Adrenal Hormone Interactions and Metabolism: A Single Sample Multi-Omics Approach.

The adrenal gland is important for many physiological and pathophysiological processes, but studies are often restricted by limited availability of sample material. Improved methods for sample preparation are needed to facilitate analyses of multiple classes of adrenal metabolites and macromolecules in a single sample. A procedure was developed for preparation of chromaffin cells, mouse adrenals, and human chromaffin tumors that allows for multi-omics analyses of different metabolites and preservation of native proteins. To evaluate the new procedure, aliquots of samples were also prepared using conventional procedures. Metabolites were analyzed by liquid-chromatography with mass spectrometry or electrochemical detection. Metabolite contents of chromaffin cells and tissues analyzed with the new procedure were similar or even higher than with conventional methods. Catecholamine contents were comparable between both procedures. The TCA cycle metabolites, cis-aconitate, isocitate, and α-ketoglutarate were detected at higher concentrations in cells, while in tumor tissue only isocitrate and potentially fumarate were measured at higher contents. In contrast, in a broad untargeted metabolomics approach, a methanol-based preparation procedure of adrenals led to a 1.3-fold higher number of detected metabolites. The established procedure also allows for simultaneous investigation of adrenal hormones and related enzyme activities as well as proteins within a single sample. This novel multi-omics approach not only minimizes the amount of sample required and overcomes problems associated with tissue heterogeneity, but also provides a more complete picture of adrenal function and intra-adrenal interactions than previously possible.

Frequency of varicella zoster virus DNA in human adrenal glands.

Varicella zoster virus (VZV) becomes latent in ganglionic neurons derived from neural crest cells. Because the adrenal gland also contains medullary chromaffin cells of neural crest origin, we examined human adrenal glands and medullary chromaffin cell tumors (pheochromocytomas) for VZV and herpes simplex virus type 1 (HSV-1). We found VZV, but not HSV-1, DNA in 4/63 (6 %) normal adrenal glands. No VZV transcripts or antigens were detected in the 4 VZV DNA-positive samples. No VZV or HSV-1 DNA was found in 21 pheochromocytomas.

Investigation of biochemical composition of adrenal gland tumors by means of FTIR.

The application of Fourier transform infrared (FTIR) microspectroscopy for the analysis of biomolecular composition of adrenal gland tumors is described. Samples were taken intraoperatively from three types of adrenal lesions: adrenal adenoma (ACA), adrenal cortical hyperplasia (ACH), both derived from adrenal cortical cells, and pheochromocytoma (Ph) derived from chromaffin cells of the adrenal medulla. The specimens were cryo-sectioned and freeze-dried. Since the investigated lesions originated from different cell types, it was predictable that they might differ in biomolecular composition. The experimental results were used to determine which absorption bands differentiate the analyzed samples the most. The main difference was observed in the lipid functional groups. The experimental results indicated that the level of lipids was higher in both the adenoma and the hyperplasia samples compared to pheochromocytomas. In contrast, the level of proteins was higher in the pheochromocytomas. Furthermore, differences within the range of nucleic acids and carbohydrates were observed in the studied adrenal gland tumor types.

Pheochromocytomas and Paragangliomas: An Update on Recent Molecular Genetic Advances and Criteria for Malignancy.

Pheochromocytomas are uncommon neuroendocrine tumors arising in the adrenal medulla, whereas paragangliomas arise from chromaffin cells in sympathetic and parasympathetic locations outside of the adrenal gland. Molecular genetic studies in the past few years have identified >10 genes involved in the pathogenesis of pheochromocytomas and paragangliomas, including RET oncogene, involved in the pathogenesis of multiple endocrine neoplasia (MEN) 2A and 2B, von Hippel-Lindau tumor-suppressor gene, neurofibromatosis type 1 gene, succinate dehydrogenase, THEM127, and several others. The presence of genetic alterations in some of these genes such as in MEN 2A and 2B can be used to diagnose these disorders clinically, and other mutations such as succinate dehydrogenase can be used in the pathologic prediction of benign and malignant pheochromocytomas and paragangliomas. Although it has been difficult to separate benign and malignant pheochromocytomas and paragangliomas, recent studies that may predict the behavior of these chromaffin-derived neoplasms have been reported. The Pheochromocytoma of the Adrenal Scale Score and the Grading system for Adrenal Pheochromocytoma and Paraganglioma scoring system are also discussed.

Biomolecular characterization of adrenal gland tumors by means of SR-FTIR.

