Andrea Prader Award. Clinical Steroidology. Coming of age.

The determination of steroid concentrations in urine and plasma is an indispensable part of the diagnostic work-up in endocrinology. Since the description of the color reaction for determination of 17-ketosteroids by Zimmermann in 1935, a vast amount of work has been invested in the specialty of clinical chemistry, which we called 'clinical steroidology'. Until the early fifties, color reactions for block determinations of C19 and C21 steroids in urine and plasma were the strongholds of steroid clinical chemistry. Around 1952, paper chromatography, and later on column chromatography were added, which afforded fractionation and individual determinations of steroids in body fluids. The final achievement and almost absolute quantification of almost 50 steroid metabolites ('profile') in urine was achieved by capillary glass column chromatography-mass spectrometry (GC-MS), which came into clinical usage around the mid-eighties. For estimation of plasma steroids radioimmunoassays (RIA) were introduced during the sixties. A whole body of physiology and pathology was erected on the basis of steroid RIAs. Recent investigation, however, revealed that steroid RIAs yield unreliable results at low concentrations due to cross reactions and interferences by impurities of samples. The newly (nineties) developed method of isotope dilution/GC-MS produced highly accurate values of small amounts of steroids and became the gold standard of plasma steroid determinations. Thus 'clinical steroidology' has certainly come of age.

Combined pituitary deficiencies of growth hormone, thyroid stimulating hormone and prolactin due to Pit-1 gene mutation: a case report.

UNLABELLED: A child exhibited postnatal obstipation and icterus together with severe growth failure during the 1st year of life, a small facial skull and a prominent forehead. Endocrine work-up established the diagnosis of combined pituitary deficiencies of growth hormone, TSH and prolactin. Subsequently, the Pit-1 gene was analysed in the patient and both parents. A single point mutation was detected in exon 6 of the child: a C to G transversion on one allele, causing arginine in position 271 to be substituted by tryptophan (R271 W). This position is known as a "hot spot" for mutations. The inheritance is autosomal-dominant, as the mutated gene product interferes with DNA-binding of the wild-type protein. In contrast, other mutations in the PIT-1 gene are inherited in an autosomal-recessive mode. CONCLUSION: Diagnosing Pit-1 gene mutations as a rare cause of combined pituitary deficiency is important both for genetic counselling as well as for predicting the future course in the patient (spontaneous puberty, no glucocorticoid substitution necessary during stress periods).

Contribution of androgens to the gender difference in leptin production in obese children and adolescents.

Recent studies demonstrated significantly higher serum leptin concentrations in females as compared with males, even after correction for differences in body fat mass. The aim of our study was to measure serum leptin concentrations in a large group of obese children and adolescents to determine the possible role of sex steroid hormones on both leptin serum concentrations and production in human adipocytes. Obese girls were found to have significantly higher leptin concentrations than boys at the same degree of adiposity (25.2+/-14.1 vs. 17.2+/-12.6 ng/ml, P < 0.001). In a multiple regression analysis with age and body mass index (percent body fat) as fixed variables, it turned out that testosterone had a potent negative effect on serum leptin in boys, but not in girls. In vitro experiments using newly developed human adipocytes in primary culture showed that both testosterone and its biologically active metabolite dihydrotestosterone are able to reduce leptin secretion into the culture medium by up to 62%. Using a semiquantitative reverse transcriptase-PCR method, testosterone was found to suppress leptin mRNA to a similar extent. These results suggest that, apart from differences in body fat mass, the higher androgen concentrations in obese boys are responsible for the lower leptin serum concentrations compared with obese girls.

Insulin resistance with altered secretory kinetics and reduced proinsulin in cystic fibrosis patients.

