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Glucose production and oxidation were measured in ventilated preterm appropriate-for-gestational-age and small-for-gestational-age infants on the first day of life. Using a new technique of NaH13CO3infusion followed by a [U-13C]glucose infusion, we measured glucose oxidation rates without measuring the CO2production rate. Infants were studied at 18 ± 4 h (mean ± 1 SD) of life and received parenterally administered glucose only (4.2 ± 0.5 mg·kg−1· min−1). In 13 of 16 patients, the glucose production rate exceeded 1.0 mg·kg−1·min−1. Infants born from mothers who had been receiving steroids antenatally had higher glucose production rates (2.3 ± 1.1 mg·kg−1·min−1) compared with infants from mothers who had not (1.1 ± 0.8 mg · kg−1· min−1, p= 0.036). The glucose oxidized (2.9 ±1.0 mg · kg−1· min−1) was lower than the amount of glucose infused (p = 0.005) and was not different for appropriate-for-gestational-age and small-for-gestational-age infants. Plasma levels of glucose, insulin, glucagon, and total IGF-I were not correlated with glucose metabolism on the first day of life. Total IGF-II levels were negatively correlated with the rate of glucose appearance. We conclude that preterm infants on the first day of life receiving a glucose infusion of 4.2 mg · kg−1· min−1continue to produce glucose. The glucose oxidation rate is lower than the glucose infusion rate and the contribution of glucose oxidation to the total energy expenditure is limited.

Androgen insensitivity syndrome (AIS) is an X-linked disorder in which defects in the androgen receptor gene have prevented the normal development of both internal and external male structures in 46,XY individuals. This survey reports the analysis of 11 AIS subjects. The androgen receptor gene of these subjects was analyzed using polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis and sequencing or sequencing of PCR-amplified androgen receptor gene fragments alone. In total, 10 single base changes and one partial gene deletion were detected. Seven single base changes resulted in an amino acid change, one resulted in the introduction of a premature stop codon, one event represented a single base insertion resulting in a frame-shift, and one single base change affected a donor splice site. The androgen receptor protein in genital skin fibroblasts from several patients was studied with respect to molecular mass after immunoprecipitation and SDS-PAGE. Two patients expressed a truncated receptor protein in agreement with the established genomic mutation. Pedigree analysis was performed to identify possible carriers for the syndrome in families of AIS patients using single-strand conformation polymorphism and restriction site analysis of PCR products. In one case, the polymorphic (CAG)n(CAA) repeat in exon 1 encoding a polyglutamine stretch was used to identify the mutant allele in a family with X-linked partial androgen insensitivity before the identification of the actual genomic mutation. PCR-single-strand conformation polymorphism analysis proved to be a fast and reliable technique to screen for androgen receptor gene mutations and to study the androgen receptor gene of family members of AlS-affected individuals.

A procedure for the continuous flow incubation of isolated adrenal cells is described. In this way the advantages of continuous flow incubations of adrenal tissue are combined with those of isolated adrenal cells. Suspensions of isolated adrenal cells were prepared by a modification of the collagenase method. A sigmoid dose-response curve was obtained when these cells were incubated with ACTH in batch incubations. Under these conditions (in the presence of 1 mU ACTH/ml) the corticosterone production rate remained constant during at least 240 min. This production rate was linearly related to the number of cells. Pre-incubation of the cells during 3 h resulted in an increased response to ACTH. In continuous flow incubations without ACTH the corticosterone production was negligible. With 100 μU ACTH/ml corticosterone production increased sharply after a short lag period. A maximum was reached after 60–75 min followed by a slow decrease. Cells pre-incubated in the continuous flow apparatus had a slightly diminished ACTH response without loss of affinity to ACTH. The continuous flow incubation of isolated adrenal cells offers new possibilities for the dynamic study of steroid biosynthesis in vitro. The method may also be valuable to study processes in a wide variety of other tissues.