The adrenal glands are small endocrine organs located on the bottom pole of each kidney. Anatomically they are composed of cortical and medullar parts. Due to dysfunctional processes they can transform into the pathological lesions (in both cortex and medulla). The incidentally detected adrenal lesions have become an arising clinical problem nowadays. The crucial issue for an accurate treatment strategy is relevant diagnosis. Distinguishing between benign and malignant lesions is often difficult during the standard histological examination. Hence the alternative methods of differentiation are investigated. One of them is Fourier transform infrared spectroscopy which allows the analysis of the biomolecular composition of the studied tissue. In this paper we present the very preliminary FTIR studies for defining the biomolecular pattern of three types of adrenal lesions: adenoma (AA) and adrenal cortical hyperplasia (ACH) - both derived from adrenal cortex as well as pheochromocytoma (PCC) - from the medullar part of the gland. All studied cases were classified as benign lesions. The general observations show that cortically derived tissues are rich in lipids and they are rather protein depleted while for medullar pheochromocytoma there is the opposite relationship. Furthermore, the unequivocal differences were noticed within the "fingerprinting" range. In addition subtle shifts in absorption band positions were observed between studied cases.

Immunohistochemical markers of the hypoxic response can identify malignancy in phaeochromocytomas and paragangliomas and optimize the detection of tumours with VHL germline mutations.

BACKGROUND: There are no reliable markers of malignancy in phaeochromocytomas (PCC) and paragangliomas (PGL). We investigated the relevance of the mammalian target of rapamycin (mTOR)/AKT and hypoxic pathways as novel immunohistochemical markers of malignancy. METHODS: Tissue microarray blocks were constructed with a total of 100 tumours (10 metastatic) and 20 normal adrenomedullary samples. Sections were immunostained for hypoxia-inducible factor 1α (Hif-1α), vascular endothelial growth factor A (VEGF-A), mTOR, carbonic anhydrase IX (CaIX) and AKT. The predictive performance of these markers was studied using univariate, multivariate and receiver operating characteristic analyses. RESULTS: In all, 100 consecutive patients, 64% PCC, 29% familial with a median tumour size of 4.7 cm (range 1-14) were included. Univariate analyses showed Hif-1α overexpression, tumour necrosis, size >5 cm, capsular and vascular invasion to be predictors of metastasis. In multivariate analysis, Hif-1α, necrosis and vascular invasion remained as independent predictors of metastasis. Hif-1α was the most discriminatory biomarker for the presence of metastatic diffusion. Strong membranous CaIX expression was seen in von Hippel-Lindau (VHL) PCC as opposed to other subtypes. CONCLUSION: Lack of vascular invasion, tumour necrosis and low Hif-1α expression identify tumours with lower risk of malignancy. We propose membranous CaIX expression as a potential marker for VHL disease in patients presenting with PCC.

Ultrastructural findings in adrenal cortical adenomas clinically mimicking pheochromocytoma: a comparison with other adrenal tumors and tissue preparation techniques.

Adrenal cortical tumors clinically mimicking pheochromocytomas are extremely rare, with 14 cases in the literature. The authors describe 2 patients with adrenal cortical adenoma (ACA) and catecholamine elevations. The impact of tissue preparation methods on electron microscopy (EM) images was assessed in ACA mimicking pheochromocytoma, pheochromocytoma, and ACA lacking pheochromocytoma-like symptoms. Ten adrenal cortical tumors were examined using EM after a variety of tissue preparation techniques, including fixation with glutaraldehyde, formalin for varying lengths of time followed by glutaraldehyde, and/or formalin followed by paraffin embedding. Electron micrographs were assessed for image quality and the presence of dense secretory granules and eccentric, norepinephrine (NE)-type granules. Images created from tissue fixed in glutaraldehyde and/or formalin and embedded in resin were of good quality, while those derived from paraffin-embedded specimens were poor with disrupted cellular architecture. When pheochromocytoma was fixed in glutaraldehyde for 24 h or in formalin for 8 days, eccentric granules were identified. These granules were absent when tissue was fixed in formalin for 20 days or was obtained from a paraffin block. ACA without pheochromocytoma-like symptoms and ACA mimicking pheochromocytoma both had noneccentric dense-core granules on EM regardless of tissue preparation, and eccentric NE-type granules were absent. ACA is a rare cause of pheochromocytoma-like symptoms. These tumors lack eccentric, NE-type dense-core granules present in pheochromocytoma. Glutaraldehyde alone or formalin fixation followed by glutaraldehyde produces electron micrographs that may aid in the diagnosis of adrenal cortical tumors, whereas formalin-fixed, paraffin-embedded tissue results in images that are inadequate.

