Nicotinamide Nucleotide Transhydrogenase Is Essential for Adrenal Steroidogenesis: Clinical and In Vitro Lessons.

CONTEXT: Nicotinamide nucleotide transhydrogenase (NNT) acts as an antioxidant defense mechanism. NNT mutations cause familial glucocorticoid deficiency (FGD). How impaired oxidative stress disrupts adrenal steroidogenesis remains poorly understood. OBJECTIVE: To ascertain the role played by NNT in adrenal steroidogenesis. METHODS: The genotype-phenotype association of a novel pathogenic NNT variant was evaluated in a boy with FGD. Under basal and oxidative stress (OS) induced conditions, transient cell cultures of the patient's and controls' wild-type (WT) mononuclear blood cells were used to evaluate antioxidant mechanisms and mitochondrial parameters (reactive oxygen species [ROS] production, reduced glutathione [GSH], and mitochondrial mass). Using CRISPR/Cas9, a stable NNT gene knockdown model was built in H295R adrenocortical carcinoma cells to determine the role played by NNT in mitochondrial parameters and steroidogenesis. NNT immunohistochemistry was assessed in fetal and postnatal human adrenals. RESULTS: The homozygous NNT p.G866D variant segregated with the FGD phenotype. Under basal and OS conditions, p.G866D homozygous mononuclear blood cells exhibited increased ROS production, and decreased GSH levels and mitochondrial mass than WT NNT cells. In line H295R, NNT knocked down cells presented impaired NNT protein expression, increased ROS production, decreased the mitochondrial mass, as well as the size and the density of cholesterol lipid droplets. NNT knockdown affected steroidogenic enzyme expression, impairing cortisol and aldosterone secretion. In human adrenals, NNT is abundantly expressed in the transition fetal zone and in zona fasciculata. CONCLUSION: Together, these studies demonstrate the essential role of NNT in adrenal redox homeostasis and steroidogenesis.

Clinical, Molecular, Functional, and Structural Characterization of CYP17A1 Mutations in Brazilian Patients with 17-Hydroxylase Deficiency.

17-Hydroxylase-deficiency (17OHD) is a rare form of congenital adrenal hyperplasia. The aim of the work was to study clinical, biochemical, and the follow up of 17OHD patients and evaluate the function and structure of CYP17A1 mutations. Brazilian patients (three 46, XX and four 46, XY; 17±1.9 years) with combined 17-hydroxylase/17,20-lyase deficiency were evaluated. CYP17A1 gene was sequenced. Functional analysis was performed transfecting COS7 cells, which were exposed to progesterone or 17α-hydroxypregnolone substrates. Hormones were determined by RIA or LC-MS/MS. Three-dimensional structural modeling was performed by Modeller software. All patients presented prepubertal female external genitalia, primary amenorrhea, hypergonadotrophic hypogonadism, hypokalemic hypertension, decreased cortisol, and increased ACTH and corticosterone levels. Five patients presented previously described mutations: p.W406R/p.W406R, IVS2-2A>C/p.P428L, and p.P428L/p.P428L. Two patients presented the compound heterozygous p.G478S/p.I223Nfs*10 mutations, whose CYP17A1 activity and the three dimensional structural modeling are originally studied in this paper. CYP17A1 activity of p.G478S was 13 and 58% against progesterone and 17-hydroxypregnenolone, respectively. The p.I223Nfs*10 caused a truncated inactive protein. Three-dimensional p.G478S structural modeling showed different internal hydrophobic interaction with W313 and created an additional chain side contact with L476 residue. Due to the rarity of 17OHD, the long term follow up (15.3±3.1 years) of our patients will help endocrinologists on the management of patients with 17OHD. The mutation p.G478S/pI223Nfs*10 led to severe 17OHD and impaired CYP17A1 structure and function. The integration of in silico and in vitro analysis showed how the amino acid changes affected the CYP17A1 activity and contributed to clarify the molecular interactions of CYP17A1.

Procedimento operacional padronizado para a dosagem de alumínio sérico em hospital do interior paulista

Por ser um metal muito abundante na crosta terrestre, estamos em contato com o alumínio a todo momento, pela alimentação, medicamentos, materiais industrializados e até mesmo o ar que respiramos. Nosso organismo elimina rapidamente o alumínio, porém pacientes em hemodiálise tem a eliminação dificultada e ocorre acúmulo do metal em vários órgãos. Nesses deve-se ter uma monitorização biológica constante do alumínio de acordo com a Portaria 2042 do MS. O objetivo do trabalho é desenvolver um Procedimento Operacional Padronizado para dosagem de Alumínio sérico em um Espectrofotômetro de Absorção Atômica, equipado com Forno de Grafite Zeeman 640Z - Varian do Laboratório de Pediatria, Setor de Metais do HCFMRP- USP. Relavamos todo o material utilizado no processo, com HNO3 a 30% overnight e enxaguamos abundantemente com água ultrapura no dia das dosagens. As leituras foram realizadas em um Ambiente Clean, com filtro de alta eficiência (HEPA), em sala com classe certificada de 10000 partículas. Testamos os parâmetros comprimento de onda, rampa de aquecimento, tubo pirolítico e curvas de calibração. Foram analisadas soluções para preparo prévio de amostras e modificadores de matriz. Utilizou-se amostras de soro de um grupo controle de pessoas saudáveis, doadores voluntários do próprio laboratório e outro grupo com uma fração de amostras de soros de pacientes da Unidade de Terapia Renal, dosagens rotineiramente terceirizadas pelo hospital, anualmente, para validação da metodologia. O método desenvolvido proposto como adequado para a dosagem de Alumínio sérico tem comprimento de onda 396,2 rampa de aquecimento com 55,0 segundos de secagem 27,0 de pirólise e 1,7 de atomização com tubo de grafite plateau. A solução para preparo prévio das amostras foi HNO3 a 6%, Triton X-100 a 0,1% e Antifoam a 0,1% diluindo-se os soros um para dois e centrifugando dez minutos a 13.000 rpm. O modificador de Matriz foi o Fostato... .