Graeme Eisenhofer

Catecholamine Metabolism: A Contemporary View with Implications for Physiology and Medicine

This article provides an update about catecholamine metabolism, with emphasis on correcting common misconceptions relevant to catecholamine systems in health and disease. Importantly, most metabolism of catecholamines takes place within the same cells where the amines are synthesized. This mainly occurs secondary to leakage of catecholamines from vesicular stores into the cytoplasm. These stores exist in a highly dynamic equilibrium, with passive outward leakage counterbalanced by inward active transport controlled by vesicular monoamine transporters. In catecholaminergic neurons, the presence of monoamine oxidase leads to formation of reactive catecholaldehydes. Production of these toxic aldehydes depends on the dynamics of vesicular-axoplasmic monoamine exchange and enzyme-catalyzed conversion to nontoxic acids or alcohols. In sympathetic nerves, the aldehyde produced from norepinephrine is converted to 3,4-dihydroxyphenylglycol, not 3,4-dihydroxymandelic acid. Subsequent extraneuronal O-methylation consequently leads to production of 3-methoxy-4-hydroxyphenylglycol, not vanillylmandelic acid. Vanillylmandelic acid is instead formed in the liver by oxidation of 3-methoxy-4-hydroxyphenylglycol catalyzed by alcohol and aldehyde dehydrogenases. Compared to intraneuronal deamination, extraneuronal O-methylation of norepinephrine and epinephrine to metanephrines represent minor pathways of metabolism. The single largest source of metanephrines is the adrenal medulla. Similarly, pheochromocytoma tumor cells produce large amounts of metanephrines from catecholamines leaking from stores. Thus, these metabolites are particularly useful for detecting pheochromocytomas. The large contribution of intraneuronal deamination to catecholamine turnover, and dependence of this on the vesicular-axoplasmic monoamine exchange process, helps explain how synthesis, release, metabolism, turnover, and stores of catecholamines are regulated in a coordinated fashion during stress and in disease states.

Pheochromocytoma and Paraganglioma: An Endocrine Society Clinical Practice Guideline

OBJECTIVE The aim was to formulate clinical practice guidelines for pheochromocytoma and paraganglioma (PPGL). PARTICIPANTS The Task Force included a chair selected by the Endocrine Society Clinical Guidelines Subcommittee (CGS), seven experts in the field, and a methodologist. The authors received no corporate funding or remuneration. EVIDENCE This evidence-based guideline was developed using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system to describe both the strength of recommendations and the quality of evidence. The Task Force reviewed primary evidence and commissioned two additional systematic reviews. CONSENSUS PROCESS One group meeting, several conference calls, and e-mail communications enabled consensus. Committees and members of the Endocrine Society, European Society of Endocrinology, and Americal Association for Clinical Chemistry reviewed drafts of the guidelines. CONCLUSIONS The Task Force recommends that initial biochemical testing for PPGLs should include measurements of plasma free or urinary fractionated metanephrines. Consideration should be given to preanalytical factors leading to false-positive or false-negative results. All positive results require follow-up. Computed tomography is suggested for initial imaging, but magnetic resonance is a better option in patients with metastatic disease or when radiation exposure must be limited. (123)I-metaiodobenzylguanidine scintigraphy is a useful imaging modality for metastatic PPGLs. We recommend consideration of genetic testing in all patients, with testing by accredited laboratories. Patients with paraganglioma should be tested for SDHx mutations, and those with metastatic disease for SDHB mutations. All patients with functional PPGLs should undergo preoperative blockade to prevent perioperative complications. Preparation should include a high-sodium diet and fluid intake to prevent postoperative hypotension. We recommend minimally invasive adrenalectomy for most pheochromocytomas with open resection for most paragangliomas. Partial adrenalectomy is an option for selected patients. Lifelong follow-up is suggested to detect recurrent or metastatic disease. We suggest personalized management with evaluation and treatment by multidisciplinary teams with appropriate expertise to ensure favorable outcomes.

