentation. An evening cortisol level was then 2.5 µg/dL, potassium was 4.9 mEq/L, and ACTH was 1014 pg/mL. She was given an empiric trial of hydrocortisone and fludrocortisone with remarkable resolution of her symptoms.
Another woman (DO) who carried a diagnosis of adrenal insufficiency was retrospectively discovered to have originally had a normal cortisol response to exogenous ACTH. Although her cortisol response was blunted in this study protocol, she had been taking hydrocortisone up to the time of admission.
Clinical symptoms and signs suggesting adrenal insufficiency were not
unusual in this subject population (Table 1A). No subject had weight
loss. No subject appeared Cushingoid on physical examination. The
subjects’ mean age, weight, body mass index, and maximum and minimum
systolic and diastolic blood pressures are included in Table 1A. Serum
sodium, potassium, chloride, and bicarbonate and serum urea nitrogen
and creatinine concentrations were normal (Table 1B). Fasting glucose
values were all within normal limits, except for one woman who had an
asymptomatic value of 35 mg/dL.
Hormonal parameters
Among the 67 subjects who had 24-h urine collections made for free
cortisol determinations (Table 1C), 6 had values above and 3 had values
below the normal range. Of the 6 who had values above the normal range,
2 were the women taking hydrocortisone, 1 had depression, and 2 were
obese. Three subjects had 24-h UFC values that were minimally below
normal (37, 38, and 43 µg/24 h). Two of these women had no other
hormonal abnormalities, but 1 (SG) also had an impaired aldosterone
response to ACTH and an elevated basal ACTH level, described below.
The range of basal 0800 h cortisol values, excluding the subjects who were previously taking glucocorticoids, was 6.8–39 µg/dL. The mean peak cortisol after ACTH stimulation was 34 µg/dL (range, 11–72 µg/dL); the range excluding the women who were previously taking glucocorticoids was 24–72 µg/dL. No women who had adequate metyrapone tests demonstrated an abnormal 11-DOC response to metyrapone. Among women with adequate metyrapone-induced hypocortisolemia, the range of 11-DOC values was 5.1–29 µg/dL.
Four subjects (6%) demonstrated subnormal aldosterone responses to
ACTH stimulation (Table 1). Three (JK, RK, and KM) were the symptomatic
subjects noted above. Their mean peak ACTH-stimulated aldosterone
concentration was 9.2 ± 5.5 vs. 42 ± 16 µg/dL
for normal subjects (P = 0.001, by Mann-Whitney rank
sum test). Three of four of these women’s peak aldosterone levels were
2 or more SD below the mean peak level (z-scores of -2.2,
-2.4, -2.0, and -1.6). These abnormal women had subnormal levels of
24-h urinary aldosterone despite normal 24-h urinary sodium levels
(range, 52–118 mEq/24 h). A fifth woman (MS), with low basal and
ACTH-stimulated aldosterone concentrations, had a high urinary sodium
level (253 mEq/24 h), suggesting that her blunted aldosterone response
may have been due to high sodium intake. Alternatively, this subject
may have had aldosterone deficiency, resulting in salt craving and
excessive sodium intake. If this were the case, then five subjects
would have subnormal aldosterone levels. None of these women was taking
an angiotensin-converting enzyme inhibitor.
Three of the 4 subjects (75%) with abnormal aldosterone responses to
ACTH were taking a nonsteroidal antiinflammatory agent [NSAID; feldene
(n = 1), toradol (n = 1), or naproxen (n = 1)], whereas
only 4 of 67 (6%) subjects with normal aldosterone responsiveness were
NSAID users (P < 0.01, by Fisher’s exact test). Thus,
NSAID use was associated with increased risk of hypoaldosteronism (odds
ratio, 50.2; 95% confidence interval, 3.3–266; P <
0.002). Of the subjects who were taking NSAIDs, 3 of 7 (43%) had
hypoaldosteronism. This is a higher prevalence than that which occurs
in subjects taking NSAIDs alone and suggests that the ALD heterozygote
state contributes to the hypoaldosteronism. None of the 4 subjects with
low aldosterone levels showed impaired cortisol responses to ACTH
(Table 1). One (SG), mentioned above, had a mildly elevated basal ACTH
level (96 pg/mL) as well as a 24-h UFC level slightly below normal (38
µg/24 h). Basal ACTH levels in the other 3 subjects were normal. All
4 subjects had normal 11-DOC responses to metyrapone testing.
Among the eight subjects who underwent stimulation testing with oCRH,
(Tables 1 and 2), five (62.5%) had abnormal responses.
Three did not meet criteria for adequate cortisol stimulation
(i.e. <20 µg/dL) and had peak ACTH levels greater than 30
pg/mL. Two other subjects had exaggerated ACTH responses with normal
cortisol levels, suggesting a blunted cortisol response to endogenous
ACTH. Of the eight subjects, one (SH) had the lowest 11-DOC response to
metyrapone (5.1 µg/dL) of all study subjects. One of these women (JK,
who was symptomatic; see above) also had a blunted aldosterone response
to ACTH stimulation. Another subject (SH) had hyperemesis gravidarum
and diarrhea postpartum. The other six subjects had no symptoms
potentially attributable to adrenal insufficiency.
