Niveles plasmáticos de vitamina D en población autóctona y en poblaciones inmigrantes de diferentes etnias menores de 6 años de edad / Plasma vitamin D levels in native and immigrant children under the age of 6 years of different ethnic origins

INTRODUCCIÓN: El raquitismo carencial es una enfermedad emergente en nuestro medio y se describe especialmente en lactantes y niños inmigrantes de raza negra o piel oscura procedentes de países en vías de desarrollo. El objetivo de este trabajo está dirigido a conocer el estado nutricional de vitamina D en lactantes y niños inmigrantes de diferentes etnias de edad inferior a 6 años y compararlo con una población infantil autóctona. Población y métodos: Estudio prospectivo efectuado en un Centro de Asistencia Primaria de la localidad de Salt (Girona). Población: 307 niños con la siguiente distribución por origen y etnia: caucásicos (n=85; 28%), subsaharianos (n=101; 32,5%); magrebíes (n=87, 28,0%); centroamericanos (n=20; 6,4%) e indopakistaníes (n=14; 4,5%). Parámetros bioquímicos evaluados: calcemia, fosforemia, fosfatasa alcalina, 25-hidroxivitamina D y parathormona. Encuesta nutricional para estimar la ingesta de calcio, el aporte de vitamina D y el grado de exposición solar. RESULTADOS: Presentan déficit de vitamina D (< 20 ng/ml) el 8% de los niños de origen caucásico, el 18% de los subsaharianos, el 20% de los centroamericanos, el 34,5% de los magrebíes y el 64% de los niños de origen indopakistaní. El 2,9% de los niños estudiados (n = 9) presentan déficit grave de vitamina D (< 10 ng/ml), de los que tan solo un niño de origen subsahariano reúne criterios bioquímicos de raquitismo clásico. La prevalencia de la deficiencia de vitamina D es significativamente más elevada en los niños sin suplementación con vitamina D durante el primer año de vida. CONCLUSIONES: El 22,5% de los niños menores de 6 años de edad presenta concentraciones plasmáticas en rango deficitario de vitamina D, siendo más prevalente en los niños de origen indopakistaní y magrebí

INTRODUCTION: Nutritional rickets is an emergent disease in Spain, and occurs particularly in black and dark-skinned infants and children from immigrant populations. The aim of this work was to ascertain the vitamin D reserve in a population of native and immigrant children under the age of 6 years. Population and methods: A prospective study was conducted at a Primary Healthcare Centre in Salt (Girona). PATIENTS: 307 children with the following origin and race distribution: Caucasian (n = 85; 28%), Sub-Saharan (n = 101; 32.5%); Maghrebí (n = 87, 28.0%); Central-American (n = 20; 6.4%) and Indo-Pakistani (n = 14; 4.5%). The biochemistry blood parameters studied were: calcium, phosphorus, alkaline phosphatase, 25-hydroxivitamin D, and parathormone. A nutritional survey was used to estimate calcium and vitamin D intake and degree of sun exposure. RESULTS: Vitamin D deficiency (< 20 ng/ml) was detected in Caucasians (8%), Sub-Saharans (18%), Central-Americans (20%), Maghrebís (34.5%), and Indo-Pakistanis (64%). Of the children studied (n = 9), 2.9% had serious vitamin D deficiency (< 10 ng/ml); only one child of Sub-Saharan origin met the biochemical criteria for classical rickets. The prevalence of vitamin D deficiency was significantly higher in children not receiving vitamin D supplements in the first year of life. CONCLUSIONS: Plasma vitamin D concentrations were deficient in 22.5% of children under the age of six, being more prevalent in children of Indo-Pakistani and Maghrebí origin

Evaluación de la anemia ferropénica en niños menores de 6 años de edad de diferentes etnias / Evaluation of iron deficiency anemia in a population of different ethnic groups under 6 years

