Characterizing the steroidal milieu in amniotic fluid of mid-gestation: A GC-MS study.

Intact steroid hormone biosynthesis is essential for growth and development of the human fetus and embryo. In the present study, gas chromatography-mass spectrometry was employed to characterize the steroidal milieu in amniotic fluid (n = 65; male: female = 35: 30) of mid-gestation (median: 18.8th week, range: 16.0th - 24.6th week) by a comprehensive targeted steroid hormone metabolomics approach. The levels of 52 steroids including pregnenolone and 17-OH-pregnenolone metabolites, dehydroepiandrosterone (DHEA) and its metabolites, progesterone and 17-OH-progesterone metabolites, sex hormones as well as corticosterone and cortisol metabolites were measured. The dominating steroids were the group of pregnenolone and 17-OH-pregnenolone metabolites (mean ± SD: 138.0 ± 59.3 ng/mL), followed by the group of progesterone and 17-OH-progesterone metabolites (107.3 ± 44.3 ng/mL), and thereafter DHEA and its metabolites (97.1 ± 56.5 ng/mL). With respect to sex steroids, only testosterone showed a significantly higher value in male fetuses (p < 0.0001). Of all estrogen metabolites, estriol showed by far the highest concentrations (33.2 ± 26.1 ng/mL). Interestingly, cortisol metabolites were clearly present (59.6 ± 13.6 ng/mL) though fetal de novo synthesis of cortisol is assumed to start from gestational 28th week onwards. Our comprehensive characterization of the steroidal milieu in amniotic fluid of mid-gestation shows presence of all relevant classes of steroid hormones and provides reference data. We conclude that the steroidal milieu in amniotic fluid mirrors the steroidome of the feto-placental unit.

Characterizing the steroidal milieu in amniotic fluid of mid-gestation: A LC-MS/MS study.

Growth and development of an embryo or fetus during human pregnancy mainly depend on intact hormone biosynthesis and metabolism in maternal amniotic fluid (AF). We investigated the hormonal milieu in AF and developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of 14 sulfated and 6 unconjugated steroids in AF. 65 A F samples (male: female = 35: 30) of mid-gestation ranging from 16th week of gestation to 25th week of gestation were analyzed. Reference data of 20 steroid levels in AF of healthy women were provided. 13 sulfated and 3 unconjugated steroids were for the first time quantified in AF by LC-MS/MS. Highest concentrations were found for pregnenolone sulfate (PregS: mean ±â€¯SD, 8.6 ±â€¯3.7 ng/mL), 17α-hydroxypregnenolone sulfate (17OHPregS: 4.9 ±â€¯2.0 ng/mL), epitestosterone sulfate (eTS: 7.3 ±â€¯3.6 ng/mL), 16α-hydroxydehydroepiandrosterone sulfate (16OH-DHEAS: 21.5 ±â€¯10.7 ng/mL), androsterone sulfate (AnS: 9.2 ±â€¯7.4 ng/mL), estrone sulfate (E1S: 3.0 ±â€¯3.0 ng/mL), estriol 3-sulfate (E3S: 8.1 ±â€¯4.0 ng/mL) and estriol (E3: 1.2 ±â€¯0.4 ng/mL). Only testosterone (T) showed a significant sex difference (p < 0.0001). Correlations between AF steroids mirrored the steroid metabolism of the feto-placental unit, and not only confirmed the classical steroid pathway, but also pointed to a sulfated steroid pathway.

Maxillary tumour-induced osteomalacia.

Tumour-induced osteomalacia (TIO) is a rare paraneoplastic form of renal phosphate wasting that results in severe hypophosphatemia, defective vitamin D metabolism, and osteomalacia. In the case reported here, maxillary TIO was not diagnosed for 6years, although initial complaints were reported when the patient was 12years old. Meanwhile she suffered from profound growth limitation, pain, weakness, and spontaneous multiple bone fractures, culminating in complete loss of ambulatory ability and severe limitation in daily activities. At age 18years, she finally received an accurate diagnosis and definitive treatment was administered. She underwent a partial maxillectomy with complete removal of the tumour, resulting in a full cure. Shortly afterwards the patient regained the ability to walk, no longer needing the wheelchair to which she had been confined. This definitive diagnosis was based on three modalities: (1) fibroblast growth factor 23 analysis (high levels of the secreted hormone were found on the left side of the maxilla in the facial vein and pterygoid plexus, pinpointing the tumour location), (2) octreotide scan, and (3) 68Ga-DOTA-NOC-PET/CT. TIO removal via partial maxillectomy led to a complete reversal of this patient's health condition, restoring her ability to walk and function. The importance of prompt employment of these diagnostic modalities and the high level of clinical suspicion required in such cases are clear.

