Roberta Valentini

Growth hormone treatment in osteogenesis imperfecta with quantitative defect of type I collagen synthesis.

OBJECTIVES We studied growth rate, bone density, and bone metabolism in patients affected by type I osteogenesis imperfecta (OI) with quantitative defect in type I collagen synthesis during treatment with human growth hormone (hGH), being aware of its collagen-stimulating synthesis activity in vitro. STUDY DESIGN Fourteen patients (6 boys; ages 4.8 to 10.8 years) were studied. Any structural alteration in the collagen chains was excluded, and reduced production of structurally normal type I collagen (increase in type III/type I collagen; reduction in the messenger ribonucleic acid alpha 1 (I)/ alpha 2 (I) ratio) was demonstrated. The patients were divided into two groups comparable in sex, age, height, and clinical severity of OI; seven patients (three boys) were treated for 12 months with hGH at a dosage of 0.2 mg/kg per week (0.6 IU/kg per week), in six injections subcutaneously, and seven were followed as control subjects. Auxologic data were measured every 3 months, and bone age was determined at the start, after 1 year of treatment, and 1 year after its completion. Every 3 months, serum insulin-like growth factor type I, osteocalcin, carboxyterminal propeptide of type I procollagen, alkaline phosphatase, calcium, and phosphorus levels and urinary hydroxyproline and calcium levels were determined. Bone mass measurements were carried out at the start of the study in all patients and repeated after 12 months in treated patients at the lumbar spine by dual-energy x-ray absorptiometry and by anteroposterior (second, third, and fourth lumbar vertebrae) and lateral (third lumbar vertebra) scan. Results were expressed as areal (anteroposterior and lateral) bone density (in milligrams per square centimeter) and as calculated true density (in milligrams per cubic centimeter). RESULTS After 12 months, linear growth velocity in treated patients increased significantly in comparison with the pretreatment period (from 3.57 +/- 0.55 to 6.04 +/- 0.69 cm/yr; p < 0.05) and with the untreated group (p < 0.05). Bone age did not advance faster than chronologic age. The fracture index per year was low before treatment, and during therapy no patient had any fractures. Serum osteocalcin levels were statistically lower than in control subjects before treatment and increased significantly after 12 months (3.3 +/- 1.0 vs 2.1 +/- 0.9 nmol/L; p < 0.05). Serum levels of carboxyterminal propeptide of type I procollagen were significantly lower than normal values before treatment (164.6 +/- 46.7 vs 310.3 +/- 97.6 ng/ml; p < 0.05) and rose, but not significantly, during and after treatment. Before therapy, patients with OI had significantly lower lumbar anteroposterior, lateral, and calculated true bone density than the normal population of the same sex compared for both age and height. After hGH treatment, bone density increased significantly in the lumbar spine, in anteroposterior and lateral scans (+2.6 +/- 2.5% and +9.8% +/- 14.0%, respectively; p < 0.05). CONCLUSIONS From our results, we conclude that hGH treatment in moderate OI does not increase the fracture risk in treated patients in the short term, significantly increases the rate of linear growth velocity, and increases bone turnover and mineral content in trabecular bone at the lumber spine.

Improved method for carbohydrate-deficient transferrin determination in human serum by capillary zone electrophoresis