BACKGROUND: Impaired glucose tolerance and secondary diabetes are frequent in older patients with cystic fibrosis (CF), associated with increased frequency of infections and reduced life expectancy. Studies on the pathophysiology of islet cell secretion in CF are a prerequisite for a scientifically based therapeutic approach. METHODS: Oral glucose tolerance tests were performed in 71 patients (14.2 +/- 0.5 years; mean +/- SE) and 56 control subjects (16.5 +/- 0.9 years). Glucose, insulin, C-peptide, and proinsulin were measured every 30 min. RESULTS: Glucose tolerance in CF patients was classified as normal (NGT, n = 48), impaired (IGT, n = 14), or diabetic (DM, n = 9). Even in CF patients with NGT, blood glucose was significantly elevated at 30, 60, and 90 min of the test. Surprisingly, the secretory responses of insulin and C-peptide were not reduced in CF patients with IGT or DM compared with both healthy controls or CF patients with normal glucose tolerance. However, peak insulin concentration was reached at 90 min in CF-IGT or CF-DM patients compared with 30 min in controls. The ratio of glucose to insulin, an indicator of insulin resistance, increased in CF patients with progression of carbohydrate intolerance. Proinsulin was significantly reduced in all CF patients compared with controls (p < 0.001; Wilcoxon's rank sum test). CONCLUSIONS: In CF patients with impaired glucose tolerance or diabetes, integrated insulin release is not diminished, indicating that insulin resistance is likely to contribute to hyperglycemia in CF patients with IGT or DM. Reduced proinsulin levels in CF patients are compatible either with enhanced conversion of proinsulin to insulin in compensation for reduced beta-cell mass, or enhanced clearance of proinsulin.

[Body composition in type 1 diabetes mellitus. Bio-impedance measurements in 274 diabetic children, adolescents and young adults]. / Körperzusammensetzung bei Typ-I-Diabetes mellitus. Bioimpedanzmessungen bei 274 diabetischen Kindern, Jugendlichen und jungen Erwachsenen.

BACKGROUND AND OBJECTIVE: Weight gain is a common undesirable side effect of insulin treatment in type 1 diabetics. This study tested the assumption that this is due to an increase in fat mass. PATIENTS AND METHOD: Bioelectric impedance measurements were performed on 157 young male and 117 female diabetics (age 17.6 +/- 4.9 years; diabetes duration 8.9 +/- 5.7 years) and the fat-free mass (FFM) calculated according to the equation of Schfer et al. The data of the diabetics were compared with those of healthy controls and normal values published by Barlett et al. RESULTS: The average weight of the diabetic cohort was 2.0 +/- 0.7 kg higher than in the reference groups, adjusted for sex and age. FFM was higher by 2.9 +/- 0.7 kg in diabetics than in the healthy cohort of Barlett et al (P < 0.005), being equally high in males and females (+2.9 +/- 0.7 kg and 2.9 +/- 0.6 kg, respectively). But compared with the values in metabolically normal controls the percentage fat proportion was lower in the diabetics than the controls, but not significantly (-1.3 +/- 0.6%). Weight gain was greater in females than males (+3.8 +/- 0.9 kg vs +1.2 +/- 0.9 kg, P < 0.05). After correcting for age, there was a partial correlation between good metabolic control and FFM in males. The form of treatment had no effect on body composition. CONCLUSION: These data indicate that weight gain in young diabetics is due not to an increase in fatty tissue but in muscle mass. This is probably the result of peripheral hyperinsulinism combined with hyperglycaemia.

Angiotensin I-converting enzyme-gene-polymorphism: relationship to albumin excretion and blood pressure in pediatric patients with type-I-diabetes mellitus.

The purpose of this study was the evaluation of a predictive genetic marker for nephropathy and hypertension in patients with type-I-diabetes mellitus (IDDM). The study was performed on 247 pediatric patients with IDDM. The mean age was 15.5 years (range 3.1-29.3), the mean duration of diabetes was 7.6 years (range 0.1-25.7). Age-related blood pressure and nocturnal albumin excretion rate were compared with the insertion/deletion-(I/D) polymorphism of the angiotensin-I converting enzyme gene. The genotype distribution did not differ significantly between IDDM patients (ID 48%, D 28%, I 24%) and the control group (ID 44%, D 37%, I 19%). Neither in the entire group, nor in patients with IDDM for more than 5 years, was a correlation found bet-ween allele distribution and albumin excretion rate. No correlation was found between genotype and blood pressure. When patients with a chronological age above 12 years were analysed separately, the genotype distribution between the groups with normal and elevated blood pressure showed no significant difference. The previously reported association of the I/D-polymorphism with nephropathy could not be confirmed in this study. The development of microalbuminuria, nephropathy and hypertension will be followed in our pediatric patients.

[Diagnosis of the adrenogenital syndrome caused by 11beta-hydroxylase deficiency using gas chromatographic-mass spectrometric analysis of the urinary steroid profile]. / Diagnostik des adrenogenitalen Syndroms vom Typ des 11beta-Hydroxylase-Mangels mit der gaschromatographisch-massenspektrometrischen Harnsteroidrofilanalyse.