Introducción: La alta prevalencia de ferropenia en nuestro medio, así como el número elevado de población inmigrante con hábitos y culturas alimentarias diferentes, fundamentan un estudio para evaluar el estado nutricional y documentar la presencia de ferropenias secundarias a las diversas pautas de alimentación. Objetivos: Evaluar los parámetros antropométricos y las concentraciones plasmáticas de hemoglobina, ferritina y sideremia de cada etnia. Población y métodos: Estudio prospectivo realizado durante los años 2008-2010 en una consulta de pediatría de atención primaria, en una población de 307 niños de Salt (Girona), con una edad inferior a 6 años, de diferentes etnias: caucásicos (n= 85; 27,4%), magrebíes (n= 87; 28%), subsaharianos (n= 101; 32,5%), centroamericanos (n= 20; 6,4%) e indopakistaníes (n= 14; 4,5%). Los parámetros bioquímicos estudiados fueron la hematimetría, la sideremia y la ferritina. Resultados: El análisis de los parámetros antropométricos no demuestra diferencias significativas entre la población autóctona y la inmigrante. En el análisis bioquímico se pone de manifiesto un déficit de hierro (sideremia <50 µg/dL) (caucásicos 38,5%, magrebíes 51%, subsaharianos 43%, centroamericanos 35%, indopakistaníes 79%), un déficit de ferritina (<20 ng/mL) (caucásicos 10,5%, magrebíes 49%, subsaharianos 29%, centroamericanos 15% e indopakistaníes 85%) y un déficit de hemoglobina (<10,5 mg/mL) (caucásicos 3,7%, magrebíes 7%, subsaharianos 15%, centroamericanos 5% e indopakistaníes 21%). Conclusiones: No se ha detectado desnutrición con repercusión auxológica en las poblaciones evaluadas. Pero se aprecia un déficit de hierro, ferritina y hemoglobina en las poblaciones infantiles magrebí, subsahariana y, de forma más acusada, indopakistaní (AU)

Introduction: Due to the high prevalence of ferropenic diseases and the increase of different immigrant populations with their own eating habits, a population-based study was considered necessary in order to evaluate the nutritional status and document possible iron deficiencies secondary to each food culture. Study purpose: To evaluate the anthropometric parameters, the hemoglobin and ferritin plasmatic concentrations and the sideremy values in each ethnic group. Population and methods: Prospective study carried out from 2008 to 2010, in a paediatrician consultation working area. Population: 307 children from Salt (Girona) under the age of 6, Caucasian (n= 85; 27.4%), Moroccan (n= 87; 28%), Sub-Saharan African (n= 101; 32.5%), Central American (n= 20; 6.4%) and Indian-Paquistan (n= 14; 4.5%). Biochemical parameters: blood count, sideremy and ferritin values. Results: Analysis of anthropometric parameters did not show any relevant difference between the native and the immigrant population. The biochemical data showed a sideremy deficiency (<50 µg/dL) (Caucasian population 38.5%, Moroccan 51%, Sub-Saharan African 43%, Central American 35%, Indian-Paquistan 79%), a ferritin deficiency (<20 ng/mL) (Caucasian population 10.5%, Moroccan 49%, Sub-Saharan African 29%, Central American 15%, Indian-Paquistan 85%) and an hemoglobin deficiency (<10.5 mg/mL) (Caucasian population 3.7%, Moroccan 7%, Sub-Saharan African 15%, Central American 5%, Indian-Paquistan 21%). Conclusions: No auxologic malnutrition was detected in the studied population; however, there was a clear deficiency in sideremy, ferritin and hemoglobin in the child populations from Morocco and Sub-Saharan Africa, this being even more evident among the Indian-Paquistan population (AU)

[Plasma vitamin D levels in native and immigrant children under the age of 6 years of different ethnic origins]. / Niveles plasmáticos de vitamina D en población autóctona y en poblaciones inmigrantes de diferentes etnias menores de 6 años de edad.

INTRODUCTION: Nutritional rickets is an emergent disease in Spain, and occurs particularly in black and dark-skinned infants and children from immigrant populations. The aim of this work was to ascertain the vitamin D reserve in a population of native and immigrant children under the age of 6 years. POPULATION AND METHODS: A prospective study was conducted at a Primary Healthcare Centre in Salt (Girona). PATIENTS: 307 children with the following origin and race distribution: Caucasian (n=85; 28%), Sub-Saharan (n=101; 32.5%); Maghrebí (n=87, 28.0%); Central-American (n=20; 6.4%) and Indo-Pakistani (n=14; 4.5%). The biochemistry blood parameters studied were: calcium, phosphorus, alkaline phosphatase, 25-hydroxivitamin D, and parathormone. A nutritional survey was used to estimate calcium and vitamin D intake and degree of sun exposure. RESULTS: Vitamin D deficiency (<20 ng/ml) was detected in Caucasians (8%), Sub-Saharans (18%), Central-Americans (20%), Maghrebís (34.5%), and Indo-Pakistanis (64%). Of the children studied (n=9), 2.9% had serious vitamin D deficiency (< 10 ng/ml); only one child of Sub-Saharan origin met the biochemical criteria for classical rickets. The prevalence of vitamin D deficiency was significantly higher in children not receiving vitamin D supplements in the first year of life. CONCLUSIONS: Plasma vitamin D concentrations were deficient in 22.5% of children under the age of six, being more prevalent in children of Indo-Pakistani and Maghrebí origin.

