Benefit of early commencement of growth hormone therapy in children with Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a chromosomal disorder and growth failure is a common presentation. Growth hormone (GH) treatment is beneficial in PWS although the optimal age for starting GH is unknown. We investigated whether GH response in PWS was associated with the age of GH commencement by comparing 16 children who commenced GH before 3 years of age (early group) with 40 children who commenced GH after 3 years of age (late group) from the Ozgrow database. Height SDS, body mass index (BMI) SDS, bone age (BA)-chronological age (CA) ratio, change in height (delta Ht) SDS and change in BMI during 4 years of GH treatment were compared between the groups. The early group had better height SDS and delta Ht SDS. BA delay was more pronounced in the early group but BA did not mature beyond CA with GH therapy in either group. Although the initial GH dose for the early group was lower than that of the late group, the former had better height outcome. The starting GH dose seen in the database is lower than the dose used by international centres.

21-hydroxylase genotyping in Australasian patients with congenital adrenal hyperplasia.

Mutations in CYP21 (21-hydroxylase) lead to congenital adrenal hyperplasia (CAH). We genotyped 26 probands with CAH by PCR-sequencing the entire CYP21 gene. 25/26 had homozygous or compound heterozygous mutations. The frequencies of mutations were similar to other populations with deletion/hybrid, I2 G splice and I172N the most common. Five patients with a I172N allele predicting simple-virilising CAH had a salt-wasting phenotype. Two other probands also had a more severe phenotype than predicted by genotype. Two families had both non-classic and salt-wasting phenotypes arising from combinations of three deleterious alleles. Two novel CYP21 alleles were detected: D106N and a large deletion encompassing CYP21 and adjacent pseudogene. Two rare CYP21 alleles were also found. Three of these four novel/rare alleles were only detected as a result of sequencing the entire CYP21 gene. Entire CYP21 sequencing will increase the number of mutations detected in CAH, and in combination with functional studies should contribute a greater understanding of phenotype-genotype correlations.

Alanine in HI: a silent mutation cries out!

It is a widely held paradigm in molecular biology that a change in the third base of a codon is silent in terms of expression. In this investigation, results are presented that challenge that paradigm, at least in terms of one polymorphism in KCNJ11, which is one of five genes that have been implicated in the disorder Hyperinsulinism of Infancy. In two cohorts of Australian patients, an uneven distribution of KCNJ11 SNP's was observed. A silent polymorphism at codon 190 was over-represented in the patients who responded well to medical treatment and under-represented in those that required radical surgical intervention. In an attempt to investigate this polymorphism, it was expressed in vitro and western blot analysis showed that there were virtually no bands from the homozygous variant samples, while strong bands were seen in normal controls. The human genome is highly redundant in terms of tRNA species for each amino acids but enigmatically under-represents a number of specific codons. The polymorphism in question occurs within one such codon. We propose that the presence of a base change at the third position of codon that is not represented by a corresponding anti-codon within the human nuclear tRNA leads to a decreased rate of expression of the protein.

Androgen receptor genotyping in a large Australasian cohort with androgen insensitivity syndrome; identification of four novel mutations.

We genotyped the androgen receptor (AR) gene in 31 Australasian patients with androgen insensitivity syndrome (AIS). The entire coding region of AR was examined including analysis of polymorphic CAG and GGN repeats in all patients. AR defects were found in 66.7% (6/9) of patients with complete AIS (CAIS) and 13.6% (3/22) of patients with partial AIS (PAIS). A novel deletion (N858delG) leading to a premature stop codon was found in CAIS patient P1. CAIS patient P2 has a novel deletion (N2676delGAGT) resulting in a stop at codon 787. These mutations would result in inactivation of AR protein. A novel insertion of a cysteine residue in the first zinc finger of the AR DNA-binding domain (N2045_2047dupCTG) was found in CAIS patient P3. PAIS patient P4 has a novel amino acid substitution (Arg760Ser) in the AR ligand binding domain, which may impair ligand binding. Five patients were found to have previously reported AR mutations and no mutations were identified in the remaining patients.

The role of ATP sensitive channels in insulin secretion and the implications in persistent hyperinsulinemic hypoglycaemia of infancy (PHHI).

