Foetal virilisation caused by overproduction of non-aromatisable 11-oxygenated C19 steroids in maternal adrenal tumour.

It is widely believed that adrenal tumours and ovarian luteomas in pregnant women cause virilisation of female foetuses through overproduction of testosterone and/or androstenedione. However, this notion raises a fundamental question as to how these classic androgens pass through the placenta without being converted by aromatase into oestrogens. Here, we report a case of maternal adrenal tumour, in which overproduction of 11-oxygenated C19 steroids (11ox C19s), newly characterised non-aromatisable androgens in humans, caused foetal virilisation. The female proband presented with severely virilised external genitalia at birth. The mother exhibited hirsutism, hyperglycaemia and hypertension and was diagnosed as having adrenal tumour. The mother was subjected to comprehensive steroid measurement. Serum levels of 11ox C19s were markedly elevated. In contrast, testosterone and androstenedione levels remained within the normal range, and levels of most other steroids in the conventional and backdoor androgenic pathways were normal or only mildly elevated. After tumour removal, levels of 11ox C19s were markedly reduced. These results provide the first evidence that 11ox C19s can be synthesised in adrenal adenomas and, due to their non-aromatisable nature, can pass through the placental barrier to cause foetal virilisation. These findings highlight a unique pathogenic property of these newly specified androgens in humans.

Change of the 5α/5ß ratio of urinary steroid metabolites in benign prostatic hyperplasia patients treated with dutasteride.

BACKGROUND: The effects of the administration of dutasteride (DUT) on steroid metabolite pathways in BPH patients have not been examined. METHODS: Urine and blood samples as well as clinical parameters were prospectively collected after the administration of DUT to 60 BPH patients, and after its withdrawal in another set of 25 BPH patients. Urine samples were assessed using gas chromatography/mass spectrometry for the urinary steroid profile (USP), which simultaneously measures 63 steroid metabolites. We examined pharmacological changes in the 5α/5ß ratio of urinary metabolites and their relationships with clinical parameters in patients treated with DUT. RESULTS: The mean urinary androsterone/etiocholanolone (An/Et) ratio in sex-steroid pathways significantly decreased from 1.39 to 0.02 (p < 0.01). Urinary metabolites in other steroid pathways such as 5αTHF/5ßTHF in the glucocorticoid pathway and 5αTHB/5ßTHB in the mineralocorticoid pathway also significant decreased after the DUT treatment. As compared to baseline level, the mean An/Et ratios in patients with the withdrawal of DUT were 0.7%, 1.4%, 12.6%, and 82.4% at just before, one month, 3 months, and 6 months after the withdrawal of DUT, respectively. All other steroid pathways changed in a similar manner without the aggravation of urinary symptoms. The recovery ratio of An/Et in USP before and 3 months after the withdrawal of DUT correlated with the recovery ratio of serum PSA levels (ρ = 0.61, p < 0.01). CONCLUSION: Urinary 5α/5ß metabolites in all pathways were strongly suppressed after the administration of DUT for one month and the pharmacological effect of DUT prolonged even after withdrawal of DUT.

Adrenocortical carcinoma characterized by gynecomastia: A case report.

We present a 4-yr-old boy with adrenocortical carcinoma (ACC), diagnosed due to the appearance of gynecomastia as the presenting symptom. Six months prior to admission, an acute growth spurt along with the development of bilateral breast swelling was observed. He did not present any features of virilization, including enlargement of the testes, increase in testis volume, and penis size. Laboratory investigations showed gonadotropin-independent hypergonadism, with low LH/ FSH levels and elevated estradiol/testosterone levels. Abdominal computed tomography revealed a large heterogeneous mass adjacent to the right kidney and below the liver. Pathological investigations of the biopsy specimen demonstrated that the tumor was an ACC. Pre- and post-operative combination chemotherapy with mitotane was administered and surgical resection was carried out. Post-surgery, the elevated estradiol/testosterone concentrations reverted to within the reference range. Urinary steroid profile and tissue concentration analysis of estradiol and testosterone indicated the presence of estrogen in the ACC tissue. An investigation for TP53 gene aberrations revealed the presence of a germline point mutation in exon 4 (c.215C>G (p.Pro72Arg)). In ACC, the most common symptom is virilization, and feminization, characterized by gynecomastia, is very rare. However, a diagnostic possibility of ACC should be considered when we encounter patients who have developed gynecomastia without the influence of causative factors such as obesity or puberty, and do not present with the typical signs of virilization.