Recent advances in genetics, diagnosis, localization, and treatment of pheochromocytoma.

Dr. Karel Pacak (Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development [NICHD] and Clinical Neurocardiology Section, National Institute of Neurological Disorders and Stroke [NINDS], National Institutes of Health [NIH], Bethesda, Maryland): Pheochromocytomas are chromaffin cell tumors that, although rare, must be considered in patients with hypertension, autonomic disturbances, panic attacks, adrenal incidentalomas, or familial diseases featuring a predisposition to develop pheochromocytoma. Hypertension, whether sustained or paroxysmal, is the most common clinical sign, and headache, excessive truncal sweating, and palpitations are the most common symptoms (1). Pallor is also common, whereas flushing occurs less frequently. Some patients present with severe episodes of anxiety, nervousness, or panic. Patients with a familial predisposition or small incidentally discovered adrenal masses can be normotensive and asymptomatic. The low pretest prevalence of pheochromocytomaclose to 0.5% among those tested because of hypertension and suggestive symptoms (1) and as high as 4% in patients with adrenal incidentaloma (2)together with imperfect sensitivity and specificity of commonly used biochemical and imaging tests, can make diagnosis and localization of pheochromocytoma difficult. Effective methods for diagnosis and localization are important because seemingly mild stimuli can provoke the tumor to release large amounts of catecholamines, with severe or fatal consequences. Moreover, surgical removal can cure pheochromocytoma in up to 90% of cases, whereas if left untreated the tumor can prove fatal. Advances in genetic mutation analysis have greatly improved identification of patients with familial pheochromocytoma, allowing detection of tumors at an early stage, often before typical signs and symptoms occur. These advances provide new insights into the biology and natural history of the disease and highlight inadequacies of commonly used diagnostic tests. In turn, new developments have been made in the management of patients with familial pheochromocytoma and in surgical procedures for preserving normal adrenal cortical tissue in persons with bilateral adrenal tumors. In this paper, we summarize advances in the genetics, biochemical diagnosis, localization, and management of pheochromocytoma and also note key questions that remain unanswered. Molecular Genetic Abnormalities Associated with Pheochromocytoma Drs. W. Marston Linehan and McClellan M. Walther (Urologic Oncology Branch, National Cancer Institute [NCI], NIH, Bethesda, Maryland): Pheochromocytomas may be classified as sporadic or familial. Most pheochromocytomas are sporadic. Familial predisposition is seen mainly in patients with multiple endocrine neoplasia type II (MEN II), von HippelLindau disease, neurofibromatosis type 1, and familial carotid body tumors (Table 1). The exact molecular mechanisms by which the hereditary mutations predispose to tumor development remain unknown. Hereditary forms of pheochromocytoma can differ in rate of tumor growth, malignant potential, and catecholamine phenotype. Table 1. Hereditary Forms of Pheochromocytoma Cancer Genes Identification of a cancer gene can help us understand the origin of cancer, such as pheochromocytoma, and elucidate mechanisms of tumor formation and behavior. Moreover, identification of a disease gene provides a method for genetic diagnosis. Phenotypic manifestations of a hereditary cancer syndrome can vary markedly, and genetic tests can confirm the diagnosis when the clinical presentation is complex. Finally, understanding of cancer genes may provide targets for therapy. The two most studied types of cancer genes are tumor suppressor genes (Figure 1) and oncogenes (7). When mutated, a proto-oncogene becomes activated, resulting in an oncogene. This is referred to as a single hit; that is, the proto-oncogene undergoes a single activating mutation that turns it into an oncogene (8, 9). Familial predisposition to pheochromocytoma in patients with MEN II results from such a mechanism. In contrast, a tumor suppressor gene is a loss-of-function gene, in which inactivation of both copies of the gene causes unregulated cell growth and division. This loss of function can result from mutation of one allele of a tumor suppressor gene and deletion of the second copy (10). Examples of tumor suppressor genes are the retinoblastoma gene, the Wilms tumor gene, the tuberous sclerosis genes, and, in the case of pheochromocytoma, the von HippelLindau gene (11-19). Figure 1. The Knudson two-hit model. Pheochromocytoma in Multiple Endocrine Neoplasia Type II: RETGene Multiple endocrine neoplasia type IIA is characterized clinically by the familial association of medullary thyroid cancer, pheochromocytoma, and parathyroid hyperplasia. Mucosal ganglioneuromas are also found in some patients (MEN IIB). Pheochromocytoma in MEN II is associated