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Large molecular weight ACTH immunoactivity in a patient with adrenoleukomyoloneuropathy
Large Molecular Weight ACTH lmmunoactivity in a Patient with Adrenoleukomyeloneuropathy
El20 SAITO, M.D. MASAO KINOSHITA, M.D. TSUTOMU KAWAMURA, M.D. KEN KASAHARA, M.D. Tokyo, Japan
Very high ACTH immunoactivity was measured in a patient with adrenoleukomyeloneuropathy. The basal adrenocortical function was hyperactive, whereas the response to exogenous ACTH was depressed. Most of the ACTH immunoactivity was a large molecule (55,000 daltons), which weakly stimulated steroidogenesls of adrenocortical cells in vitro, but inhibiied the binding of 1251-iodoACTH to the cells and suppressed ACTH-induced steroid production. Abnormalities in the pituitary-adrenal axis In adrenoleukomyeloneuropathy have been considered to be solely attributable to destruction of the adrenal cortex. In the current case, however, large molecular weight ACTH immunoactivity is present and might have some role in the adrenocortical dysfunction. Adrenoleukodystrophy and adrenomyeloneuropathy have been proven to be different expressions of the same gene defect by a number of genetic, ultrastructural, and biochemical studies, although clinical features of each are not identical [l-3]. Recently, cases showing overlapping features of adrenoleukodystrophy and adrenomyeloneuropathy have been reported [4,5], and the term of adrenoleukomyeloneuropathy is proposed in order to integrate these conditions [3]. It is well known that adrenoleukomyeloneuropathy causes adrenal hypofunction, as well as neurologic lesions. In the majority of reported cases, however, patients have not shown any clinical evidence of adrenal insufficiency and have had normal levels of serum cortisol and urinary 17-hydroxycorticosteroids. High levels of plasma ACTH or lack of response of adrenocortical hormones to exogenous ACTH administration are regarded as signs of adrenal insufficiency in cases with subclinical or partial adrenal insufficiency. We describe an adrenoleukomyeloneuropathy patient with very high ACTH immunoactivity in whom the elevated plasma ACTH levels did not seem to be the consequence of adrenal hypofunction. Chemical and biologic characteristics of the ACTH immunoactivity in the patient’s plasma were studied. CASE REPORT
From the Fourth Department of Medicine, School of Medicine, Toho University, Tokyo, Japan. Requests for reprints should be addressed to Dr. Eizo Saito, Fourth Department of Medicine, School of Medicine, Toho University, 2-17-6 Ohashi, Meguro-ku, Tokyo 153, Japan. Manuscript submitted December 8, 1986, and accepted May 7, 1987.
The patient is a 39-year-old man with adrenoleukomyeloneuropathy. The diagnosis was confirmed by clinical observations, findings of a computerized tomographic scan and delayed technetium-99m scintiscan, and biochemical analysis. He has shown progressive spastic paraplegia and peripheral neuropathy since the age of 31. One year before admission, he also manifested rapidly progressive mental deterioration. A computerized tomographic scan of the brain showed widespread confluent low-density lesions of the white matter in the frontal and temporal areas, bilaterally. The
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ments showed no abnormalities, including urinary 17-ketosteroids and serum testosterone levels. No neoplasm has been found during a three-year follow-up period. METHODS
+ mean + SD
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minutes after ACTH injection @we 1. Serum cortisol response to exogenous ACiln injection. Synthetic alpha-ACTH-( 7-24 (250 pg) was intravenously injected into the patient three times at 9:00 A.M. on three different days. Blood samples were collected be fore ACTH injection and 30 and 60 minutes after ACTH injection. The shaded area shows the range of mean f 2 SD for data of 16 normal subjects. Bars represent the mean f SD for data of the patient.
ratio of C26:O to C22:O of sphingomyelin in the patient’s plasma was extremely high (0.022); this level is within the pathognomonic range for the diagnosis of adrenoleukomyeloneuropathy [7]. Fasting plasma ACTH levels were always very high (310 to 1,000 pg/ml; mean f SD: 660 f 196 pg/ml). Fasting serum cortisol levels were 15.5 to 20.6 pg/dl (mean f SD: 16.6 f 2.4 pg/dl) and slightly high (normal range: 3.7 to 15.0 pg/dl). Urinary 17-hydroxycorticosteroid levels were 6.1 to 11.2 mg per day (mean f SD: 9.3 f 1.7 mg per day) and also slightly elevated (normal range for adult man: 1.6 to 6.7 mg per day). ACTH immunoactivity in the cerebrospinal fluid was markedly increased (240 pg/ml). Despite slightly high levels on basal adrenocortical function tests, increments of the serum cottisoi level 30 and 60 minutes after injection of 250 pg of synthetic alpha-ACTH-(l-24) (Daiichi Seiyaku Co., Tokyo, Japan) were 4.9 f 0.9 and 10.2 f 0.5 pg/dl, respectively, and lower than those for normal subjects (Figure 1). Treatment with 2 mg of dexamethasone at 9:00 P.M. did not suppress plasma ACTH levels or cortisol levels on the following morning (ACTH: 715 pg/ml, cortisol: 16.1 pg/dl). Other hormone measure-
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Measurement of ACT4 and Other Hormones. ACTH was measured in duplicate by radioimmunoassay using a commercial assay kit purchased from Commissariat ri j Energie Atomique (France), as previously reported [6]. ACTH immunoactivity of plasma samples was directly measured without an extraction procedure. The mean (& SD) plasma ACTH concentration of 66 normal subjects studied with this assay system was 54 f 25 pg/ml, using a plasma sample collected at 9:00 A.M. The sensitivity was 27 pg/ml. The intra-assay coefficient of variation over the entire dose-response curve averaged 6.3 percent; at 70 pg/ml, it averaged 5.0 percent. The interassay coefficient of variation averaged 11.9 percent. Serum cottisol and testosterone levels were measured by radioimmunoassay using commercial assay kits purchased from Clinical Assay Co. (Massachusetts) and Eiken Kagaku Co. (Tokyo, Japan), respectively. Levels of urinary 17-hydroxycorticosteroids were determined by the Poster-Silber reaction and those of 17-ketosteroids were determined by the Zimmerman reaction at Special Reference Laboratories Inc. (Tokyo, Japan). Sephadex 075 SF Column Chromatography. Aliquots (8 ml) of plasma from the patient and five normal subjects were applied on a Sephadex G75 SF column (2.6 X 90 cm) and equilibrated and eiuted with 0.05 M sodium phosphate buffer, pH 7.4, 