Influence of dutasteride treatment on serum hormone levels and aging male symptoms in patients with benign prostatic enlargement.

OBJECTIVES: To clarify the effects of dutasteride on serum hormone levels and aging male symptoms in patients with benign prostatic enlargement. METHODS: The present prospective study was carried out in 110 symptomatic benign prostatic enlargement patients treated with daily administration of 0.5 mg dutasteride. We analyzed serum hormonal levels and aging related symptoms using a validated Aging Male Symptom questionnaire at baseline and after 3 months of dutasteride treatment. RESULTS: The mean total testosterone, free testosterone and luteinizing hormone levels after dutasteride treatment were approximately 20% higher than those at baseline. The percentage increases in total and free testosterone levels were negatively correlated with these baseline levels. Baseline age, levels of total testosterone and free testosterone, and the changes in the rate of luteinizing hormone after dutasteride treatment tended to be correlated with an increase in the rate of total testosterone and free testosterone after dutasteride treatment. In a subgroup of 26 patients with moderate-to-severe aging male symptoms, poor morning erection and free testosterone levels <8.5 pg/mL, total aging male symptoms, and somatic symptoms scores significantly decreased after dutasteride treatment with an increase of total and free testosterone. CONCLUSIONS: The increase of endogenous free testosterone and total testosterone by dutasteride might bring additional benefits of improvement of aging male-related symptoms, especially in patients with lower free testosterone baseline levels and moderate-to-poor aging-related symptoms.

Feminizing Adrenocortical Carcinoma with Distinct Histopathological Findings.

We herein present a 60-year-old man with adrenocortical carcinoma who had gynecomastia. An endocrinological examination revealed increased levels of serum estradiol and dehydroepiandrosterone-sulfate (DHEA-S) and reduced levels of free testosterone. Magnetic resonance imaging showed an adrenal tumor with heterogeneous intensity. Iodine-131 adosterol scintigraphy showed an increased uptake at the same site. Because feminizing adrenocortical carcinoma was suspected, right adrenalectomy was performed; the pathological diagnosis was adrenocortical carcinoma. The results of immunostaining indicated a virilizing tumor. Aromatase activity was identified on RT-PCR. As disorganized steroidogenesis is pathologically present in adrenocortical carcinoma, this diagnosis should be made with caution.

Steroidogenic pathways involved in androgen biosynthesis in eumenorrheic women and patients with polycystic ovary syndrome.

The conventional Δ5 and Δ4 steroidogenic pathways mediate androgen production in females. While multiple non-conventional pathways to dihydrotestosterone (DHT) have recently been postulated in humans, the functional significance of these pathways remains to be elucidated. The aim of this study was to clarify the origin of androgens in healthy women and in patients with polycystic ovary syndrome (PCOS), a multifactorial disorder characterized by androgen overproduction. We measured 13 steroids in blood samples of 31 eumenorrheic females and 28 PCOS patients using liquid chromatography-tandem mass spectrometry and chemiluminescent enzyme immunoassay. We found that 17-hydroxy (17-OH) progesterone (17-OHP), androstenedione (Δ4A), testosterone, androstanedione, androsterone, and androstanediol levels were higher in the patient group than in the eumenorrheic group, while levels of other steroids were comparable between the two groups. In the eumenorrheic group, DHT levels were correlated with testosterone, androstanedione, and androstanediol. Quantitative correlations were also observed among 17-OH allopregnanolone, androsterone, androstanediol, and DHT, and among Δ4A, androstanedione, androsterone, and androstanediol. In the patient group, DHT levels were correlated with testosterone levels, but not with androstanedione or androstanediol levels. Δ4A and testosterone paralleled 17-OHP. Androstanedione, androsterone, androstanediol, and 17-OH allopregnanolone were quantitatively correlated. In both groups, multivariable linear regression analyses suggested relationships between androsterone and androstanedione, as well as between androsterone and 17-OH allopregnanolone. These results indicate that multiple androgen biosynthesis pathways are operating in eumenorrheic females and PCOS patients. In PCOS patients, excessive androgens are produced primarily via the conventional pathways, while two alternative pathways; i.e., an androstanedione-mediated pathway and a so-called backdoor pathway, likely serve as sources of a weak androgen and potential precursors of DHT.

