Search

(1) In the present study we determined the effects of glutaric (GA, 0.01-1 mM) and 3-hydroxyglutaric (3-OHGA, 1.0-100 microM) acids, the major metabolites accumulating in glutaric acidemia type I (GA I), on Na(+)-independent and Na(+)-dependent [(3)H]glutamate binding to synaptic plasma membranes from cerebral cortex and striatum of rats aged 7, 15 and 60 days. (2) GA selectively inhibited Na(+)-independent [(3)H]glutamate binding (binding to receptors) in cerebral cortex and striatum of rats aged 7 and 15 days, but not aged 60 days. In contrast, GA did not alter Na(+)-dependent glutamate binding (binding to transporters) to synaptic membranes from brain structures of rats at all studied ages. Furthermore, experiments using the glutamatergic antagonist CNQX indicated that GA probably binds to non-NMDA receptors. In addition, GA markedly inhibited [(3)H]kainate binding to synaptic plasma membranes in cerebral cortex of 15-day-old rats, indicating that this effect was probably directed towards kainate receptors. On the other hand, experiments performed with 3-OHGA revealed that this organic acid did not change Na(+)-independent [(3)H]glutamate binding to synaptic membranes from cerebral cortex and striatum of rats from all ages, but inhibited Na(+)-dependent [(3)H]glutamate binding to membranes in striatum of 7-day-old rats, but not in striatum of 15- and 60-day-old rats and in cerebral cortex of rats from all studied ages. We also provided some evidence that 3-OHGA competes with the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting a possible interaction of 3-OHGA with glutamate transporters on synaptic membranes. (3) These results indicate that glutamate binding to receptors and transporters can be inhibited by GA and 3-OHGA in cerebral cortex and striatum in a developmentally regulated manner. It is postulated that a disturbance of glutamatergic neurotransmission caused by the major metabolites accumulating in GA I at early development may possibly explain, at least in part, the window of vulnerability of striatum and cerebral cortex to injury in patients affected by this disorder.

Even with current so called physiologic doses of glucocorticoid replacement therapy, children with congenital adrenal hyperplasia (CAH) often show relative short stature and delayed bone maturation, an observation that suggests possible long-term effects on bone metabolism of daily transient post-absorptive hypercortisolemia. In 28 patients with 21-hydroxylase or 17 alpha-hydroxylase deficiency (16 females and 12 males, ages 4.9-22 yr) who had received oral cortisol 10-15 mg/M2/day for 4.7-22 yr, we studied cortisol bioavailability, growth, bone maturation, vertebral bone mineral density, and various markers of bone formation and resorption. Patients were grouped according to mean on-therapy serum 170H-progesterone or progesterone levels as tight control (170HP < 10 nmol/L), fair control (170HP 10-40 nmol/L or progesterone 1.0-1.5 nmol/L), or poor control (170HP > 40 nmol/L). There was no difference in peak post-absorptive serum cortisol or area under the concentration-time curve, and only three patients had a peak serum cortisol of more than 700 nmol/L. There was no difference in present height Z-score (-0.96; -0.24; -0.6), height Z-score at age 2 yr (-1.5; +0.4; -1.3), or current growth velocity Z-score (-0.1; +1.2; -2.2) between the groups, but bone maturation Z-score was significantly delayed (-1.63) in the tight control group and advanced (+0.8) in the poor control group. Present height was highly correlated (r = 0.8) with height at age 2 yr. Serum calcium, phosphorus, alkaline phosphatase, parathormone, and 25OH-vitamin D levels were all normal. There was no difference between the groups in age-corrected vertebral bone mineral density, and no difference in serum osteocalcin, procollagen peptide, or collagen C-terminal telopeptide, nor in urinary amino-terminal telopeptide. The data suggest that current methods of cortisol replacement do not significantly influence bone formation, resorption or density during childhood and therefore should not contribute to adult osteoporosis. The possibility remains that hypercortisolemia during infancy produces the short stature and delayed bone maturation that are present by the age 2 yr.