AR and SRD5A2 gene mutations in a series of 51 Turkish 46,XY DSD children with a clinical diagnosis of androgen insensitivity.

46,XY disorders of sex development (DSD) are caused by disorders of gonadal development, androgen biosynthesis and receptor (AR) defects. Although, clinical/biochemical features help in distinguishing specific aetiologies, there are overlaps which necessitate molecular analyses for the definitive diagnosis. To test precision of our clinical diagnosis of androgen insensitivity (AIS) by analysing AR and then SRD5A2 genes, patients were recruited at Marmara University Hospital and molecular analyses were performed at Vall d'Hebron Research Institute. Among 101 46,XY DSD patients, 46 index and five siblings (nine complete, 42 partial) with clinical/biochemical data suggestive of AIS and stimulated T/DHT ratio <25 were selected. AR and then SRD5A2 genes were sequenced. We detected AR mutations in 11 patients [seven index and four siblings (22% of all and 15% of index patients)] and SRD5A2 mutations in six [five index and one sibling (12% of all and 11% of index)]. AR mutation detection rate was 6/9 in all CAIS and 4/7 in the index (67 and 57% respectively) and 5/42 in all PAIS and 3/40 in the index (12 and 7.5% respectively). The eight mutations detected in the AR gene were as follows: p.Q58L, p.P392S, p.R609K, p.R775H, p.R856H, p.A871A, p.V890M and p.F892L, with p.A871A and p.F892L being novel. Further six patients had SRD5A2 mutations which were as follows: p.L73WfsX59, p.Y91H, p.R171S and p.G196S, the first being novel. Hormonal data in those with AR mutations, SRD5A2 mutations and no mutations were not statistically different. In conclusion, a significant proportion of children with presumptive diagnosis of AIS has a normal AR gene. The less severe the phenotype, the less likely is the chance of demonstrating a mutation. Furthermore, a significant number of children with presumptive diagnosis of AIS have mutations in SRD5A2 gene and are clinically and biochemically indistinguishable from AIS.

Deficiencia de hormona de crecimiento (GH) en la edad pediátrica / Growth hormone (GH) deficiency in the pediatric age

La deficiencia de hormona de crecimiento (DGH) provoca manifestaciones clínicas distintas, según su etiología y la etapa del desarrollo, pero siempre existe un denominador común: Las otras manifestaciones clínica dependerán d ela etiología (genética, adquirida o idiopática), de la intensidad de la deficienica y de si es la única hormona hipofisaria afectada o existe afectación de otras hormonas hipofisarias. Los avances de los últimos años han ampliado el conocimiento de sus bases moleculares y han caracterizado mejor las formas adquiridas. Sin embargo, la mayor parte de DGH no tienen una causa conocida y son catalogadas como idiopáticas. Mientras que los criterios clínicos y moleculares del diagnóstico de DGH están bien establecidos, los criterios hormonales continúan siendo un rompecabezas a esar de los esfuerzos realizados para armonizar las técnicas bioquímicas de análisis de GH y de IGF-1. Los diagnósticos basados en los estímulos secretores de GH han demostrado ser la escasa utilidad clínica para predecir la respueta terapéutica a la GH (AU)

Growth hormone (GH) deficiency manifests differently according to the individual´s developmental stage. During the paediatric period, one of the msot prominent clincial features is chronic skeletal growth retardation. Clinical signs also depend on the cause (genetic, acquired or idiopathic), deficiency intensity and whether GH is the only pituitary-affected hormone or is combined with that of other pituitary hormones. Growing knowledge of the genetic basis of GH deficiency continues to provide us with useful information to further characterise to provide us with useful information to further characterise mutation types and mechanisms for prevously-described and new candidate genes. Despite these advances, a high proportion of GH deficiencies with no recognisable acquired basis continue to be labelled as idiopathic. The hormonal diagnoses continue to be a conundrum despite efforts to harmonise biochemical assays for GH and IGF-1 analysis, and the diagnosis based on the so-called GH secretion stimulation tests with prove to be of limited usefulness for predicting response to GH therapy (AU)

Height gain at adult-height age in 184 short patients treated with growth hormone from prepubertal age to near adult-height age is not related to GH secretory status at GH therapy onset.