with germline mutation of the proto-oncogene RET. This proto-oncogene becomes an oncogene when an activating mutation occurs (20-25). The activating mutation in the RETgene drives the abnormal cellular proliferation that leads to adrenal medullary hyperplasia and pheochromocytoma. Several RETgermline mutations are associated with the development of pheochromocytoma, with some variation dependent on the particular mutation (3-5, 26, 27) (Table 1). Pheochromocytoma in von HippelLindau Disease: the von HippelLindau Gene Patients with von HippelLindau disease have a germline mutation of the von HippelLindau gene (28). Affected persons can develop early-onset bilateral kidney tumors and cysts, pheochromocytomas, cerebellar and spinal hemangioblastomas, retinal angiomas, pancreatic cysts and tumors, epididymal cystadenomas, and tumors in the endolymphatic sac canal of the inner ear (29-31). von HippelLindau disease has marked phenotypic heterogeneity. While patients from some families present with central neural, eye, kidney, and pancreatic tumors, patients in other families present mainly with pheochromocytoma (30, 32, 33). Some reports have described families thought to have familial pheochromocytoma who proved to have von HippelLindau disease (32, 34-37). Missense mutations in the von HippelLindau gene are associated with the development of pheochromocytoma more than twice as often as are other types of mutations (74% vs. 32%) (6, 33). Molecular Genetic Diagnosis von HippelLindau disease and MEN II have a similar prevalence (approximately 1 in 30 000 to 1 in 45 500). Mutations predisposing to pheochromocytoma have greater penetrance in MEN II than in von HippelLindau disease (38, 39). Pheochromocytoma in von HippelLindau families has been reported as familial pheochromocytoma or MEN II (40, 41). Because different kindreds can present with different phenotypes, it can be difficult to distinguish between von HippelLindau disease and MEN II in some patients with familial pheochromocytoma. Patients with bilateral adrenal, recurrent, or multifocal pheochromocytoma should undergo clinical or genetic testing for mutations of the von HippelLindau or RETgenes. The availability of germline testing for both von HippelLindau (42) and RET (15, 20, 23, 40, 43) gene mutations (at OncorMed in Gaithersburg, Maryland, and at the University of Pennsylvania in Philadelphia) has improved the clinical management of patients with hereditary pheochromocytoma. When a patient presents with a family history in which the primary manifestation is pheochromocytoma, the von HippelLindau gene is a likely cause. Some von HippelLindau families present mainly with pheochromocytoma and occult or delayed manifestations in the central nervous system, eye, or other organs. It is less likely that a member of a MEN II family will present predominantly with pheochromocytoma because most of these patients have medullary thyroid carcinoma (44). A small number of families with familial pheochromocytoma have neither von HippelLindau nor RETgermline mutations, and the genetic basis for this is currently being studied. Biochemical Diagnosis of Pheochromocytoma Dr. Graeme Eisenhofer (Clinical Neurocardiology Section, NINDS, NIH, Bethesda, Maryland): Diagnosis of pheochromocytoma usually requires biochemical evidence of excessive catecholamine production by the tumor, usually achieved from measurements of catecholamines or catecholamine metabolites in urine or plasma. These biochemical approaches, however, have several limitations. Since catecholamines are normally produced by sympathetic nerves and by the adrenal medulla, high catecholamine levels are not specific to pheochromocytoma and may accompany other conditions or disease states. In addition, sometimes pheochromocytomas do not secrete enough catecholamines to produce positive test results or typical signs and symptoms. In addition, pheochromocytomas often secrete catecholamines episodically. Between episodes, levels of catecholamines may be normal. Thus, commonly used tests of plasma or urinary catecholamines and metabolites and other biochemical tests, such as measurements of plasma chromogranin A levels, do not always reliably exclude or confirm a tumor (45-55). A recently developed biochemical test, involving measurements of plasma levels of free metanephrines (o-methylated metabolites of catecholamines), circumvents many of the above problems and offers a more effective means to diagnose pheochromocytoma than other tests (46, 56). Sensitivity of Biochemical Tests Measurements of plasma levels of normetanephrine and metanephrine have higher sensitivity than other biochemical tests for diagnosis of both sporadic and familial pheochromocytoma (46, 56). In familial pheochromocytoma, periodic screening can lead to early-stage detection before symptoms and signs, when tumors are small and are not secreting large amounts of catecholamines (6). The difficulty of biochemical diagnosis of familial pheochromocytoma is illustrated