Fatty acid ethyl esters are a family of non-oxidative metabolites of ethanol present in many tissues after ethanol consumption. In this report we demonstrate the existence in human liver of an acyl-CoA:ethanol acyltransferase activity which may be responsible in part for the synthesis of these compounds in vivo. The effects of oleoyl-CoA and ethanol concentrations, presence or absence of bovine serum albumin and detergent, pH and enzyme concentration on this activity have been determined. Acyl-CoA:ethanol acyltransferase activity is localised in the membrane-bound fraction. Using inhibitors directed against related enzyme activities, it has been shown that the activity is not related to serine-dependent carboxylesterases or acyl-CoA:cholesterol acyltransferase, but tht it may be associated with acyl-CoA hydrolase activity. We have also compared acyl-CoA:ethanol acyltransferase activity with fatty acid ethyl ester synthase activity in microsomes and cytosol from the same liver. Our data indicate that these activities are comparable in vitro (on a unit/g liver basis), and suggest that both may be significant in vivo.

Female adolescence is normally accompanied by increased adrenal and ovarian production of androgens. Indeed it is not uncommon in early to midpuberty to see typical features of adult polycystic ovary syndrome, with luteinizing hormone-driven ovarian hyperandrogenism, hyperinsulinemia, acne, anovulation, oligomenorrhea, and large, multifollicular ovaries. Unfortunately, no single prospective test can differentiate girls in whom this maturational stage is self-limited from those in whom it will progress to adult polycystic ovary syndrome with hirsutism and anovular infertility. An occasional hirsute adolescent will prove by corticotropin testing to have a nonclassical variant of adrenal 21-hydroxylase deficiency and will benefit from glucocorticoid therapy. The prevalence or even the existence of mild 11 beta-hydroxylase or 3 beta-hydroxysteroid dehydrogenase deficiency is more problematic. Given these difficulties of exact diagnosis and prognosis, therapy for the adolescent with mild hirsutism, acne, or oligomenorrhea should be conservative.

A 14-year-old boy was seen because of irritability, insomnia, lethargy, and profuse sweating, together with hypertension (blood pressure: 160/120 mm Hg), tachycardia, and a diffuse erythematous rash with desquamation of the palms and soles. Initial biochemical investigation suggested a diagnosis of pheochromocytoma, but subsequently a history of exposure to mercury vapor was obtained. This case emphasizes the clinical and biochemical similarities between mercury poisoning (acrodynia) and pheochromocytoma.

The gene for human P450(17 alpha) (CYP17) was previously mapped to chromosome 10 through analysis of somatic cell hybrids. Using a modified procedure of fluorescence in situ hybridization, this gene has now been visualized on simultaneously banded chromosomes and localized to a specific subband of chromosome 10 at q24.3. This precise assignment may facilitate the understanding of the molecular basis of 17 alpha-hydroxylase/17,20-lyase deficiency and the evolution of the CYP superfamily of genes.

The molecular defect in a reported case of isolated 17,20-lyase deficiency in a 46XY individual has been elucidated. The patient was found to be a compound heterozygote, carrying two different mutant alleles in the CYP17 gene. One allele contains a point mutation of arginine (CGC) to cysteine (TGC) at amino acid 496 in exon 8. The second allele contains a stop codon (TAG) in place of glutamine (CAG) at position 461 in exon 8 which is located 19 amino acids to the carboxy-terminal side of the P-450(17) alpha heme binding cysteine. COS-1 cells transfected with cDNAs containing one or the other of these mutations showed dramatically reduced 17 alpha-hydroxylase and 17,20-lyase activities relative to cells transfected with the wild type P-450(17) alpha cDNA. While the in vitro data in COS 1 cells can explain the patient's physical phenotype, with female external genitalia, it was somewhat discordant with the clinical expression of isolated 17,20-lyase deficiency with relative preservation of 17 alpha-hydroxylase activity in vivo. In addition to the expression studies of these two examples of mutants in the C-terminal region of cytochrome P-450(17) alpha, a third mutant cDNA construct containing a 4-base duplication at codon 480 previously found in patients with combined 17 alpha-hydroxylase/17,20-lyase deficiency was also expressed in COS-1 cells. This expressed protein was completely inactive with respect to both activities, supporting the biochemical findings in serum and in vitro biochemical data obtained using a testis from the patient. The results from these patients clearly indicate the importance of the C-terminal region of human P-450(17) alpha in its enzymatic activities.

