Primary culture and identification of cells from human aldosterone-producing adenoma / 基础医学与临床

Objective To establish the primary culture of aldosterone-producing adenoma cells. Methods The tumor tissue was digested by collagen type I and cultured in complete DMEM/F12 medium. Aldosterone concentra-tion in culture medium was detected by radioimmunoassay. The expression of aldosterone synthase in the culture cells was detected by immunofluorescence. Results Aldosterone-producing adenoma cells grew adherently in a round or approximately round shape. The cells were positively immunostained for aldosterone synthase. The aldoste-rone concentration in the culture medium at the 5th culture day was (30.0±8.9)nmol/L. During the primary cul-ture,aldosterone secretion was the strongest at the first day,and decreased afterwards. It kept stable from day 4 to 11. Conclusions We successfully established the primary culture of aldosterone-producing adenoma cells, which are important for the future studying on the mechanism and function of aldosterone-producing adenoma.

Value of Chloride Clearance Test in Differential Diagnosis of Gitelman Syndrome / 中国医学科学院学报

Objective To investigate the value of chloride clearance test in differential diagnosis of Gitelman syndrome (GS). Methods For patients with hypokalemic metabolic alkalosis and highly suspected GS,clinical data were documented and SLC12A3 gene screening was performed as gold standard to diagnose GS. Hydrochlorothiazide (HCT) test and furosemide (FUR) test were performed according to the standard process. Baseline and maximal increasement of chloride excretion fraction (FECl,the net and relative increase measured as εFECl) were compared between patients and controls to evaluated the reaction to the corresponding diuretics. Receiver operating characteristic (ROC) curve was used to evaluate the sensitivity and specificity of HCT test in GS diagnosis. Results Totally 27 patients and 20 health controls received HCT test. Among those patients,23 were diagnosed with GS genetically. When using the net and relative εFECl to diagnose GS,the areas under the ROC curve were 0.987 (95% CI:0.963～1.000,P<0.001) and 0.984 (95%CI:0.950～1.000,P<0.001),respectively. When a reasonable cutoff value for εFECl was selected,the sensitivity and specificity were both higher than 95%. Eight patients received both HCT test and FUR test. Five of them showed decreased reaction to HCT(net εFECl≤2.86% or relative εFECl≤223%),while normal reaction to FUR.SLC12A3 mutations confirmed their GS. Three patients with blunt reaction to FUR showed normal reaction to HCT,finally they were diagnosed as BS clinically because no SLC12A3 gene mutation was detected. Conclusion Comprehensive application of HCT test and FUR test to evaluate the diuretic reaction can effectively differentiate GS and BS.

Heteroplasmy level of the mitochondrial tRNaLeu(UUR) A3243G mutation in a Chinese family is positively associated with earlier age-of-onset and increasing severity of diabetes / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the mutations of mitochondrial genome in a pedigree with suspected maternally inherited diabetes and deafness and to explore the correlations between the mutations and clinical features.</p><p><b>METHODS</b>Genomic DNA was isolated from blood leucocytes of each member of the pedigree. The mitochondrial genome was amplified with 24-pair primers that could cover the entire mitochondrial DNA. Direct sequencing of PCR products was used to identify any mitochondrial DNA mutations.</p><p><b>RESULTS</b>Family members on the maternal side all harbored the tRNALeu(UUR) A3243G mutation. The paternal side family members did not have the mutation. The age-of-onset of diabetes of the 4 maternal side family members was 15, 41, 44, and 65 years old, and their corresponding heteroplasmy level of the mutation was 34.5%, 14.9%, 14.6%, and 5.9%, respectively. The age-of-onset of diabetes and heteroplasmy level of A3243G mutation were negatively correlated with a correlation coefficient of -0.980 (P = 0.02). Meanwhile, patient with high heteroplasmy level of A3243G mutation had relatively low severity of disease. Moreover, 6 reported polymorphisms and 2 new variants were found.</p><p><b>CONCLUSIONS</b>The main cause of diabetes in this pedigree is the tRNALeu(UUR) A3243G mutation. However, other gene variants may contribute to its pathogenicity. The heteroplasmy level of the tRNALeu(UUR) A3243G mutation is positively associated with earlier age-of-onset and increasing severity of diabetes.</p>

Bilateral pheochromocytoma as first presentation of von Hippel-Lindau disease in a Chinese family / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the clinical and genetic features of a Chinese family with von Hippel-Lindau (VHL) disease revealed by bilateral pheochromocytoma.</p><p><b>METHODS</b>The proband and other members in a Chinese family with familial pheochromocytoma were clinically evaluated and followed up. Genomic DNA extracted from the peripheral blood of 8 family members (including 3 patients) was amplified by polymerase chain reaction (PCR) and the PCR products were directly sequenced.</p><p><b>RESULTS</b>The first presentation in the proband, his mother, and his sister was bilateral pheochromocytoma, and the missense mutation of 695G-A (Arg161Gln) in exon 3 of VHL gene was detected in the three patients. In the follow-up study, the proband and his mother were found to have other VHL tumors, induding retinal and cerebellar hemangioblastomas and pancreatic tumor. Neither clinical presentation of VHL disease nor gene mutation was found in other family members.</p><p><b>CONCLUSION</b>VHL disease should be suspected in some patients with familial pheochromocytoma, and VHL gene screening helps to achieve early diagnosis of the disease.</p>