Global Consensus Recommendations on Prevention and Management of Nutritional Rickets

"BACKGROUND: Vitamin D and calcium deficiencies are common worldwide, causing nutritional rickets and osteomalacia, which have a major impact on health, growth, and development of infants, children, and adolescents; the consequences can be lethal or can last into adulthood. The goals of this evidence-based consensus document are to provide health care professionals with guidance for prevention, diagnosis, and management of nutritional rickets and to provide policy makers with a framework to work toward its eradication. EVIDENCE: A systematic literature search examining the definition, diagnosis, treatment, and prevention of nutritional rickets in children was conducted. Evidence-based recommendations were developed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system that describes the strength of the recommendation and the quality of supporting evidence. PROCESS: Thirty-three nominated experts in pediatric endocrinology, pediatrics, nutrition, epidemiology, public health, and health economics evaluated the evidence on specific questions within five working groups. The consensus group, representing 11 international scientific organizations, participated in a multiday conference in May 2014 to reach a global evidence-based consensus. RESULTS: This consensus document defines nutritional rickets and its diagnostic criteria and describes the clinical management of rickets and osteomalacia. Risk factors, particularly in mothers and infants, are ranked, and specific prevention recommendations including food fortification and supplementation are offered for both the clinical and public health contexts. CONCLUSION: Rickets, osteomalacia, and vitamin D and calcium deficiencies are preventable global public health problems in infants, children, and adolescents. Implementation of international rickets prevention programs, including supplementation and food fortification, is urgently required."

Hypothalamic regulation of adiposity: the role of 11beta-hydroxysteroid dehydrogenase type 1.

Following extensive suprasellar operations for excision of hypothalamic tumors, some patients develop morbid obesity despite receiving replacement doses of glucocorticoids. Urine analysis of cortisol and cortisone metabolites show that 11-OH/11-oxo ratios are significantly higher in patients with hypothalamic obesity, indicating enhanced 11beta-HSD1 activity. This correlates with the visceral-to-subcutaneous fat ratio. The consequence of increased 11beta-HSD1 activity and a shift of the steroid inter-conversion towards cortisol may contribute to the effects of the latter in adipose tissue. The message from the hypothalamus to adipocyte 11beta-HSD-1 involves hormones, the sympathetic nervous system and cytokines. CRH and ACTH downregulate 11beta-HSD-1 activity and induce lipolysis. Tumor necrosis factor-alpha and interleukin-1beta upregulate 11beta-HSD-1 expression and activity, while enhancing lipolysis. The sympathetic nervous system exerts its effects through beta-adrenergic upregulation and alpha-adrenergic downregulation of 11beta-HSD-1 activity. Inhibition of 11beta-HSD-1 suppresses preadipocyte differentiation into mature adipocytes, and may provide a therapeutic tool.

The importance of adrenocortical glucocorticoids for adrenomedullary and physiological response to stress: a study in isolated glucocorticoid deficiency.

Glucocorticoids are required for the normal functioning of chromaffin cells and their capacity to produce epinephrine. This was modeled in a unique clinical syndrome of isolated glucocorticoid deficiency due to unresponsiveness to ACTH. The working hypotheses were that in patients with isolated glucocorticoid deficiency, adrenomedullary epinephrine would be suppressed despite replacement therapy; that norepinephrine might show a compensatory response; and that the physiological response to stress would reflect these changes. Toward these hypotheses, patients with ACTH unresponsiveness on glucocorticoid replacement were subjected to three levels of acute stress: assumption of upright posture, cold pressor, and exercise. Their catecholamine and physiological response were monitored. Patients with isolated glucocorticoid deficiency of this study had severe adrenomedullary dysfunction, characterized by a minimal resting production of epinephrine (6 +/- 2 pg/ml compared with 64 +/- 22 pg/ml of the controls) and a minimal response to stress. A slight compensatory increase of norepinephrine was found in response to cold pressor test (754 +/- 200 pg/ml compared with 431 +/- 73 pg/ml of the control). The physiological response is characterized by low systolic blood pressure and high pulse rate in rest and mild stress and in a pressor response to exercise (diastolic 87 +/- 5 mm Hg, compared with 73 +/- 2 mm Hg of the control). It is concluded that intra-adrenal glucocorticoids are essential for epinephrine secretion, that norepinephrine may be compensatory, and that these result in a distinct physiological response. The implications of the pressor response to exercise, the declining pulse pressure, and the increased pulse response insinuate a lower physical fitness in patients with adrenal insufficiency.