17 alpha-Hydroxylase deficiency is characterized by defects in either or both the 17 alpha-hydroxylase/17,20-lyase activities. We have, for the first time, elucidated the molecular basis of the deficiency in a male pseudohermaphrodite with ambiguous external genitalia resulting from partial combined deficiency of both activities. The patient is found to be a compound heterozygote, carrying two different inherited mutant alleles in the cytochrome P45017 alpha (CYP17) gene. One allele, from his mother, contains a stop codon (TGA) in place of arginine (CGA) at amino acid position 239 in exon 4. Because this occurs at the N-terminal side of the heme binding sequence, the putative resultant truncated protein is nonfunctional. The second allele, from his father, contains a missense mutation encoding the substitution of proline (CCA) by threonine (ACA) at position 342 in exon 6. Reconstruction of this mutation by site-directed mutagenesis into human P45017 alpha cDNA followed by expression in COS 1 cells leads to the same amount of immunodetectable P45017 alpha protein as found with expression of the normal P45017 alpha cDNA, although both the 17 alpha-hydroxylase and 17,20-lyase activities are found to be reduced to 40-45% of those of the normal enzyme. The presence of ambiguous external genitalia in this 46 XY individual indicates that greater than 20% of the total normal 17,20-lyase activity is required for complete virilization in the male.

Allgrove syndrome (isolated glucocorticoid deficiency, achalasia and alacrima) was found in eight members of an inbred French Canadian/North American Indian pedigree. The high degree of consanguinity supports an autosomal recessive mode of inheritance for this disorder. Six patients presented with hypoglycaemia and other evidence of cortisol deficiency between 2.5 and 8 years of age; however, two others became cortisol deficient after initial testing showed normal cortisol responses to ACTH, evidence that the glucocorticoid insufficiency of this syndrome may not be congenital, but may develop as late as the third decade. No evidence of mineralocorticoid deficiency has been found during 65 patient-years of follow-up. Alacrima was the earliest and most consistent clinical sign of Allgrove syndrome. Other manifestations of peripheral or autonomic neuropathy were found in four patients. The patients showed similar facial features, and three had significant velo-pharyngeal incompetence. All showed oesophageal dysmotility even in the absence of symptomatic dysphagia. In-vitro studies of lymphocyte ACTH binding showed no differences from normal controls. If such lymphocyte binding, as has been suggested, reflects adrenal ACTH receptor activity, these data would suggest that the glucocorticoid deficiency of Allgrove syndrome is not the result of a defect in that receptor. However, the observation that ACTH does not elicit increased adenylate cyclase activity even in normal lymphocytes casts considerable doubt on the physiological significance of ACTH binding to lymphocytes. It seems likely, therefore, that true ACTH receptors are not expressed on peripheral lymphocytes, and any conclusions regarding a possible receptor defect in Allgrove syndrome must await studies of receptor expression on adrenal cell membranes.

A 2.8-year-old girl was investigated for early breast development and delayed psychomotor development. Chromosome analysis showed a fragile site at Xq27. Skeletal maturation was advanced. Computerized tomography of the head was normal. Pelvic ultrasonography showed ovaries and uterus enlarged for age. Basal serum gonadotropins were within the normal prepubertal range, but serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels rose during sleep and following stimulation by gonadotropin-releasing hormone (GnRH), consistent with true precocious puberty. The occurrence of precocious puberty in this girl, and macro-orchidism in affected males may be a reflection of an underlying disturbance of hypothalamic-pituitary-gonadal function in fragile X patients.

Studies using cultured bovine adrenocortical cells now demonstrate that the cytokines interleukin-1 (IL-1) alpha and beta, contrary to previous reports, can stimulate cortisol secretion in vitro in a dose- and time-dependent fashion. However detectable levels of IL-1 receptor could not be demonstrated in adrenal cortical, medullary, or capsular cells by membrane displacement of iodinated IL-1 alpha by unlabeled IL-1 beta, a technique that readily demonstrates specific IL-1 alpha-binding sites on 3T3 fibroblasts. The stimulatory effect of IL-1 on cortisol secretion could be totally blocked by indomethacin, an indication that this effect might be mediated indirectly via local release of prostaglandins (PGs). Subsequent investigations have confirmed that IL-1 does enhance the conversion of [3H]arachidonate to PGs by cultured adrenal cells, and that some of these PGs (PGD2, PGF2 alpha, and PGE2), in turn, can stimulate cortisol production. Taken together these observations suggest that IL-1-induced stimulation of cortisol secretion is mediated through local release of PGs by a small subpopulation of cells within the adrenal gland.

