Jacques J. Willemsen

Plasma metanephrines in the diagnosis of pheochromocytoma.

Pheochromocytoma is a tumor of chromaffin cells that usually presents as hypertension. The tumor has potentially life-threatening consequences if it is not promptly diagnosed, located, and removed. Evidence of excessive production of catecholamines is essential for diagnosis of the tumor. Traditional tests have relied on measurements of the 24-hour urinary excretion of catecholamines (norepinephrine and epinephrine) or of the products of catecholamine metabolism [1-4]. Because of the common problems of incompleteness and inconvenience associated with 24-hour urine collections, clinicians have long sought a diagnostic test based on sampling of antecubital venous blood. Measurements of plasma catecholamines are useful in this respect [4, 5]. However, patients with a pheochromocytoma can have plasma concentrations of catecholamines that fall within the range of those in patients with essential hypertension [4, 6] (that is, false-negative results). In addition, emotional distress or pathologic conditions other than pheochromocytoma (such as heart failure) can produce abnormally high catecholamine concentrations [7, 8] (that is, false-positive results). Glucagon stimulation and clonidine suppression testing can enhance the accuracy of plasma catecholamine determinations in the diagnosis of pheochromocytoma [9, 10]. These tests, however, can still yield false-negative or false-positive results [9-11]; they also require considerable time and effort. The search has continued for a single simple, highly sensitive and specific blood test with which to confirm the presence of the tumor in patients with pheochromocytoma. We studied the diagnostic accuracy of tests for specific catecholamine metabolites for this purpose, notably the metanephrinesnormetanephrine and metanephrine. An understanding of why plasma metanephrines may be particularly useful for diagnosis of pheochromocytoma requires an understanding of catecholamine metabolism. Norepinephrine and epinephrine are first metabolized intraneuronally by deamination to dihydroxyphenylglycol or extraneuronally by o-methylation to the metanephrines [12]. Because most dihydroxyphenylglycol is formed from norepinephrine leaking from neuronal stores and little is formed from circulating catecholamines [13, 14], plasma levels of this metabolite are relatively insensitive to the release of catecholamines into the circulation from a pheochromocytoma [6, 15]. The formation of most methoxyhydroxyphenylglycol from dihydroxyphenylglycol [14] and the formation of most vanillylmandelic acid from methoxyhydroxyphenylglycol within the liver [16] explains why a test for vanillylmandelic acid is also a poorer marker for pheochromocytoma than other tests [17]. In contrast, preferential metabolism of circulating catecholamines compared with neuronal catecholamines by extraneuronal pathways [14] suggests that the metanephrinesas extraneuronal metabolitesmay provide good markers for release of catecholamines from a pheochromocytoma. Furthermore, substantial production of metanephrines within adrenal tissue [18] suggests that metanephrines may be produced within the tumor itself. In humans, metanephrines are extensively sulfate-conjugated [18, 19]. Assays of metanephrines in urine depend on measurements after deconjugation to free metanephrines [19] so that measurements represent the sum of free and conjugated metabolites (total metanephrines). In contrast, good sensitivity of the assay for plasma metanephrines [20] enables measurements of both free and total metanephrines. We compared the sensitivity, specificity, and positive and negative predictive values of tests for plasma free and total metanephrines with those of tests for plasma catecholamines and urinary total metanephrines. Study participants included a relatively large sample of patients with pheochromocytoma, patients with essential hypertension or secondary hypertension from causes other than pheochromocytoma, and patients with either heart failure or angina pectoris in whom sympathetically mediated catecholamine release would be expected to be increased. Methods Patients Fifty-two patients with a histologically proven pheochromocytoma were studied. Thirty patients were studied retrospectively, and 22 were studied before the final diagnosis was made. The pheochromocytoma was benign in 39 patients and malignant in 13. Sixty-seven healthy, normotensive persons and 51 patients with essential hypertension served as a reference group. Blood samples were obtained from 23 patients with secondary hypertension (12 patients with renal artery stenosis, 2 with kidney disease, 1 with Cushing disease, 1 with primary hyperaldosteronism, and 7 with cyclosporine-induced hypertension) and from 50 patients with either heart failure or angina pectoris. The age, sex, and specialty center where the patients were studied for each of the five groups are shown in Table 1. Except for the few patients who were