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.

Effects of pre- and post-pubertal dihydrotestosterone treatment on penile length in 5α-reductase type 2 deficiency.

Steroid 5α-reductase type 2 deficiency (5αRD2) is a congenital disorder of sex development caused by impairment of conversion from testosterone (T) to 5α-dihydrotestosterone (DHT). DHT deficiency leads to various degrees of undervirilized external genitalia including micropenis, primarily correlated with mutations of the SRD5A2 gene that encodes 5α-reductase type 2. Four Japanese boys with isolated micropenis were diagnosed as 5αRD2 by elevated ratios of serum T/DHT, and decreased ratios of urinary 5α/5ß-reduced steroid metabolites. Genetic analyses for SRD5A2 identified that the four patients shared a hypomorphic mutation R227Q that has a residual activity related to the mild-form of 5αRD2. For prepubertal micropenis, DHT was transdermally applied to the four patients at the ages of 4-11 year, increasing a median of stretched penile lengths (SPLs) from 2.6 cm (-2.5 SD) to 4.4 cm (-0.2 SD). Nevertheless, the post-pubertal penile growth was apparently retarded, despite normal levels of T secreted from well-developed testes. The second course of DHT treatment underwent at ages of 12-18 year, but unable to normalize SPLs at a range of 6.0 to 7.0 cm (-3.4 to -2.4 SD). The prostate volumes of two patients were variable at 8.1 and 21 cm3, and a sperm cell count of one patient was normal as young adult. DHT treatment contributes to development of the penis and prostate, which are favorable for the potential fertility of 5αRD2 adults. Meanwhile, the retarded penile growth and a risk of prostate overgrowth may complicate the post-pubertal management with DHT for 5αRD2 males.

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.

Longitudinal serum and urine steroid metabolite profiling in a 46,XY infant with prenatally identified POR deficiency.

Although POR deficiency (PORD) is assumed to be accompanied by excessive placental androgen accumulation and enhanced adrenal and testicular androgen production via the backdoor pathway as well as compromised testicular androgen production via the frontdoor pathway, there is no direct evidence for the flux of excessive placental androgens into the fetal circulation and for the production of dihydrotestosterone (DHT) via the backdoor pathway. We examined longitudinal serum and urine steroid metabolite profiles in a 46,XY infant with PORD who was prenatally identified because of the progressive fetal masculinization and maternal virilization from the mid-gestation and the presence of fetal radio-humeral synostosis and was confirmed to have compound heterozygous mutations of POR (p.Q201X and p.R457H). The results showed (1) markedly and inappropriately elevated serum androstenedione and testosterone (T) values at birth, (2) a markedly increased serum DHT value with a normal DHT/T ratio at birth, (3) transient elevation of serum T and DHT values accompanied by a normal DHT/T ratio and concomitant elevations of intermediate steroid metabolites on both the frontdoor and backdoor pathways at 30 days of age, and (4) persistent PORD-compatible urine steroid profiles. Although the data obtained from a single infantile patient are too premature to be generalized, they imply: (1) the transfer of excessive placental androgens into the fetal as well as the maternal circulations from the mid-gestation, (2) lack of a clinically discernible amount of DHT production via the adrenal backdoor pathway around birth, and (3) the activation of both the frontdoor and backdoor pathways in the testis around the mini-puberty, with no production of a clinically discernible amount of DHT via the testicular backdoor pathway.

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.

Development of a Risk Assessment Assistance Tool for Robotic Care Devices.

Aiming to eliminate a labor shortage caused by the aging of society, many kinds of service robots are under development. Nursing care for the elderly is considered as a typical application of service robots, especially in Japan. Elderly care robots or robotic care device must be designed with safety, but not a few manufacturers are inexperienced in designing and manufacturing them on the basis of safety. In this paper, we introduce a risk assessment assistance tool for robotic care device targeted for inexperienced manufacturers of risk assessment.

Genetic defects in pediatric-onset adrenal insufficiency in Japan.