0.1 percent bovine serum albumin, and 0.5 percent mercaptoethanol at 4’C. Fractions (7 ml) were collected and assayed directly by ACTH radioimmunoassay. For measurement of ACTH immunoactivity in eluates, 50 pg of dexamethasone-suppressed plasma was added to each tube, because this assay kit is prepared for direct assay of plasma ACTH immunoactivity and the bound to free ratio becomes extremely low without plasma. A portion of the eluates was concentrated with Minicon B15 (Amicon Co., Massachusetts) to 20 times and used for the next anti-ACTH immunocolumn study. Antl-ACTH Immunocolumn. Anti-ACTH immunocoiumn was prepared using cyanogen bromide-activated Sepharose 48 (Pharmacia Fine Chemical Co., Uppsaia, Sweden), as previously reported [8]. Anti-ACTH antiserum for this purpose was developed in rabbits against purified porcine ACTH (67 i.U./mg; Sigma Chemical Co., Missouri) conjugated to bovine serum albumin. The antisera showed no cross-reaction with thyroid-stimulating hormone, iuteinizing hormone, follicle-stimulating hormone, and beta meianocyte-stimulating hormone. Samples were applied on antiACTH immunocolumns (0.6 X 5 cm) and control columns (normal rabbit gamma-globulin columns) and thoroughly washed with 0.05M sodium phosphate’ buffer, pH 7.4, 0.1 percent bovine serum albumin, and 0.5 percent mercaptoethanol. Materials absorbed to columns were elutad with glycin-hydrochloride buffer, pH 2.5. The pH of eluates was immediately adjusted to 7.4 with TRIS base, and ACTH
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and ACTH bioactivity were directly measured. The eiuates were also used for the ACTH binding study. Anti-ACTH antibody was not separated from columns through these procedures, i.e., non-specific binding in the radioimmunoassay was the same in eiuates of both antiACTH immunocolumns and control columns. ACTH Bioactlvlty. ACTH bioactivity was measured using dispersed rat adrenocortical cells according to the method of Sayers [9]. Two hundred microliters of sample-as incubated with 2 X lo5 adrenocottical cells in a total of 1 ml of RPMI 1640 medium with 0.5 percent bovine serum aibumin for 120 minutes at 37’C under 5 percent carbon dioxide and 95 percent oxygen. Steroids secreted into the culture medium were measured by spectrofluorometry [lo]. Porcine 1-39 ACTH (100 IU/mg; Sigma Chemical Co., Missouri) was used as standard ACTH. Sensitivity of the assay was 25 pg/ml. The intra-assay coefficient of variation averaged 11.3 percent at 100 pg/ml, and the interassay coefficient of variation averaged 14.2 percent. ACTH Binding Study. The ACTH binding study was performed by the method previously reported [IO]. Porcine l39 ACTH (100 iU/ml) was radioiodinated by the iactoperoxidase method of Thorell and Johanson [ 1 l] and purified by the QUSO adsorption method [ 121. Dispersed rat adrenocortical ceils (1 X 105) and 1251-iodoACTHin 10 pi of 20 percent acetone with 0.25 percent acetic acid were incubated with or without 100 pi of immunocoiumn eluates in a total of 1 ml RPM 1640 medium and 0.5 percent bovine serum albumin at O°C for 120 minutes. Bound and free fractions were separated by centrifugation at 3,000 revolutions per minute for 10 minutes and the radioactivity of each fraction was counted with an Aioca ARC-251 well deitasystem. Statistical analysis was performed by the Student t test.
immunoactivity
RESULTS Figure 2 Sephadex 675 SF Column Chromatography. shows the representative profile of ACTH immunoactivity in the patient’s plasma after eiution through a Sephadex G75 SF column. Most of the ACTH immunoactivity was eluted in a single peak just after that of bovine serum albumin. The estimated molecular weight is about 55,000 daltons. In contrast, no ACTH immunoactivity appeared in this range of eiution volume when the plasma from normal subjects was applied to this column. Eiuates in the range of 150 to 190 ml were concentrated with Minicon 615 to 20 times. ACTH immunoactivity of concentrated eluates of the patient’s plasma was 2,540 f 723 pg/mi, whereas those of normal subjects’ plasma were below the sensitivity of the ACTH radioimmunoassay. Anti-ACTH Immunocolumn. When concentrated eluates of the patient’s plasma were applied to anti-ACTH immunocolumns, no ACTH immunoactivity passed through the columns. By glycin-hydrochloride buffer, pH 2.5, 69.7 f 4.3 percent of ACTH immunoactivity was recovered. After the pH was adjusted to 7.4 with TRIS base, this fraction was used to study ACTH bioactivity and in the binding study as the large molecular weight ACTH
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Void Volume 1Bovine Serum ChymoAlbumin trypsinogen 5 4
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Figure 2. Gel filtration profiles of ACTH immunoactivity in the patient’s plasma (0 -0) and the plasma from five normal subjects (0 -0). ACTH immunoactivity in the patient’s plasma applied to this column was 427 pg/ml, and recovery of ACTH immunoactivity in the first peak was 103 percent. ACTH immunoactivities in the plasma from normal subjects ranged between 27 and 56 pg/ml.
fraction. The corresponding eiuates of normal subjects’ plasma were used as the control fraction. When concentrated eiuates of the patient’s plasma were applied to control columns, 82.5 f 2.9 percent of ACTH immunoactivity passed through the columns. A small portion (7.6 f 5.4 percent) of ACTH immunoactivity was absorbed to these columns and eiuted by glycine-hydrochloride buffer, pH 2.5. ACTH Bioactivity. The large molecular weight ACTH fraction stimulated steroidogenesis of dispersed rat adrenocortical ceils. Estimated ACTH bioactivity was 165 f 25 pg/ml. ACTH immunoactivity of this fraction was 2,375 f 262 pg/mi, and, therefore, the ratio of ACTH bioactivity to ACTH immunoactivity was 6.9 percent. The control fraction did not stimulate steroidogenesis of these cells (Table I). Although the large molecular weight ACTH fraction possessed steroidogenic activity, it strongly inhibited ACTH-induced steroidogenesis (Table I). In the presence of 100 pg/mi of standard ACTH, cells secreted 3.1 f 0.4 ng of steroids per lo5 ceils. When the large molecular weight ACTH fraction was added to the medium plus 100 pg/ml of ACTH, the amount of steroids secreted by the ceils was significantly decreased to 1.5 f 0.4 ng per 1O5 ceils. The control fraction did not show the inhibitory effect on ACTH-induced steroidogenesis (Table I). The ceil viability after incubation was not changed by the presence of the large molecular weight ACTH fraction nor the control fraction in the medium (Table II).