being treated with phenoxybenzamine, no patients with pheochromocytoma had been receiving medication for at least 2 weeks at the time of blood sampling. No patients with essential hypertension had been receiving medication for at least 2 weeks at the time of blood sampling. Medications taken by the other patient groups included digoxin, calcium channel blockers, diuretics, acetylsalicylic acid, dipyridamole, and cyclosporine. Procedures used in our study were approved by the hospital ethics committee or intramural research board of each of the three centers where patients were studied. Table 1. Patient Characteristics* Blood and Urine Samples All patients refrained from ingesting methylxanthine-containing food products and from smoking after midnight on the day before blood sampling. Blood was collected from an indwelling catheter in an antecubital vein after the patients had rested supine for 20 minutes. In 39 patients with heart failure and 15 with secondary hypertension, arterial blood was obtained through an indwelling arm arterial catheter. Blood samples were collected into precooled tubes containing heparin or EGTA and glutathione and were centrifuged within 30 minutes to separate the plasma, which was stored frozen until assayed. All plasma catecholamine and urinary metanephrine assays were done within 2 weeks of sample collection. Seven of the 52 pheochromocytoma samples were assayed for plasma metanephrines after being stored at 80C for more than 2 years (range, 2 to 8 years), whereas the remaining 45 samples were assayed within 2 years of collection (22 samples within 4 weeks). In 46 of the 52 patients with pheochromocytoma, a 24-hour urine collection was obtained, with 30 mL of 6-M hydrochloric acid used as a preservative. Analytic Methods Plasma metanephrines were assayed at the National Institutes of Health (NIH) using liquid chromatography with electrochemical detection [20]. Concentrations of total metanephrines (the sum of concentrations of free and sulfoconjugated metanephrines) were measured after incubation of 0.25 mL of plasma with 0.1 units of sulfatase (Sigma Chemical Company, St. Louis, Missouri) at 37 C for 30 minutes. The detection limits were 0.013 nmol/L for normetanephrine and 0.019 nmol/L for metanephrine. At a plasma normetanephrine concentration of 0.31 nmol/L and a metanephrine concentration of 0.21 nmol/L, the interassay coefficients of variation were 12.2% for normetanephrine and 11.2% for metanephrine. As previously reported [20], the presence of acetaminophen in samples of plasma can substantially interfere with measurements of plasma normetanephrine concentrations. Therefore, this analgesic must not be used by patients for several days before blood samples are collected. No analytic interference of various other drugs with this assay has been shown [20]. Plasma catecholamines were assayed using liquid chromatography. Electrochemical detection was used for quantification at the NIH [21], and fluorometric detection was used at St. Radboud University Hospital, Nijmegen, the Netherlands [22]. At the NIH, the detection limits were 0.006 nmol/L for norepinephrine and 0.010 nmol/L for epinephrine. At a plasma norepinephrine concentration of 2.4 nmol/L and an epinephrine concentration of 0.39 nmol/L, the interassay coefficients of variation were 6.5% for norepinephrine and 11.4% for epinephrine. At St. Radboud University Hospital, the detection limits for norepinephrine and epinephrine were 0.002 nmol/L and 0.003 nmol/L, respectively. At plasma concentrations of 1.02 nmol/L for norepinephrine and 0.15 nmol/L for epinephrine, interassay coefficients of variation were 8.5% for norepinephrine and 7.2% for epinephrine. Urinary concentrations of metanephrines were measured according to a previously described method [23]; the upper reference limit of the normal range for the 24-hour urinary output of metanephrines was 6.8 mol/d. Data Analysis Because plasma concentrations of catecholamines and metanephrines were not normally distributed, only medians and ranges are presented for these concentrations. Differences in plasma concentrations of metanephrines and catecholamines among patients with pheochromocytoma and other groups were tested using the Kruskal-Wallis test. We assessed relations among variables using the Spearman rank correlation coefficient. Normal distributions of plasma concentrations of catecholamines and metanephrines were obtained after logarithmic transformation of the data. Thus, upper reference limits, defined as the 97.5th percentile, were determined after logarithmic transformation of individual values for the combined data from normotensive persons and those with essential hypertension (118 persons). The 97.5th percentiles were calculated from the antilogarithm of the mean plus 2 standard deviations of the transformed data. A false-negative result of a test for plasma metanephrines in a patient with pheochromocytoma was defined as plasma concentrations of both normetanephrines and metanephrines that were