PROBLEM AND OBJECTIVE: In 5-7% of all cases of congenital adrenogenital hyperplasia (AGH) there is 11 beta-hydroxylase deficiency (11 beta-HM). Its clinical picture is characterised by hyperandrogenism and, in some cases, arterial hypertension. The diagnosis of the enzyme deficiency depends on a reliable method of analysing the hormone in plasma and urine. As little is known and data often contradictory about the pattern of urinary steroid excretion in 11 beta-HM, these steroid metabolites were measured by a highly specific method. PATIENT AND METHOD: The pattern of urinary excretion of steroids was determined by gas chromatography and mass spectrometry (GC/MS) in 16 children and adults (11 males, 5 females: mean age 9(8)/12 [2/12-20(3)/12] years) with 11 beta-HM. RESULTS: In all patients there was greatly increased excretion of tetrahydrated (TH) and hexahydrated (HH) metabolites of 11-desoxycortisol (S) and desoxycorticosterone (DOC). The excretion of THS and THDOC was extremely increased in all patients. The metabolites 5 alpha-THS as well as 20 alpha- and 20 beta-isomers of HHS, not normal found in healthy persons, were present in 15 patients (94%), while the 20 alpha- and 20 beta-isomers of 5 alpha-HHS were demonstrated in 14 (88%). For the first time, 20 alpha- and 20 beta-isomers of 5 alpha-HHS were shown to be typical urinary steroid metabolites in 11-HM. The excretion of cortisol metabolites is typically decreased in 11 beta-HM. No corticosterone metabolites were found. CONCLUSION: The urinary steroid excretion pattern, measured by GC/MS, is a noninvasive, highly specific and nonselective method in the differential diagnosis of abnormal steroid metabolism.

Insulin-like growth factors and their binding proteins before and after weight loss and their associations with hormonal and metabolic parameters in obese adolescent girls.

OBJECTIVE: To investigate serum concentrations of insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) in obese adolescent girls before and after weight reduction and to examine their associations with hormonal and metabolic parameters. PATIENTS AND METHODS: 73 girls (age: 15.0 +/- 1.1 y, BMI: 31.1 +/- 3.8 kg/m2) participated in this 6 w intervention study. RESULTS: (1) Compared to 100 normal-weight girls of same age the obese subjects had increased levels of IGF-I, IGFBP-1, IGFBP-2, and IGFBP-3 and normal levels of IGF-II. (2) After weight loss (8.1 +/- 2.0 kg) IGF-II and IGFBP-2 increased and IGF-I, IGFBP-1, IGFBP-3 and the ratio IGF-I/IGFBP-3 decreased significantly. (3) Correlation analysis revealed that fasting insulin levels of the patients were positively correlated with IGF-I but inversely with IGFBP-1 and IGFBP-2. IGFBP-2 was associated with several metabolic parameters: it showed an inverse correlation with uric acid and triglyceride levels. The ratio IGFBP-2 to IGFBP-1 was inversely correlated with total cholesterol and LDL-cholesterol. In addition, IGFBP-2 was inversely correlated with the waist-to-hip ratio of the girls. CONCLUSIONS: These studies of a homogeneous group of patients give further insight into possible physiological regulations and roles of IGFs and IGFBPs. The main conclusion is that obesity has to be considered for interpretation of serum concentrations of IGF-I, IGFBP-1, IGFBP-2, and IGFBP-3. Furthermore, out results suggest that (i) the decrease in IGF-I/IGFBP-3 after weight loss could be partly responsible for the impaired growth velocity seen in obese children during hypocaloric feeding and (ii) low IGFBP-2 and high IGFBP-1 levels are associated with an unfavorable atherogenic risk factor profile.

[Gas chromatography-mass spectrometry determination of plasma 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 alpha,17 beta-diol glucuronide in children with premature and normal puberty ]. / Gaschromatographisch-Massenspektrometrische Bestimmung von 5 alpha-Androstan-3 alpha,17 beta-diol und 5 alpha-Androstan-3 alpha, 17 beta-diol-glucuronid im Plasma von Kindern mit prämaturer und normaler Pubarche.