CONTEXT: Most patients with pediatric-onset primary adrenal insufficiency (PAI), such as 21-hydroxylase deficiency, can be diagnosed by measuring the urine or serum levels of steroid metabolites. However, the etiology is often difficult to determine in a subset of patients lacking characteristic biochemical findings. OBJECTIVE: To assess the frequency of genetic defects in Japanese children with biochemically uncharacterized PAI and characterize the phenotypes of mutation-carrying patients. METHODS: We enrolled 63 Japanese children (59 families) with biochemically uncharacterized PAI, and sequenced 12 PAI-associated genes. The pathogenicities of rare variants were assessed based on in silico analyses and structural modeling. We calculated the proportion of mutation-carrying patients according to demographic characteristics. RESULTS: We identified genetic defects in 50 (85%) families: STAR in 19, NR0B1 in 18, SAMD9 in seven, AAAS in two, NNT in two, MC2R in one and CDKN1C in one. NR0B1 defects were identified in 78% of the male patients that received both glucocorticoid and mineralocorticoid replacement therapy and had normal male external genitalia. STAR defects were identified in 67% of female and 9% of male patients. Seven of the 19 patients with STAR defects developed PAI at age two or older, out of whom, five did not have mineralocorticoid deficiency. CONCLUSIONS: Molecular testing elucidated the etiologies of most biochemically uncharacterized PAI patients. Genetic defects such as NR0B1 defects are presumed based on phenotypes, while others with broad phenotypic variability, such as STAR defects, are difficult to diagnose. Molecular testing is a rational approach to diagnosis in biochemically uncharacterized PAI patients.

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.

Classic and non-classic 21-hydroxylase deficiency can be discriminated from P450 oxidoreductase deficiency in Japanese infants by urinary steroid metabolites.

We previously reported a two-step biochemical diagnosis to discriminate classic 21-hydroxylase deficiency (C21OHD) from P450 oxidoreductase deficiency (PORD) by using urinary steroid metabolites: the pregnanetriolone/tetrahydrocortisone ratio (Ptl / the cortisol metabolites 5α- and 5ß-tetrahydrocortisone (sum of these metabolites termed THEs), and 11ß-hydroxyandrosterone (11OHAn). The objective of this study was to investigate whether both C21OHD and non-classic 21OHD (C+NC21OHD) could be biochemically differentiated from PORD. We recruited 55 infants with C21OHD, 8 with NC21OHD, 16 with PORD, 57 with transient hyper-17α-hydroxyprogesteronemia (TH17OHP), and 2,473 controls. All infants were Japanese with ages between 0-180 d. In addition to Ptl, THEs, and 11OHAn, we measured urinary tetrahydroaldosterone (THAldo) and pregnenediol (PD5). The first step: by Ptl with the age-specific cutoffs 0.06 mg/g creatinine (0-10 d of age) and 0.3 mg/g creatinine (11-180 d of age), we were able to differentiate C+NC21OHD and PORD from TH17OHP and controls (0-10 d of age: 0.065-31 vs. < 0.001-0.052, 11-180 d of age: 0.40-42 vs. < 0.001-0.086) with 100% sensitivity and specificity. The second step: by the 11OHAn/THAldo or 11OHAn/PD5 ratio with a cutoff of 0.80 or 1.0, we were able to discriminate between C+NC21OHD and PORD (1.0-720 vs. 0.021-0.61 or 1.8-160 vs. 0.005-0.32, respectively) with 100% sensitivity and specificity. Ptl, 11OHAn/THAldo, and 11OHAn/PD5 could differentiate between C+NC21OHD and PORD in Japanese infants.

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.

Distinguishing primary from secondary Δ(4) -3-oxosteroid 5ß-reductase (SRD5B1, AKR1D1) deficiency by urinary steroid analysis.