Effects of Insulin on Vascular Tone and Sympathetic Nervous System in NIDDM

Chronic activation of the sympathetic nervous system may be a pathogenetic mechanism by which hyperinsulinemia induces cardiovascular damage in insulin-resistant NIDDM patients. The influence of physiological hyperinsulinemia (∼ 700 pmol/l) on basal and stimulated sympathetic outflow was studied in 12 lean normotensive subjects with well-controlled NIDDM without complications and in 13 matched control subjects. Forearm blood flow (FBF) was measured with forearm plethysmography; sympathetic nervous system activity was assessed by the [3H]norepinephrine (NE) tracer method. NIDDM patients were insulin resistant (glucose infusion rates 31.8 ± 3.8 vs. 48.7 ± 2.0 mumol·kg−1 · min−1 in control subjects, P < 0.01). After a mixed meal, NIDDM patients showed a hyperinsulinemic response (2-h insulin levels: NIDDM patients 324 ± 34 pmol/l, control subjects 165 ± 19 pmol/l, P < 0.001). Insulin infusion induced a vasodilator response (not significantly different between the groups). Arterial plasma NE levels and total-body NE spillover increased significantly (total spillover in NIDDM patients from 0.77 ± 0.09 to 1.18 ± 0.16 nmol · m−2 · min−1, in control subjects from 0.98 ± 0.14 to 1.23 ± 0.18 nmol · m−2 · min−1 P < 0.01 for all, not different between groups). Total-body NE clearance did not change. Sympathetic stimulation (lower-body negative pressure [LBNP] 15 mmHg) induced forearm vasoconstriction and increased arterial and venous plasma NE and total NE spillover. Responses of FBF and NE kinetics to LBNP were not significantly different between groups and were not altered by hyperinsulinemia. Although these nonobese subjects with uncomplicated NIDDM showed postprandial hyperinsulinemia and resistance to the effect of insulin on glucose metabolism, this group was not resistant to the vasodilator and sympathetic stimulant effects of insulin. Responses to sympathetic stimuli (LBNP) were normal and unaffected by physiological hyperinsulinemia. Therefore, because of daily life hyperinsulinemia, chronic sympathetic stimulation could be operative in these patients and may explain the increased incidence of hypertension and/or cardiovascular complications.

Combinatorial code of growth factors and neuropeptides define neuroendocrine differentiation in PC12 cells.