Urinary steroid profiling: a powerful method for the diagnosis of abnormal steroidogenesis.

In this review, we will focus on urinary steroid profiling by gas chromatography mass spectrometry (GC/MS) and summarize its contribution to the diagnosis of abnormal steroidogenesis; congenital enzyme deficiency of steroid synthesis and metabolism, adrenal carcinoma and other steroid related diseases. Mass spectrometry technique, such as GC/MS and liquid chromatography tandem mass spectrometry (LC-MS/MS), has become the main tool for steroid measurement and GC/MS is mainly used for urine sampling. We will discuss the pros and cons of urinary steroid profiling by GC/MS and LC-MS/MS. Although GC/MS analysis needs intricate pretreatment, time and expenses, sensitive and simultaneous measurement of whole pathway steroid measurements have improved the accuracy of diagnosis.

A 68-year-old phenotypically male patient with 21-hydroxylase deficiency and concomitant adrenocortical neoplasm producing testosterone and cortisol.

The steroidogenic enzyme 21-hydroxylase is necessary for the synthesis of both glucocorticoids and mineralocorticoids. 21-hydroxylase is a cytochrome P-450 enzyme and is encoded by the gene CYP21A2. Here we report a 68-year-old phenotypically 'male' but genetically female patient with 21-hydroxylase deficiency (21OHD) and the concomitant virilizing adrenocortical carcinoma. This patient grew up as a male and has not encountered any episodes of adrenal insufficiency without glucocorticoid replacement in his lifetime. A chromosome test at admission, however, identified the 46, XX karyotype, and serum 17-hydroxyprogesterone and urine pregnanetriolone and 11ß-hydroxyandrostendione were all elevated, consistent with 21OHD. Moreover, serum testosterone was 1.90 ng/ml, much higher than the female standard levels, and serum cortisol was 5.7 µg/ml, slightly lower than standard levels. Genetic analysis identified the patient as a heterozygote of the two pathogenic mutations in the CYP21A2 gene: IVS2-13C(A)>G and R356W. Magnetic resonance imaging (MRI) revealed the presence of left adrenal tumor measuring 6 cm, which was subsequently diagnosed as adrenocortical carcinoma based on the criteria of Weiss. Immunohistochemical analysis of the tumor specimens revealed the expression of various enzymes involved in testosterone production, including 3ß-hydroxysteroid dehydrogenase, 17α-hydroxylase/17,20-lyase, and 17ß-hydroxysteroid dehydrogenase. Importantly, the expression of immunoreactive 21-hydroxylase was detected in these tumor cells. The levels of adrenal tumor-derived steroid metabolites were all markedly decreased following the surgery. This is the first report on a virilized 21OHD patient associated with the adrenocortical tumor that produces testosterone. Moreover, the concomitant adrenocortical tumor may ameliorate adrenocortical insufficiency by producing cortisol.

Measurement of reference intervals for urinary free adrenal steroid levels in Japanese newborn infants by using stable isotope dilution gas chromatography/mass spectrometry.