BACKGROUND: GH release after stimuli classifies short children as severe idiopathic isolated GH deficiency (IIGHD), mild IIGHD, dissociated GH release (DGHR) and normal GH release (NGHR) and anthropometric birth data as adequate for gestational age (AGA) or small for gestational age (SGA). GH release after stimuli classifies AGA patients as IIGHD or as idiopathic short stature (ISS). AIM: To compare height gain induced by GH therapy (31.8 ± 3.5 µg/kg/day, 7.7 ± 1.6 years) started at prepubertal age and stopped at near adult-height age. METHODS: A retrospective longitudinal multicenter study including 184 short patients classified as severe IIGHD n = 25, mild IIGHD n = 75, DGHR n = 55 and NGHR n = 29; or as IIGHD n = 78, ISS n = 57 and SGA n = 49. Height gain was evaluated throughout GH therapy and at adult-height age. RESULTS: Height-SDS gain at adult-height age was similar among severe IIGHD (1.8 ± 0.8 SDS), mild IIGHD (1.6 ± 0.6 SDS), DGHR (1.7 ± 0.7 SDS) and NGHR (1.6 ± 0.7 SDS), or among IIGHD (1.7 ± 0.7 SDS), ISS (1.7 ± 0.6 SDS) and SGA (1.6 ± 0.8 SD). CONCLUSION: GH-release stimuli are of little help for deciding on GH therapy in the clinical management of prepubertal children with IIGHD, ISS or SGA.

Growth hormone deficiency: an update.

Growth hormone (GH) deficiency (GHD) in humans manifests differently according to the individual developmental stage (early after birth, during childhood, at puberty or in adulthood), the cause or mechanism (genetic, acquired or idiopathic), deficiency intensity and whether it is the only pituitary-affected hormone or is combined with that of other pituitary hormones or forms part of a complex syndrome. Growing knowledge of the genetic basis of GH deficiency continues to provide us with useful information to further characterise mutation types and mechanisms for previously described and new candidate genes. Despite these advances, a high proportion of GH deficiencies with no recognisable acquired basis continue to be labelled as idiopathic, although less frequently when they are congenital and/or familial. The clinical and biochemical diagnoses continue to be a conundrum despite efforts to harmonise biochemical assays for GH and IGF-1 analysis, probably because the diagnosis based on the so-called GH secretion stimulation tests will prove to be of limited usefulness for predicting therapy indications.

Ten novel mutations in the NR5A1 gene cause disordered sex development in 46,XY and ovarian insufficiency in 46,XX individuals.

CONTEXT: Steroidogenic factor-1 (SF-1/NR5A1) is a nuclear receptor that regulates adrenal and reproductive development and function. NR5A1 mutations have been detected in 46,XY individuals with disorders of sexual development (DSD) but apparently normal adrenal function and in 46,XX women with normal sexual development yet primary ovarian insufficiency (POI). OBJECTIVE: A group of 100 46,XY DSD and two POI was studied for NR5A1 mutations and their impact. DESIGN: Clinical, biochemical, histological, genetic, and functional characteristics of the patients with NR5A1 mutations are reported. SETTING: Patients were referred from different centers in Spain, Switzerland, and Turkey. Histological and genetic studies were performed in Barcelona, Spain. In vitro studies were performed in Bern, Switzerland. PATIENTS: A total of 65 Spanish and 35 Turkish patients with 46,XY DSD and two Swiss 46,XX patients with POI were investigated. MAIN OUTCOME: Ten novel heterozygote NR5A1 mutations were detected and characterized (five missense, one nonsense, three frameshift mutations, and one duplication). RESULTS: The novel NR5A1 mutations were tested in vitro by promoter transactivation assays showing grossly reduced activity for mutations in the DNA binding domain and variably reduced activity for other mutations. Dominant negative effect of the mutations was excluded. We found high variability and thus no apparent genotype-structure-function-phenotype correlation. Histological studies of testes revealed vacuolization of Leydig cells due to fat accumulation. CONCLUSIONS: SF-1/NR5A1 mutations are frequently found in 46,XY DSD individuals (9%) and manifest with a broad phenotype. Testes histology is characteristic for fat accumulation and degeneration over time, similar to findings observed in patients with lipoid congenital adrenal hyperplasia (due to StAR mutations). Genotype-structure-function-phenotype correlation remains elusive.

SRD5A2 gene mutations and polymorphisms in Spanish 46,XY patients with a disorder of sex differentiation.