CuCoFe Layered double hydroxides as laccase mimicking nanozymes for colorimetric detection of pheochromocytoma biomarkers.

A laccase catalyzed colorimetric biosensing approach is promising for the detection of pheochromocytoma biomarkers, yet suffers from the poor stability of enzymes and high cost for production. Here we report for the first time an easy to produce, cheap, stable and reliable laccase-mimicking CuCoFe-LDHzyme, which can catalyze the oxidation of pheochromocytoma biomarkers to form a chromogenic product for smartphone-based colorimetric detection.

Molecular investigations on the high-affinity nerve growth factor receptor.

Nerve growth factor (NGF) receptors have been investigated by means of affinity labeling with 125I-NGF and chemical cross-linking. Two distinct NGF-receptor complexes are detected on PC12 cells; these correspond to 100 kd and 158 kd for the low-affinity (LNGFR) and the high-affinity (HNGFR) receptors, respectively. Interestingly, three different antibodies directed against distinct epitopes on the LNGFR immunoprecipitate the low-but not the high-affinity NGF-receptor complex. Although the identities of the signaling molecules in the HNGFR are unknown, antibodies to the src, ras, raf-1, and yes products fail to immunoprecipitate either receptor complex, suggesting that these molecules are not a part of, or tightly coupled to, either receptor type. Phosphotyrosine residues are found exclusively on the HNGFR complex, suggesting that tyrosine phosphorylation may be one of the initiating events in the NGF-induced signal transduction cascade.

A case of multiple endocrine neoplasia type II accompanied by thyroid medullary carcinoma and pheochromocytomas expressing corticotropin-releasing factor and urocortins.

A 38-year-old woman with RET gene mutation presented with tumors in her thyroid and bilateral adrenal glands. I-metaiodobenzylguanidine scintigraphy revealed accumulation of the radioisotope in both adrenal glands. Both plasma adrenaline and noradrenaline levels were elevated. The circadian rhythms for plasma adrenocorticotropic hormone (ACTH) and cortisol levels were disturbed. Plasma ACTH and cortisol levels failed to be suppressed by an overnight dexamethasone test, suggesting autonomic secretion of ACTH and cortisol, although the patient had no typical Cushingoid features, hypertension, or impaired glucose tolerance. Pathological examination showed that these tumors were pheochromocytoma and thyroid medullary carcinoma, respectively, both of which highly expressed corticotropin-releasing factor, urocortin1, and urocortin3. Together with the endocrinological and pathological observations, the patient was diagnosed as multiple endocrine neoplasia type II with corticotropin-releasing factor- and urocortin-producing tumors that stimulated ACTH and glucocorticoid secretion.

Neuropeptide Y expression in phaeochromocytomas: relative absence in tumours from patients with von Hippel-Lindau syndrome.

Phaeochromocytomas are rare neuroendocrine tumours that produce catecholamines and numerous secretory proteins and peptides, including neuropeptide Y (NPY), a vasoactive peptide with influences on blood pressure. The production of catecholamines and NPY by phaeochromocytomas is highly variable. This study examined influences of hereditary factors and differences in catecholamine production on tumour expression of NPY, as assessed by quantitative PCR, enzyme immunoassay and immunohistochemistry. Phaeochromocytomas included hereditary adrenaline-producing tumours (adrenergic phenotype) in multiple endocrine neoplasia type 2 (MEN 2), predominantly noradrenaline-producing tumours (noradrenergic phenotype) in von Hippel-Lindau (VHL) syndrome, and other adrenergic and noradrenergic tumours where there was no clear hereditary syndrome. NPY levels in phaeochromocytomas from VHL patients were lower (P<0.0001) than in those from MEN 2 patients for both mRNA (84-fold difference) and the peptide (99-fold difference). These findings were supported by immunohistochemistry. NPY levels were also lower in VHL tumours than in those where there was no hereditary syndrome. Relative absence of expression of NPY in phaeochromocytomas from VHL patients when compared with other groups appears to be largely independent of differences in catecholamine production and is consistent with a unique phenotype in VHL syndrome.

Expression of urocortin 3/stresscopin in human adrenal glands and adrenal tumors.