BACKGROUND: In newborn infants, there are no reference intervals for urinary free steroids, which are thought to reflect the bioavailable fraction of steroids in the blood. We establish a method for simultaneous measurement of urinary free adrenal steroids such as pregnenolone, progesterone, 16α-hydroxyprogesterone, 17α-hydroxyprogesterone, 21-deoxycortisone, 21-deoxycortisol, dehydroepiandrosterone, androstenedione, and 11ß-hydroxyandrostenedione by using stable isotope dilution gas chromatography/mass spectrometry (SID-GC/MS) and determined the reference intervals for urinary levels of free adrenal steroids in Japanese newborn infants. METHODS: Newborn pooled urine was used for validation. Spot urine samples were collected from 67 full-term Japanese newborn infants (34 male and 33 female infants) at 3-4 days of age to determine reference intervals. The extracted and purified free steroids were delivered with heptafluorobutyric anhydride and analyzed by SID-GC/MS. RESULTS: We validated a SID-GC/MS method with good repeatability and recovery rate. The preliminary reference intervals (median [range], µmol/mol creatinine) were as follows: pregnenolone, 4.2 (0.7-31.6); progesterone, 0.5 (not detected (n.d.)-0.6); 16α-hydroxyprogesterone, 1.4 (n.d.-10.3); 17α-hydroxyprogesterone, 1.1 (n.d.-1.9); 21-deoxycortisone, n.d. (n.d.-n.d.); 21-deoxycortisol, n.d. (n.d.-n.d.); dehydroepiandrosterone, 2.2 (0.6-27.3); androstenedione, 0.7 (n.d.-5.2); and 11ß-hydroxyandrostenedione, 2.9 (n.d.-26.7). CONCLUSIONS: We established a reliable SID-GC/MS method and were able to determine preliminary reference intervals for 9 urinary free adrenal steroids in newborn infants.

Clinicopathological features, biochemical and molecular markers in 5 patients with adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a very rare malignant tumor with poor prognosis. To gain insight into the pathogenic significance of ACC, we studied clinicopathological features and gene expression profile in ACC. We analyzed five ACC cases (two men and three women) with the median age of 45-year-old who underwent adrenalectomy at our institute. Endocrine studies revealed that two cases had subclinical Cushing's syndrome (SCS) and one with concomitant estrogen-secreting tumor, while the rest of three cases had non-functioning tumors. Analysis of urinary steroids profile by gas chromatography/mass spectrometry showed increased metabolites of corticosteroid precursors, such as 17-OH pregnenolone, 17-OH progesterone, dehydroepiandorosterone (DHEA), and 11-deoxycortisol in all five cases. The pathological diagnosis of ACC was based on Weiss's criteria with its score ≥ 3. The mean size of the resected tumors was 87 mm and Ki67/MIB1 labeling index, a proliferative marker, was 3-27%. Immunohistochemical analysis revealed a disorganized expression of several steroidogenic enzymes, such as 3ß-hydroxysteroid dehydrogenase, 17α-hydroxylase, and DHEA-sulfotransferase. Among several genes determined by RT-PCR, insulin-like growth factor (IGF)-II mRNA was consistently and abundantly expressed in all 5 tumor tissues. Postoperatively, two cases with SCS developed local recurrence and liver metastasis. The present study suggests that the disorganized expression of steroidogenic enzymes and the overexpression of IGF-II by the tumor are hallmarks of ACC, which could be used as biochemical and molecular markers for ACC.

Unilateral adrenalectomy can be an alternative therapy for infantile onset Cushing' s syndrome caused by ACTH-independent macronodular adrenal hyperplasia with McCune-Albright syndrome.

We report herein the case of a 1-year-old boy with McCune-Albright syndrome (MAS) who presented with infantile-onset Cushing' s syndrome caused by ACTH independent macronodular adrenal hyperplasia (AIMAH). Abdominal CT, MRI, and adrenal scintigraphy with (131)I-adosterol identified bilateral adrenal involvement with the left adrenal gland being larger and functionally more active. Unilateral adrenalectomy of the left gland was performed and ameliorated many clinical symptoms, such as Cushingoid appearance and height restriction, and it also normalized many endocrinological data, such as diurnal rhythms of ACTH and cortisol, ACTH and cortisol responses to CRH, and urinary 24 hr free cortisol. Glucocorticoid was replaced for the first 1 year and 6 months after the operation. One adrenal crisis episode occurred at 3 weeks after the operation, but none have occurred since. These results suggest that unilateral adrenalectomy of the larger gland can be an alternative therapy for infantile onset Cushing' s syndrome caused by AIMAH with MAS, when asymmetric involvement is evident and the smaller gland is not markedly enlarged.