One hundred and forty-six index patients with 46,XY DSD in whom gonads were confirmed as testes were consecutively studied for a molecular diagnosis during the period 2002-2010. AR gene was analysed in all patients as the first candidate gene, yielding a mutation in 42.5% of cases and SRD5A2 gene was analysed as the second candidate gene, resulting in the characterization of 10 different mutations (p.Y91D, p.G115D, p.Q126R, p.R171S, p.Y188CfsX9, p.N193S, p.A207D, p.F219SfsX60, p.R227Q and p.R246W) in nine index patients (6.2% of the total number of 46,XY DSD patients). One of the mutations (p.Y188CfsX9) has never been reported. In addition, we genotyped SRD5A2 gene p.V89L and c.281+15T>C polymorphisms in 46,XY DSD and in 156 normal adult males and found that patients with SRD5A2 mutations or without a known molecular diagnosis presented a higher frequency of homozygous p.L89, homozygous TT and combined CCTT genotypes compared with controls. This result suggests that 46,XY DSD patient phenotypes may be influenced by SRD5A2 polymorphism genotypes. SRD5A2 gene mutations may not be as infrequent as previously considered in 46,XY DSD patients with variable degrees of external genitalia virilization at birth and normal T production and appears to be the second aetiology in our series.

Growth hormone secretory status evaluated by growth hormone peak after two pharmacological growth hormone release stimuli did not significantly influence the two-year catch-up growth induced by growth hormone therapy in 318 prepubertal short children with idiopathic growth retardation.

BACKGROUND/AIMS: In prepubertal short children with idiopathic growth retardation, growth hormone (GH) peak after GH release stimuli classifies patients as growth hormone- deficient (GHD) or non-GHD. This study compared a 2-year growth response to GH therapy in 318 prepubertal short children. METHODS: Patients were classified as: severe GHD (GH peaks <5 ng/ml after 2 stimuli; n = 54), mild GHD (GH peaks <10 ng/ml, but one or two between 5 and 10 ng/ml; n = 140), dissociated GH release (GH peak ≥ 10 ng/ml after 1 stimulus and <10 ng/ml after the other; n = 89), and normal GH release (GH peaks ≥ 10 ng/ml after 2 stimuli; n = 35). RESULTS: Two-year height gain did not differ statistically among the 4 groups: 1.39 ± 0.51 SD, 16.4 ± 2.3 cm; 1.23 ± 0.56 SD, 15.8 ± 2.1 cm; 1.18 ± 0.53 SD, 15.3 ± 2.0 cm, and 1.14 ± 0.53 SD, 15.4 ± 2.0 cm, respectively, as was also the case for bone age gain: 2.5 ± 0.6, 2.4 ± 0.7, 2.6 ± 0.7 and 2.3 ± 0.5 years, respectively. CONCLUSIONS: Our results suggest that GH release stimuli are of little help for deciding on GH therapy in the clinical management of prepubertal short children with idiopathic growth retardation, while well-defined anthropometric and biochemical criteria may be useful.

Novel (60%) and recurrent (40%) androgen receptor gene mutations in a series of 59 patients with a 46,XY disorder of sex development.

BACKGROUND: Androgen receptor (AR) gene mutations are the most frequent cause of 46,XY disorders of sex development (DSD) and are associated with a variety of phenotypes, ranging from phenotypic women [complete androgen insensitivity syndrome (CAIS)] to milder degrees of undervirilization (partial form or PAIS) or men with only infertility (mild form or MAIS). OBJECTIVE: The aim of the study was to characterize the contribution of the AR gene to the molecular cause of 46,XY DSD in a series of Spanish patients. SETTING: We studied a series of 133 index patients with 46,XY DSD in whom gonads were differentiated as testes, with phenotypes including varying degrees of undervirilization, and in whom the AR gene was the first candidate for a molecular analysis. METHODS: The AR gene was sequenced (exons 1 to 8 with intronic flanking regions) in all patients and in family members of 61% of AR-mutated gene patients. RESULTS: AR gene mutations were found in 59 individuals (44.4% of index patients), of whom 46 (78%) were CAIS and 13 (22%) PAIS. Fifty-seven different mutations were found: 21.0% located in exon 1, 15.8% in exons 2 and 3, 57.9% in exons 4-8, and 5.3% intronic. Twenty-three mutations (40.4%) had been previously described and 34 (59.6%) were novel. CONCLUSIONS: AR gene mutation is the most frequent cause of 46,XY DSD, with a clearly higher frequency in the complete phenotype. Mutations spread along the whole coding sequence, including exon 1. This series shows that 60% of mutations detected during the period 2002-2009 were novel.