Urocortin 3 (Ucn 3)/stresscopin (SCP) is a novel peptide of the corticotropin-releasing factor (CRF) family and is a specific ligand for the CRF type 2 receptor. In the present study, we studied expression of Ucn3/SCP in the normal adrenal and adrenal tumors by radioimmunoassay and reverse transcriptase-polymerase chain reaction (RT-PCR). High concentrations of immunoreactive (IR)-Ucn3 were present in the normal portions of adrenal glands (4.2+/-0.51 pmol/g wet weight, mean+/-S.E.M., n = 14), and the levels were higher than those in the brain. IR-Ucn3 was also detected in the tumor tissues of aldosterone-secreting adenomas (6.2+/-0.6 pmol/g wet weight, n = 10), cortisol-secreting adenomas (5.0+/-1.2 pmol/g wet weight, n = 4), and pheochromocytomas (1.9+/-0.4 pmol/g wet weight, n = 7). Reverse phase high performance liquid chromatography showed that IR-Ucn3 in normal portions of adrenal glands and aldosterone-secreting adenomas was eluted mainly in the positions of Ucn3 and SCP with several minor peaks eluting earlier. The RT-PCR showed expression of Ucn3 mRNA in normal portions of adrenal gland (positive ratio; 4/4), aldosterone-secreting adenomas (3/4), cortisol-secreting adenomas (1/3) and pheochromocytomas (6/7). These findings indicate that Ucn3 is produced in normal adrenal and adrenal tumors (both adrenocortical tumors and pheochromocytomas), and suggest that Ucn3 acts as an autocrine or paracrine regulator in normal adrenal and adrenal tumors.

Cutaneous metastasis of pheochromocytoma in multiple endocrine neoplasia IIB.

Pheochromocytoma is a rare tumor originating from neuroectodermic cells. Only 10% of these tumors are malignant. There are many familial forms of this tumor, including multiple endocrine neoplasia type II, Von Hippel-Lindau syndrome, and neurofibromatosis type I. Skin manifestations of pheochromocytoma are rare, and cutaneous metastasis in patients with multiple endocrine neoplasia IIB has never been described. The case of a patient with multiple endocrine neoplasia IIB who presented malignant pheochromocytoma with multiple cutaneous metastasis is described.

Histopathology and immunohistochemistry of adrenal medullary tumors and paragangliomas.

Paragangliomas arise from sympathetic or parasympathetic paraganglia and should now be defined by their site and type. The term pheochromocytoma is reserved for intra-adrenal tumors. This short review discusses the gross and microscopic features, the immunohistochemical profile, the problem of recognizing malignant potential, and the rare instances where a differential diagnosis has to be considered.

Somatostatin receptor imaging for neuroendocrine tumors.

Tumors and metastases that express the somatostatin receptor subtypes sst2 sst3 or sst5 can be visualized in vivo after injection of radiolabeled octapeptide somatostatin analogs, like (111)In-pentetreotide. (111)In-pentetreotide scintigraphy also allows for more accurate staging of the disease by demonstrating tumor sites, which were not shown by conventional imaging. (111)In-pentetreotide scintigraphy may also detect resectable tumors that would have remained unrecognized using conventional radiological imaging techniques; it may prevent surgery with curative intent in those patients whose tumors have metastasized to a greater extend than could be detected with conventional radiological imaging and it may be used to select patients for treatment with the currently available octapeptide somatostatin analogs or with tumor targeted radioactive treatment with radiolabelled somatostatin analogs. (111)In-pentetreotide scintigraphy has also been used to select patients with pituitary tumors for medical treatment with octapeptide analogs, but its clinical usefulness for this purpose seems to be limited. It further allows scar tissue to be differentiated from tumor recurrence after the pituitary surgery or radiotherapy. However, a large variety of lesions in and around the pituitary region also express somatostatin receptors and, therefore, can be visualized by (111)In-pentetreotide scintigraphy.

Pheochromocytoma and tetralogy of Fallot: Coincidence or a rare association?

We report the case of a 25-year-old woman with tetralogy of Fallot who presented with chest pain and hypertension, and on further investigation, was diagnosed with a pheochromocytoma in the right adrenal gland. She underwent surgical excision of the tumor. While the simultaneous occurrence of these two diseases is extremely rare, the suspicion of a possible relationship has been raised in the past.

High plasma levels of human chromogranin A and adrenomedullin in patients with pheochromocytoma.