Adrenal chromaffin cells constitute one of the first cell types to have been defined as a neuroendocrine cell type. Since they produce dopamine, these cells have been proposed for the treatment of neuronal deficits in human Parkinsons disease. However, the factors involved in the development of chromaffin cells are still poorly understood. Based on recent insights from stem cell research, we decided to study the role of extracellular matrices, growth factors and neuropeptides on the neuroendocrine differentiation in a serum-free medium of PC12 cells. Employing immunohistochemistry, quantitative PCR and HPLC analysis, neuroendocrine differentiation was determined by evaluating neurite outgrowth, catecholamine biosynthesis and release as well as neuropeptide and vesicular protein mRNA expression. The combination of bFGF, NGF and PACAP could prevent the inhibition of neurite process development induced by dexamethasone in PC12 cells cultured on ECM. Whereas glucocorticoids were essential in the regulation of enzymes of catecholamine biosynthesis and metabolism, growth factors and PACAP were more efficient in inducing neuropeptide and chromogranin B expression as well as release of dopamine and 3-methoxytyramine. Therefore, in addition to glucocorticoids, chromaffin cells need a gradient of matrix, growth factors, and neuropeptides to develop the full functional phenotype of a neuroendocrine cell.

Neurohumoral antecedents of vasodepressor reactions

Abstract. Vasodepressor (vasovagal) syncope, the most common cause of acute loss of consciousness, can occur in otherwise vigorously healthy people during exposure to stimuli decreasing cardiac filling. Antecedent physiological or neuroendocrine conditions for this dramatic syndrome are poorly understood. This study compared neurocirculatory responses to non‐hypotensive lower body negative pressure (LBNP) in subjects who subsequently developed vasodepressor reactions during hypotensive LBNP with responses in subjects who did not. In 26 healthy subjects, LBNP at ‐15 and ‐40mmHg was applied to inhibit cardiopulmonary and arterial baroreceptors. All the subjects tolerated 30min of LBNP at ‐15 mmHg, but during subsequent LBNP at ‐40 mmHg 11 subjects had vasodepressor reactions, with sudden hypotension, nausea, and dizziness. In these subjects, arterial plasma adrenaline responses to LBNP both at ‐15 and at ‐40 mmHg exceeded those in subjects who did not experience these reactions. In 16 of the 26 subjects, forearm noradrenaline spillover was measured; in the eight subjects with a vasodepressor reaction, mean forearm noradrenaline spillover failed to increase during LBNP at ‐15mmHg (Δ= ‐0.06±(SEM) 0.04pmol min‐1 100mL‐1), whereas in the eight subjects without a vasodepressor reaction, mean forearm noradrenaline spillover increased significantly (Δ=0.31±0.13pmolmin‐1100mL‐1). Plasma levels of β‐endorphin during LBNP at ‐15 mmHg increased in some subjects who subsequently had a vasodepressor reaction during LBNP at ‐40 mmHg. The findings suggest that a neuroendocrine pattern including adre‐nomedullary stimulation, skeletal sympathoinhibition, and release of endogenous opioids can precede vasodepressor syncope.

Blood pressure and both venous and urinary catecholamines after cerebral infarction.

Blood pressure, both venous and urinary catecholamines and plasma renin activity (PRA) were studied in 10 patients (6 men and 4 women, mean age 70 +/- 10 years) on the first three days after cerebral infarction. Blood pressure fell significantly (p less than 0.02) on the second and third day after stroke. There was a small but significant (p less than 0.01) decrease in plasma epinephrine concentration on the third day. The norepinephrine values remained constant on the three days. The PRA showed a significant (p less than 0.01) rise on the third day. No significant correlation was detected between the course of the blood pressure and the plasma catecholamines or PRA. When blood pressure was correlated with the urinary catecholamines, however, a significant correlation with epinephrine (r = 0.45; p less than 0.05) and with norepinephrine (r = 0.44; p less than 0.05) was found. We conclude that the changes in blood pressure after stroke are at least partly mediated by the changes in catecholamine production.