Vitamin D and growth hormone regulate growth hormone/insulin-like growth factor (GH-IGF) axis gene expression in human fetal epiphyseal chondrocytes.

OBJECTIVE: Cell proliferation and gene expression regulation were studied in human fetal epiphyseal chondrocytes to ascertain the involvement of GH-IGF axis components in human fetal growth regulation by 1,25-dihydroxyvitamin D(3) (VitD) and growth hormone (GH). DESIGN: Chondrocytes from primary cultures were plated in serum-free medium for 48 h and incubated for a further 48 h with VitD (10(-11) to 10(-6)M) and/or IGF-I (100 ng/ml) and/or GH (500 ng/ml). We analyzed (3)H-thymidine incorporation into DNA and IGF-I, IGFBP-3, GHR, SOX9, COL2A1, aggrecan and COMP gene expression by real-time quantitative PCR. RESULTS: VitD dose-dependently and significantly inhibited (3)H-thymidine incorporation whereas GH had no effect on proliferation and, when combined with VitD, the same inhibition was observed as with VitD alone. IGF-I (100 ng/ml) significantly stimulated proliferation and opposed inhibition by VitD. VitD dose-dependently stimulated IGF-I (11.1+/-19.8 at VitD10(-6)M), IGFBP-3 (2.6+/-0.9), GHR (3.8+/-2.8) and COMP (1.5+/-0.6) expression whereas it inhibited SOX9 (0.7+/-0.2), COL2A1 (0.6+/-0.3) and aggrecan (0.6+/-0.2) expression and had no significant effect on IGF-II. IGF-I stimulated IGF-I, IGFBP-3, SOX9, COL2A1 and aggrecan expression and opposed COL2A1 and aggrecan gene expression inhibition by VitD. GH alone had no effect on gene expression whereas, in the presence of VitD, significantly-increased IGF-I expression stimulation was observed above values obtained with VitD alone (17.5+/-7.4). CONCLUSIONS: Our results suggest that VitD regulation of fetal growth cartilage could have consisted of parallel enhancing of cell differentiation and conditioning to a phenotype more sensitive to regulation by other hormones such as GH as shown by increased GHR and IGF-I expression, but not by IGF-II expression which was not regulated.

IGF-I and not IGF-II expression is regulated by glucocorticoids in human fetal epiphyseal chondrocytes.

OBJECTIVE: To elucidate the involvement of IGF axis components and the potential effects of glucocorticoids (GCs) in human fetal growth regulation. DESIGN: We studied the regulation by dexamethasone (Dx) and IGF-I of proliferation and IGF axis components and matrix protein gene expression in human fetal epiphyseal chondrocytes. RESULTS: High Dx concentration (10(-7)-10(-6)M) inhibited (3)H-thymidine incorporation, mifepristone (MF) 10(-6)M limited inhibition by Dx, and IGF-I (100 ng/ml) significantly stimulated proliferation and completely opposed inhibition by Dx. Dx dose-dependently (10(-9)-10(-6)M) inhibited IGF-I, IGFBP3 and SOX9 gene expression and expression of GHR, COL2A1 and aggrecan from 10(-7)M to 10(-6)M whereas it stimulated IGF-IR expression. By contrast, Dx had no significant effect on IGF-II expression. IGF-I stimulated IGF-I, IGFBP3, SOX9, COL2A1 and aggrecan expression whereas it inhibited IGF-IR expression. IGF-I could oppose COL2A1 and aggrecan gene expression inhibition by Dx. CONCLUSIONS: We demonstrated for the first time by real-time quantitative PCR that human fetal epiphyseal chondrocytes expressed IGF axis components, such as IGF-I, IGF-II, IGFBP3, IGF-IR and GHR and SOX9, COL2A1 and aggrecan, and that their expression was regulated by Dx and IGF-I. Among IGFs, IGF-I and not IGF-II expression was demonstrated to be down-regulated by GCs whereas IGF-I expression was up-regulated by itself.

The exon 3-deleted/full-length growth hormone receptor polymorphism does not influence the effect of puberty or growth hormone therapy on glucose homeostasis in short non-growth hormone-deficient small-for-gestational-age children: results from a two-year controlled prospective study.