Using stable isotope dilution/gas chromatography-mass spectrometry, we determined the plasma concentrations of 5 alpha-androstane-3 alpha,17 beta-diol (AD) and 5 alpha-androstane-3 alpha,17 beta-diol-glucuronide (ADG) in 20 patients with premature pubarche (16 patients with idiopathic premature pubarche, 4 patients with late onset 21-hydroxylase deficiency) and in 55 healthy children with Tanner stages P1 to P4. No differences between sexes were found in healthy children with Tanner stages P1 and P2. Patients with idiopathic premature pubarche (median, range, nmol/1: 0.22; 0.12-0.31) or late onset 21-hydroxylase deficiency (0.27; 0.23-0.29) had higher plasma AD concentrations than healthy prepubertal children (0.09; 0.00-0.17). Regarding ADG, patients with idiopathic precocious puberty (1.35; 0.25-4.74) or late onset 21-hydroxylase deficiency (4.01; 3.50-4.58) had also higher plasma concentrations than healthy prepubertal children (0.35; 0.00-0.75). Thus, AD and ADG, which both represent end metabolites of peripheral androgen metabolism, can be regarded as markers of androgenicity. Steroid analysis by mass spectrometry is recommended, whenever uncertainties of immunological determinations are to be avoided.

Insulin and cortisol promote leptin production in cultured human fat cells.

The aim of this study was to investigate the regulation of leptin expression and production in cultured human adipocytes using the model of in vitro differentiated human adipocytes. Freshly isolated human preadipocytes did not exhibit significant leptin mRNA and protein levels as assessed by reverse transcriptase (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). However, during differentiation induced by a defined adipogenic serum-free medium, cellular leptin mRNA and leptin protein released into the medium increased considerably in accordance with the cellular lipid accumulation. In fully differentiated human fat cells, insulin provoked a dose-dependent rise in leptin protein. Cortisol at a near physiological concentration of 10(-8) mol/l was found to potentiate this insulin effect by almost threefold. Removal of insulin and cortisol, respectively, was followed by a rapid decrease in leptin expression, which was reversible after readdition of the hormones. These results clearly indicate that both insulin and cortisol are potent and possibly physiological regulators of leptin expression in human adipose tissue.

Mitogenic and antiadipogenic properties of human growth hormone in differentiating human adipocyte precursor cells in primary culture.

Children with GH deficiency have enlarged fat cells but a reduced number of fat cells compared with healthy children. After treatment with human GH (hGH) both fat cell volume and number are shifted toward normal. To clarify the role of hGH in fat cell formation in human adipose tissue, we investigated the effect of hGH on the proliferation and the differentiation of cultured human adipocyte precursor cells obtained from five children and 10 adults. In a chemically defined serum-free medium treatment of adipocyte precursor cells with hGH led to an increase in IGF-I production and a stimulation of cell proliferation, which could be blocked by a MAb raised against human IGF-I. hGH dose-dependently reduced the number of differentiating cells and suppressed the expression of glycerol-3-phosphate dehydrogenase (GPDH), a marker of adipose differentiation. No significant differences in the hGH effects on proliferation and differentiation capacities were seen between cultures obtained from children and adults. In newly differentiated adipocytes, hGH inhibited glucose uptake and lipogenesis, and stimulated lipolysis. Scatchard analysis of hGH competition experiments using 125I-labeled hGH yielded a linear plot with an apparent Kd of 1.08 nM and an estimated number of 7000 hGH receptors per cell. These data suggest that hGH is able to enlarge the human adipocyte precursor pool via induction of IGF-I synthesis but exhibits a direct antiadipogenic activity. hGH is also able to reduce fat cell volume by reducing lipogenesis and increasing lipolysis.

Body composition in 5-18-y-old obese children and adolescents before and after weight reduction as assessed by deuterium dilution and bioelectrical impedance analysis.

The aim of the present study was to develop an equation for the prediction of total body water (TBW) from bioelectrical impedance analysis (BIA) in obese children and adolescents before and after weight reduction. In 146 obese subjects with a mean age of 12.7 +/- 3.0 y (5.5-17.8 y), TBW was measured by using deuterium dilution as well as the resistance index (RI; ht2/resistance) using BIA before and after weight loss. Initially, the RI correlated well with measured TBW (r2 = 0.92, P < 0.001). A multiple-regression analysis using forward stepwise selection of the variables RI, sex, age, weight, height, and waist-hip ratio revealed that the equation TBW = 0.35 x RI + 0.27 x age + 0.14 x weight - 0.12 predicts most accurately individual values of TBW before weight loss (adjusted r2 = 0.96, SEE = 1.9 L) with a mean error of predicted TBW of 1.40 +/- 1.38 L. This equation was validated in 1000 random samples (bootstrap-sampling method), giving a mean r2 of 0.95. During the weight-reduction program, which included an energy-restricted diet and an extensive exercise program, the patients lost 7.7 +/- 3.2 kg, leading to a small decrease in TBW of 0.4 +/- 1.5 L. When the developed prediction equation was applied to the data after weight loss, an r2 value of 0.94 between measured and calculated TBW and a mean error of 2.18 +/- 1.89 L was obtained. Validation of the equation in 1000 random samples after weight loss again gave a mean r2 value of 0.95. Individual changes in predicted TBW correlated only weakly with those of measured TBW (r = 0.21, P < 0.05). Thus, individual TBW values before and after weight loss can be predicted by BIA with acceptable accuracy by using the developed equation. However, prediction of small individual changes in TBW during weight loss is not possible by BIA.