Adrenomedullary Secretion of Epinephrine Is Increased in Mild Essential Hypertension

To assess whether patients with mild essential hypertension have excessive activities of the sympathoneuronal and adrenomedullary systems, we examined total body and forearm spillovers and norepinephrine and epinephrine clearances in 47 subjects with mild essential hypertension (25 men, 22 women, aged 38.1 +/- 6.7 years) and 43 normotensive subjects (19 men, 24 women, aged 36.5 +/- 5.9 years). The isotope dilution method with infusions of tritiated norepinephrine and epinephrine was used at rest and during sympathetic stimulation by lower body negative pressure at -15 and -40 mm Hg. Hypertensive subjects had a higher arterial plasma epinephrine concentration (0.20 +/- 0.01 nmol.L-1: mean +/- SE) than normotensive subjects (0.15 +/- 0.01) (P < .01). The increased arterial plasma epinephrine levels appeared to be due to a higher total body epinephrine spillover rate in the hypertensive subjects (0.23 +/- 0.02 nmol.min-1.m-2) than the normotensive subjects (0.18 +/- 0.01) (P < .05) and not to a decreased plasma clearance of epinephrine. The arterial plasma norepinephrine level, total body and forearm norepinephrine spillover rates, and plasma norepinephrine clearance were not altered in the hypertensive subjects. The responses of the catecholamine kinetic variables to lower body negative pressure were not consistently different between normotensive and hypertensive individuals. These data indicate that individuals with mild essential hypertension (1) have elevated arterial plasma epinephrine concentrations that are due to an increased total body epinephrine spillover rate, indicating an increased adrenomedullary secretion of epinephrine; (2) have no increased generalized sympathoneuronal activity and no increased forearm norepinephrine spillover; and (3) have similar responses of both the sympathoneuronal and adrenomedullary systems to sympathetic stimulation by lower body negative pressure.

Adenosine attenuates the response to sympathetic stimuli in humans.

The effect of adenosine on the forearm vasoconstrictor response to a-adrenergic and sympathetic stimulation was studied in healthy volunteers. During a predilated state achieved by infusion of sodium nitroprusside into the brachial artery, subsequent infusion of norepinephrine induced a mean increase in forearm vascular resistance of 571%, whereas this response was only 270% when an equipotent vasodilator dose of adenosine was used instead of sodium nitroprusside (nitroprusside versus adenosine,p<0.05, n=6). A comparable difference was found when the endogenous release of norepinephrine was stimulated by the local infusion of tyramine, with tyramine-induced increments in forearm vascular resistance of 438% during nitroprusside versus 93% during adenosine (n=6, p<0.05). During these tyramine infusions a similar increase in the calculated forearm norepinephrine overflow occurred in the adenosine and the nitroprusside tests. In a third experiment, we demonstrated that adenosine also reduced the vasoconstrictor response to lower body negative pressure, an endogenous stimulus, of the sympathetic nervous system. During nitroprusside, lower body negative pressure induced an increase in forearm vascular resistance of 135%, whereas this was 39% during adenosine (n=6, p<0.05). We conclude that adenosine attenuates the response to sympathetic nervous system-mediated vasoconstriction in humans, and that this effect may at least partly be explained by a postsynaptic inhibition of or-adrenergic vasoconstriction. Therefore, we think that adenosine may be an important endogenous modulator of sympathetic nervous system activity in humans.

Value of the plasma norepinephrine/3,4-dihydroxyphenylglycol ratio for the diagnosis of pheochromocytoma