AIMS AND BACKGROUND: The aim of our study was to investigate the plasma chromogranin A (CgA) and adrenomedullin (AM) levels in patients with pheochromocytomas. METHODS AND STUDY DESIGN: We collected blood samples for measurement of plasma CgA and AM in 21 patients with pheochromocytomas, 43 healthy subjects and 26 patients with solid non-functioning adrenocortical adenomas. In 11 patients with pheochromocytomas plasma CgA and AM were measured again four weeks after tumor removal. CgA and AM were measured by means of a novel solid-phase two-site immunoradiometric assay based on monoclonal antibodies (CgA-RIA CT, CIS bio international) and by a specific radioimmunoassay (RIA, Phoenix Pharm. Inc.), respectively. RESULTS: The mean plasma CgA level (+/- SD) in patients with pheochromocytomas (204 +/- 147.9 ng/mL) was significantly higher (P < 0.001) than that in healthy subjects (41.6 +/- 10.7 ng/mL) and in patients with non-functioning adrenocortical adenomas (47.3 +/- 17.6 ng/mL). The mean plasma AM concentration (+/- SD) in patients with pheochromocytomas (27.5 +/- 10.4 pg/mL) was significantly higher (P < 0.001) than that in HS (13.8 +/- 4.5 pg/mL) and in patients with non-functioning adrenocortical adenomas (16.6 +/- 7.3 pg/mL). Plasma CgA levels correlated with plasma AM levels (r = 0.501; P < 0.02) and with plasma metanephrine levels (r = 0.738; P < 0.0001) in patients with pheochromocytomas. In 11 patients with pheochromocytomas plasma CgA and AM concentrations significantly decreased after tumor removal (P < 0.001 for both). Circulating CgA and AM had a sensitivity of 76.2% and 81%, a specificity of 97.7% and 90.7%, and an accuracy of 91% and 88%, respectively. CONCLUSION: This study demonstrates that circulating CgA and AM levels are increased in pheochromocytoma patients compared with healthy subjects and patients with non-functioning adrenocortical adenomas. Moreover, at the time of diagnosis plasma CgA levels correlated with plasma AM levels and with plasma metanephrine levels in all patients with pheochromocytomas. In conclusion, plasma CgA and AM concentrations may represent additional biochemical parameters for clinical monitoring of patients with pheochromocytomas.

[Correlation between CT image presentations and biochemical indexes in adrenal adenomas and pheochromocytomas].

OBJECTIVE: To investigate the correlation between CT image presentations and biochemical indexes in adrenal adenomas and pheochromocytomas. METHODS: We retrospectively analyzed the CT features, cortisol rhythm, supine and orthostatic hypertension and 24-h urine methoxy in 209 patients with benign adrenal tumors. The relationship between CT findings and the biochemical indexes were analyzed in patients with different benign adrenal tumors. RESULTS: The 209 cases analyzed included 53 cases of cortisol adenoma, 65 cases of aldosterone adenoma, 45 cases of non-functional adenoma and 46 cases of pheochromocytomas. The plain CT scan values of the 4 groups were 17.25 ± 1.81, 14.52 ± 1.57, 12.20 ± 2.05, 42.42 ± 0.97 HU, enhanced CT values (arterial phase) were 47.82 ± 3.07, 39.23 ± 2.37, 45.35 ± 6.46, and 104.93 ± 5.84 HU, respectively, and the differences between CT scan and enhanced CT values were 30.58 ± 2.29, 24.71 ± 1.55, 33.15 ± 5.18, and 62.51 ± 5.73 HU, respectively. In cortisol adenoma group, cortisol levels measured at 16:00 and 24:00 were positively correlated with plain CT scan value (r=0.506, P=0.0001; r=0.504, P=0.0001) and enhanced CT value (r=0.514, P=0.0001; r=0.554, P=0.0001). In pheochromocytoma group, plain scan CT value and the difference between plain and enhanced CT scan value were correlated with 24-h urine methoxy adrenaline (Rho;=0.342, 0.350; P=0.020, 0.017, respectively) and norepinephrine (Rho;=0.419, 0.412; P=0.004, 0.004, respectively). CONCLUSION: Plain and enhanced CT scan values and their combination have important value in differential diagnosis of adenoma and pheochromocytoma tumor, CT values combine with biochemical indexes can reduce misdiagnosis and missed diagnosis of pheochromocytoma.

Diverse proportion in composite pheochromocytoma-ganglioneuroma may induce varied clinical symptom: comparison of two cases.

Composite pheochromocytoma-ganglioneuroma is extremely rare. We described two cases of composite pheochromocytomas in the adrenal medullar. Case 1 was a 70-year-old male presenting with lower abdominal pain and normal blood electrolytes. Case 2 was a 48-year-old female with palpitation and back tenderness. Biochemical investigations showed hypocalcium, hypokalemia and high level of vma. The histological images and the immunohistochemical staining demonstrated the two cases composed of pheochromocytoma and ganglioneuromoma components. Ganglioneuroma component in case 2 accounted for more proportion than that in case 1. We speculated that the varied clinical symptoms were related with the diverse proportions in composite pheochromocytome-ganglioneuroma.