Semilente-insulin at bedtime is superior to NPH-insulin for the suppression of the dawn-phenomenon in adolescents with type-I-diabetes.

The dawn-phenomenon causes high fasting glucose values in IDDM patients during puberty. Even a bedtime injection of intermediate-acting insulin does not reliably suppress glucose rises during the morning hours. We therefore examined whether Semilente, an amorphous zinc insulin with kinetics different from NPH insulin, is better suited to alleviate the dawn-phenomenon in adolescent patients with long-standing diabetes. This prospective study included 15 adolescent patients (age 15.5 +/- 0.4 years; mean +/- SE) well beyond the remission phase of diabetes (mean duration: 7.5 +/- 0.8 years). On an inpatient basis, blood glucose profiles following bedtime injections of NPH or semilente insulin were compared, using a sequential cross-over design for intra-patient comparison. Fasting blood glucose was significantly lower following bedtime injections of Semilente (183 +/- 21 mg/dL [10.2 +/- 1.1 mmol/L]) compared to nights where NPH had been injected (235 +/- 22 mg/dL [13.1 +/- 1.2 mmol/L]). In addition, the morning postprandial blood glucose was significantly improved. The frequency of nocturnal hypoglycemia was not different, and the dose of Semilente insulin was slightly lower compared to the dose of NPH-insulin injected. For adolescent IDDM patients with suboptimal metabolic control due to a marked dawn-phenomenon, with high fasting glucose concentrations despite a bedtime injection of NPH insulin, bedtime injection of Semilente insulin may result in reduced fasting hyperglycemia on the next morning.

5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 alpha, 17 beta-diol-glucuronide in plasma of normal children, adults and patients with idiopathic hirsutism: a mass spectrometric study.

We investigated the developmental patterns of 5 alpha-androstane-3 alpha, 17 beta-diol (AD) and 5 alpha-androstane-3 alpha, 17 beta-diol-glucuronide (ADG) in plasma of normal children and adults of both sexes and in patients with idiopathic hirsutism using a physicochemical method: high-resolution gas chromatography/mass spectrometry (HRGC/MS). In children below the age of 11 years, AD and ADG increased with age showing no differences between sexes (mean +/- SD, nmol/l): normal subjects 3-6 years: AD in females 0.08 +/- 0.03, in males 0.07 +/- 0.03; ADG in females 0.15 +/- 0.05, in males 0.14 +/- 0.04; normal subjects 7-10 years; AD in females 0.17 +/- 0.03, in males 0.17 +/- 0.07; ADG in females 0.59 +/- 0.12, in males 0.47 +/- 0.14. Thereafter, AD and ADG showed a greater increase in males (normal subjects 11-15 years: AD in females 0.24 +/- 0.06, in males 0.41 +/- 0.14; ADG in females 1.47 +/- 0.36, in males 3.36 +/- 1.22). In adults, plasma levels did not overlap between females and males (AD in females 0.24 +/- 0.07, in males 0.99 +/- 0.31; ADG in females 2.32 +/- 0.68, in males 13.01 +/- 3.05). 5 alpha-Androstane-3 alpha, 17 beta-diol-glucuronide discriminated better between sexes than AD. In idiopathic hirsutism, mean plasma concentrations of AD and ADG were higher than those of healthy females (ages 11-15 years: AD 0.31 +/- 0.10, ADG 3.48 +/- 2.00; ages > 16 years: AD 0.44 +/- 0.27, ADG 6.46 +/- 3.11), but 54% of patients had normal plasma concentrations of AD and 29% had normal ADG values. Thus, ADG reflected androgenicity better than AD. However, both metabolites were imperfect markers of androgenicity in idiopathic hirsutism. Therefore, our findings do not support the concept of increased 5 alpha-reductase activity in all patients with idiopathic hirsutism.