Pheochromocytoma: Recommendations for clinical practice from the First International Symposium

The First International Symposium on Pheochromocytoma, held in October 2005, included discussions about developments concerning these rare catecholamine-producing tumors. Recommendations were made during the symposium for biochemical diagnosis, localization, genetics, and treatment. Measurement of plasma or urinary fractionated metanephrines, the most accurate screening approach, was recommended as the first-line test for diagnosis; reference intervals should favor sensitivity over specificity. Localization studies should only follow reasonable clinical evidence of a tumor. Preoperative pharmacologic blockade of circulatory responses to catecholamines is mandatory. Because approximately a quarter of tumors develop secondary to germ-line mutations in any one of five genes, mutation testing should be considered; however, it is not currently cost effective to test every gene in every patient. Consideration of tumor location, presence of multiple tumors, presence of metastases, and type of catecholamine produced is useful in deciding which genes to test. Inadequate methods to distinguish malignant from benign tumors and a lack of effective treatments for malignancy are important problems requiring further resolution.

Cardiac Sympathetic Nerve Function in Congestive Heart Failure

BACKGROUND Increased availability of norepinephrine (NE) for activation of cardiac adrenoceptors (increased cardiac adrenergic drive) and depletion of myocardial NE stores may contribute to the pathophysiology and progression of congestive heart failure. This study used a comprehensive neurochemical approach to examine the mechanisms responsible for these abnormalities. METHODS AND RESULTS Subjects with and without congestive heart failure received intravenous infusions of [(3)H]NE. Cardiac spillover, reuptake, vesicular-axoplasmic exchange, and tissue stores of NE were assessed from arterial and coronary venous plasma concentrations of endogenous and [(3)H]-labeled NE and dihydroxyphenylglycol. Tyrosine hydroxylase activity was assessed from plasma dopa, and NE turnover was assessed from measurements of NE metabolites. NE release and reuptake were both increased in the failing heart; however, the efficiency of NE reuptake was reduced such that cardiac spillover of NE was increased disproportionately more than neuronal release of NE. Cardiac NE stores were 47% lower and the rate of vesicular leakage of NE was 42% lower in the failing than in the normal heart. Cardiac spillover of dopa and NE turnover were increased similarly in congestive heart failure. CONCLUSIONS Increased neuronal release of NE and decreased efficiency of NE reuptake both contribute to increased cardiac adrenergic drive in congestive heart failure. Decreased vesicular leakage of NE, secondary to decreased myocardial stores of NE, limits the increase in cardiac NE turnover in CHF. Decreased NE store size in the failing heart appears to result not from insufficient tyrosine hydroxylation but from chronically increased NE turnover and reduced efficiency of NE reuptake and storage.