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TABLE I
ACTH Concentration (Pglml) 0 100 333
ET AL
Inhibition of ACTH-Induced Steroid Production by Large Molecular Weight ACTH Fraction Steroid Production (ng/lOs Wiih Large Without Molecular Weight Column Eiuete ACTH Fraction 0 3.1 f 7.3 f
1.2 f 1.5 f 2.4 f
0.4 0.4
,rP
ceils)
0.04-
0
2 2 tL ii
3.1 f 0.5 8.3 f 0.7
Dispersed rat adrenocorticai ceils (2 X 105) were incubated with 0, 100, or 333 pg/mi of ACTH in the presence or absence of the large molecular weight ACTH fraction or the control fraction. Steroids secreted into the incubation medium were measured by spectrofiuorometry. * p
0.030.02O.Ol-
without column
&ate
TABLE II
Effect of Column Eluates on Cell Vlabllity after Incubation Cell Viability (Percent)
Without column eiuate With large molecular ACTH fraction With control fraction
weight
93.6
f
5.2”
92.6
f
5:4
95.8
f
3.5
Dispersed rat adrenocorticai ceils (2 X 105) were incubated in the presence or absence of the large molecular weight ACTH fraction or the control fraction. Ceil viability was studied using trypan blue staining. * The mean f SD for analysis of triplicate cultures.
ACTH Binding Study. In incubations of dispersed rat adrenocortical cells with ‘251-iodoACTH, the cells bound 5.3 f 0.3 percent of the total activity. When the large molecular weight ACTH fraction was added to the incubation medium, binding of 1251-iodoACTHto the cells was strongly suppressed to 1.9 f 0.4 percent (Figure 3). The control fraction did not affect the binding of labeled ACTH to the cells. COMMENTS This patient showed typical clinical manifestations of adrenoleukomyeloneuropathy. Although the plasma ACM immunoactivity was always very high, it did not seem to be the consequence of adrenocortical hypofunction. Fasting serum cortisol levels and urinary 17-hydroxycorticosteroid levels were slightly elevated, and pretreatment with 2 mg of dexamethasone at 9:00 P.M. did not suppress plasma ACTH levels or serum cortisol levels on the following morning. The possibility of artifacts in the ACTH radioimmunoassay is unlikely, because the ACTH immunoactivity in the patient’s plasma speclfically bound to anti-ACTH immunocolumns. This strongly indicates that 780
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Figure 3. Inhibition of 1251-iodoACTHbinding to rat adrs nocortical cells by the large molecular weight ACTH fraction. Dispersed rat adrenocortical c@ls ( 1 X 705) and 125/iodoACTH were incubated in the presence or absence of the large molecular weight ACTH fraction ( 100 ~lj or the control fraction ( 100 EL/).ACTH immunoactivity of the large molecular weight ACTH fraction was 2,150 pg/ml, whereas that of the control fraction was below the sensitivity of the ACTH radioimmunoassay. The open bar shows the binding of 1251-iodoACTHto adrenocortical cells in incubations without the large molecular weight ACTH fraction or the control fraction. The hatched bar shows binding in incubations with the large molecular weight ACTH fraction, and the dotted bar shows binding in incubations with the control fraction. Vefttcal bars represent the mean f SD for analysis of triplicate incubations.
the ACTH immunoactivity actually shares the same antigenie site with native ACTH. Moreover, the slope of the dose-response curve of the ACTH immunoactivity in the patient’s plasma was identical to that of standard ACTH, and the addition of trypsin inhibitors did not modify the level of ACTH immunoactivity (data not shown). Sephadex G75 SF column chromatography revealed that most of the ACTH lmmunoactivity in the patient’s plasma was a larger molecule than native ACTH. Although the elution profile of the ACTH immunoactivity is similar to that reported in Yalow and Berson’s article [ 131, it is not clear whether this material is the same as the “big ACTH” previously reported to be found in the plasma of patients with neoplasms or bilateral adrenal hyperplasia [ 13,141. In the current case, no neoplasm has been found during the last three-year follow-up period. On the basis of molecular weight, this ACTH immunoactivity seems to be different from the normal pituitary ACTH precursor (ACTH/lipotropin common precursor), as previously described by Eipper and Mains [ 151. However, the possibility that the ACTH immunoactivity in the patient’s plasma is the precursor form of native ACTH could not be ruled out. 83
ACTH
This material might be a pm-precursor of native ACTH or the polymerized form of ACTH precursors. We tried trypsin treatment of the ACTH immunoactivity, but we could not get reproducible results, probably due to the damaging effect of the trypsin treatment on ACTH immunoactivity (data not shown). Therefore, at the current time, we cannot answer this question. The reason why no peak of ACTH immunoactivity appeared in the range of native ACTH by Sephadex G75 SF column chromatography is that the concentration of ACTH immunoactivity of this portion of eluate was too low to be detected by the radioimmunoassay used in this study. Although the large molecular weight ACTH immunoactivity weakly stimulated steroidogenesis of dispersed rat adrenocortical cells in vitro, it strongly suppressed ACTHinduced steroidogenesis of these cells. This material inhibited the binding of ‘251-iodoACTH to adrenocottical cells, and, therefore, it might be possible to assume that the large molecular weight ACTH immunoactivity binds to ACTH receptors of adrenocortical cells and shows weak steroidogenic activity. This binding might inhibit the binding of native ACTH to its receptors and depress ACTHinduced steroidogenesis. It is interesting to note that the patient showed a depressed serum cortisol response to exogenous ACTH administration despite hyperactive basal adrenocortical function. According to the in vitro study, large molecular weight ACTH immunoactivity possesses 6.9 percent of the bioactivity of standard ACTH. On the assumption that in vivo bioactivity is the same as in vitro bioactivity, bioactivity of the large molecular weight ACTH immunoactivity in the patient’s plasma is estimated to be 20 to 70 pg/ml. Because large molecular weight ACTH immunoactivity is consistently present in the plasma, this amount may be enough to cause the patient’s slightly elevated basal adrenocortical function. Consequently, the large molecular weight ACTH immunoactivity might have some role in the adrenocottical dysfunction in this patient with adrenoleukomyeloneuropathy. Although there have been a relatively small number of adrenoleukomyeloneuropathy cases reported in which endocrine function was fully documented, patients who showed normal basal adrenocortical function despite very high plasma ACTH levels have occasionally been seen [2,16]. It should be noted that plasma ACTH levels reported in the literature were sometimes as high as 2,000 pg/
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ml in patients whose serum cortisol levels were within normal limits [3,17]. The level of 2,000 pg/ml of ACTH is rarely reached even in the case of well-documented Addison’s disease [ 18,191. Moreover, patients who showed poor or no response to exogenous ACTH stimulation despite high basal serum cortisol levels, like the current case, have also been reported [2,17]. The pathologic changes of the adrenal gland in adrenoleukomyeloneuropathy have been well documented, and abnormalities in the pituitary-adrenal axis in adrenoleukomyeloneuropathy have been considered to be solely attributed to the destruction of the adrenal gland. Our results and those of others suggest that large molecular weight ACTH immunoactivity may be widely present and affect adrenocortical functions in adrenoleukomyeloneuropathy. The origin of large molecular weight ACTH immunoactivity is unknown. ACTH-like materials are normally present in brain [20,21], probably in the form of ACTH/ lipotropin precursor [22]. Accordingly, the large molecular weight ACTH immunoactivity may have leaked from his brain because of damage to the blood brain barrier. However, the level of plasma ACTH immunoactivity seems to be too high as compared with ACTH-like materials normally present in brain to support this possibility. It has been reported that the level of ACTH immunoactivity in the cerebrospinal fluid does not reflect the plasma ACTH immunoactivity or the activity of ACTH production at the pituitary level [23], and that it might reflect the content of brain ACTH-like activity. Because ACTH content in the cerebrospinal fluid was very high in this patient, the production of ACTH immunoactivity might be augmented in his central nervous system. Recently, we reported that ACTH immunoactivity is much more widespread in extrapituitary tissues than ever thought [24]. Hence, it is also possible that ACTH immunoactivity in this case is produced by tissues other than pituitary or brain. ACKNOWLEDGMENT We are indebted to Shoji Tsuji, M.D., Department of Biochemistry and Metabolism, the Tokyo Metropolitan Institute of Medical Science, for analysis of fatty acid composition of sphingomyelin in the patient’s plasma, and Mrs. Sumiko Kawamata for her invaluable technical assistance.