Biological effects of human growth hormone in rat adipocyte precursor cells and newly differentiated adipocytes in primary culture.

The effects of human growth hormone (hGH) on proliferation and differentiation of primary adipocyte precursor cells isolated from rat epididymal fat pads were studied under serum-free culture conditions. hGH markedly reduced the formation of new fat cells and the expression of glycerophosphate dehydrogenase activity, a marker enzyme of adipose differentiation, in a dose-dependent manner. To find an explanation for this inhibitory effect, we investigated the action of GH on (1) cell proliferation and on (2) lipid accumulation, the latter in the absence and presence of corticosterone. In undifferentiated cells, 5 nmol/L hGH increased both cell number and [3H]-thymidine incorporation (1.3- and 2.6-fold over basal, respectively). This effect was mediated by insulin-like growth factor-I (IGF-I), since hGH stimulated IGF-I production in undifferentiated cells by 12-fold and addition of an anti-IGF-I monoclonal antibody (IGF-I MAb) abolished the mitogenic effect of hGH but did not prevent hGH-induced suppression of adipose differentiation. In developing fat cells, hGH significantly reduced cellular 2-deoxyglucose uptake and glucose incorporation into lipids. In addition, hGH exhibited a lipolytic action in the presence of insulin and triiodothyronine. These effects were not prevented by IGF-I MAb. Specific binding of [125I]-hGH to precursor cells increased significantly during adipose conversion. In differentiated cells Scatchard analysis yielded linear plots with an apparent Kd of 0.16 nmol/L and 8,400 sites per cell. Taken together, these data show that hGH reduces adipose conversion in primary cultures of rat adipocyte precursor cells while promoting cell proliferation through an increase in IGF-I production.

Body fat distribution and steroid hormone concentrations in obese adolescent girls before and after weight reduction.

The aim of this study was to investigate the role of body fat distribution on steroid hormone serum concentrations in obese adolescent girls before and after weight reduction. Ninety-two girls (age, 15.1 +/- 0.7 yr) with a mean body mass index of 31.2 +/- 4.6 kg/m2 participated in this 6-week intervention study. Initially, girls with abdominal obesity (waist to hip ratio, > 0.86; n = 30) had higher levels of total and free testosterone and lower levels of sex hormone-binding globulin as well as lower morning levels of total and free cortisol than girls with gluteal-femoral obesity (waist to hip ratio, < 0.80; n = 31) independent of their body mass index. After a mean weight loss of 8.3 +/- 2.6 kg by a standardized weight loss program, significant reductions were observed in estradiol, total and free testosterone, dehydroepiandrosterone sulfate, and the ratio of LH to FSH, whereas sex hormone-binding globulin and free cortisol levels increased significantly. Decreases in total and free testosterone and increases in total and free cortisol were significantly greater in the girls with abdominal obesity than in the girls with gluteal-femoral obesity. Our results suggest that obese girls with an abdominal pattern of fat distribution exhibit more pronounced steroid hormone aberrations, in particular a high androgenic activity, than girls with a gluteal-femoral pattern of fat distribution. The reduction of excess body weight by a conventional treatment regimen is associated with a remarkable improvement of steroid hormone abnormalities in this particular subtype of obese adolescent girls.

Expression of osteoblastic markers in cultured human bone and fracture callus cells.

We compared the expression of osteoblastic markers in cultured human cells isolated from fracture calluses of various histological states of development with that in cells from adult and fetal bone. Adult osteoblasts and all callus cells produced almost exclusively type I collagen, whereas fetal osteoblasts produced also considerable amounts of type III collagen in vitro. 1,25-Dihydroxyvitamin D3 induced the synthesis of osteocalcin in all bone and callus cells but to varying extents. Fetal bone cells and early-stage callus cells synthesized less than 10% the amount of osteocalcin produced by adult bone cells. Late-stage callus cells produced intermediate levels of osteocalcin. Fetal bone cells and early-stage callus cells responded to parathyroid hormone with a less pronounced increase in intracellular cAMP than did adult bone cells. Late-stage callus cells showed the best response to parathyroid hormone. The activity of alkaline phosphatase was highest in fetal bone cells. These observations show that cells isolated from fetal bone and from fracture callus tissues express a pattern of markers clearly relating them to the osteoblastic lineage. On the basis of the different patterns of osteoblastic markers expressed in vitro we conclude that functionally distinct subtypes of osteoblasts do exist in different mineralized tissues and at different developmental stages.