Carbohydrate-deficient transferrin (CDT) is a reliable marker of chronic or repeated alcohol abuse. It indicates a group of isoforms of human transferrin (Tf), the main iron transport serum protein, deficient in sialic acid residues (asialo-, monosialo- and disialo-Tf) in comparison to the main isotransferrin which contains four sialic acid groups (tetrasialo-Tf). The aim of the present work was to develop a capillary electrophoretic method suitable for rapid determination of CDT components in serum. Serum samples (0.1 ml) were saturated with iron by incubation with 10 mM FeCl3 (2 microl) and 500 mM NaHCO3 (3 microl) for 30 min, then diluted 1:10 in water and injected by positive pressure (0.5 p.s.i. for 10 s). Separation was performed with a capillary zone electrophoretic method using bare fused-silica capillaries (57 cm x 20 microm I.D.) and a buffer composed of 100 mM sodium tetraborate adjusted with 6 M HCl to pH 8.3 added with 1.5 mM diaminobutane. Applied voltage was 20 kV and temperature 25 degrees C. Detection was by UV absorption at 200 nm wavelength. Under the described conditions, asialo-, monosialo-, disialo-, trisialo- and tetrasialo-transferrin were baseline separated. The limit of detection (signal-to-noise ratio of 2) was about 0.3% for disialo-Tf, and 0.5% of trisialo-Tf, expressed as percentages of the terasialo-Tf peak area. Day-to-day RSDs of relative migration times were < or = 0.2%. Quantitation showed day-to-day RDSs < or = 6.9% and < or = 10.9% for disialo- and trisialo-Tf, respectively. The results from 79 control subjects, including social drinkers, and 23 alcoholics showed disialo- and trisialo-Tf significantly increased in patients (P<0.0001 and <0.01, respectively). A clear interference from trisialo-Tf in an immunoassay for CDT was demonstrated. The present method is suitable for confirmation of CDT immunoassays by independent technique.

Hair analysis by using radioimmunoassay, high-performance liquid chromatography and capillary electrophoresis to investigate chronic exposure to heroin, cocaine and/or ecstasy in applicants for driving licences

The present paper describes an integrated diagnostic strategy to check the physical fitness of subjects, formerly users of illicit drugs, to obtain a driving license, after having quit their addiction. According to the Italian law, applicants for a driving license with a history of drug abuse must give evidence to have quit this behaviour and to show no risk of relapse in the future. To prove this, at our institute, they undergo medical examination, hair analysis and a urinalysis program on eight seriate samples, collected over about 40 days. About 700 subjects per year are investigated with this strategy. The hair samples are screened for opiates (morphine), cocaine and ecstasy, the most abused illicit substances in our region, by using commercial radioimmunoassays adopting cut-off levels of 0.1 ng/mg. All positive samples and about 10% of negatives are confirmed by high-performance liquid chromatography. Further confirmation of results can be carried out by capillary electrophoresis (and/or GC/MS or MS/MS). In 1998, the prevalence of positives for morphine, cocaine and ecstasy was 4.8, 11.3 and 2.6%, respectively. In this year, for the first time, the percentage of hair samples positive for cocaine was greater than that for opiates. The results of this integrated diagnostic strategy are presented and discussed, with particular emphasis on the comparison between hair analysis on a single sample and seriate urinalyses (on eight samples).

Growth Hormone-Binding Proteins and Insulin-Like Growth Factor-Binding Proteins in Protein-Energy Malnutrition, before and after Nutritional Rehabilitation

To clarify the influence of nutrition on the GH-IGF axis in protein-energy malnutrition (PEM), we determined the serum levels of GH, GH-binding proteins(BP) (GHBPs), IGF-I, and IGFBPs in nine children with kwashiorkor and 13 with marasmus, before and after nutritional rehabilitation. In a basal condition, the GH level was significantly higher in the two malnourished groups than in controls (p < 0.01); in contrast, the second fraction of GHBP was lower and seemed to be related to the high GH and to a reduction in GH receptors. After refeeding, the GH level increased and the second fraction of GHBP decreased. The IGF-I basal level was higher in kwashiorkor than in marasmus subjects (p < 0.05), but in both groups it was significantly lower than in controls (p < 0.01); after refeeding it increased. IGFBP-3, measured by RIA and Western blotting techniques, was in the control range in the kwashiorkor group but in the marasmic group it was significantly lower than in controls; after refeeding it decreased in kwashiorkor (p < 0.01 versus basal values) and increased in marasmus (p < 0.05 versus prerefeeding level). When sera of malnourished patients were mixed with adult control sera, incubated for 5 h at 37°C, and assessed by ligand blotting, a low IGFBP-3 level in marasmus was found to be due to increased adaptive proteolysis of IGFBP-3; in contrast, in kwashiorkor the IGFBP-3 proteolytic activity was very low, probably because of inhibition by aflatoxins. These findings confirm that malnutrition affects the GH-IGF axis.

Analysis of morphine by RIA and HPLC in fingernail clippings obtained from heroin users.