REFERENCES 1.
2.
Schaumburg HH, Powers JM, Raine C, et al: Adrenomyeloneuropathy: a probable variant of adrenoleukodystrophy. II. General pathologic, neuropathologic, and biochemical aspects. Neurology 1977; 27: 1114-l 119. Davis LE, Snyder RD, Orth DN, Nicholson WE, Kornfeld M, Seelinger DF: Adrenoleukodystrophy and adrenomyeloneuropathy associated with partial adrenal insufficiency in three generations of a kindred. Am J Med 1979; 66: 342347.
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3.
4. 5.
6.
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O’Neill BP, Marmion CC, Feringa ER: The adrenoleukomyeloneuropathy complex: expression in four generations. Neurology 1981; 31: 151-156. Case Records of the Massachusetts General Hospital (Case 18-1979). N Engl J Med 1979; 300: 1037-1045. Weiss GM, Nelson RL, O’Neill BP, Carney JA, Edis AJ: Use of adrenal biopsy in diagnosing adrenoleukomyeloneuropathy. Arch Neurol 1980; 37: 634-636. Saito E, lchikawa Y, Homma M: Direct inhibitory effect of
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7.
8.
9.
10.
11.
12. 13.
14.
15.
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dexamethasone on steroidogenesis of human adrenal in vivo. J Clin Endocrinol Metab 1979; 48: 861-863. Tsuji S, Suzuki M, Sano T, Ariga T, Miyatake T: Abnormality of very long-chain fatty acids in erythrocyte membrane and plasma, and increased synthesis of these fatty acids by cultured skin fibroblasts in adrenoleukodystrophy and adrenomyeloneuropathy. International Symposium on the Leukodystrophy and allied diseases. Sasaki Shuppan (Kyoto), 1981: 247-253. Saito E, Odell WD: CorticotropirVlipotropin common precursor-like material in normal rat extrapituitary tissues. Proc Natl Acad Sci USA 1983; 80: 3792-3796. Sayers G: Bioassays of ACTH using isolated cortex cells. Applications: structure activity relationship for ACTH and analogues, assay of corticotropin-releasing factor, and assay of plasma ACTH. Ann NY Acad Sci 1977; 297: 220-24 1. Saito E, Mukai M, Muraki T, lchikawa Y, Homma M: Inhibitory effects of corticosterone on cell proliferation and steroidogenesis in the mouse adrenal tumor cell line Y-l. Endocrinology 1979; 104: 487-492. Thorell JI, Johanson BG: Enzymatic iodination of polypeptides with 1251 to high specific activity. Biochim Biophys Acta 1971; 251: 363-369. Berson SA, Yalow RS: Radioimmunoassay of ACTH in plasma. J Clin Invest 1968; 47: 2725-275 1. Yalow RS, Berson SA: Characteristics of “big ACTH” in human plasma and pituitary extracts. J Clin Endocrinol Metab 1973; 36: 415-423. Yamada Y, Ito S, Miyashita M: Clinical studies of “big ACTH”: its physico-chemical characteristics. Acta Endocrinol (Copenh) 1978; 89: 221-231. Eipper BA, Mains RE: Structure and biosynthesis of proa-
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16.
17.
18.
19.
20.
21.
22.
23.
24.
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drenocorticotropin/endorphin and related peptides. Endoer Rev 1980; 1: l-27. Martin JJ, Lowenthal A, Ceuterick C, Gacoms H: Adrenomyeloneuropathy. A report of two families. J Neurol 1982; 226: 221-232. Yoshida T, Ono 2, Takayama Y, Suzuki T, Yanagisawa H: Adrenoleukomyeloneuropathy presenting as hyperpigmentation, neuropathy, visual disturbance and hearing loss. Clin Neurol (Tokyo) 1982; 22: 343-350. Besser GM, Gullen DR, Irvine WJ, Ratcliffe JG, Landon J: lmmunoactive corticotrophin levels in adrenocortical insufficiency. Br Med J [Clin Res] 1971; 1: 374-376. West CD, Dolman LI: Plasma ACTH radioimmunoassays in the diagnosis of pituitary-adrenal dysfunction. Ann NY Acad Sci 1977; 297: 205-219. Krieger DT, Liotta A, Brownstein WJ: Presence of corticotropin in limbic system of normal and hypophysectomired rats. Brain Res 1977; 128: 575-579. Moldow RL, Yalow RS: Extrahypophysial distribution of corticotropin as a function of brain size. Proc Natl Acad Sci USA 1978; 75: 994-998. Krieger DT, Liotta AS, Brownstein MJ, Zimmerman EA: ACTH, P-lipotropin, and related peptides in brain, pituitary, and blood. Recent Prog Horm Res 1980; 36: 277344. Nakao K, Oki S, Tanaka I, Horii K, Nakai Y, Furii T, et al: lmmunoreactive /!I-endorphin, and adrenocorticotropin in human cerebrospinal fluid. J Clin Invest 1980; 66: 13831390. Saito E, lwasa S, Odell WD: Widespread presence of large molecular weight adrenocorticotropin-like substances in rat extrapituitary tissues. Endocrinology 1983; 113: 1010-1019.