Effect of hypokalemia on glucose metabolism in primary hyperaldosteronism / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effect of potassium deficiency on glucose and insulin metabolism in primary hyperaldosteronism, including aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA).</p><p><b>METHODS</b>Totally 178 patients who were diagnosed as primary hyperaldosteronism (103 patients with APA and 75 with IHA) were divided into hypokalemia group and normal potassium group according to their serum potassium levels. All patients received 3 hours of oral glucose tolerance test and aldosterone test to observe the relationship among glucose, insulin and serum potassium.</p><p><b>RESULTS</b>Area under curve of serum potassium, area under curve of plasma insulin, and fasting serum insulin were significantly lower in the hypokalemia group than in the normal potassium group (P <0. 05, P <0. 01); area under curve of glucose and aldosterone level were significantly higher in the hypokalemia group than in the normal potassium group ( P < 0. 05 ) . The prevalence of metabolic syndrome was significantly higher in IHA than in APA (57. 3% vs 38. 8% ; P < 0. 05).</p><p><b>CONCLUSION</b>Hypokalemia may play an important role in inhibiting insulin secretion in primary hyperaldosteronism.</p>

Expression of renin-angiotensin-aldosterone system in human adipose tissues / 中国医学科学院学报

<p><b>OBJECTIVE</b>To compare the mRNA expression of renin-angiotensin-aldosterone system in human subcutaneous and visceral adipose tissues.</p><p><b>METHODS</b>Total RNA was extracted from 12 human subcutaneous adipose tissues, 12 perirenal adipose tissue and 9 periadrenal adipose tissues. The expressions of angiotensinogen ( AGT) , renin, angiotensin converting enzyme ( ACE) , angiotensin converting enzyme 2 (ACE2), angiotensin I1 receptor type 1 (AT1), angiotensin II receptor type 2 (AT2 ), CYP11 B2, and their internal reference glyceraldehyde phosphate (GAPDH) were studied by reverse transcription-polymerase chain reaction. The ratios of each target genes were used to evaluate the expression levels of AGT, renin, ACE, ACE2, AT1, AT2, and CYP11B2 in different adipose tissues.</p><p><b>RESULTS</b>The mRNA expressions of AGT, ACE, ACE2, AT1, and AT2 were detected in human subcutaneous, perirenal, and periadrenal adipose tissues. However, CYPI B2 mRNA expression was not found in these three adipose tissues. The mRNA expressions of renin was only detected in perirenal and periadrenal adipose tissues, which was significantly higher in perirenal adipose tissues than in periadrenal adipose tissues ( P < 0. 05 ). The mRNA expressions of ACE and ACE2 in perirenal adipose tissues were significantly higher than that in subcutaneous adipose tissues ( P < 0. 05). The mRNA expressions of ACE were significantly higher than that of ACE2 in subcutaneous, perirenal, and periadrenal adipose tissues (P <0. 05). The mRNA expressions of AT1 were significantly lower than that of AT2 in periadrenal adipose tissues (P < 0. 05).</p><p><b>CONCLUSION</b>Local renin-angiotensin system exists in the adipose tissues; however, aldosterone is not synthesized in the adipose tissues.</p>

Expression of adrenomedullin and its receptor mRNA in the tissues of normal adrenal medulla and pheochromocytoma / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the expression of human adrenomedullin (ADM) and its receptor-receptor activity modifying protein 2/calcitonin receptor-like receptor (RAMP2/CRLR) mRNA in the tissues of normal adrenal medulla and pheochromocytoma.</p><p><b>METHODS</b>Total RNA was extracted from normal adrenal medulla and pheochromocytomas. The expression of ADM and RAMP2/CRLR mRNA were studied by reverse transcription-polymerase chain reaction. The ratios of ADM/GAPDH, RAMP2/ GAPDH, CRLR/GAPDH were used to evaluate the expression levels of ADM, RAMP2 and CRLR mRNA.</p><p><b>RESULTS</b>Expressions of ADM and its receptor- RAMP2/CRLR mRNA were detected in normal adrenal medulla and pheochromocytoma tissues. ADM/GAPDH were 0.48+/-0.09 and 0.75+/-0.24, RAMP2/ GAPDH 0.79+/-0.12 and 1.29+/-0.30, CRLR/GAPDH 0.40+/-0.08 and 0.87+/-0.22 in normal adrenal medulla and pheochromocytomas, respectively (P < 0.05).</p><p><b>CONCLUSION</b>ADM exerts a possible autocrine or paracrine effect in the adrenal. ADM may be involved in the pathogenesis of pheochromocytoma.</p>