CONTEXT: The exon 3-deleted/full-length (d3/fl) GH receptor polymorphism (d3/fl-GHR) has been associated with responsiveness to GH therapy in short small-for-gestational-age (SGA) patients, although consensus is lacking. However, its influence on glucose homeostasis, at baseline or under GH therapy, has not been investigated. OBJECTIVE: Our objective was to evaluate whether the d3/fl-GHR genotypes influence insulin sensitivity in short SGA children before or after puberty onset or during GH therapy. DESIGN: We conducted a 2-yr prospective, controlled, randomized trial. SETTING: Thirty Spanish hospitals participated. Auxological, GH secretion, and glucose homeostasis evaluation was hospital based, whereas molecular analyses and data computation were centralized. PATIENTS: Patients included 219 short SGA children [body mass index sd score (SDS) < or = 2.0]; 159 were prepubertal (group 1), and 60 had entered puberty (group 2). INTERVENTION: Seventy-eight patients from group 1 were treated with GH (66 microg/kg.d) for 2 yr (group 3). MAIN OUTCOME MEASURES: Previous and 2-yr follow-up auxological and biochemical data were recorded, d3/fl-GHR genotypes determined, and data analyzed. RESULTS: In groups 1 and 2, fasting glucose, insulin, homeostasis model assessment (HOMA), and quantitative insulin sensitivity check index (QUICKI) were similar in each d3/fl-GHR genotype. Group 2 glucose, insulin, and HOMA were significantly higher and QUICKI lower than in group 1. In group 3 GH-treated patients, height SDS, growth velocity SDS, fasting glucose, insulin, and HOMA significantly increased as did body mass index SDS at the end of the second year, and QUICKI decreased during the first and second years, with no differences among the d3/fl-GHR genotypes. CONCLUSION: In short SGA patients, the d3/fl-GHR genotypes do not seem to influence prepubertal or pubertal insulin sensitivity indexes or their changes over 2 yr of GH therapy (66 mug/kg.d).

The exon 3-deleted/full-length growth hormone receptor polymorphism did not influence growth response to growth hormone therapy over two years in prepubertal short children born at term with adequate weight and length for gestational age.

CONTEXT: Consensus is lacking as to whether the exon 3-deleted (d3)/full-length (fl) GH receptor (GHR) polymorphism is associated with responsiveness to GH therapy. OBJECTIVE: Our objective was to evaluate, in short, prepubertal, appropriate-for-gestational age (AGA) patients, 2-yr growth response to GH therapy (31.7+/-3.5 microg/kg.d) according to exon 3-deleted/full-length GHR genotypes. DESIGN: We conducted a retrospective study. PATIENTS: We studied 106 short AGA children, 58 boys and 48 girls, 7.8+/-2.3 yr, (d3/d3 n=18, d3/fl n=42, and fl/fl n=46). The GH response to two provocative stimuli were under 10 ng/ml in 65 and one or both over 10 ng/ml in 41 patients. MAIN OUTCOME MEASURES: Patients were followed by a single clinical team and remained prepubertal during the study. The exon 3-deleted/full-length GHR genotypes were determined and analyzed in the same hospital. RESULTS: Growth velocity significantly (P<0.0001) increased during the first and second years of therapy, as did height sd score (SDS). These increases were similar in each exon 3-deleted/full-length GHR genotype. Total 2-yr height gain (SDS) did not differ statistically among genotypes: 15.5+/-2.2 cm and 1.2+/-0.5 SDS in d3/d3, 15.9+/-2.0 cm and 1.3+/-0.4 SDS in d3/fl, and 15.4+/-2.1 cm and 1.1+/-0.3 SDS in fl/fl. No significant differences among the three genotypes were found in both sexes or in patients with different GH peak response to provocative stimuli for these parameters. An analysis of previously published studies was also performed. CONCLUSIONS: These results confirm in AGA patients those previously found by us and others in small-for-gestational-age patients and suggest that neither sex nor GH peaks after provocative stimuli might influence significantly the responsiveness to GH therapy according to the exon 3-deleted/full-length GHR genotypes.

The d3/fl-growth hormone (GH) receptor polymorphism does not influence the effect of GH treatment (66 microg/kg per day) or the spontaneous growth in short non-GH-deficient small-for-gestational-age children: results from a two-year controlled prospective study in 170 Spanish patients.