El20 SAITO, M.D. MASAO KINOSHITA, M.D. TSUTOMU KAWAMURA, M.D. KEN KASAHARA, M.D. Tokyo, Japan
Very high ACTH immunoactivity was measured in a patient with adrenoleukomyeloneuropathy. The basal adrenocortical function was hyperactive, whereas the response to exogenous ACTH was depressed. Most of the ACTH immunoactivity was a large molecule (55,000 daltons), which weakly stimulated steroidogenesls of adrenocortical cells in vitro, but inhibiied the binding of 1251-iodoACTH to the cells and suppressed ACTH-induced steroid production. Abnormalities in the pituitary-adrenal axis In adrenoleukomyeloneuropathy have been considered to be solely attributable to destruction of the adrenal cortex. In the current case, however, large molecular weight ACTH immunoactivity is present and might have some role in the adrenocortical dysfunction. Adrenoleukodystrophy and adrenomyeloneuropathy have been proven to be different expressions of the same gene defect by a number of genetic, ultrastructural, and biochemical studies, although clinical features of each are not identical [l-3]. Recently, cases showing overlapping features of adrenoleukodystrophy and adrenomyeloneuropathy have been reported [4,5], and the term of adrenoleukomyeloneuropathy is proposed in order to integrate these conditions [3]. It is well known that adrenoleukomyeloneuropathy causes adrenal hypofunction, as well as neurologic lesions. In the majority of reported cases, however, patients have not shown any clinical evidence of adrenal insufficiency and have had normal levels of serum cortisol and urinary 17-hydroxycorticosteroids. High levels of plasma ACTH or lack of response of adrenocortical hormones to exogenous ACTH administration are regarded as signs of adrenal insufficiency in cases with subclinical or partial adrenal insufficiency. We describe an adrenoleukomyeloneuropathy patient with very high ACTH immunoactivity in whom the elevated plasma ACTH levels did not seem to be the consequence of adrenal hypofunction. Chemical and biologic characteristics of the ACTH immunoactivity in the patient’s plasma were studied. CASE REPORT
From the Fourth Department of Medicine, School of Medicine, Toho University, Tokyo, Japan. Requests for reprints should be addressed to Dr. Eizo Saito, Fourth Department of Medicine, School of Medicine, Toho University, 2-17-6 Ohashi, Meguro-ku, Tokyo 153, Japan. Manuscript submitted December 8, 1986, and accepted May 7, 1987.
The patient is a 39-year-old man with adrenoleukomyeloneuropathy. The diagnosis was confirmed by clinical observations, findings of a computerized tomographic scan and delayed technetium-99m scintiscan, and biochemical analysis. He has shown progressive spastic paraplegia and peripheral neuropathy since the age of 31. One year before admission, he also manifested rapidly progressive mental deterioration. A computerized tomographic scan of the brain showed widespread confluent low-density lesions of the white matter in the frontal and temporal areas, bilaterally. The
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ments showed no abnormalities, including urinary 17-ketosteroids and serum testosterone levels. No neoplasm has been found during a three-year follow-up period. METHODS
+ mean + SD
0-~
do
6’0
minutes after ACTH injection @we 1. Serum cortisol response to exogenous ACiln injection. Synthetic alpha-ACTH-( 7-24 (250 pg) was intravenously injected into the patient three times at 9:00 A.M. on three different days. Blood samples were collected be fore ACTH injection and 30 and 60 minutes after ACTH injection. The shaded area shows the range of mean f 2 SD for data of 16 normal subjects. Bars represent the mean f SD for data of the patient.
ratio of C26:O to C22:O of sphingomyelin in the patient’s plasma was extremely high (0.022); this level is within the pathognomonic range for the diagnosis of adrenoleukomyeloneuropathy [7]. Fasting plasma ACTH levels were always very high (310 to 1,000 pg/ml; mean f SD: 660 f 196 pg/ml). Fasting serum cortisol levels were 15.5 to 20.6 pg/dl (mean f SD: 16.6 f 2.4 pg/dl) and slightly high (normal range: 3.7 to 15.0 pg/dl). Urinary 17-hydroxycorticosteroid levels were 6.1 to 11.2 mg per day (mean f SD: 9.3 f 1.7 mg per day) and also slightly elevated (normal range for adult man: 1.6 to 6.7 mg per day). ACTH immunoactivity in the cerebrospinal fluid was markedly increased (240 pg/ml). Despite slightly high levels on basal adrenocortical function tests, increments of the serum cottisoi level 30 and 60 minutes after injection of 250 pg of synthetic alpha-ACTH-(l-24) (Daiichi Seiyaku Co., Tokyo, Japan) were 4.9 f 0.9 and 10.2 f 0.5 pg/dl, respectively, and lower than those for normal subjects (Figure 1). Treatment with 2 mg of dexamethasone at 9:00 P.M. did not suppress plasma ACTH levels or cortisol levels on the following morning (ACTH: 715 pg/ml, cortisol: 16.1 pg/dl). Other hormone measure-
778
Measurement of ACT4 and Other Hormones. ACTH was measured in duplicate by radioimmunoassay using a commercial assay kit purchased from Commissariat ri j Energie Atomique (France), as previously reported [6]. ACTH immunoactivity of plasma samples was directly measured without an extraction procedure. The mean (& SD) plasma ACTH concentration of 66 normal subjects studied with this assay system was 54 f 25 pg/ml, using a plasma sample collected at 9:00 A.M. The sensitivity was 27 pg/ml. The intra-assay coefficient of variation over the entire dose-response curve averaged 6.3 percent; at 70 pg/ml, it averaged 5.0 percent. The interassay coefficient of variation averaged 11.9 percent. Serum cottisol and testosterone levels were measured by radioimmunoassay using commercial assay kits purchased from Clinical Assay Co. (Massachusetts) and Eiken Kagaku Co. (Tokyo, Japan), respectively. Levels of urinary 17-hydroxycorticosteroids were determined by the Poster-Silber reaction and those of 17-ketosteroids were determined by the Zimmerman reaction at Special Reference Laboratories Inc. (Tokyo, Japan). Sephadex 075 SF Column Chromatography. Aliquots (8 ml) of plasma from the patient and five normal subjects were applied on a Sephadex G75 SF column (2.6 X 90 cm) and equilibrated and eiuted with 0.05 M sodium phosphate buffer, pH 7.4, 0.1 percent bovine serum albumin, and 0.5 percent mercaptoethanol at 4’C. Fractions (7 ml) were collected and assayed directly by ACTH radioimmunoassay. For measurement of ACTH immunoactivity in eluates, 50 pg of