We have analyzed the lower leg growth using a knemometer and the height growth using a stadiometer of 90 healthy children aged 3-16 years, for one year. The intra- and inter-individual monthly lower leg growth varied up to 4-fold, which was not accounted for by age or sex. The correlation between short term and annual lower leg growth rates increased with longer observation periods. There was no month-to-month consistency in the ratio of lower leg growth and height growth. There was no correlation between 1 month lower leg growth and annual height growth. The correlation increased with time. The 6-month observation interval was the interval with the highest predictive value for annual lower leg growth (R2 = 0.727) and annual height growth (R2 = 0.732). We conclude that growth of different parts of the skeleton and variable interval growth rates limits the ability of knemometry to predict long term growth.

Changes in gonadotropins, progesterone, cortisol, DHA, and DHAS were monitored in 10 female rhesus monkeys (Days 20-23 of the menstrual cycle) subjected to cage restraint with or without ketamine anesthesia for successive venipunctures. All animals were bled without sedation for 2 hr at 30-min intervals. Then 4 of the animals were anesthetized with ketamine-HCl and bleedings in all animals were continued for an additional 2.5 hr. FSH and progesterone were not appreciably affected by either restraint technique. LH declined steadily for the duration of the bleedings (P less than 0.05). Serum levels of cortisol and the adrenal androgens increased twofold (P less than 0.05). Anesthesia with ketamine had no effect on any of the six variables when compared with saline controls. Cortisol and dehydroepiandrosterone (DHA) levels tended to plateau (P less than 0.01) after 2 hr in both treated and control groups. In contrast, dehydroepiandrosterone sulfate (DHAS) levels increased continuously throughout the entire study period. These data indicate that ketamine anesthesia does not alter endocrine responses to venipuncture when administered following cage restraint of conscious animals. These findings further confirm the difficulties in obtaining estimates of basal levels of hormones which are responsive to stress and suggest that the first sample may provide the best estimate.

Mixed cord sera (27 male, 28 female) and sera from 105 male and 93 female children aged 5 days to 4 yr were assayed for FSH, LH and hCG. Cord hCG was similar in both sexes (median 58 mIU/ml; range 20-9000), and fell to less than 5 mIU/ml by 5 days of life, a value which is below the limit of detectable cross reactivity in the LH radioimmunoassay. Cord FSH was less than 5.5 mug LER-907/100 ml in both sexes. In boys there was a rapid rise of FSH in early postnatal life, with peak levels up to 55 mug/100 ml between 1 week and 3 months, followed by a decline by 4 months reaching the low values seen in older prepubertal subjects. This postnatal FSH rise was both more marked in females with peak values at 2-3 months up to 169 mug/100 ml, and also more sustained with levels staying above those of older prepubertal children until 4 yr of age. Serum LH levels in the boys were in the adolescent range by 1 week of age, peaked at 1 month and then declined to the usual childhood range by 4 months. A similar pattern, though with lower peak LH values, was seen in the female infants. A longitudinal study of serum FSH and LH values in one male and one female chimpanzee from 17 to 456 days of age showed patterns in serum gonadotropins which paralleled those seen in the human cross-sectional study.

Serum concentrations of dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHA-sulfate), cortisol, prolactin, and growth hormone were measured at half-hour intervals for 24 hr in five healthy children aged 8--13 yr. Their adolescent development was assessed by clinical staging, plus determinations of serum FSH, LH, testosterone and estradiol during both wakefulness and sleep. Correlative analysis indicates that there was synchronous secretion of DHA and cortisol, implying regulation of both by ACTH. With advancing age and sexual maturation, there was a progressive rise in mean serum DHA and DHA-sulfate levels, but no similar change in serum cortisol concentrations. There was evidence for enhanced secretion of both growth hormone and prolactin during sleep in all subjects (including one who was hyperprolactinemic), but there was no obvious relationship between levels of these pituitary hormones and the early pubertal rise in adrenal androgens.

This paper details the experiences of the Princess Mary's Royal Air Force Hospital, Akrotiri (TPMH), during the last 6 months of 1974. TPMH was at that time a small hospital (120 beds) with a clinical staff of 17 situated in the south of Cyprus (fig. 1). The series comprises 71 patients injured by bullets, shrapnel, bomb-blasts or mines, drawn from British personnel, United Nations Forces and the local Cypriot military and civilian population, resulting from the coup d'état of 15 July 1974 and the subsequent Turkish invasion and air attacks. The casualties were often severely wounded beyound the capabilities of the local hospitals. They were mostly admitted during a 2-week period, but many required multiple operations extending over the next 3 or 4 months, and altogether 119 operations were carried out on 41 patients at TPMH. The organization of the limited resources of the medical, nursing and supporting staff is outlined. The correct treatment of missile wounds is emphasized. Resuscitation, wound excision, splintage and delayed suture or grafting are essential. Minimal morbidity and mortality are gained by a practised approach to the compounded results of violence.