A case of pediatric virilizing adrenocortical tumor resulting in hypothalamic-pituitary activation and central precocious puberty following surgical removal.

We present a 6-year-old boy with a virilizing adrenocortical tumor who initially presented with peripheral precocious puberty. Development of facial acne, pubic hair and a growth spurt were noted at the age of five. A low-pitched voice as well as maturation of external genitalia was noted at the age of six. Both serum and urinary levels of adrenal androgens were elevated. Abdominal computed tomography revealed a large right suprarenal mass and he underwent surgical resection without any complications. The histological diagnosis was adrenocortical carcinoma according to the criteria of Weiss. Following surgical removal of the androgen-producing tumor, the patient subsequently developed hypothalamic-pituitary activation and demonstrated central precocious puberty. He was treated with a gonadotropin-releasing hormone agonist in order to delay further pubertal progression. Clinical follow-up of potential secondary effects of excess hormone secretion after removal is important in some pediatric patients with virilizing adrenocortical tumor.

Cytochrome P450 oxidoreductase deficiency: identification and characterization of biallelic mutations and genotype-phenotype correlations in 35 Japanese patients.

CONTEXT: Cytochrome P450 oxidoreductase (POR) deficiency is a rare autosomal recessive disorder characterized by skeletal dysplasia, adrenal dysfunction, disorders of sex development (DSD), and maternal virilization during pregnancy. Although multiple studies have been performed for this condition, several matters remain to be clarified, including the presence of manifesting heterozygosity and the underlying factors for clinical variability. OBJECTIVE: The objective of the study was to examine such unresolved matters by detailed molecular studies and genotype-phenotype correlations. PATIENTS: Thirty-five Japanese patients with POR deficiency participated in the study. RESULTS: Mutation analysis revealed homozygosity for R457H in cases 1-14 (group A), compound heterozygosity for R457H and one apparently null mutation in cases 15-28 (group B), and other combinations of mutations in cases 29-35 (group C). In particular, FISH and RT-PCR sequencing analyses revealed an intragenic microdeletion in one apparent R457H homozygote, transcription failure of apparently normal alleles in three R457H heterozygotes, and nonsense mediated mRNA decay in two frameshift mutation-positive cases examined. Genotype-phenotype correlations indicated that skeletal features were definitely more severe, and adrenal dysfunction, 46,XY DSD, and pubertal failure were somewhat more severe in group B than group A, whereas 46,XX DSD and maternal virilization during pregnancy were similar between two groups. Notable findings also included the contrast between infrequent occurrence of 46,XY DSD and invariable occurrence of 46,XX DSD and pubertal growth pattern in group A mimicking that of aromatase deficiency. CONCLUSIONS: The results argue against the heterozygote manifestation and suggest that the residual POR activity reflected by the R457H dosage constitutes the underlying factor for clinical variability in some features but not other features, probably due to the simplicity and complexity of POR-dependent metabolic pathways relevant to each phenotype.

Close examination of steroidogenesis disorders in a DOC- and progesterone-producing adrenocortical carcinoma.

We report a case of hypertension, hypokalemia, and amenorrhea accompanying an adrenocortical carcinoma. A 27-year-old woman was admitted to our hospital because of a left adrenal incidentaloma. She presented with hypertension, hypokalemia, and amenorrhea; her plasma renin activity was low, but her plasma aldosterone concentration was normal, as were cortisol and androgens. By contrast, her serum concentrations of deoxycorticosterone (DOC), 18-hydroxydeoxycorticosterone, and progesterone were high, and her urinary steroid profile showed elevated secretion of 17-deoxysteroids and 11-deoxysteroids (progesterone, DOC, 11-dehydrocorticosterone, and 11-deoxycortisol), and 3beta-hydroxy 5-en steroids (pregnenolone, 17-hydroxypregnenolone, and DHEA). Decreased ratios of metabolites of (1) 17-OHpregnenolone to pregnenolone and 17-OHprogesterone to progesterone, (2) corticosterone to DOC and cortisol to 11-deoxycortisol, and (3) progesterone to pregnenolone, 17-OHprogesterone to 17-OHpregnenolone and androstenedione to DHEA suggested the impairment of 17alpha-hydroxylase, 11beta-hydroxylase, and 3beta-HSD activities, respectively. After the tumor was removed, levels of all adrenal steroids were normalized. Based on the Weiss criteria, the tumor was diagnosed as an adrenocortical carcinoma, and immunohistochemical analysis of steroidogenic enzymes revealed disorganized steroidogenesis in the tumor tissue. With adrenocortical carcinomas, heterogeneity of individual steroid producing enzymes within tumor cells can lead to hypersecretion of various steroid intermediates, even when steroid end products are within the normal range.