dexamethasone-suppressed plasma was added to each tube, because this assay kit is prepared for direct assay of plasma ACTH immunoactivity and the bound to free ratio becomes extremely low without plasma. A portion of the eluates was concentrated with Minicon B15 (Amicon Co., Massachusetts) to 20 times and used for the next anti-ACTH immunocolumn study. Antl-ACTH Immunocolumn. Anti-ACTH immunocoiumn was prepared using cyanogen bromide-activated Sepharose 48 (Pharmacia Fine Chemical Co., Uppsaia, Sweden), as previously reported [8]. Anti-ACTH antiserum for this purpose was developed in rabbits against purified porcine ACTH (67 i.U./mg; Sigma Chemical Co., Missouri) conjugated to bovine serum albumin. The antisera showed no cross-reaction with thyroid-stimulating hormone, iuteinizing hormone, follicle-stimulating hormone, and beta meianocyte-stimulating hormone. Samples were applied on antiACTH immunocolumns (0.6 X 5 cm) and control columns (normal rabbit gamma-globulin columns) and thoroughly washed with 0.05M sodium phosphate’ buffer, pH 7.4, 0.1 percent bovine serum albumin, and 0.5 percent mercaptoethanol. Materials absorbed to columns were elutad with glycin-hydrochloride buffer, pH 2.5. The pH of eluates was immediately adjusted to 7.4 with TRIS base, and ACTH
October 1987 The American Journal of Medicine Volume 83
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and ACTH bioactivity were directly measured. The eiuates were also used for the ACTH binding study. Anti-ACTH antibody was not separated from columns through these procedures, i.e., non-specific binding in the radioimmunoassay was the same in eiuates of both antiACTH immunocolumns and control columns. ACTH Bioactlvlty. ACTH bioactivity was measured using dispersed rat adrenocortical cells according to the method of Sayers [9]. Two hundred microliters of sample-as incubated with 2 X lo5 adrenocottical cells in a total of 1 ml of RPMI 1640 medium with 0.5 percent bovine serum aibumin for 120 minutes at 37’C under 5 percent carbon dioxide and 95 percent oxygen. Steroids secreted into the culture medium were measured by spectrofluorometry [lo]. Porcine 1-39 ACTH (100 IU/mg; Sigma Chemical Co., Missouri) was used as standard ACTH. Sensitivity of the assay was 25 pg/ml. The intra-assay coefficient of variation averaged 11.3 percent at 100 pg/ml, and the interassay coefficient of variation averaged 14.2 percent. ACTH Binding Study. The ACTH binding study was performed by the method previously reported [IO]. Porcine l39 ACTH (100 iU/ml) was radioiodinated by the iactoperoxidase method of Thorell and Johanson [ 1 l] and purified by the QUSO adsorption method [ 121. Dispersed rat adrenocortical ceils (1 X 105) and 1251-iodoACTHin 10 pi of 20 percent acetone with 0.25 percent acetic acid were incubated with or without 100 pi of immunocoiumn eluates in a total of 1 ml RPM 1640 medium and 0.5 percent bovine serum albumin at O°C for 120 minutes. Bound and free fractions were separated by centrifugation at 3,000 revolutions per minute for 10 minutes and the radioactivity of each fraction was counted with an Aioca ARC-251 well deitasystem. Statistical analysis was performed by the Student t test.
immunoactivity
RESULTS Figure 2 Sephadex 675 SF Column Chromatography. shows the representative profile of ACTH immunoactivity in the patient’s plasma after eiution through a Sephadex G75 SF column. Most of the ACTH immunoactivity was eluted in a single peak just after that of bovine serum albumin. The estimated molecular weight is about 55,000 daltons. In contrast, no ACTH immunoactivity appeared in this range of eiution volume when the plasma from normal subjects was applied to this column. Eiuates in the range of 150 to 190 ml were concentrated with Minicon 615 to 20 times. ACTH immunoactivity of concentrated eluates of the patient’s plasma was 2,540 f 723 pg/mi, whereas those of normal subjects’ plasma were below the sensitivity of the ACTH radioimmunoassay. Anti-ACTH Immunocolumn. When concentrated eluates of the patient’s plasma were applied to anti-ACTH immunocolumns, no ACTH immunoactivity passed through the columns. By glycin-hydrochloride buffer, pH 2.5, 69.7 f 4.3 percent of ACTH immunoactivity was recovered. After the pH was adjusted to 7.4 with TRIS base, this fraction was used to study ACTH bioactivity and in the binding study as the large molecular weight ACTH
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IN ADRENOLEUKOMYELONEUROPATHY-SAITO
Void Volume 1Bovine Serum ChymoAlbumin trypsinogen 5 4
150
200
RiboA nuclease 4
250
ELuTION
300 VOLUME
ET AL
4500MW ACTH .I
A
350
400
450
(me)
Figure 2. Gel filtration profiles of ACTH immunoactivity in the patient’s plasma (0 -0) and the plasma from five normal subjects (0 -0). ACTH immunoactivity in the patient’s plasma applied to this column was 427 pg/ml, and recovery of ACTH immunoactivity in the first peak was 103 percent. ACTH immunoactivities in the plasma from normal subjects ranged between 27 and 56 pg/ml.
fraction. The corresponding eiuates of normal subjects’ plasma were used as the control fraction. When concentrated eiuates of the patient’s plasma were applied to control columns, 82.5 f 2.9 percent of ACTH immunoactivity passed through the columns. A small portion (7.6 f 5.4 percent) of ACTH immunoactivity was absorbed to these columns and eiuted by glycine-hydrochloride buffer, pH 2.5. ACTH Bioactivity. The large molecular weight ACTH fraction stimulated steroidogenesis of dispersed rat adrenocortical ceils. Estimated ACTH bioactivity was 165 f 25 pg/ml. ACTH immunoactivity of this fraction was 2,375 f 262 pg/mi, and, therefore, the ratio of ACTH bioactivity to ACTH immunoactivity was 6.9 percent. The control fraction did not stimulate steroidogenesis of these cells (Table I). Although the large molecular weight ACTH fraction possessed steroidogenic activity, it strongly inhibited ACTH-induced steroidogenesis (Table I). In the presence of 100 pg/mi of standard ACTH, cells secreted 3.1 f 0.4 ng of steroids per lo5 ceils. When the large molecular weight ACTH fraction was added to the medium plus 100 pg/ml of ACTH, the amount of steroids secreted by the ceils was significantly decreased to 1.5 f 0.4 ng per 1O5 ceils. The control fraction did not show the inhibitory effect on ACTH-induced steroidogenesis (Table I). The ceil viability after incubation was not changed by the presence of the large molecular weight ACTH fraction nor the control fraction in the medium (Table II).