CONTEXT: The d3/fl-GH receptor (d3/fl-GHR, exon 3-deleted/full-length GHR) has recently been associated with responsiveness to GH therapy. OBJECTIVE: The objective of the study was to evaluate whether the d3/fl-GHR genotypes influence the intensity of spontaneous and/or GH therapy-stimulated growth in small-for-gestational-age (SGA) patients. DESIGN: This was a 2-yr prospective, controlled, randomized trial. SETTING: Thirty Spanish hospitals participated. Auxologic and GH secretion evaluation was hospital based, whereas molecular analyses and auxologic data computation were centralized. PATIENTS: Patients included 170 short SGA children: 140 remained prepubertal and 30 entered puberty during the second follow-up year. INTERVENTION: Eighty-six were treated with GH (66 microg/kg.d) for 2 yr and 84 were not treated. MAIN OUTCOME MEASURES: Previous and 2-yr follow-up auxologic data were recorded at each hospital, d3/fl-GHR genotypes determined, and data analyzed for patients who remained prepubertal (group 1, 68 GH treated and 72 non-GH treated) and for all the patients (group 2). RESULTS: In group 1 GH-treated patients, growth velocity, and height-sd score during the first and second years, total 2-yr height gain (18.5 +/- 2.4 cm in d3/d3; 18.4 +/- 2.6 in d3/fl; 19.5 +/- 2.3 in fl/fl), Delta 2-yr height increase (9.1 +/- 2.4 cm in d3/d3; 9.4 +/- 3.0 in d3/fl; 10.4 +/- 2.1 in fl/fl), first-year growth prediction and studentized residual values (0.08 +/- 1.26 in d3/d3; 0.28 +/- 1.21 in d3/fl; 0.67 +/- 0.95 in fl/fl) did not differ among the d3/fl-GHR genotypes. In group 1 non-GH-treated patients, neither growth velocity nor height-sd score changed significantly, and values were similar in each d3/fl-GHR genotype. Results in all patients (group 2) were similar to those in group 1. CONCLUSIONS: In short non-GH-deficient SGA children, both spontaneous growth rate and responsiveness to 66 microg/k.d GH therapy were similar for each d3/fl-GHR genotype carried.

Disgenesias gonadales y pseudohermafroditismo masculino / No disponible

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Structural requirements of benzodiazepines for the inhibition of pig brain nitric oxide synthase.

Nitric oxide synthases (NOS) are heme-containing enzymes which catalyse the oxidation of L-arginine to nitric oxide and L-citrulline. Some nitrogenated compounds have been reported to coordinate with the iron atom from the heme group, thus inhibiting NOS. 1,4-Benzodiazepines are nitrogenated compounds which have many physiological effects such as antianxiety, antiepileptic, hypnotic, and muscle relaxation properties. The aim of this paper was to measure the effect of different benzodiazepines on NOS activity in pig brain extracts. Medazepam, pinazepam, diazepam, oxazepam and alprazolam competitively inhibited NOS with IC(50) in the micromolar range. Other benzodiazepines showed no effect at concentrations as high as 200 microM. Due to the structural similarity of the benzodiazepine ring nucleus with L-arginine, we propose a benzodiazepine-enzyme interaction to explain the competitive inhibitions. By comparing benzodiazepine effects and their structures, the inhibitory effect of benzodiazepines on NOS is related to the absence of substituents on N4 and to the absence of a halogen substituent on C5 phenyl group. Although benzodiazepine's inhibitions observed in this study are not in the physiological range in normal cases, these inhibitions could be significant in drug abuse situations and should be taken into account for the rational design of drugs which specifically inhibit NOS.

Sources of interference in the use of 2,3-diaminonaphthalene for the fluorimetric determination of nitric oxide synthase activity in biological samples.

The use of 2,3-diaminonaphthalene (DAN) for the fluorimetric determination of nitric oxide synthase (NOS) activity in rat brain extracts has been re-examined. Two types of interference were observed, due either to components of the reaction mixture or to the enzymatic sample itself. One of the substrates (NADPH) and some cofactors (FADH(2), FMNH(2)) required for the enzyme activity interfere in the assay by quenching the fluorescence produced. Interference was minimized by using lower FADH(2), FMNH(2) and NADPH concentrations (1 micromol/l) and a NADPH recycling system in the reaction mixture. The addition of bovine serum albumin or hemoglobin to the sample quenched fluorescence intensity, but these protein interferences could be reduced by filtering the samples after reaction. We conclude that the DAN fluorimetric assay as originally described is not suitable for the determination of NOS activity in crude extracts such as rat brain cytosolic fraction, due to the presence of interfering substances. Nevertheless, DAN could be used for the determination of enzyme activity after reducing protein interference by filtering, or in less complex samples such as cell cultures (e.g. activated macrophages), or in chromatographic fractions obtained during the purification of the enzyme. A careful use of the commercial kits based on the use of DAN for the determination of NOS activity is recommended.