Persistent Hyperinsulinemic Hypoglycaemia of Infancy (PHHI) is a metabolic syndrome of unregulated insulin secretion. It is a heterogenous disease with causes linked to mutations of the ATP sensitive potassium channels of the beta cell, as well as to metabolism in the beta cell. 5 candidate genes--ABCC8, KCNJ11, GCK, GLUD1 and SCHAD have been implicated in the disease so far, however the aetiology of the disease remains unknown in up to 50% of all patients. We genotyped 43 subjects with PHHI (20 surgically treated and 23 medically treated) for disease associated mutations in the candidate genes. Mutations on ABCC8 were identified in 16 of the 20 (80%) of the surgically treated patients. One putative mutation was identified in the medically treated cohort. The polymorphism E23K on KCNJ11 that is associated with NIDDM was differentially distributed in the 2 cohorts. We discuss the mutations identified, emphasise the importance of the K-ATP channel in physiological processes and discuss the possibility that the disease is caused by mutations in other genes associated with insulin release, glucose metabolism in the beta cell or beta cell apoptosis and survival. We propose that these processes must be explored in order to further our understanding of PHHI.

Parental pre-pregnancy BMI is a dominant early-life risk factor influencing BMI of offspring in adulthood.

OBJECTIVE: We examined parental and early-life variables in order to identify risk factors for adulthood overweight and obesity in offspring. We report here on the longitudinal prevalence of overweight and obesity in Australian children born between 1989 and 1991 and followed from birth to age 22. METHODS: Data were analysed on 1355 participants from the Western Australian Pregnancy Cohort (Raine) Study, with anthropometry collected during pregnancy, at birth, one year and at three yearly intervals thereafter. Multivariate analyses and cross-sectional logistic regression quantified the timing and contribution of early-life risk factors for overweight and obesity in young-adulthood. RESULTS: At five years of age 12.6% of children were overweight and 5.2% were obese. By early adulthood, the prevalence of obesity had increased to 12.8%, whilst overweight remained relatively stable at 14.2% (range from early childhood to adulthood 11-16%). Parental pre-pregnancy body mass index (BMI) was the strongest determinant of adult offspring BMI. Although rapid first year weight gain was associated with increased offspring BMI, the impact of first year weight-gain diminished over childhood, whilst the impact of parental BMI increased over time. CONCLUSIONS: Parental pre-pregnancy BMI and rapid early-life weight gain predispose offspring to obesity in adulthood.

Regulation of the growth hormone-independent growth factor-binding protein in children.

Regulation of the diurnal variation of the GH-independent insulin-like growth factor-binding protein (BP-28) was studied in 53 children who underwent various investigations for possible endocrine abnormalities. The plasma BP-28 levels increased 12-fold from 8 +/- 2 (+/-SE) micrograms/L at 2100 h to a peak level of 109 +/- 15 micrograms/L between 0600 and 0800 h. This rise was inversely related to plasma insulin levels and was unrelated to plasma cortisol levels. The overnight rise of plasma BP-28 was significantly altered in children who ate a light meal at 0130 h; in them BP-28 levels started to fall after 0300 h, reached nadir levels at 0400 h, began to rise again by 0700 h, and returned to control levels by 0800 h. Such changes did not occur in children given water alone. From the peak early morning level, plasma BP-28 fell to basal levels in children given oral glucose at 0800 h; the t1/2 of the fall was 55 +/- 9 (+/-SE) min. In children who continued to fast, plasma BP-28 did not fall but, rather, increased from 144 +/- 12 micrograms/L at 0800 h after 10 h of fasting to 239 +/- 30 micrograms/L by 1600 h. The induction of hypoglycemia by insulin given at 0945 h after an overnight fast caused a similar but more rapid rise in plasma BP-28 to 668 +/- 317 micrograms/L (range, 208-1763 micrograms/L) by 1230 h. These results suggest that the diurnal variation of plasma BP-28 concentrations in children is not due to an intrinsic rhythm, but is regulated by the metabolic status of the child.

The insulin-like growth factor (IGF)-binding proteins and IGF bioactivity in Laron-type dwarfism.