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ACTH
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TABLE I
ACTH Concentration (Pglml) 0 100 333
ET AL
Inhibition of ACTH-Induced Steroid Production by Large Molecular Weight ACTH Fraction Steroid Production (ng/lOs Wiih Large Without Molecular Weight Column Eiuete ACTH Fraction 0 3.1 f 7.3 f
1.2 f 1.5 f 2.4 f
0.4 0.4
,rP
ceils)
0.04-
0
2 2 tL ii
3.1 f 0.5 8.3 f 0.7
Dispersed rat adrenocorticai ceils (2 X 105) were incubated with 0, 100, or 333 pg/mi of ACTH in the presence or absence of the large molecular weight ACTH fraction or the control fraction. Steroids secreted into the incubation medium were measured by spectrofiuorometry. * p
0.030.02O.Ol-
without column
&ate
TABLE II
Effect of Column Eluates on Cell Vlabllity after Incubation Cell Viability (Percent)
Without column eiuate With large molecular ACTH fraction With control fraction
weight
93.6
f
5.2”
92.6
f
5:4
95.8
f
3.5
Dispersed rat adrenocorticai ceils (2 X 105) were incubated in the presence or absence of the large molecular weight ACTH fraction or the control fraction. Ceil viability was studied using trypan blue staining. * The mean f SD for analysis of triplicate cultures.
ACTH Binding Study. In incubations of dispersed rat adrenocortical cells with ‘251-iodoACTH, the cells bound 5.3 f 0.3 percent of the total activity. When the large molecular weight ACTH fraction was added to the incubation medium, binding of 1251-iodoACTHto the cells was strongly suppressed to 1.9 f 0.4 percent (Figure 3). The control fraction did not affect the binding of labeled ACTH to the cells. COMMENTS This patient showed typical clinical manifestations of adrenoleukomyeloneuropathy. Although the plasma ACM immunoactivity was always very high, it did not seem to be the consequence of adrenocortical hypofunction. Fasting serum cortisol levels and urinary 17-hydroxycorticosteroid levels were slightly elevated, and pretreatment with 2 mg of dexamethasone at 9:00 P.M. did not suppress plasma ACTH levels or serum cortisol levels on the following morning. The possibility of artifacts in the ACTH radioimmunoassay is unlikely, because the ACTH immunoactivity in the patient’s plasma speclfically bound to anti-ACTH immunocolumns. This strongly indicates that 780
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0.1’ 0.4” 0.3”
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with large MW AC-C-I fra&n
with control fraction
Figure 3. Inhibition of 1251-iodoACTHbinding to rat adrs nocortical cells by the large molecular weight ACTH fraction. Dispersed rat adrenocortical c@ls ( 1 X 705) and 125/iodoACTH were incubated in the presence or absence of the large molecular weight ACTH fraction ( 100 ~lj or the control fraction ( 100 EL/).ACTH immunoactivity of the large molecular weight ACTH fraction was 2,150 pg/ml, whereas that of the control fraction was below the sensitivity of the ACTH radioimmunoassay. The open bar shows the binding of 1251-iodoACTHto adrenocortical cells in incubations without the large molecular weight ACTH fraction or the control fraction. The hatched bar shows binding in incubations with the large molecular weight ACTH fraction, and the dotted bar shows binding in incubations with the control fraction. Vefttcal bars represent the mean f SD for analysis of triplicate incubations.
the ACTH immunoactivity actually shares the same antigenie site with native ACTH. Moreover, the slope of the dose-response curve of the ACTH immunoactivity in the patient’s plasma was identical to that of standard ACTH, and the addition of trypsin inhibitors did not modify the level of ACTH immunoactivity (data not shown). Sephadex G75 SF column chromatography revealed that most of the ACTH lmmunoactivity in the patient’s plasma was a larger molecule than native ACTH. Although the elution profile of the ACTH immunoactivity is similar to that reported in Yalow and Berson’s article [ 131, it is not clear whether this material is the same as the “big ACTH” previously reported to be found in the plasma of patients with neoplasms or bilateral adrenal hyperplasia [ 13,141. In the current case, no neoplasm has been found during the last three-year follow-up period. On the basis of molecular weight, this ACTH immunoactivity seems to be different from the normal pituitary ACTH precursor (ACTH/lipotropin common precursor), as previously described by Eipper and Mains [ 151. However, the possibility that the ACTH immunoactivity in the patient’s plasma is the precursor form of native ACTH could not be ruled out. 83
ACTH
This material might be a pm-precursor of native ACTH or the polymerized form of ACTH precursors. We tried trypsin treatment of the ACTH immunoactivity, but we could not get reproducible results, probably due to the damaging effect of the trypsin treatment on ACTH immunoactivity (data not shown). Therefore, at the current time, we cannot answer this question. The reason why no peak of ACTH immunoactivity appeared in the range of native ACTH by Sephadex G75 SF column chromatography is that the concentration of ACTH immunoactivity of this portion of eluate was too low to be detected by the radioimmunoassay used in this study. Although the large molecular weight ACTH immunoactivity weakly stimulated steroidogenesis of dispersed rat adrenocortical cells in vitro, it strongly suppressed ACTHinduced steroidogenesis of these cells. This material inhibited the binding of ‘251-iodoACTH to adrenocottical cells, and, therefore, it might be possible to assume that the large molecular weight ACTH immunoactivity binds to ACTH receptors of adrenocortical cells and shows weak steroidogenic activity. This binding might inhibit the binding of native ACTH to its receptors and depress ACTHinduced steroidogenesis. It is interesting to note that the patient showed a depressed serum cortisol response to exogenous ACTH administration despite hyperactive basal adrenocortical function. According to the in vitro study, large molecular weight ACTH immunoactivity possesses 6.9 percent of the bioactivity of standard ACTH. On the assumption that in vivo bioactivity is the same as in vitro bioactivity, bioactivity of the large molecular weight ACTH immunoactivity in the patient’s plasma is estimated to be 20 to 70 pg/ml. Because large molecular weight ACTH immunoactivity is consistently present in the plasma, this amount may be enough to cause the patient’s slightly elevated basal adrenocortical function. Consequently, the large molecular weight ACTH immunoactivity might have some role in the adrenocottical dysfunction in this patient with adrenoleukomyeloneuropathy. Although there have been a relatively small number of adrenoleukomyeloneuropathy cases reported in which endocrine function was fully documented, patients who showed normal basal adrenocortical function despite very high plasma ACTH levels have occasionally been seen [2,16]. It should be noted that plasma ACTH levels reported in the literature were sometimes as high as 2,000 pg/
IN ADRENOLEUKOMYELONEUROPATHY-SAITO
ET AL
ml in patients whose serum cortisol levels were within normal limits [3,17]. The level of 2,000 pg/ml of ACTH is rarely reached even in the case of well-documented Addison’s disease [ 18,191. Moreover, patients who showed poor or no response to exogenous ACTH stimulation despite high basal serum cortisol levels, like the current case, have also been reported [2,17]. The pathologic changes of the adrenal gland in adrenoleukomyeloneuropathy have been well documented, and abnormalities in the pituitary-adrenal axis in adrenoleukomyeloneuropathy have been considered to be solely attributed to the destruction of the adrenal gland. Our results and those of others suggest that large molecular weight ACTH immunoactivity may be widely present and affect adrenocortical functions in adrenoleukomyeloneuropathy. The origin of large molecular weight ACTH immunoactivity is unknown. ACTH-like materials are normally present in brain [20,21], probably in the form of ACTH/ lipotropin precursor [22]. Accordingly, the large molecular weight ACTH immunoactivity may have leaked from his brain because of damage to the blood brain barrier. However, the level of plasma ACTH immunoactivity seems to be too high as compared with ACTH-like materials normally present in brain to support this possibility. It has been reported that the level of ACTH immunoactivity in the cerebrospinal fluid does not reflect the plasma ACTH immunoactivity or the activity of ACTH production at the pituitary level [23], and that it might reflect the content of brain ACTH-like activity. Because ACTH content in the cerebrospinal fluid was very high in this patient, the production of ACTH immunoactivity might be augmented in his central nervous system. Recently, we reported that ACTH immunoactivity is much more widespread in extrapituitary tissues than ever thought [24]. Hence, it is also possible that ACTH immunoactivity in this case is produced by tissues other than pituitary or brain. ACKNOWLEDGMENT We are indebted to Shoji Tsuji, M.D., Department of Biochemistry and Metabolism, the Tokyo Metropolitan Institute of Medical Science, for analysis of fatty acid composition of sphingomyelin in the patient’s plasma, and Mrs. Sumiko Kawamata for her invaluable technical assistance.