Measurement of Serum 17α-hydroxyprogesterone in Newborn Infants by Stable Isotope Dilution-Gas Chromatography/Mass Spectrometry.

Immunochemical measurement of serum 17α-hydroxyprogesterone (17OHP), the most important parameter for diagnosis of classical 21-hydroxylase deficiency (21OHD) in newborn infants, is known to be inaccurate due to the cross-reactivity of antibodies with a large quantity of fetal adrenal steroids. The aims of this study were 1) to establish reference values for the serum 17OHP level in Japanese newborn infants using non-immunochemical stable isotope dilution -gas chromatography/mass spectrometry (SID-GC/MS) and 2) to compare the serum 17OHP levels determined by SID-GC/MS with those determined by radioimmunoassay (RIA). The first study subjects were used for determination of reference values and included 57 healthy full-term newborn infants (4-5 d of age). The second study subjects were used for comparison of SID-GC/MS with RIA and included 27 healthy full-term newborn infants (3-6 d of age) and two subjects with neonatal transient hyper 17OHPnemia; these two subjects were 16 and 27 d of age, respectively. In the first study subjects, the intra-assay coefficient of variation for SID-GC/MS was 3% (n=5), the recovery rate was 98%, the sensitivity was 0.2 ng/ml, and the range of linearity was 0.5-200 ng/ml. The reference values for the serum 17OHP level determined by SID-GC/MS ranged from 0.3-1.5 (0.6) (ng/ml) (median). In the second study subjects, the serum 17OHP levels determined by SID-GC/MS were lower in one of the 27 subjects and both of the two subjects with neonatal transient hyper 17OHPnemia compared with the levels determined by RIA. Measurement of the serum 17OHP level using SID-GC/MS may be clinically useful for definitive diagnosis of classical 21OHD in newborn infants.

Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration.

Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into two components: (1) RGC survival, and (2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased four- to five-fold at the protein level by 3-5 days after nerve injury. Subsequently, Bcl-2 levels increased 1.7-fold, accompanied by a slight reduction in caspase-3 activity 10-20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2-3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury.

The circadian variation of cortisol secretion in patients with anorexia nervosa in childhood and adolescence after recovery of body weight by treatment using gas chromatography/mass spectrometry in selected ion monitoring.

Anorexia nervosa (AN) is a chronic psychiatric disorder which is characterized by patient-induced weight loss. Complications in many organ systems can be seen in AN such as cardiovascular, gastrointestinal, and endocrine system including hypothalamic-pituitary-adrenal axis, even after recovery of body weight by treatment. Urinary steroid profile analysis using gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM) has been reported to be useful for the diagnosis of abnormal steroidogenesis in newborn infants, childhood, and adults. The aim of this study was to analyze the circadian variation of cortisol secretion in patients with anorexia nervosa (AN) in childhood and adolescence after recovery of body weight by treatment using GC/MS in SIM. The subjects were 7 healthy young adults (20-23 yr of age, BMI 19.7-24.8 kg/m(2)) and 5 AN patients in childhood and adolescence (13-19 yr of age), who had recovered body weight by treatment (BMI 15.4-19.3 kg/m(2); 3(rd)-25(th) to 50(th) percentile). Urine samples were collected for 26 hours (from 21:00 to 23:00 next day) at each urination. In each sample, the cortisol metabolites were measured by GC/MS in SIM. The sum of all cortisol metabolites was calculated as mg/g creatinine. In all 5 AN patients in childhood and adolescence, the circadian variation of the sum of cortisol metabolites was observed and was similar to that in healthy young adults. Although our data are preliminary, in patients with AN in childhood and adolescence, who have recovered body weight by treatment, the circadian variation of cortisol secretion may be conserved.