OBJECTIVE: Deficiency of Δ(4) -3-oxosteroid 5ß-reductase (5ß-reductase), a bile acid synthesis disorder, presents findings of neonatal cholestasis and hyper-3-oxo-Δ(4) bile aciduria. The 5ß-reductase enzyme participates in not only bile acid synthesis but also hepatic steroid metabolism. Deficiency of 5ß-reductase includes 2 types: primary deficiency, with an SRD5B1 gene mutation; and secondary deficiency, lacking a mutation. Secondary deficiency is caused by fulminant liver failure from various aetiologies including neonatal hemochromatosis (NH). Distinguishing primary from secondary deficiency based on γ-glutamyltransferase (GGT), serum total bile acids (TBA), and urinary bile acid analysis using gas chromatography-mass spectroscopy (GC-MS) is very difficult. SRD5B1 gene analysis is the only reliable method. We examined urinary steroid analysis as a way to distinguish primary from secondary 5ß-reductase deficiency. DESIGN, PATIENTS AND MEASUREMENTS: We examined 12 patients with cholestatic jaundice, normal or slightly elevated GGT, and hyper-3-oxo-Δ(4) bile aciduria using urinary steroid analysis by GC-MS of both cortisol and cortisone compounds, such as 5ß-tetrahydrocortisol (5ß-THF) and 5ß-tetrahydrocortisone (5ß-THE). Patients previously were diagnosed with primary 5ß-reductase deficiency (n = 3), deficiency secondary to NH (n = 3) and deficiency secondary to other liver disorders (n = 6). RESULTS: Urinary steroid analysis in 3 primary deficiency and 3 NH patients showed low 5ß-THE and elevated 5α/5ß-THE ratios, making distinction difficult without also considering the clinical course and abdominal magnetic resonance imaging (MRI) findings, such as a very low signal intensity in liver and/or pancreas, especially in T2 -weighted images. In the six patients with other secondary deficiencies, urinary 5ß-THF and 5α/5ß-THF differed from those in primary deficiency (P < 0·05). CONCLUSIONS: Urinary steroid analysis can distinguish primary and NH-related deficiencies from other secondary deficiencies.

Discordant genotype-phenotype correlation in familial hyperaldosteronism type III with KCNJ5 gene mutation: a patient report and review of the literature.

BACKGROUND: Familial hyperaldosteronism type III (FH-III) is a rare autosomal dominant disease for which five missense mutations in KCNJ5 have been identified. FH-III has a wide phenotypic variability from spironolactone-responsive hyperaldosteronism to massive adrenal hypertrophy with drug-resistant hypertension. This variation has mainly been attributed to genotype, because, in contrast to other genotypes (G151R, T158A, I157S, and Y152C), (1) FH-III patients with G151E have shown milder phenotype, and (2) G151E-harboring cells were found to have rapid lethality due to much larger sodium conductance of the encoded channel (Kir3.4), which prevents adrenal hypertrophy. METHODS: Here we describe the clinical course of a sporadic case of FH-III, with de novo G151R mutation. RESULTS: The patient developed polyuria at around 1.5 years of age and developed hypertension and hypokalemia by 4 years of age. Thereafter, spironolactone treatment successfully ameliorated hyperaldosteronism for 7 years with no discernible adrenal enlargement. CONCLUSION: Diverse clinical severity in FH-III cannot be defined solely by KCNJ5 genotype.

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.

Backdoor pathway for dihydrotestosterone biosynthesis: implications for normal and abnormal human sex development.

We review the current knowledge about the "backdoor" pathway for the biosynthesis of dihydrotestosterone (DHT). While DHT is produced from cholesterol through the conventional "frontdoor" pathway via testosterone, recent studies have provided compelling evidence for the presence of an alternative "backdoor" pathway to DHT without testosterone intermediacy. This backdoor pathway is known to exist in the tammar wallaby pouch young testis and the immature mouse testis, and has been suggested to be present in the human as well. Indeed, molecular analysis has identified pathologic mutations of genes involved in the backdoor pathway in genetic male patients with undermasculinized external genitalia, and urine steroid profile analysis has argued for the relevance of the activated backdoor pathway to abnormal virilization in genetic females with cytochrome P450 oxidoreductase deficiency and 21-hydroxylase deficiency. It is likely that the backdoor pathway is primarily operating in the fetal testis in a physiological condition to produce a sufficient amount of DHT for male sex development, and that the backdoor pathway is driven with a possible interaction between fetal and permanent adrenals in pathologic conditions with increased 17-hydroxyprogesterone levels. These findings provide novel insights into androgen biosynthesis in both physiological and pathological conditions.

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.

Two neonatal cholestasis patients with mutations in the SRD5B1 (AKR1D1) gene: diagnosis and bile acid profiles during chenodeoxycholic acid treatment.