REFERENCES 1.
2.
Schaumburg HH, Powers JM, Raine C, et al: Adrenomyeloneuropathy: a probable variant of adrenoleukodystrophy. II. General pathologic, neuropathologic, and biochemical aspects. Neurology 1977; 27: 1114-l 119. Davis LE, Snyder RD, Orth DN, Nicholson WE, Kornfeld M, Seelinger DF: Adrenoleukodystrophy and adrenomyeloneuropathy associated with partial adrenal insufficiency in three generations of a kindred. Am J Med 1979; 66: 342347.
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3.
4. 5.
6.
1987
O’Neill BP, Marmion CC, Feringa ER: The adrenoleukomyeloneuropathy complex: expression in four generations. Neurology 1981; 31: 151-156. Case Records of the Massachusetts General Hospital (Case 18-1979). N Engl J Med 1979; 300: 1037-1045. Weiss GM, Nelson RL, O’Neill BP, Carney JA, Edis AJ: Use of adrenal biopsy in diagnosing adrenoleukomyeloneuropathy. Arch Neurol 1980; 37: 634-636. Saito E, lchikawa Y, Homma M: Direct inhibitory effect of
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7.
8.
9.
10.
11.
12. 13.
14.
15.
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ET AL
dexamethasone on steroidogenesis of human adrenal in vivo. J Clin Endocrinol Metab 1979; 48: 861-863. Tsuji S, Suzuki M, Sano T, Ariga T, Miyatake T: Abnormality of very long-chain fatty acids in erythrocyte membrane and plasma, and increased synthesis of these fatty acids by cultured skin fibroblasts in adrenoleukodystrophy and adrenomyeloneuropathy. International Symposium on the Leukodystrophy and allied diseases. Sasaki Shuppan (Kyoto), 1981: 247-253. Saito E, Odell WD: CorticotropirVlipotropin common precursor-like material in normal rat extrapituitary tissues. Proc Natl Acad Sci USA 1983; 80: 3792-3796. Sayers G: Bioassays of ACTH using isolated cortex cells. Applications: structure activity relationship for ACTH and analogues, assay of corticotropin-releasing factor, and assay of plasma ACTH. Ann NY Acad Sci 1977; 297: 220-24 1. Saito E, Mukai M, Muraki T, lchikawa Y, Homma M: Inhibitory effects of corticosterone on cell proliferation and steroidogenesis in the mouse adrenal tumor cell line Y-l. Endocrinology 1979; 104: 487-492. Thorell JI, Johanson BG: Enzymatic iodination of polypeptides with 1251 to high specific activity. Biochim Biophys Acta 1971; 251: 363-369. Berson SA, Yalow RS: Radioimmunoassay of ACTH in plasma. J Clin Invest 1968; 47: 2725-275 1. Yalow RS, Berson SA: Characteristics of “big ACTH” in human plasma and pituitary extracts. J Clin Endocrinol Metab 1973; 36: 415-423. Yamada Y, Ito S, Miyashita M: Clinical studies of “big ACTH”: its physico-chemical characteristics. Acta Endocrinol (Copenh) 1978; 89: 221-231. Eipper BA, Mains RE: Structure and biosynthesis of proa-
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Journal
of Medicine
Volume
16.
17.
18.
19.
20.
21.
22.
23.
24.
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drenocorticotropin/endorphin and related peptides. Endoer Rev 1980; 1: l-27. Martin JJ, Lowenthal A, Ceuterick C, Gacoms H: Adrenomyeloneuropathy. A report of two families. J Neurol 1982; 226: 221-232. Yoshida T, Ono 2, Takayama Y, Suzuki T, Yanagisawa H: Adrenoleukomyeloneuropathy presenting as hyperpigmentation, neuropathy, visual disturbance and hearing loss. Clin Neurol (Tokyo) 1982; 22: 343-350. Besser GM, Gullen DR, Irvine WJ, Ratcliffe JG, Landon J: lmmunoactive corticotrophin levels in adrenocortical insufficiency. Br Med J [Clin Res] 1971; 1: 374-376. West CD, Dolman LI: Plasma ACTH radioimmunoassays in the diagnosis of pituitary-adrenal dysfunction. Ann NY Acad Sci 1977; 297: 205-219. Krieger DT, Liotta A, Brownstein WJ: Presence of corticotropin in limbic system of normal and hypophysectomired rats. Brain Res 1977; 128: 575-579. Moldow RL, Yalow RS: Extrahypophysial distribution of corticotropin as a function of brain size. Proc Natl Acad Sci USA 1978; 75: 994-998. Krieger DT, Liotta AS, Brownstein MJ, Zimmerman EA: ACTH, P-lipotropin, and related peptides in brain, pituitary, and blood. Recent Prog Horm Res 1980; 36: 277344. Nakao K, Oki S, Tanaka I, Horii K, Nakai Y, Furii T, et al: lmmunoreactive /!I-endorphin, and adrenocorticotropin in human cerebrospinal fluid. J Clin Invest 1980; 66: 13831390. Saito E, lwasa S, Odell WD: Widespread presence of large molecular weight adrenocorticotropin-like substances in rat extrapituitary tissues. Endocrinology 1983; 113: 1010-1019.