ABSTRACT: The aim of this longitudinal study was to examine vitamin D metabolism in exclusively breast-fed infants. The four common vitamin D metabolites—25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D [1,25(OH)2D], 24,25-dihydroxyvitamin D [24,25(OH)2D], and 25,26-dihydroxyvitamin D [25,26(OH)2D]—as well as vitamin D binding protein (DBP) were determined simultaneously in mothers and their children from delivery to several months of age. Maternal blood samples, drawn approximately 6 wk before the expected date of delivery, were also analyzed. At delivery, total vitamin D metabolites in maternal and fetal plasma were closely correlated, maternal levels being higher. Unbound (free) vitamin D metabolite concentrations were higher in fetal than in maternal plasma, with the exception of free 1,25(OH)2D levels, which were equal. This suggests a rapid placental transfer of 1,25(OH)2D. 24,25(OH)2D and 25,26(OH)2D levels both in mothers and children were closely correlated with the precursor sterol 25OHD. For 1,25(OH)2D, no correlation could be demonstrated with any of the other vitamin D metabolites. DBP concentrations in maternal plasma at the time of delivery were about twice the mean adult reference value. In cord blood, DBP levels were in the lower part of the adult reference range. Maternal total 1,25(OH)2D levels, which were twice the reference mean during pregnancy, fell sharply after delivery but free 1,25(OH)2D levels much less. Analogous to the biochemical changes in the mother, the infants' DBP levels fell after birth, as a result of the sudden disappearance of the estrogen stimulus. At the same time, the mineral supply via the placenta was cut off. These two factors are probably responsible for a stimulus to 1,25(OH)2D synthesis (and/ or inhibition of 1,25(OH)2D degradation), resulting in a sharp increase of 1,25(OH)2D and an even stronger increase of free 1,25(OH)2D. Concentrations of 25OHD and 1,25(OH)2D in breast milk were low. Such water-soluble metabolites as 25OHD-glucuronides were not detected. Judged by plasma 25OHD levels, the vitamin D stores of most children born to mothers with normal vitamin D status are depleted approximately 8 wk after delivery. Therefore, supplementation with an appropriate dose of vitamin D shortly after birth seems advisable, especially in winter.

Measurements of whole-body protein turnover in preterm infants have been made using different stable isotope methods. Large variation in results has been found, which could be due to different clinical conditions and/or the use of different tracers. We studied 14 appropriate for gestational age and nine small for gestational age orally fed preterm infants using [15N]glycine and [1-13C]leucine simultaneously, which allowed us to make a comparison of commonly used methods to calculate whole-body protein turnover. Whole-body protein turnover was calculated from 15N enrichment in urinary ammonia and urea after [15N]-glycine administration and from the 13C enrichment in expired CO2after administration of [1-13C]leucine. Enrichment of α-ketoisocaproic acid after [1-13C]leucine constant infusion was measured as a direct parameter of whole-body protein turnover. Group means for whole-body protein turnover using [15N]glycine or [1-13C]leucine ranged from 10 to 14 g.kg-1.d-1, except when using the end product method that assumes a correlation between leucine oxidation and total nitrogen excretion. We found very low 15N enrichment of urinary urea in the majority of small for gestational age infants. These infants also had a lower nitrogen excretion in urine and oxidized less leucine. Nitrogen balance was higher in small for gestational age infants (416 pm 25 mg±kg-1d-1) compared with appropriate for gestational age infants (374 pm 41 mg±kg-1d-1, p= 0.003). [15N]Glycine does not seem to exchange its label with the body nitrogen pool to a significant degree and is therefore not always suitable as a carrier for 15N in protein turnover studies in premature infants.

This study provides evidence that 22S-OH-cholesterol inhibits the conversion of 25-OH-cholesterol but has no effect on the conversion of 22R-OH-cholesterol. The latter sterol is an intermediate in the cholesterol side-chain cleavage, whereas for the conversion of 25-OH-cholesterol into pregnenolone the complete side-chain cleaving enzyme system is necessary. This complements a previous study in which it was shown, that 22S-OH-cholesterol has an inhibitory effect on the ACTH-induced conversion of cholesterol into corticosterone in isolated rat adrenal cells.The available evidence thus suggests an inhibition by 22S-OH-cholesterol of the first step in the cholesterol side-chain cleavage.The results, obtained from the experiments with rat adrenal cells and with bovine adrenal mitochondria, allow the hypothesis, that a causal relationship exists between conversion of 22S-OH-cholesterol and production of corticosterone, respectively pregnenolone.We conclude, that 22S-OH-cholesterol is a substrate for steroid production in the adrenal cell. This sterol inhibits the ACTH-stimulated corticosterone production. The site of this inhibition is located at one of the first steps in the cholesterol side-chain cleavage, probably the binding of cholesterol to the cytochrome P450-complex.

Several effects of hydroxylated sterols on cell cultures are known. Most of these can be explained by an inhibition of the cholesterol synthesis at the level of the 3-hydroxy-3-methylglutaryl CoA reductase.When studying cholesterol metabolism in rat adrenal cells, an inhibitory action of some sterols on the ACTH-stimulated corticosterone production was observed. The effects of one sterol, 22S-OH-cholesterol, were investigated further.The sterol had no effect on the ACTH-stimulated cyclic AMP production, suggesting an intact receptor-adenylate cyclase complex and cellular membrane. In the presence of ACTH and 22S-OH-cholesterol particularly the free cholesterol concentration was elevated; 22S-OH-cholesterol therefore probably exerts its inhibitory effect at a step located after hydrolysis of the cholesterol esters. 22S-OH-cholesterol had no effect on the conversion of exogenous pregnenolone into corticosterone.These results make it probable, that the inhibitory effect of 22S-OH-cholesterol on the ACTH-stimulated corticosterone production is situated at the level of the cholesterol side-chain cleavage.