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) in the mean or median
levels of plasma VLCFA (C26:0, C24/22 ratios, or C26/22 ratios) between
all subjects and those with either abnormal ACTH-stimulated aldosterone
or oCRH stimulation tests (P > 0.05, by ANOVA analysis
and Kruskall-Wallis test, respectively) in either C26:0 levels or the
levels of the ratios of C24/22 and C26/22. Subject ST, who had adrenal
insufficiency diagnosed at age 9 yr, had a C26/22 value equivalent to
the mean heterozygote value for C26/22, but her C26:0 and C24/22 values
were intermediate between the heterozygote and homozygote values for
C26:0 and C24/22. Discussion
Adrenal cortical dysfunction is relatively common in women who are heterozygous carriers of ALD. Although clinical adrenal insufficiency is rare, subtle abnormalities of adrenal function are often present when sensitive tests of glandular reserve are employed. Among 71 subjects in our study, 1 woman (1.4%) had clinical and biochemical evidence of adrenal insufficiency, which was effectively treated with glucocorticoid and mineralocorticoid therapy. Furthermore, 13% (9 of 71 subjects) had 1 or more abnormalities of adrenal cortical function testing.
Four subjects (6%) had isolated partial mineralocorticoid deficiency with an impaired aldosterone response to ACTH. Isolated mineralocorticoid deficiency has been reported to occur in patients with autoimmune adrenal failure (10) and in patients infected with human immunodeficiency virus (12) as the first abnormality in adrenal function. It is interesting to note that 75% of the subjects in our study who had mineralocorticoid deficiency were taking NSAID, which has been associated with mineralocorticoid deficiency in other disease states (22, 23). We postulate that these women have subtle adrenal cortical dysfunction that renders them vulnerable to NSAID-induced mineralocorticoid deficiency. These findings are consistent with the historical observation that adrenal cortical striation is present in asymptomatic ALD heterozygotes as well as homozygotes (8).
oCRH stimulation testing revealed the highest proportion of abnormal laboratory values in our subjects (62.5%). The adrenal response to oCRH stimulation (11) is a more sensitive test than conventional ACTH stimulation because the ACTH levels achieved are in the physiological range. Only one of seven subjects tested with oCRH (14.3%) had an inadequate 11-DOC response to metyrapone; all had normal 1-h ACTH stimulation tests. This suggests that among the three tests performed, metyrapone, ACTH stimulation, and oCRH, the oCRH test is the most sensitive indicator of subclinical glucocorticoid insufficiency. Of the eight subjects tested, only one (JK) had persistent symptoms potentially attributable to adrenal dysfunction. This finding is consistent with the results of a study by Boscaro et al. (11), in which subjects with organ-specific adrenal antibodies and abnormal oCRH stimulation tests were asymptomatic.
Although VLCFA have been shown to decrease cortisol release in vitro from zona fasciculata cells stimulated with ACTH (24), we found no association between VLCFA levels and aldosterone deficiency using the ACTH stimulation test or cortisol deficiency using the oCRH test. Thus, levels of circulating VLCFA do not explain the predisposition to NSAID-induced mineralocorticoid deficiency in these subjects.
In conclusion, although complete adrenocortical insufficiency, with both clinical and laboratory abnormalities, is uncommon in ALD heterozygotes, subtle laboratory evidence of adrenal dysfunction is commonly demonstrable. Clinical vigilance for signs and symptoms is appropriate in this patient population, especially for those related to mineralocorticoid deficiency. NSAID use should be considered a risk factor for mineralocorticoid deficiency in these patients. When adrenal function testing is clinically indicated in these women, both mineralocorticoid and glucocorticoid deficiencies should be sought during ACTH stimulation testing. Women with subclinical adrenal dysfunction should be closely monitored for the development of and potentially treated for overt adrenal insufficiency to prevent adrenal crisis during times of stress.
Acknowledgments
We thank Ms. Mary Beth Yablonski, Ms. Barbara Ann Bradford, and the General Clinical Research Center nurses and staff for their contributions to this study and the care of these women.
Footnotes
1 This work was supported by the General Clinical Research Center of
the Johns Hopkins Hospital (NIH Grant RR-00035) and NIH Grant RO-1 NSDK
46877–02. 
Received July 10, 1996.
Revised November 5, 1996.
Accepted November 12, 1996.
References
This article has been cited by other articles:
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  R. I. Dorin, C. R. Qualls, and L. M. Crapo Diagnosis of Adrenal Insufficiency Ann Intern Med, August 5, 2003; 139(3): 194 - 204. [Abstract] [Full Text] [PDF]
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G. N. O'Neill, M. Aoki, and R. H. Brown Jr. ABCD1 translation-initiator mutation demonstrates genotype-phenotype correlation for AMN Neurology, December 11, 2001; 57(11): 1956 - 1962. [Abstract] [Full Text] [PDF]
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 P. I. Rosebush, S. Garside, A. J. Levinson, and M. F. Mazurek The Neuropsychiatry of Adult-Onset Adrenoleukodystrophy J Neuropsychiatry Clin Neurosci, August 1, 1999; 11(3): 315 - 327. [Abstract] [Full Text]
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