Laron-type dwarfism (LTD) is caused by a variable defect in the GH receptor gene and is, therefore, an ideal model to study the physiology of the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in the complete absence of GH action. In this study we examined the overnight variation of the IGFs, IGFBPs, and IGF bioactivity in two prepubertal subjects with LTD. Subject 1 was a 14-yr-old female, 103 cm tall (-8.3 SD), and subject 2 was a 11.5-yr-old male, 103.6 cm tall (-5.9 SD). Both had serum IGF-I levels below 0.07 U/mL and low constant serum IGF-II levels overnight (185 +/- 10 and 232 +/- 8 micrograms/L), despite high serum GH levels [mean GH, 65 (32.5 micrograms/L) and 53 mU/L (26.5 micrograms/L)]. Serum IGFBP-1 levels increased overnight (from 24 and 22 micrograms/L at 2000 h to 83 and 110 micrograms/L at 0800 h) as serum insulin levels fell [from 19 (136 pmol/L) and 17 mU/L (122 pmol/L) at 2000 h to less than 2 (less than 14 pmol/L) and 5 mU/L (36 pmol/L) at 0800 h] in subjects 1 and 2, respectively. Serum IGFBP-2 levels remained constant overnight, as assessed on Western Ligand blotting and, despite the changes in IGFBP-1, remained the most prominent IGFBP throughout. On size separation, most of the IGF-II (greater than 60%) eluted with IGFBP-2 and the other low mol wt IGFBPs. Serum IGFBP-3 levels were reduced, and IGFBP-3 was not the major IGF carrier in LTD serum, in contrast to normal serum. An IGFBP-3-specific protease that was heat sensitive and cation dependent was identified as the cause of an apparent overnight rise of serum IGFBP-3 levels. No IGFBP-3 variation and no proteolytic activity was seen in normal serum or rapidly separated LTD plasma. Serum IGF bioactivity, measured in a porcine cartilage bioassay, was 0.18 and 0.55 U/mL in subjects 1 and 2; differences in bioactivity between subjects did not relate to serum IGF-II levels, but, rather, to differences in IGFBP-3 levels. Serum IGF bioactivity was not constant overnight and varied in a similar fashion in both subjects 1 and 2, with reduction in bioactivity between 0600-0800 h by 55% and 32%, suggesting the presence of inhibitory factors in the LTD serum; this decrease coincided with the rise in serum IGFBP-1 levels.(ABSTRACT TRUNCATED AT 400 WORDS)

The short-term effects of growth hormone therapy on height velocity and cardiac ventricular wall thickness in children with Noonan's syndrome.

Noonan's syndrome (NS) is associated with short stature and cardiac defects. Small studies reported linear growth increases with recombinant human GH (rhGH) therapy, but also raised concerns related to the anabolic effects of rhGH and the possible progression of ventricular hypertrophy. We report a multicenter study examining the efficacy and safety of rhGH (4 IU/m2.day, sc) in children with NS. Entry criteria were: NS confirmed by single observer, height SD score less than -2(UK Height Standards 1990), prepubertal, and normal maximal left ventricular (LV) wall thickness less than 1 cm by 2-dimensional echocardiography. Thirty subjects were recruited (19 males and 11 females), aged 8.9 +/- 0.5 yr (range, 4.8-13.7 yr). Growth was monitored for 12 months before and at 3-month intervals during therapy. Measurements of maximal LV wall thickness were taken at 0 and 12 months. Serum insulin-like growth factor I(IGF-I), IGF-II, and IGF-binding protein-3 levels were determined at 0, 3, 6, 9, and 12 months. Ten subjects with NS (4 females and 6 males), aged 8.8 +/- 0.7 yr (range, 6.3-11.8 yr), were monitored over the same period as a comparison group. In the treatment group, 27 subjects completed 12 months of therapy. Height SD score increased from -3.01 +/- 0.10 to -2.36 +/- 0.10 (P < 0.0001) after 12 months; height velocity (HV) increased from 4.9 +/- 0.2 to 8.9 +/- 0.3 cm/yr at 6 months and 8.1 +/- 0.4 cm/yr (P < 0.0001) from 6-12 months. The HV SD score increased from -0.7 +/- 0.15 to +2.42 +/- 0.32 over 12 months (P < 0.0001). The increase in HV was more than 2 cm/yr in 24 patients. IGF-I increased from 121 +/- 13 to 240 +/- 22 micrograms/L at 12 months (P < 0.0001), and IGF-binding protein-3 increased from 2.65 +/- 0.20 to 4.01 +/- 0.42 mg/L at 12 months (P = 0.0009). In the comparison group, there was no change in height SD score (-2.03 +/- 0.19), HV (4.4 +/- 0.24 CM/yr), or HV SD score (- 1.08 +/- 0.21). There was no increase in mean maximal LV wall thickness during the study in either the treatment group (12 month values were 0.63 +/- 0.02 cm at the mitral valve level and 0.66 +/- 0.02 cm at the papillary muscle level) or in the comparison group (0.63 +/- 0.04 cm at the mitral valve level and 0.61 +/- 0.03 cm at the papillary muscle level). In conclusion, rhGH was effective in 24 of the treated patients; these subjects achieved a significant increase in height SD score and HV over 1 yr. Abnormal anabolic effects of rhGH on myocardial thickness were not confirmed, and no patient developed features of hypertrophic cardiomyopathy.