A case of estrogen-secreting adrenocortical carcinoma with subclinical Cushing's syndrome.

A 25-year-old man was found to have a large right adrenal mass detected by abdominal echography and computed tomography, and presented with a mild gynecomastia. Endocrine study showed increased serum concentrations and urinary excretion of estrogens and dehydroepiandorosterone sulfate (DHEA-S). The patient had no Cushingoid features but autonomous cortisol secretion, compatible with the diagnosis of subclinical Cushing's syndrome. Surgical removal of the adrenal tumor led to normalization of serum and urinary excretion of estrogens and DHEA-S. Histopathological examination revealed a high-grade adrenocortical carcinoma (ACC). The disorganized expression of all the steroidogenic enzymes in individual tumor cells was demonstrated by immunohistochemical analysis, and the abundant expression of both aromatase mRNA and insulin-like growth factor (IGF)-II mRNA was shown by RT-PCR. These data suggest the excessive secretion of estrogen as well as the ineffective steroidogenesis by the adrenal tumor. This is a very rare case of estrogen-secreting ACC associated with subclinical Cushing's syndrome.

Activation of cell survival signals in the goldfish retinal ganglion cells after optic nerve injury.

Generally, nerve injury of adult mammalian CNS neurons leads to a retrograde neuronal degeneration and cell death. The retinal ganglion cells (RGCs) of rat fail to regenerate and become apoptotic after optic nerve injury. In contrast, goldfish RGCs can survive and regrow their axons after injury. Focusing on this different response of RGCs in both species to optic nerve injury, we compared cell death and cell survival signals in the rat and goldfish RGCs after optic nerve injury. In goldfish retina, levels of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) first rapidly increased at 3-5 days after optic nerve injury. Subsequently, levels of Bcl-2 increased and caspase-3 activity decreased at 10 days after nerve injury. In rat retina, levels of p-Akt and p-Bad first rapidly decreased at 1-2 days after optic nerve injury. Subsequently, levels of Bax and caspase-3 activity increased 6 days after optic nerve crush. These changes after optic nerve injury were all morphologically localized only in the RGCs. The data suggest that goldfish RGCs are warranted the cell survival by rapid p-Akt and subsequent Bcl-2 activations during the optic nerve regeneration, whereas rat RGCs are made a progress of the cell death by rapid inactivation of p-Akt and subsequent activation of Bax after optic nerve crush.

A Case of a Preterm Infant with 21-Hydroxylase Deficiency: Implications of the Biochemical Diagnosis with Urinary Pregnanetriolone by Gas Chromatography/Mass Spectrometry in Selected Ion Monitoring (GCMS-SIM).

The biochemical diagnosis of 21-hydroxylase deficiency (21-OHD) is difficult in preterm infants. To date, no marker for the biochemical diagnosis of 21-OHD has been found. Seventeen α-hydroxyprogesterone (17-OHP), is not useful because of interference by delta 5 steroids from the fetal adrenal cortex. A 5-d-old female infant, born at 31 wk of gestation, was suspected of having 21-OHD based on physical findings (mild clitoromegaly, pigmentation of the tongue and gingiva) as well as laboratory data (17-OHP >93.5 ng/ml by ELISA 7 prime extractive method in filter paper-dried blood spot and 718.3 ng/ml by RIA after high performance liquid chromatography extraction in serum; plasma ACTH 690 pg/ml; and serum testosterone 3,169 ng/dl). We examined her urinary steroid profiles by gas chromatography/mass spectrometry in selected ion monitoring (GCMS-SIM) at 8 d of age. The pregnanetriolone (Ptl) level was noticeably high (0.80 mg/g creatinine), which was strongly suggestive of 21-OHD. Gene analysis of CYP21A2 showed compound heterozygosity, one allele having a cluster mutation in exon 6 and the other having a large deletion including CYP21A2, confirming the diagnosis of 21-OHD. This case suggested that, in preterm infants, urinary Ptl by GCMS-SIM can be useful for the biochemical diagnosis of 21-OHD.