BACKGROUND AND AIMS: In two Japanese infants with neonatal cholestasis, 3-oxo-Δ(4)-steroid 5ß-reductase deficiency was diagnosed based on mutations of the SRD5B1 gene. Unusual bile acids such as elevated 3-oxo-Δ(4) bile acids were detected in their serum and urine by gas chromatography-mass spectrometry. We studied effects of oral chenodeoxycholic acid treatment. PATIENTS AND METHODS: SRD5B1 gene analysis used peripheral lymphocyte genomic DNA. Diagnosis and treatment of these two patients were investigated retrospectively and prospectively investigated. RESULTS: With respect to SRD5B1, one patient was heterozygous (R266Q, a novel mutation) while the other was a compound heterozygote (G223E/R261C). Chenodeoxycholic acid treatment was effective in improving liver function and decreasing unusual bile acids such as 7α-hydroxy- and 7α,12α-dihydroxy-3-oxo-4-cholen-24-oic acids in serum and urine. CONCLUSION: Primary bile acid treatment using chenodeoxycholic acid was effective for these patients treated in early infancy before the late stage of chronic cholestatic liver dysfunction.

Two novel mutations of the CYP11B2 gene in a Japanese patient with aldosterone deficiency type 1.

Isolated hypoaldosteronism is a rare and occasionally life-threatening cause of salt wasting in infancy. A 2-month-old Japanese boy of unrelated parents was examined for failure to thrive and poor weight gain. Laboratory findings were hyponatremia, hyperkalemia, high plasma renin and low aldosterone levels. Spot urine analysis by gas chromatography-mass spectrometry (GC-MS) showed that urinary excretion of corticosterone metabolites was elevated. Whereas excretion of 18-hydroxycortricosterone metabolites was within the normal range, excretion of aldosterone metabolites was undetectable. The patient was therefore suspected to have aldosterone synthase deficiency type 1. Sequence analysis of CYP11B2, the gene encoding aldosterone synthase (CYP11B2), showed that the patient was a compound heterozygote for c.168G>A, p.W56X in exon 1 and c.1149C>T, p.R384X in exon 7. p.W56X was inherited from his mother and p.R384X was from his father. Since both alleles contain nonsense mutations, a lack of CYP11B2 activity was speculated to cause his condition. To our knowledge, this is the first Japanese patient in which the molecular basis of aldosterone synthase deficiency type 1 has been clarified. This case also indicates that spot urinary steroid analysis is useful for diagnosis.

Two-step biochemical differential diagnosis of classic 21-hydroxylase deficiency and cytochrome P450 oxidoreductase deficiency in Japanese infants by GC-MS measurement of urinary pregnanetriolone/ tetrahydroxycortisone ratio and 11ß-hydroxyandrosterone.

BACKGROUND: The clinical differential diagnosis of classic 21-hydroxylase deficiency (C21OHD) and cytochrome P450 oxidoreductase deficiency (PORD) is sometimes difficult, because both deficiencies can have similar phenotypes and high blood concentrations of 17α-hydroxyprogesterone (17OHP). The objective of this study was to identify biochemical markers for the differential diagnosis of C21OHD, PORD, and transient hyper 17α-hydroxyprogesteronemia (TH17OHP) in Japanese newborns. We established a 2-step biochemical differential diagnosis of C21OHD and PORD. METHODS: We recruited 29 infants with C21OHD, 9 with PORD, and 67 with TH17OHP, and 1341 control infants. All were Japanese and between 0 and 180 days old; none received glucocorticoid treatment before urine sampling. We measured urinary pregnanetriolone (Ptl), the cortisol metabolites 5α- and 5ß-tetrahydrocortisone (sum of these metabolites termed THEs), and metabolites of 3 steroids, namely dehydroepiandrosterone, androstenedione (AD4), and 11ß-hydroxyandrostenedione (11OHAD4) by GC-MS. RESULTS: At a cutoff of 0.020, the ratio of Ptl to THEs differentiated C21OHD and PORD from TH17OHP and controls with no overlap. Among metabolites of DHEA, AD4, and 11OHAD4, only 11ß-hydroxyandrosterone (11HA), a metabolite of 11OHAD4, showed no overlap between C21OHD and PORD at a cutoff of 0.35 mg/g creatinine. CONCLUSIONS: A specific cutoff for the ratio of Ptl to THEs can differentiate C21OHD and PORD from TH17OHP and controls. Additionally, the use of a specific cutoff of 11HA can distinguish between C21OHD and PORD.