Heroin is abused around the world and is frequently reported as the cause of death in overdose cases. Analysis of morphine in hair has been used in the past in forensic toxicology to study the addiction history of heroin addicts. The purpose of the present study was to evaluate the usefulness of the nail as an analytical specimen in the identification and quantification of morphine in fingernail clippings of known heroin users. Fingernail clippings were obtained from 26 consenting patients of the Glasgow Drug Problem Service. At the time of sampling, the participants provided answers to a questionnaire regarding their drug use patterns. Samples were decontaminated by sonication in SDS, deionized water and methanol, and the methanolic washes were screened for analyte presence. The washed nail clippings were then hydrolyzed and extracted. RIA was used for the screening and HPLC for the confirmation of morphine. Positive RIA results were obtained with nail clippings from 25 of the 26 heroin users. The levels ranged from 0.06 to 4.69 ng/mg with a mean morphine concentration of 1.67 ng/mg. HPLC results were positive for 22 of the 26 nail samples. The mean morphine level by HPLC was 2.11 ng/mg with a range from 0.14 to 6.90 ng/mg. Based on these results, we suggest that nails have the potential of becoming a powerful alternative to hair for the detection of past heroin use in forensic cases.

Growth hormone-binding proteins, IGF-I and IGF-binding proteins in children and adolescents with type 1 diabetes mellitus.

Growth hormone binding proteins, insulin-like growth factor I and insulin-like growth factor binding proteins were determined in 54 children and adolescents affected by type 1 diabetes mellitus (25 prepubertal and 29 pubertal) showing reduced height velocity and the results were compared to those of 104 matched controls. Growth hormone binding proteins were similar in prepubertal and pubertal subjects but were significantly lower in the prepubertal diabetic group than in controls. Insulin-like growth factor I was low both in prepubertal and pubertal diabetic subjects. Insulin-like growth factor binding protein 3 was similar to controls, while insulin-like growth factor binding protein 1 and 2 were always high in diabetic children. Insulin-like growth factor binding protein 4 was high only in the prepubertal diabetic group. In conclusion, a low insulin-like growth factor I in diabetic children seems to depend on a GH receptor and/or a postreceptor defect. A low insulin-like growth factor I together with a normal insulin-like growth factor binding protein 3 and high levels of insulin-like growth factor binding proteins 1, 2 and 4 results in a reduced bioavailability of insulin-like growth factor I to target tissues. This could be a possible contributing factor to the reduced height velocity seen in our diabetic children.

Determination of thyroxine in the hair of newborns by radioimmunoassay with high-performance liquid chromatographic confirmation

Hair analysis is often used in forensic toxicology to study, retrospectively, chronic exposure of individuals to drugs, and consequently newborn hair may become an ideal sample to study intrauterine exposure to xenobiotics as well as to endogenous compounds. As a tool to investigate a supposed maternal thyroxine (T4) supply to the congenital hypothyroid fetus, we devised to use the analysis of T4 extracted from newborn hair. In the present paper, the analytical method based on T4 extraction from hair followed by a radioimmunoassay is described. To verify the nature of the T4-like immunoreactive material present in newborn hair, it was further studied by HPLC fractionation with radioimmunoassay of the eluted fractions. On the basis of a clear correspondence between retention times of T4 standard and T4-immunoreactive compound extracted from hair, we assigned this immunoreactive material to T4. Then, we determined T4 hair concentrations in 19 control newborns at birth and 12 congenital hypothyroid infants at 22 days of life. Values obtained from hypothyroid infants (31.47+/-8.8 pg/mg(hair), mean+/-S.D.) were not significantly lower than those obtained from healthy newborns at birth (36.10+/-13.2 pg/mg(hair)). Such results are in agreement with the hypothesis of a maternal supply of thyroxine to the fetus through placental crossing.