Clinical features and endocrine status in patients with growth hormone insensitivity (Laron syndrome).

Twenty-seven patients with GH insensitivity were identified from 44 possible cases, using a scoring system based on height standard deviation score (SDS), basal GH, insulin-like growth factor-I (IGF-I), IGF-I response to IGF-I generation test, and GH-binding protein (GH-BP) determinations. The 27 cases were from 8 European countries and Australia. Clinical features were as follows: age 2.8-22.6 yr; 12 male, 15 female, 19 prepubertal. Birth weight was median -0.72 SDS (1.75(-)-3.29) and birth length, median -1.59 SDS (0.63(-)-3.63). Hypoglycemia had been documented in 33% of the cases, and micropenis was present in 58% of the males. At assessment, height was median -6.1 SDS (-3.8(-)-10.2), weight was median -3.2 SDS (-0.1 to -5.2), and percentage weight for height, median 111.3 (72-271). Puberty was absent in 2 boys aged 15 yr and in 3 girls aged 13 yr. Bone age was delayed in 19 of the 27 patients. Endocrine investigations showed basal serum GH median 17 micrograms/L (0.5-79), IGF-I values less than 5th percentile, and all except 2, age less than 8 yr, less than 0.1 percentile for age. Percentage increment of IGF-I during IGF-I generation test (hGH 0.1 U/kg body weight daily x 4) did not exceed twice the intraassay coefficient of variation, being less than 0.1 percentile for age. IGF-II was median 135.0 micrograms/L (62-232), all values being less than 5th percentile for age. Insulin-like growth factor binding protein-3 (IGFBP-3) values were median 0.53 mg/L (0.10-1.17 mg/L), all being less than 5th percentile for age. IGFBP-3 values after hGH remained less than 5th percentile. IGFBP-1 values showed the normal fall with age, some being above the normal range; IGFBP-2 values were normal. There was a positive correlation between height SDS and IGF-II SDS (r = 0.66, P < 0.001) and IGFBP-3 (r = 0.64, P < 0.001). Specific binding of [125I]hGH to GH-BP was undetectable in 18 patients and extremely low (< or = 5.6%) in 2. GH-BP was normal (14.2-45.9% radioactivity) in 7 subjects, all female, demonstrating that normal GH-BP does not exclude GH insensitivity.

Placental growth hormone (GH), GH-binding protein, and insulin-like growth factor axis in normal, growth-retarded, and diabetic pregnancies: correlations with fetal growth.

We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases ofnoninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFBP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28-30 weeks gestation (K28) and 36-38 weeks gestation (K36). The mean concentration (+/-SEM) of total PGH increased significantly from K28 to K36 (30.0 +/- 2.2 to 50.7 +/- 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 +/- 2.3 to 43.7 +/- 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-3 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGH at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r = -0.52; P < .001), free PGH and total PGH correlated very closely (r = 0.98). The results are consistent with an inhibitory function for GHBP in vivo and support a critical role for placental GH and IGF-I in driving normal fetal growth.

Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay.

Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUNT beads and the whole blood "Lyse/No-Wash" protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 microl of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 h after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained.

Cyclopropamitosenes, novel bioreductive anticancer agents. Synthesis, electrochemistry, and biological activity of 7-substituted cyclopropamitosenes and related indolequinones.

The synthesis of the indolequinones 8 and 9 starting from methyl 4-(benzyloxy)-5-methoxy-indole-2-carboxylate (10) is described. The methoxy group in the indolequinones 1, 2, 4, 5, and 7-9 can be displaced by various nitrogen nucleophiles (ammonia, 2-methoxyethylamine, aziridine, 2-methylaziridine, pyrrolidine) in 22-88% yield. The resulting amino-substituted quinones, together with their methoxy precursors, were studied by cyclic voltammetry to determine their reduction potentials, which, in DMF solution, lie in the range -1.355 to -1.597 V (vs ferrocene). The cytotoxicity of the compounds towards aerobic and hypoxic mammalian cells was also determined; in general, under aerobic conditions, the cyclopropamitosenes are more toxic than the corresponding pyrrolo[1,2-a]indolequinones, which are in turn more toxic than the simple 1,2-dimethylindolequinones, with many of the compounds in each series showing greater toxicity toward hypoxic cells.

Indolequinone antitumor agents: correlation between quinone structure, rate of metabolism by recombinant human NAD(P)H:quinone oxidoreductase, and in vitro cytotoxicity.

A series of indolequinones bearing various functional groups has been synthesized, and the effects of substituents on the metabolism of the quinones by recombinant human NAD(P)H:quinone oxidoreductase (NQO1) were studied. Thus 5-methoxyindolequinones were prepared by the Nenitzescu reaction, followed by functional group interconversions. The methoxy group was subsequently displaced by amine nucleophiles to give a series of amine-substituted quinones. Metabolism of the quinones by NQO1 revealed that, in general, compounds with electron-withdrawing groups at the indole 3-position were among the best substrates, whereas those with amine groups at the 5-position were poor substrates. Compounds with a leaving group at the 3-indolyl methyl position generally inactivated the enzyme. The toxicity toward non-small-cell lung cancer cells with either high NQO1 activity (H460) or no detectable activity (H596) was also studied in representative quinones. Compounds which were good substrates for NQO1 showed the highest selectivity between the two cell lines.

Insulin and variation in glucose levels modify the secretion rates of the growth hormone-independent insulin-like growth factor binding protein-1 in the human hepatoblastoma cell line Hep G2.

The plasma level of the GH-independent insulin-like growth factor binding-protein-1 (IGFBP-1) is regulated inversely by insulin. In this study the effect of insulin and changes in the glucose concentration on in-vitro IGFBP-1 secretion by the Hep G2 cell line was studied. Media from confluent cells in 12 replicates were collected for consecutive periods: initial control (20 h), study (6 h) and recovery (20 h). Insulin suppressed IGFBP-1 secretion maximally at 100 mU/l (-32%) within 6 h. The secretion of IGFBP-1 was stimulated by a decrease in the glucose concentration in the medium, maximally (+25%) with a decrease from 24 to 6 mmol/l. Stimulation by varying glucose levels and suppression by insulin of IGFBP-1 secretion persisted on return to control conditions after the removal of physiological concentrations of glucose (4-12 mmol/l) and insulin (50-500 mU/l). The findings in the Hep G2 cell line that a variation in the physiological concentrations of glucose and insulin each independently regulate IGFBP-1 secretion suggest that this cell line may be a suitable model for further in-vitro studies of the regulation of secretion of IGFBP-1.

Nutritional modulation of canine insulin-like growth factors and their binding proteins.