Spectrum of mutations of the AAAS gene in Allgrove syndrome: lack of mutations in six kindreds with isolated resistance to corticotropin.

Familial glucocorticoid deficiency due to corticotropin (ACTH) resistance consists of two distinct genetic syndromes that are both inherited as autosomal recessive traits: isolated ACTH resistance (iACTHR), which may be caused by inactivating mutations of the ACTH receptor (the MC2R gene) or mutations in an as yet unknown gene(s), and Allgrove syndrome (AS). The latter is also known as triple-A syndrome (MIM 231550). In three large cohorts of AS kindreds, the disease has been mapped to chromosome 12; most recently, mutations in the AAAS gene on 12q13 were found in these AS families. AAAS codes for the WD-repeat containing ALADIN (for alacrima-achalasia-adrenal insufficiency-neurologic disorder) protein. We investigated families with iACTHR (n = 4) and AS (n = 6) and a Bedouin family with ACTHR and a known defect of the TSH receptor. Four AS families were of mixed extraction from Puerto Rico (PR); most of the remaining six families were Caucasian families from North America (NA). Sequencing analysis found no MC2R genetic defects in any of the kindreds. No iACTHR kindreds, but all of AS families, had AAAS mutations. The previously reported IVS14+1G-->A splice donor mutation was found in all PR families, apparently due to a founder effect; one NA kindred was heterozygous for this mutation. In the latter family, long-range PCR failed to identify a deletion or other rearrangements of the AAAS gene. No other heterozygote or transmitting parent had any phenotype that could be considered part of AS. The IVS14+1G-->A mutation results in a premature termination of the predicted protein; although it was present in all PR families (in the homozygote state in three of them), there was substantial clinical variation between them. One PR family also carried a novel splice donor mutation of the AAAS gene in exon 11, IVS11+1G-->A; the proband was a compound heterozygote. A novel point mutation, 43C-->A(Gln15Lys), in exon 1 of the AAAS gene was identified in the homozygote state in a Canadian AS kindred with a milder AS phenotype. The predicted amino acid substitution in this family is located in a sequence that may participate in the preservation of stability of ALADIN beta-strands, whereas the splicing mutation in exon 11 may interfere with the formation of WD repeats in this molecule. We conclude that 1) AAAS does not appear to be frequently mutated in families with iACTHR; 2) AAAS is mutated in AS families from PR (that had previously been mapped to 12q13) and NA; and, 3) there is significant clinical variability between patients with the same AAAS defect.

Endocrine complications of thalassemia.

Growth, sexual development, fertility, bone mineral density, diabetes mellitus, hypothyroidism, hypoparathyroidism, and hypoadrenalism are the main issues to be addressed in the long-term follow-up of patients with thalassemia. During childhood, growth may be affected by anemia, and other potential endocrine complications. Puberty is the stage of the maximal growth insult. Beta thalassemia is associated with bone abnormalities characterized by bone marrow expansion of the medullary cavity, and osteopenia with cortical thickening and trabecular coarsening. Good nutrition with adequate vitamins and trace elements intake, along with calcium and vitamin D supplementation, can increase bone density and prevent bone loss. Endocrine abnormalities should be monitored carefully and a thorough endocrine evaluation should be carried out yearly in every patients to detect subclinical endocrinopaties.

The role of the vitamin D receptor in regulating vitamin D metabolism: a study of vitamin D-dependent rickets, type II.