Expression of urotensin II and G-protein coupled receptor 14 mRNA in human pheochromocytoma tissues / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the expression of urotensin II (U II) and G-protein coupled receptor 14 (GPR14) mRNA in human pheochromocytoma tissues.</p><p><b>METHODS</b>Total RNA from normal adrenal and pheochromocytoma tissues was extracted. The reverse transcription-polymerase chain reaction method was used to evaluate the levels of U II and GPR14 mRNA expression in human pheochromocytoma tissues.</p><p><b>RESULTS</b>There was no significant difference of U II and GPR14 mRNA expression between normal adrenal cortex and medulla. The expression of U II and GPR14 mRNA in pheochromocytoma was significantly lower than that in normal adrenal cortex and medulla (P < 0.05). The expression of GPR14 mRNA in adrenal pheochromocytomas was significantly lower than that of extra-adrenal pheochromocytomas (P < 0.05).</p><p><b>CONCLUSION</b>U II and GPR14 may play a role in the pathogenesis and hypertension regulating of pheochromocytoma.</p>

Expression of transforming growth factor alpha, tumor necrosis factor alpha, and vascular endothelial growth factor of human pheochromocytoma tissues / 中国医学科学院学报

<p><b>OBJECTIVE</b>To compare the expressions of transforming growth factor alpha (TGFalpha), tumor necrosis factor alpha (TNFalpha), and vascular endothelial growth factor (VEGF) between pheochromocytoma (PHEO) tissues and normal adrenal medulla tissues.</p><p><b>METHODS</b>The mRNA expressions of TGFalpha, TNFalpha, and VEGF detected by RT-PCR, were compared between 22 PHEO tissues and 18 normal adrenal medulla tissues (according with the principle of medical ethnics). Immunohistochemistry staining was performed on 27 PHEO tissues and 14 normal adrenal medulla tissues. The comparisons of the protein expression of TGFalpha, TNFalpha, and VEGF were analyzed in both of PHEO tissues and normal adrenal medulla tissues.</p><p><b>RESULTS</b>Compared with normal adrenal medulla tissues, the expressions of TGFalpha and TNFalpha mRNA and protein were higher in PHEO tissues, and VEGF145 mRNA expression was also higher in PHEO tissues, while there was no significant difference of the mRNA expression of VEGF121 and VEGF165 between these two tissues. Positive staining rates for VEGF of endothelial cells and tumor cells were higher in PHEO tissues than in normal adrenal medulla tissues. Expressions of the TGFalpha, TNFalpha, and VEGF protein were higher in extra-adrenal PHEO than in adrenal PHEO. The TNFalpha immunohistochemistry staining rate was higher in the malignant or multiple PHEO than in the benign or single PHEO.</p><p><b>CONCLUSIONS</b>The mRNA and protein expressions of TGFalpha, TNFalpha, and VEGF are higher in PHEO tissues than those in normal adrenal medulla tissues. Expressions of these cytokines vary in PHEO with different characteristic.</p>

Insulin resistance and abnormal glucose metabolism in patients with primary aldosteronism / 中华内分泌代谢杂志

Objective To analysis the relationship of primary aldosteronism to insulin resistance and abnormal glucose metabolism in patients with primary aldosteronism.Methods One hundred and three patients were diagnosed as aldosterone-producing adenoma(APA),75 cases as idiopathic hyperaldosterenism(IHA),56 patients as metabolic syndrome(MS)in Peking Union Medical College Hospital from September 2003 to December 2005.All patients were accepted 3 h oral glucose tolerance test and calculated HOMA-insulin resistance index (HOMA-IR)by Homeostasis Model.Results The Insulin area under curve[INS_(AUC) 270.8(192.7,370.4)mU?L~(-1)?h~(-1)]、HOMA-IR[3.2(2.4,4.7)]and prevalence of insulin resistance(64.3%)were significantly higher in patients with MS as compared with APA[113.2(81.5,193.6)mU?L~(-1)?h~(-1),1.4(1.0,2.2),16.5%]and IHA[186.9(116.6,243.3)mU?L~(-1)?h~(-1),2.0(1.4,3.1),32.0%]groups (all P＜0.01);The INS_(AUC), HOMA-IR and prevalence of insulin resistance were significantly higher in patients with IHA than those with APA (P＜0.05 or P＜0.01).There was no difference between the prevalence of impaired glucose regulation(41.7%, 34.7%),diabetes mellitus(15.5%,16.0%)in patients with APA,IHA and those with MS(41.1%, 26.8%).Conclusion Insulin resistance is present in primary aldosteronism patients,and the prevalence of insulin resistance is higher in patients with IHA.