The response of canine insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) to moderate nutritional restriction followed by refeeding has not previously been studied in detail. The purpose of these studies was to examine the effects of nutritional restriction on the IGF system of adult dogs. Normal serum IGF values were established after validation of heterologous RIAs for measuring canine IGFs-I and -II. Canine serum IGFBP profiles were examined by Western ligand blotting (WLB), using radiolabelled recombinant human (rh) IGF-I as the ligand, and were found to be similar to those of other species. IGF-I and IGFBP-3 concentrations correlated with body weight, thus reflecting breed size as previously shown, whereas IGF-II concentrations did not. IGFBP-2 serum concentrations and band intensity on WLB were increased compared with normal human serum IGFBP-2. Overnight fasting had no effect on IGF or IGFBP concentrations, including IGFBP-1, nor did refeeding. Prolonged restriction to 56% and then 42.5% of maintenance energy requirements for 2 weeks decreased IGF-I concentrations by 20.4% and 32.7% respectively. Feeding of the same diet ad libitum for 2 weeks normalised IGF-I concentrations. There were no changes in IGF-II or insulin levels. Serum IGFBP-2 concentrations increased with 56% restriction of maintenance energy (P = 0.03). We conclude that serum IGF-I is potentially a useful marker of short-term change in nutritional status in the adult dog.

The insulin-like growth factors and their binding proteins in a case of non-islet-cell tumour-associated hypoglycaemia.

Non-islet-cell tumours which induce hypoglycaemia are rare. Insulin-like growth factor-II (IGF-II) produced by some tumours is thought to be responsible for the hypoglycaemia and other systemic effects, despite normal or even low serum IGF-II levels. We studied a 44-year-old woman presenting with symptomatic hypoglycaemia associated with a large intraabdominal haemangiopericytoma. The serum IGF-II level was 455 micrograms/l when measured after acid-ethanol extraction (normal range (NR) 450-750 micrograms/l) and 1063 micrograms/l after acid chromatography (normal human serum pool 1068 micrograms/l). Levels of fasting plasma insulin, C-peptide, glucose and serum IGF-I levels were low before the operation (less than 2 mU/l (NR less than 2-14), 0.23 nmol/l (NR 0.4-1.2), 3.1 mmol/l, (NR 3.7-5.9) and 0.02 U/ml respectively). After tumour removal, the symptoms resolved rapidly and the patient made a full recovery. Secretion of both insulin and growth hormone was suppressed before the operation in response to a 75 g glucose meal and to an infusion of 100 micrograms GH-releasing hormone (GHRH) respectively in comparison with studies after the operation. Serum IGF-II levels 6 weeks and 12 weeks after the operation fell to 385 micrograms/l (777 micrograms/l; acid chromatography) and 280 micrograms/l (647 micrograms/l; acid chromatography) and serum IGF-I levels increased to 0.35 U/ml and 0.26 U/ml. Serum before the operation and tumour extract contained chiefly a large molecular weight precursor IGF-II (molecular weight 15,000-20,000) which disappeared from the serum after the operation. The IGF-binding proteins (IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-4) were examined.(ABSTRACT TRUNCATED AT 250 WORDS)

The induction of a specific protease for insulin-like growth factor binding protein-3 in the circulation during severe illness.

The insulin-like growth factors (IGF-I and IGF-II) are almost completely bound in the circulation to specific binding proteins (IGFBPs). These IGFBPs appear to play a pivotal role in maintaining circulating levels and modulating the delivery of the IGFs to the tissues. A large proportion of the circulating IGFs are bound with high affinity to one of the binding proteins. IGFBP-3. The mechanism by which these IGFs are transferred from the circulatory pool to the tissue receptors is at present unclear. Recent studies in late pregnancy have demonstrated the presence of specific proteases which may modify the IGFBPs such that their affinities for the IGFs are reduced. In this paper, we have demonstrated the presence of a heat-sensitive cation-dependent proteolytic enzyme specific for IGFBP-3 in the serum of five severely ill patients. The activity of this protease was found to vary in these patients, becoming more apparent during fasting than when studied after commencement of parenteral nutrition, indicating that one of the influencing factors in the activity of this protease is the nutritional intake of the patient. Age- and sex-matched healthy adults were also studied in a similar protocol, but no proteolytic modification of any of the IGFBPs was found in any of the samples examined. As the levels of both IGF-I and IGF-II were found to be low in the patients, the presence of a circulatory protease suggests that this may be an adaptive response to increase the bioavailability of the IGFs and possibly to improve the nitrogen retention and counter the catabolic state in severe illness.