Simultaneous measurement of serum concentrations of estrone (E1), estradiol (E2), estriol (E3), and progesterone were carried out in multiple serial blood samples obtained during the last 3-10 weeks of pregnancy, labor, and the immediate postpartum period in 5 normal women. Estrogen and progesterone levels showed a small, but statistically significant diurnal variation during pregnancy. They did not change during labor; however, with the exception of E1 levels, all declined following delivery. Individual patterns preceding labor, derived from calculated moving mean values, showed no consistent decline in progesterone levels nor a surge in E1 and E2 concentrations whereas estriol levels showed a steady rise starting 14-28 days prior to the onset of labor. Whether this E3 elevation reflects fetal maturation and/or plays a role in the triggering mechanism of labor is unknow. Failure to detect changes in E1, E2, and progesterone levels in the maternal peripheral circulation does not preclude the possibility that alterations of metabolism of these hormones in the fetal or uterine compartments might be involved in the initiation of human labor.

The effects upon production of cortisol and dehydroepiandrosterone (DHA) by human fetal adrenal cells in tissue culture were studied using commercial hCG (0.5 and 5 IU/ml), purified hCG (0.7-6.7 IU/ml), the alpha-subunit of hCG (200 and 1000 ng/ml), human GH (50 and 200 ng/ml), human PRL (0.1-100 ng/ml), alpha-MSH (0.1-10 ng/ml), corticotropin-like intermediate lobe peptide (200 ng/ml), human beta-lipotropin (0.1 and 0.2 ng/ml), and beta-endorphin (100 ng/ml). Although each peptide was added to the culture medium in a concentration either similar to that observed in the fetal circulation or (where such information was not available) in amounts several times greater than those effective for ACTH in this system, none demonstrated any significant stimulation of steroid production. In particular, repeated studies with hCG showed that this hormone had no stimulating effect upon DHA production, neither in cultures of whole adrenals nor in cultures of separated fetal zone and definitive zone cells. Furthermore, none of these peptides showed a synergistic effect upon DHA production when they were added to cultures together with concentrations of alpha-ACTH-(1-24) (10(2)-10(3) pg/ml) previously demonstrated to represent the middle of the dose-response curve. Indeed, the only significant interactions with alpha-ACTH-(1-24) observed in these studies were a slight reduction in cortisol production produced by corticotropin-like intermediate lobe peptide and apparent inhibition of DHA production by beta-lipotropin and GH. The data do not lend credence to the suggestion that any of these peptides plays an important role in vivo in stimulating fetal adrenal steroidogenesis.

Using the Valk knemometer, lower leg length (LLL) was assessed relative to changes in the positioning of the upper leg. Lowering the chair height of the knemometer resulted in a more acute angle between the upper and lower leg and a decrease in LLL. This decrease in measurement was attributed to changes in the anatomical surface of the knee underlying the measuring platform as a result of increasing the acuity of the leg angle. Based on four different leg positions, the average change in LLL per centimeter change in chair height was 0.607 mm in a child sample of 50, and 0.655 mm in an adult sample of 20. The difference in chair height with the leg angle at 90 degrees and the lowest chair height possible, ranged from 12.3 to 30.3 mm, relative to lower leg length. This meant the longest leg in the study had a LLL measurement differing by 19.8 mm between these two positions. Due to the effect of leg position, we advised the use of a standard method of measuring LLL with respect to leg angle. Given the difficulties in accurately measuring leg angle with current available tools, we advise the most acute angle.