The role of neuronal and extraneuronal plasma membrane transporters in the inactivation of peripheral catecholamines.

Catecholamines are translocated across plasma membranes by transporters that belong to two large families with mainly neuronal or extraneuronal locations. In mammals, neuronal uptake of catecholamines involves the dopamine transporter (DAT) at dopaminergic neurons and the norepinephrine transporter (NET) at noradrenergic neurons. Extraneuronal uptake of catecholamines is mediated by organic cation transporters (OCTs), including the classic corticosterone-sensitive extraneuronal monoamine transporter. Catecholamine transporters function as part of uptake and metabolizing systems primarily responsible for inactivation of transmitter released by neurons. Additionally, the neuronal catecholamine transporters, recycle catecholamines for rerelease, thereby reducing requirements for transmitter synthesis. In a broader sense, catecholamine transporters function as part of integrated systems where catecholamine synthesis, release, uptake, and metabolism are regulated in a coordinated fashion in response to the demands placed on the system. Location is also important to function. Neuronal transporters are essential for rapid termination of the signal in neuronal-effector organ transmission, whereas non-neuronal transporters are more important for limiting the spread of the signal and for clearance of catecholamines from the bloodstream. Besides their presynaptic locations, NET and DAT are also present at several extraneuronal locations, including syncytiotrophoblasts of the placenta and endothelial cells of the lung (NET), stomach and pancreas (DAT). The extraneuronal monoamine transporter shows a broad tissue distribution, whereas the other two non-neuronal catecholamine transporters (OCT1 and OCT2) are mainly localized to the liver, kidney, and intestine. Altered function of peripheral catecholamine transporters may be involved in disturbances of the autonomic nervous system, such as occurs in congestive heart failure and hypernoradrenergic hypertension. Peripheral catecholamine transporters provide important targets for clinical imaging of sympathetic nerves and diagnostic localization and treatment of neuroendocrine tumors, such as neuroblastomas and pheochromocytomas.

Increased Cardiac Adrenergic Drive Precedes Generalized Sympathetic Activation in Human Heart Failure

BACKGROUND Previous studies with radiotracer methods have indicated increases in cardiac norepinephrine (NE) and renal NE spillover in patients with severe congestive heart failure (CHF). However, data on the regional sympathetic profile in early stages of CHF are limited. In this study, sympathetic function in the heart, kidneys, and skeletal muscle was evaluated in patients with mild-to-moderate CHF and compared with that in patients with severe CHF and healthy subjects. METHODS AND RESULTS Total body and regional NE spillover from the heart and kidney was assessed with isotope dilution with steady state infusions of [3H]NE. Sympathetic nerve traffic to the skeletal muscle vascular bed (MSA) was recorded intraneurally. Cardiac NE spillover in patients with mild-to-moderate CHF (n = 21) was increased threefold versus that in healthy subjects (n = 12, P < .05), whereas total body and renal NE spillover and MSA did not differ from those in healthy subjects. In the severe CHF group (n = 12), cardiac NE spillover was increased fourfold (P < .05), and total body and renal NE spillover and MSA were high compared with both mild-to-moderate CHF subjects and healthy subjects (P < .05 for both). Fractional extraction of [3H]NE across the heart was reduced by approximately 40% in both CHF groups versus control subjects (P < .05). CONCLUSIONS These results indicate a selective increase in cardiac adrenergic drive (increased amounts of transmitter available at neuroeffector junctions) in patients with mild-to-moderate CHF. This increase appears to precede the augmented sympathetic outflow to the kidneys and skeletal muscle found in advanced CHF.