In vitro studies and animal experiments suggest that the production of 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] and 24,25-(OH)(2)D is reciprocally controlled by 1,25-(OH)(2)D. To investigate the role of the vitamin D receptor (VDR) in controlling vitamin D metabolism in humans, we studied 10 patients with vitamin D-dependent rickets type II due to a defective VDR. After a period of high dose calcium therapy, 7 of the patients had normal serum calcium, phosphorus, alkaline phosphatase, and plasma PTH levels (PTH-N), and 3 showed increased serum alkaline phosphatase and plasma PTH (PTH-H). Serum calcium, phosphorus, alkaline phosphatase, PTH, vitamin D metabolites, urinary calcium/creatinine, and renal phosphate threshold concentration were compared with unaffected family members that comprised the control group. Vitamin D metabolites were measured before and after an oral load of 50,000 U/m(2) cholecalciferol. Compared with the control group, 1,25-(OH)(2)D levels were significantly higher and 24,25-(OH)(2)D levels were lower in the PTH-N group and even more so in the PTH-H group. 1alpha-Hydroxylase (1-OHase) and 24-OHase activities were estimated by the product/substrate ratio. In the PTH-N group, 1-OHase activity was higher and 24-OHase activity was lower than in controls. In the PTH-H group, 1-OHase activity was even higher, probably due to an additive effect of PTH. Thus, 1,25-(OH)(2)D-liganded VDR is a major control mechanism for vitamin D metabolism, and PTH exerts an additive effect. Assessment of the influence of 1,25-(OH)(2)D shows reciprocal control of enzyme activity in man, suppressing 1-OHase and stimulating 24-OHase activity.

The hypothyroidism in an inbred kindred with congenital thyroid hormone and glucocorticoid deficiency is due to a mutation producing a truncated thyrotropin receptor.

Growth and function of the thyroid and adrenal glands are maintained and controlled by thyrotropin (TSH) and adrenocorticotrophic hormone (ACTH), respectively. The action of these trophic hormones requires the presence of functional TSH and ACTH receptors. We describe a large inbred Bedouin kindred in which profound congenital hypothyroidism and hypoadrenocortisolism occurred alone or together in eight family members belonging to four nuclear families. The high serum TSH and ACTH levels in the presence of normal or hypoplastic thyroid glands and low glucocorticoid, but not mineralocorticoid concentrations, are characteristic of resistance to TSH and ACTH. Linkage analysis, using specific polymorphic markers, excluded the involvement of the ACTH receptor but not thyrotropin receptor (TSHR). A novel point mutation was identified in exon 10 of the TSHR that replaces the normal cytosine in nucleotide 2024 with a thymidine. As a result the normal arginine in codon 609 (CGA) is replaced with a stop codon (TGA). This mutation produces a truncated TSHR lacking the third intracellular and extracellular loops, the sixth and seventh transmembrane segments, and the intracytoplasmic tail. The presence of hypothyroidism did not affect the timing, severity, and manner of clinical manifestation of hypoadrenocortisolism.

Evidence for oligogenic inheritance of type 1 diabetes in a large Bedouin Arab family.

Based on a genomic search for linkage, a locus contributing to type 1 diabetes in a large Bedouin Arab family (19 affected relatives) maps to the long arm of chromosome 10 (10q25; nonparametric linkage = 4.99; P = 0.00004). All affected relatives carry one or two high-risk HLA-DR3 haplotypes that are rarely found in other family members. One chromosome 10 haplotype, the B haplotype, was transmitted from a heterozygous parent to 13 of 13 affected offspring compared to 10 of 23 unaffected siblings. Recombination events occurring on this haplotype place the susceptibility locus in an 8-cM interval between markers D10S1750 and D10S1773. Two adjacent markers, D10S592 and D10S554, showed evidence of linkage disequilibrium with the disease locus. A 273-bp allele at D10S592 was transmitted to 8 of 10 affected offspring compared to 3 of 14 unaffected siblings, and a 151-bp allele at D10S554 was transmitted to 15 of 15 affected offspring compared with 10 of 24 unaffected siblings. D10S554 and D10S592 and the closest flanking markers are contained in a 1,240-kb yeast artificial chromosome, a region small enough to proceed with positional cloning.

Melatonin replacement corrects sleep disturbances in a child with pineal tumor.

A child with a germ cell tumor involving the pineal region had marked suppression of melatonin secretion associated with severe insomnia. Exogenous melatonin (3 mg in the evening) for 2 weeks restored sleep continuity, as demonstrated by objective monitoring of rest-activity cycles. This case report provides direct evidence of the essential role of melatonin in normal sleep.

Pseudo-precocious puberty in a male patient and the melatonin-testosterone relationship.