In order to assess a possible influence of endogenous opioids upon gonadotrophin secretion in women, we examined the effects of i.v. administration of 10 mg naloxone, a specific opiate antagonist, in ten normal menstruating women, in thirteen women with amenorrhoea and/or hyperprolactinaemia and in two women with putative deficiency of gonadotrophin-releasing hormone (GnRH). In thirteen subjects, a saline vehicle control study (randomized order of administration) was also performed. In the normal women, naloxone failed to elicit changes in serum gonadotrophin levels when administered during the early follicular phase of the menstrual cycle. However, significant increments of LH were observed from 30 to 165 min following naloxone administration during the late follicular phase. Similar LH responses occurred in the amenorrhoeic and hyperprolactinaemic women. There was a tendency towards a concomitant increment in FSH levels, which reached statistical significance variably from 60 to 105 min post-naloxone. The LH response to naloxone in individual subjects showed a significant (P less than 0.01) quadratic (U-shaped) relationship to the log basal oestradiol concentration. No response to naloxone was observed in the two patients with GnRH deficiency despite a brisk response to an exogenous GnRH bolus. Taken together, these data suggest that central nervous system inhibitory opioid pathways may be involved in the regulation of LH secretion in normal women and that excessive production of endogenous opioids may play a role in the pathophysiology of some amenorrhoeic conditions.

Heterologous double-antibody radioimmunoassay methods are described for the measurement of circulating levels of rhesus monkey (Macaca mulatta) FSH and LH; the latter assay is also applicable to rhesus chorionic gonadotropin (CG) estimations. The FSH assay utilizes purified rat FSH for trace, either of two anti-human FSH antisera and a semipurified rhesus pituitary standard. The LH assay utilizes purified ovine LH for trace, an anti-human CG antiserum and the same rhesus pituitary standard. The use of these systems obviates the necessity of purifying rhesus gonadotropins which are required for the development of homologous radioimmunoassay systems.

Concentrations of human chorionic gonadotropin (HCG) and progesterone were measured in the peripheral sera of 101 normal pregnant women between 25 and 41 weeks' gestation. HCG levels rose significantly with advancing gestation in the 43 female-bearers (r equals 0.516, p less than 0.001), whereas the 58 male-bearers showed no change (r equals 0.168, p greater than 0.1). Mean HCG levels were significantly higher in female- than in male-bearers (10.7 plus or minus standard error 1.0 versus 8.0 plus or minus 0.9 International Units per milliliter; p less than 0.05). Progesterone levels rose significantly in both female- and male-bearers. The calculated regression lines and mean levels (female-bearers 9.1 plus or minus 0.5; male-bearers 9.8 plus or minus 0.4 mug per deciliter) were not significantly different. There was no correlation between HCG and progesterone levels in either sex or in the entire group independent of gestational age. It is postulated that the lower HCG levels observed at term in male-bearers may result from an inhibitory influence of the higher progesterone and/or androgen concentrations in the male umbilical arterial circulation.

A technique of monolayer tissue culture of human fetal adrenal cells was developed in order to study steroidogenic responses to factors such as ACTH. The daily production of 12 steroids [pregnenolone, 17-hydroxy pregnenolone, dehydroepiandrosterone (DHA), DHA sulfate, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone, corticosterone, 11-desoxycortisol, cortisol, and aldosterone) was measured by RIA. Initially, fresh fetal adrenal cells produced DHA, DHA sulfate, 17-hydroxypregnenolone, and small amounts of cortisol, but in the absence of ACTH, the production of all steroids declined during culture to low levels. The addition of physiological amounts (1-10(4) pg/ml) of either alpha ACTH-1(1-24) or alpha ACTH-(1-39) or coculture with fetal pituitary cells elicited a progressive rise in steroid production during the first 4-6 days of incubation. The lowest ACTH doses elicited a proportionately greater adrenal androgen response (as reflected in the DHA to cortisol ratio), but with increasing ACTH dosage, there was greater stimulation of cortisol production, which equalled or exceeded that of DHA. The data demonstrate that fetal adrenal cells may be maintained in short term culture and can respond to physiological amounts of ACTH. The progressive increase in the production of cortisol and other delta 4, 3-ketosteroids in vitro suggests that the characteristic fetal pattern of steroidogenesis may result from the interaction of ACTH with some circulating inhibitor of adrenal 3 beta-hydroxysteroid dehydrogenase.