The induction of specific proteases for insulin-like growth factor-binding proteins following major heart surgery.

Insulin-like growth factors (IGF-I and IGF-II) circulate bound to specific high-affinity binding proteins (IGFBPs). Recent evidence has shown that in pregnancy and severe illness, specific proteases modify these binding proteins, reducing their affinity for IGFs. We have studied 12 patients, undergoing elective coronary artery vein-bypass graft surgery, for the appearance of these proteases and have demonstrated the induction of two independent, heat-labile, cation-dependent proteases. Proteolytic activity directed against IGFBP-3 was detected in all patients between 24 h and 5 days after surgery; the second IGFBP-4 specific protease was active 1 h after sternotomy. The total IGF-I levels were found to decrease following surgery, with the IGF-I distribution in the plasma being radically altered from that seen prior to the operation. One day after the operation the majority of the IGF-I, instead of being bound in the relatively inert 150 kDa complex, was associated with the smaller binding proteins which are more readily accessible to the tissues. These findings are in contrast to pregnancy where, despite similar proteases, the majority of the IGF-I remains in the 150 kDa complex. The alteration seen in IGF-I distribution after surgery did not appear to be a direct result of the IGFBP-3 proteolytic activity or an effect of the addition of heparin to the circulation. The potential increase in bioavailability of IGFs caused by the alteration in carrier protein may play a pivotal role in countering the catabolic state induced by surgery.

Girls with virilisation in childhood: a diagnostic protocol for investigation.

AIM: To analyse critically a protocol for the investigation of girls presenting with virilisation in childhood. METHODS: Twenty five girls aged 1.6-8.7 years with features of virilisation were evaluated. Twenty four had presented with pubic hair, eight with auxilliary hair, seven with facial acne, four with clitoromegaly, and 10 with tall stature. They underwent clinical assessment (height, weight, height velocity, staging of puberty, physical examination for acne, body odour, and clitoromegaly) and laboratory assessment comprising basal concentrations of cortisol, 17 OH-progesterone (17 OHP), androstenedione, dehydroepiandrosteronesulphate (DHEAS), testosterone, and oestradiol. The above steroids were also measured during the short synacthen test (0.25 mg intramuscularly) in 16 subjects and low dose dexamethasone suppression tests (0.5 mg at six hourly intervals over 48 hours). Pelvic ultrasound, computed tomography and magnetic resonance imaging of adrenals were carried out when the biochemical findings suggested that there might be an autonomous source of androgen secretion. RESULTS: Clinical and laboratory assessments differentiated the patients into three diagnostic categories: adrenarche (18 cases), congenital adrenal hyperplasia (five cases), and adrenocortical tumour (two cases). The last had elevated concentrations of DHEAS, 1.5 and 19.1 mumol/l (normal value < 0.5 mumol/l), androstenedione, 24.6 and 21.8 nmol/l (normal < 1 nmol/l), and testosterone, 4.5 and 2.4 nmol/l (normal < 0.8 nmol/l), with none suppressing on dexamethasone suppression. Congenital adrenal hyperplasia subjects had elevated basal serum concentrations of 17 OHP (n = 4): 250, 140, 14, and 14.1 nmol/l (normal < 10 nmol/l) and elevated peak values of 17 OHP after synacthen (n = 3): 76, 179.5, and 175 nmol/l. Adrenarche patients had elevated basal concentrations of DHEAS (median: 2.3 mumol/l; n = 17) and androstenedione (median 2.6 nmol/l; n = 17). Nine patients also had elevated basal serum testosterone concentrations (median 0.9 nmol/l). Peak values of 17 OHP after synacthen were significantly different from baseline (n = 12) and were < 50% of the lowest value in congenital adrenal hyperplasia. Serum DHEAS, androstenedione, and testosterone suppressed following dexamethasone suppression (n = 16), thereby distinguishing adrenarche patients from adrenal tumour patients. Clinical details did not distinguish patients, except for clitoromegaly which was present only in the tumour and congenital adrenal hyperplasia patients. CONCLUSIONS: This protocol proved useful and practical in cases of virilisation presenting particular diagnostic difficulty.