Medium-chain triglycerides (MCTs) are included in the fat blend of several preterm formulas because of their complete absorption and rapid oxidation. The effects of two different fat blend compositions on calcium (Ca), phosphorus (P), and magnesium (Mg) balances and plasma levels and on plasma levels of parathyroid hormone (PTH), alkaline phosphatase (AP), and 1,25-dihydroxyvitamin D [1,25-(OH)2D] were investigated in 28 healthy very-low-birth weight infants at 4 weeks of age. A preterm formula with a traditional corn oil/MCT blend containing 38 MCTs (MCT group) was compared to a new fat blend, designed to resemble human milk more, containing 6 MCTs (LCT group). There was a higher absorption of Ca in the MCT group (73 vs. 60. p< 0.005), and an equal absorption of P (both 92). The excretion of Ca correlated with the excretion of fat (p< 0.00005). The LCT group showed a higher median PTH level (MCT: 2.1 pmol/L, LCT: 4.7 pmol/L, p< 0.01) and a higher urinary P excretion (p< 0.001). Mg absorption was also lower with LCT, but retention of Mg exceeded intrauterine values in both groups. Mineral plasma levels were in the normal range in both groups. AP was not different between groups and in the upper part of the reference range, whereas 1,25-(OH)2D levels were above the normal range and also not different between groups. We conclude that with the LCT formula, Ca absorption is slightly lower than with the MCT formula, whereas P absorption is unaffected. The resulting imbalance in absorption is compensated for by an increased urinary excretion of P. We conclude that current recommendations for the maximum Ca/P ratio in preterm formulas (2:1) might be too low in formulas containing only LCT.

Extract: Testosterone, both in a free and in a conjugated form, hydrolyzable by snail ß-glucuronidase and sulfatase, was extracted from the urine of children and measured by forming 14C-acetate derivatives. Administration of 3H-labeled testoterone permitted estimation of rates of production. Study of 10 normal prepubertal children (group A), varying in age from 8/12to 109/12years, yielded excretion values of 0.3–6.3 µg/24 h (mean 2.1 µg/24 h) and production rates of 16–880 µg/24 h (mean 274 µg/24 h). Mean ratio of excretion to production was 0.6%. A linear correlation between testosterone excretion and age could be demonstrated.These values were compared with those obtained from 13 children suffering from congential adrenal hyperplasia (CAH) caused by a deficiency of 21-hydroxylase (group B) and with those of 4 children with idiopathic precocious puberty (PP) (group C). In children of group B, increased andregenic activity could be demonstrated. Excretion values of testosterone varied from 11 to 261 µg/24 h (mean 98 µg/24 h). Mean production-to-excretion ratio was 0.6%. It could be calculated, however, that free testosterone secreted by the adrenals represented only a small percentage of the total production. By far the largest part was derived from two other adrenal hormones: androstenedione and dehydroepiandrosterone. In these cases, however, rates of testosterone production and excretion can still give a valuable indication of the total androgenic activity.Children in group C showed elevated production values as well: 540–1,900 µ/24 h (mean 1,180 µg/24 h). After receiving HCG (human chorionic gonadotropin) for 3 days (3,000 U/24 h) the range was 1,250–3,400 µg/24 h (mean 2,460 µg/24 h).Speculation: A linear correlation exists between rates of excretion of testosterone and age in prepubertal children. Very young children (less than 12 months of age) may have higher levels. Rates of production of testosterone in urine are more useful than is generally thought.

Enzyme preparations were isolated from post-mortem adrenal tissue of a patient with lipoid adrenal hyperplasia and normal human- and bovine adrenal tissue. Cholesterol-cleaving properties were studied in incubation experiments with the addition of appropriate substrates. Cholesterol-cleaving activity of enzyme preparations of adrenal tissue from the patient was extremely low as compared with normal controls. There was no difference in 20α-hydroxycholesterol-cleaving activity. There is thus evidence for a deficient 20α-cholesterol-hydroxylase activity in the adrenal tissue of this patient with lipoid adrenal hyperplasia.

Using isotope dilution methods the secretion rates of cortisol, aldosterone, deoxycorticosterone, corticosterone and 18-hydroxycorticosterone were determined in two brothers (76/12 and 108/12 years old) with dwarfism, chronic hypokalaemia alkalosis, tendency to hyponatraemia and normal blood pressure. Both patients showed a decreased response of the blood pressure to infused angiotensin.The urinary excretion of cortisol, cortisone, corticosterone, 11-dehydrocorticosterone and their tetrahydrometabolites as well as of aldosterone was determined. In general, results were in agreement with those of the secretion rate studies.The results suggest a relatively small mineralocorticoid excess (mainly corticosterone) to high plasma-renin concentrations. The differences in both the plasma-renin concentrations and the steroid secretory rates during low and high sodium intake indicate that the renin-angiotensin-mineralocorticoid system is still under regular control, although the normal regulatory process is severely disturbed.The primary defect in these patients is probably not a relative insensitivity of the arterial walls to the pressor effect of angiotensin as suggested by Bartter et al.(1962), but rather a renal tubular defect in the re-absorption of sodium.