Serum concentrations of 17OH-progesterone were studied serially over 24 hours in 13 treated and untreated patients with the C21 hydroxylase form of congenital adrenal hyperplasia. The results were correlated with measurements of plasma renin activity, serum electrolytes, and urinary 17-ketosteroids and pregnanetriol. In 500 healthy subjects from birth to adult life, serum 17OH-pregesterone levels ranged from 5 to 315 ng/dl. In untreated CAH, serum 17OH-progesterone was markedly elevated (2,000 to 80,000 ng/dl). Treatment with cortisol (20 to 30 mg/m2/day in 3 doses) resulted in normal serum 17OH-progesterone levels in both non-salt-losing and salt-losing patients receiving adequate mineralocorticoid. Even slightly inadequate mineralocorticoid therapy (shown by high plasma renin activity with normal serum electrolytes) was associated with elevated 17OH-progesterone (to 65,000 ng/dl) in spite of usually effective doses of cortisol. Some patients showed isolated 17OH-progesterone elevations (usually early morning), a situation which requires only revision of the cortisol dosage schedule without an increase in total dosage. The data confirm the value of 17OH-progesterone assays in both the diagnosis and management of CAH. Taken together with determinations of plasma renin activity, serum 17OH-progesterone assays can permit more exact control of CAH without excessive doses of glucocorticoid.

Prolactin concentrations were measured in 161 amniotic fluid specimens from 8-40 weeks fetal age and the levels compared with those observed in 45 fetal and neonatal cord sera and in 42 fetal pituitary specimens. Amniotic fluid prolactin levels rose steeply between 12-16 weeks gestation, and then declined to term; the calculated total amniotic fluid content of prolactin showed a similar pattern, but the peak was later, at about 26 weeks gestation. Amniotic fluid concentrations consistently exceeded fetal serum prolactin levels, even during the last trimester, when fetal serum and pituitary levels were highest. The data are compatible with a fetal origin for amniotic fluid prolactin, but only if one assumes that flux of prolactin out of amniotic fluid compartment is negligible, that the fetal kidney in mid-pregnancy clears prolactin at a rate virtually equal to the glomerular filtration rate, and the fetal pituitary shows secretion characteristics quite different from those of the adult gland.

To study the effect of 1-deamino-8D-arginine vasopressin (DDAVP) on the factor VIII response in nephrogenic diabetes insipidus (NDI), 0.30 microgram/kg DDAVP was given to 2 unrelated NDI patients, 3 obligate carriers, and 20 controls. Factor VIII coagulant activity (FVIIIC) and factor VIII related antigen (FVIIIR:Ag) responses were absent in both NDI patients and were decreased by approximately 50% in the carriers by comparison with controls. These results show that the vasopressin receptor defect in NDI is not confined to the kidney but is equally expressed in other tissues including the vascular endothelium and hepatic sinusoids, the respective sites of FVIIIR:Ag and FVIIIC production. A decreased factor VIII response may help in identifying carriers in families at risk.

Pituitary gonadotropin reserve was evaluated in 8 normal pregnant women (13-35 weeks gestation) by measuring serum concentrations of FSH and LH (betaLH assay) before and after an IV bolus of 100 mug LHRH. Basal levels of FSH and LH were low or undetectable. LHRH administration failed to stimulate FSH release but did result in a small short-lived rise in LH levels. These findings provide further evidence that pituitary gonadotropin synthesis and release are inhibited during pregnancy.

Steroid 17 alpha-hydroxylase (cytochrome P-450 17 alpha) catalyzes both 17 alpha-hydroxylation of pregnenolone and progesterone and 17,20-lysis of 17 alpha-hydroxypregnenolone and 17 alpha-hydroxyprogesterone. In the course of undertaking detailed investigation of the structure-function relationships which exist within this enzyme we have begun to elucidate the molecular basis of human deficiencies in either or both of these activities. Consequently we have determined the exonic structure of the human P-450 17 alpha gene as well as the sequences at the exon/intron boundaries and at the site of initiation of transcription. A single gene in the human genome encodes this protein, being the sole member of a unique gene family (P450XVII) within the P-450 supergene family. A protocol for exonic sequencing of the P-450 17 alpha gene has been established which permits structural analysis of the gene from patients having 17 alpha-hydroxylase and/or 17,20-lyase deficiency. This procedure has been applied to the mutant gene from one individual having combined 17 alpha-hydroxylase/17,20-lyase deficiencies. A four-base duplication is found in exon 8 producing a protein with an altered C-terminal amino acid sequence which results in loss of both enzymatic activities.