We describe a 14 year-old boy with a pineal germ cell tumor which secreted beta HCG. Serum testosterone levels were markedly elevated with concomitant decreased LH secretion. 24-h serum melatonin levels were suppressed and lacked the normal nocturnal rise. Pineal radiation therapy was followed by tumor regression and the diminution of beta HCG stimulated testosterone, which in turn inhibited melatonin and LH. When beta HCG and testosterone were normalized after tumor radiation, a recovery of normal melatonin and LH secretory pattern occurred. These results indicate that circulating testosterone down-regulates pineal melatonin.

Calcium therapy for calcitriol-resistant rickets.

Ten patients with calcitriol-resistant rickets caused by a defect in the ligand-binding domain of the vitamin D receptor are described. Eight patients, 1.7 to 13.8 years of age, received high doses of elemental calcium (range, 0.4 to 1.4 gm/m2) through indwelling intracaval catheters for periods of 1.8 to 3.8 years. Two other patients, aged 1.1 and 2.2 years, were given oral calcium therapy as the sole mode of treatment. In five of the intravenously treated patients, oral calcium therapy was initiated after radiologic evidence of healing of the rickets. To maintain normal serum calcium concentration, the patients required daily doses of elemental calcium of 3.5 to 9 gm/m2 body surface area. Clinical improvement was observed within a week of the start of intravenous therapy, with disappearance of bone pain; several of the younger patients started to walk for the first time. Growth velocity increased within 2 to 3 months, from a pretreatment rate of -0.8 to -6.3 standard deviation score (SDS), to a posttreatment rate of +0.1 to +5.1 (SDS). Serum calcium, parathyroid hormone, phosphorus, and alkaline phosphatase values returned to normal within a year. Radiologic signs of healing occurred more rapidly in the intravenous treatment groups and in younger patients. Episodes of septicemia occurred frequently in those receiving parenteral therapy and required replacement of the catheter. We recommend that in the treatment of calcitriol-resistant rickets, oral calcium therapy be started at the youngest possible age, in doses to the limit of intestinal tolerance. When rickets is present, calcium should be infused through a large vessel in doses high enough to produce normocalcemia, normophosphatemia, and suppression of parathyroid hormone. Only after radiologic healing has been observed can oral calcium therapy be introduced.

The molecular basis of hereditary 1,25-dihydroxyvitamin D3 resistant rickets in seven related families.

Hereditary 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant rickets (HVDRR) is an autosomal recessive disease caused by target organ resistance to the action of 1,25(OH)2D3, the active form of the hormone. The defect in target cells is heterogenous and commonly appears to be a mutation in the gene encoding the vitamin D receptor (VDR). We have studied cultured skin fibroblasts and Epstein-Barr virus transformed lymphoblasts of seven family branches of an extended kindred having eight children affected with HVDRR. We have previously shown that cells from three affected children in this group contain an "ochre" nonsense mutation coding for a premature stop codon in exon 7 within the steroid-binding domain of the VDR gene. In the current studies, we found that cells from affected children failed to bind [3H]1,25(OH)2D3 and had undetectable levels of VDR as determined by immunoblots using an anti-VDR monoclonal antibody. Measurement of VDR mRNA by hybridization to a human VDR cDNA probe showed undetectable or decreased abundance of steady-state VDR mRNA. Parents, expected to be obligate heterozygotes, showed approximately half the normal levels of [3H]1,25(OH)2D3 binding, VDR protein, and mRNA. The mutation at nucleotide 970 (counting from the mRNA CAP site) results in the conversion of GTAC to GTAA, which eliminates an Rsa I restriction enzyme site and facilitates identification of the mutation. We found that polymerase chain reaction (PCR) amplification of exons 7 and 8 from family members and subsequent Rsa I digestion allows detection of the specific genotype of the individuals. When Rsa I digests of PCR-amplified DNA are subjected to polyacrylamide gel electrophoresis, children with HVDRR exhibit a homozygous banding pattern with loss of an Rsa I site. Parents exhibit a heterozygotic DNA pattern with detection of both normal and mutant alleles. In summary, our data show that the genetic abnormality is a point mutation within the steroid-binding domain of the VDR in all seven related families with HVDRR. Analysis of restriction fragment length polymorphism at the 970 locus of PCR-amplified DNA fragments can be used to diagnose this mutation in both affected children and parents carrying the disease.