Plasma Normetanephrine and Metanephrine for Detecting Pheochromocytoma in von Hippel–Lindau Disease and Multiple Endocrine Neoplasia Type 2

BACKGROUND The detection of pheochromocytomas in patients at risk for these tumors, such as patients with von Hippel-Lindau disease or multiple endocrine neoplasia type 2 (MEN-2), is hindered by the inadequate sensitivity of commonly available biochemical tests. In this study we evaluated measurements of plasma normetanephrine and metanephrine for detecting pheochromocytomas in patients with von Hippel-Lindau disease or MEN-2. METHODS We studied 26 patients with von Hippel-Lindau disease and 9 patients with MEN-2 who had histologically verified pheochromocytomas and 50 patients with von Hippel-Lindau disease or MEN-2 who had no radiologic evidence of pheochromocytoma. Von Hippel-Lindau disease and MEN-2 were diagnosed on the basis of germ-line mutations of the appropriate genes. The plasma concentrations of normetanephrine and metanephrine were compared with the plasma concentrations of catecholamines (norepinephrine and epinephrine) and urinary excretion of catecholamines, metanephrines, and vanillylmandelic acid. RESULTS The sensitivity of measurements of plasma normetanephrine and metanephrine for the detection of tumors was 97 percent, whereas the other biochemical tests had a sensitivity of only 47 to 74 percent. All patients with MEN-2 had high plasma concentrations of metanephrine, whereas the patients with von Hippel-Lindau disease had almost exclusively high plasma concentrations of only normetanephrine. One patient with von Hippel-Lindau disease had a normal plasma normetanephrine concentration; this patient had a very small adrenal tumor (<1 cm). The high sensitivity of measurements of plasma normetanephrine and metanephrine was accompanied by a high level of specificity (96 percent). CONCLUSIONS Measurements of plasma normetanephrine and metanephrine are useful in screening for pheochromocytomas in patients with a familial predisposition to these tumors.

Sympathetic cardioneuropathy in dysautonomias.

Background The classification of dysautonomias has been confusing, and the pathophysiology obscure. We examined sympathetic innervation of the heart in patients with acquired, idiopathic dysautonomias using thoracic positron-emission tomography and assessments of the entry rate of the sympathetic neurotransmitter norepinephrine into the cardiac venous drainage (cardiac norepinephrine spillover). We related the laboratory findings to signs of sympathetic neurocirculatory failure (orthostatic hypotension and abnormal blood-pressure responses associated with the Valsalva maneuver), central neural degeneration, and responsiveness to treatment with levodopa–carbidopa (Sinemet). Methods Cardiac scans were obtained after intravenous administration of 6-[18F]fluorodopamine in 26 patients with dysautonomia. Fourteen had sympathetic neurocirculatory failure — three with no signs of central neurodegeneration (pure autonomic failure), two with parkinsonism responsive to treatment with levodopa–carbidopa, and nine wit...

Simultaneous measurements of cardiac noradrenaline spillover and sympathetic outflow to skeletal muscle in humans.

1. Muscle sympathetic nerve activity (MSA) was recorded in the peroneal nerve at the knee by microneurography in ten healthy subjects and determinations were made simultaneously of intra‐arterial blood pressure, and whole‐body and cardiac noradrenaline spillover to plasma. Measurements were made at rest, during isometric handgrip at 30% of maximum power and during stress induced by forced mental arithmetic. 2. At rest there were significant positive correlations between spontaneous MSA (expressed as number of sympathetic bursts min‐1) and both spillover of noradrenaline from the heart and concentration of noradrenaline in coronary sinus venous plasma. 3. Both isometric handgrip and mental arithmetic led to sustained increases of blood pressure, heart rate and MSA. Plasma concentrations of noradrenaline and spillover of noradrenaline (total body and cardiac) increased. In general the effects were more pronounced during handgrip than during stress. 4. When comparing effects during handgrip and stress the ratio between the fractional increases of MSA and cardiac noradrenaline spillover were significantly greater during handgrip. 5. The data suggest (a) that there are proportional interindividual differences in the strength of resting sympathetic activity to heart and skeletal muscle which are determined by a common mechanism and (b) that handgrip and mental stress are associated with differences in balance between sympathetic outflows to heart and skeletal muscle.