Two sisters (28 and 30 years) were investigated for primary infertility and milk hirsutism. Both had normal puberty, were having regular menses and had normal female sexual characteristics. Studies revealed elevated urinary 17-ketosteroid levels (15.8, 18.8 mg/24 hours) and increased serum levels of 17-OH-progesterone (2,756, 1,121 ng/dl), 21-desoxycortisol (1,882, 1,090 ng/dl), progesterone (300, 346 ng/dl), dehydroepiandrosterone (DHA) (1,600, 1,700 ng/dl), and androstenedione (402, 366 ng/dl) and testosterone (100, 104 ng/dl), together with a slight increase in serum 11-desoxycortisol (1,180, 1,560 ng/dl). Blood pressure, serum sodium/potassium plasma renin and serum aldosterone, corticosterone, 11-desoxycorticosterone and cortisol levels were normal. The administration of ACTH caused a further increase in 21-hydroxylase precursors; the administration of dexamethasone normalized hormone levels and produced ovulatory cycles. Similar studies in two siblings were normal. The affected sisters were HLA identical and did not share any HLA antigens with their healthy siblings. The data suggest that these patients have a mild form of 21-hydroxylase deficiency which was insufficient to cause prenatal virilization. The gene for this disorder may be allelic with that for typical congenital adrenal hyperplasia.

Preparations of dispersed human fetal adrenal cells from the inner third of the gland and from the subcapsular area were maintained in culture, and their ultrastructure and steroid production were studied. The former type of preparation contained only fetal zone cells, while the latter contained definitive zone cells together with varying numbers of fetal zone cells. Both types could be cultured with equal ease, but during short term culture, fetal and definitive zone cells became morphologically indistinguishable. The patterns of steroid production and, in particular, the relative production of delta 4,3-ketosteroids and delta 5,3 beta-hydroxysteroids were similar in both preparations, as were their dose-response relationships during incubation with alpha ACTH-(1-24). Although considerable variability in total steroid production was observed between cells from different adrenal glands, in no specimen was any evidence for functional zonation of the fetal adrenal cortex observed in vitro. The results suggest that the apparently unique histological appearance and function of the fetal adrenal cortex may only reflect intense stimulation by ACTH secondary to the combined influences of a rapid cortisol MCR and of some inhibitor of fetal adrenal 3 beta-hydroxysteroid dehydrogenase activity.

The inhibitory effects of varying concentrations of steroids upon 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) kinetics were studied in human adrenal microsomes. Each enzyme assay was conducted in triplicate at five different concentrations of three substrates (dehydroepiandrosterone, pregnenolone, and 17OH-pregnenolone), using microsomes from at least three donors. Each steroid was screened for possible inhibition at concentrations of 10(-8) and 10(-6) M and then studied in more detail at five different concentrations. The type of inhibition and the inhibition constant (Ki) were determined by analysis of Lineweaver-Burk and Dixon plots, together with replots of the slopes from the Dixon plots. The mean Km (Michaelis-Menten constant) for the three substrates was 0.42 +/- 0.04 (SE) mumol/liter (n = 73). Each steroid tested, including delta 5-3 beta-hydroxysteroids, estrogens, and several delta 4-3-ketosteroids, with the exception of cortisol, caused significant inhibition of 3 beta-HSD activity, and in each case the steroid appeared to behave as a competitive inhibitor. In most cases the Ki value was approximately 10(-7) M. At micromolar concentrations several steroids, notably estrone and estradiol, caused almost total inhibition of adrenal 3 beta-HSD activity. Comparison of the calculated Ki values with available data concerning changes in intra-adrenal steroid concentrations during childhood suggests that these changes would be sufficient to cause a relative decline in 3 beta-HSD activity during adrenarche. Although postnatal circulating steroid concentrations would appear to be insufficient to influence adrenal steroidogenesis, the high serum levels of placental steroids during fetal life would be expected to cause marked 3 beta-HSD inhibition.

Serum determinations of follicle-stimulating hormone and luteinizing hormone have been carried out in 13 prepubertal and adult patients who had been treated with courses of either oral or intravenous cyclophosphamide. All results were within the normal range for the patients' ages and sexual development. Although these results establish that gonadal endocrine function and pituitary-gonadal feedback relations may not be destroyed by cyclophosphamide, the possibility remains that prolonged cyclophosphamide therapy in young patients will result in some impairment of future fertility.

Concentrations of unconjugated dehydroepiandrosterone, estradiol, and estriol were measured in samples of amniotic fluid from uneventful pregnancies of 9-40 weeks conceptual age. There was no apparent influence of fetal sex upon the levels of these steroids. Dehydroepiandrosterone concentrations rose slightly from 9-20 weeks, and then showed little further change. Estradiol concentrations declined slightly from 9-20 weeks; after 32 weeks gestation, there was a 2-fold rise to term. Estriol levels rose in almost exponential fashion throughout gestation.