HIGH LEVELS OF GH-BP IN KWASHIORKOR AND MARASMUS

Severe malnutritions are characterised by low IGF-I and high GH plasma levels. The reasons why high levels of GH do not stimulate IGF-I synthesis are not clear. With the aim of improving our knowledge of this problem, we assayed plasma basal GH (by RIA), GH-BP(by HPLC gel filtration), IGF-I (by RIA) and IGFBP-3 (by RIA) in 20 children (age range: 1-2.2 yrs.). Of these, 6 were affected by kwashiorkor, 8 by marasmus and 6 were controls of the same ethnic group and age, hospitalised for minimal pathologies. At the same time the same parameters were evaluated in 5 acromegalic adults for comparison. All blood samples were taken after informed consent in occasion of routine samples. High basal GH levels were observed in kwashiorkor (14.6 ±5.3 ng/ml)(M±SD), marasmus (13. 5 ±2.5 ng/ml) and acromegalic patients (52.7 ±32.4 ng/ml); as expected in kwashiorkor and marasmus low levels of IGF-I (62.8 ±14.8 and 47.2 ±12.6 ng/ml respectively) and IGFBP-3 (1.37 ±0.8 and 0.81 ±0.6 mg/l respectively) were also found. In normal controls IGF-I was significantly higher (165.2 ±34.8 ng/ml, p<0.05 vs. both) and IGFBP-3 was higher but not in a significant way (3.3 ±1.6 mg/l). The specific binding of [125]-hGH to peak II GH-BP was 22.17 ±2.53 % of radioactivity in normal controls, was significantly higher in marasmus (28.92 ±1.65 %; p<0.05), kwashiorkor (31.8 ±2.3 %; p<0.01) and acromegaly (27.3 ±2.2 %; p<0.05). Circulating GH-BP is known to be a part of GH cell receptor and his amount is correlated and controlled by GH levels. The high levels of GH present in acromegalic serum are known to decrease the specific binding of the added [125]hGH during HPLC gel nitration. Marasmus and kwashiorkor have high levels of GH but also significantly higher % levels of specific bound radioactivity. Therefore we can conclude that in these conditions the presence of high amounts of circulating GHBP is possible.

BONE MINERAL METABOLISM IN PRECOCIOUS PUBERTY DURING TREATMENT

Gonadal steroids are known to play an important role in the control of GH-IGF-I axis and in bone mineral increase that occur during puberty. To better know the importance of estrogens on bone mineral metabolism during growth and puberty we studied ten girls (age range 6-7.2 yrs.) with true precocious puberty, before and after 1 year of LHRH analogs treatment. In these patients, before starting treatment with Leuprolide acetate (Enantone depot-Takeda), 0.2 mg/kg administered i.m. every 28 days, we performed: a clonidin stimulation test for GH; a 1, 25dihydroxyvitaminD3 llad (Rocaltrol-Roche) giving a dose of 2 μg/day for four consecutive days with the dosage before and after of serum Ca, P, IGF-I and Osteocalcin; a densitometric evaluation (dual photon absorptiometry) of BMC and BD (BMC/BW) in the distal and ultradistal region of the nondominant radius, After 1 year of therapy we repeated the same evaluations. During therapy serum estradiol levels decreased from pubertal (35.7 ± 3.5 pg/ml) (M± DS) to prepubertal levels (<20 pg/ml in each patient) as expected. GH peak after clonidin stimulation test after treatment was significantly lower than before (18.7 ± 2.2 vs 6.8 ± 3.5 ng/ml; p=0.005). IGF-I levels decreased not significantly at 12 months (from 278 ± 38 to 223 ± 53 ng/ml) and were not influenced by 1, 25(OH)2D3 load, while Osteocalcin levels were in the pubertal range before therapy (35, 8 ± 6, 2 ng/ml), decreased not significantly after therapy, and showed a slight increase after 1, 25(OH)2D3 load both before and after LHRHa therapy. Before therapy, BMC and BD were increased for chronological age, but appropriate for bone age. After 12 months of LHRH analogs treatment, BMC and BD showed a significant decrease (−4, 9 ± 1, 0 and −5, 1 ± 0, 9 %; p<0.05) in the ultradistal region (measuring trabecular bone), while there was a not significant difference in the distal region (measuring cortical bone). These results indicate that contemporary decrease in estrogen and GH-IGF-I levels may reduce osteoblastic activity and produce a decrease in bone mass.