PURPOSE The purpose of this study was to assess whether the plasma norepinephrine/3,4-dihydroxyphenylglycol ratio (NE/DHPG) is of diagnostic relevance for patients with a pheochromocytoma. SUBJECTS AND METHODS In 18 patients with a histologically proven pheochromocytoma and in nine patients with congestive heart failure, plasma levels of NE, epinephrine (EPI), and DHPG (radioenzymatic method) were determined after 20 minutes of supine rest. In 10 healthy subjects, the plasma catecholamine responses to active standing (5 minutes) and mental arithmetic (5 minutes) were measured. From the plasma NE and DHPG levels, the plasma NE/DHPG ratio was calculated. In order to analyze whether NE or EPI was the major secreted catecholamine, the patients with a pheochromocytoma were divided into two groups based on the increase of plasma NE above normal relative to that of EPI: Group 1 included patients with increased plasma NE or increased plasma NE and EPI. Group 2 included patients with increased plasma EPI in combination with a nearly normal NE. RESULTS Both active standing and mental arithmetic increased the plasma NE/DHPG ratio by 105% and 13.6%, respectively, but in all subjects the ratio did not exceed 1.0. Patients with heart failure demonstrated a threefold higher plasma NE/DHPG ratio than did healthy subjects, and the ratio also did not exceed 1.0. The plasma NE/DHPG ratio was about seven to eight times higher in Group 1 (mean: 1.62, range: 0.81 to 2.84) than in Group 2 (mean: 0.24, range: 0.12 to 0.68). Nearly all patients in Group 1 had a NE/DHPG ratio that was higher than 1.0. In contrast, five of six samples of Group 2 demonstrated a NE/DHPG ratio within the normal range. The calculated positive and negative predictive values of a basal plasma catecholamine level were higher than that for the plasma NE/DHPG ratio. CONCLUSIONS In contrast to earlier reports, a normal plasma NE/DHPG ratio does not exclude the presence of a pheochromocytoma. In patients with a pheochromocytoma that produces EPI predominantly, this ratio may be normal. On the other hand, in patients with congestive heart failure, the plasma NE/DHPG ratio is increased, although there is no clear overlap with values of patients with a pheochromocytoma. Although the prevalence of pure EPI-producing pheochromocytomas is low, the plasma NE/DHPG ratio should be used with caution in the diagnostic evaluation of patients with a suspected pheochromocytoma.

Sympathoadrenal activation and the dumping syndrome after gastric surgery

Dumping symptoms suggest concomitant sympathoadrenal activation. To evaluate the relation between dumping symptoms and postprandial plasma catecholamine changes, standardized dumping-provocation tests with use of oral glucose were performed for 16 gastric surgery patients with dumping, for 14 gastric surgery patients without dumping, and for 14 healthy control patients. Early dumping symptoms were present for all patients with dumping, and late symptoms developed in three patients with dumping after glucose ingestion. Patients without dumping and healthy control patients had slight complaints or no complains. Systolic and diastolic blood pressure remained unaffected for the three groups. Positive breath-hydrogen tests, heart rate increments, and reactive plasma glucose decrements were present for patients with dumping and for patients without dumping, but not for control patients. Plasma noradrenaline and adrenaline increased for patients with dumping and for patients without dumping, but not for control patients. The noradrenaline increment was higher for patients with dumping (98%) than for patients without dumping (78%; p<0.05). The noradrenaline increment was related to the dumping score and to the heart rate increment for the first hour after glucose ingestion, whereas the adrenaline increment was related to the plasma glucose decrement for the third hour. Therefore, dumping symptoms clearly are accompanied by postprandial sympathoadrenal activation, but sympathoadrenal activation cannot account completely for development of dumping symptoms.Dumping symptoms suggest concomitant sympathoadrenal activation. To evaluate the relation between dumping symptoms and postprandial plasma catecholamine changes, standardized dumping-provocation tests with use of oral glucose were performed for 16 gastric surgery patients with dumping, for 14 gastric surgery patients without dumping, and for 14 healthy control patients. Early dumping symptoms were present for all patients with dumping, and late symptoms developed in three patients with dumping after glucose ingestion. Patients without dumping and healthy control patients had slight complaints or no complains. Systolic and diastolic blood pressure remained unaffected for the three groups. Positive breath-hydrogen tests, heart rate increments, and reactive plasma glucose decrements were present for patients with dumping and for patients without dumping, but not for control patients. Plasma noradrenaline and adrenaline increased for patients with dumping and for patients without dumping, but not for control patients. The noradrenaline increment was higher for patients with dumping (98%) than for patients without dumping (78%; p<0.05). The noradrenaline increment was related to the dumping score and to the heart rate increment for the first hour after glucose ingestion, whereas the adrenaline increment was related to the plasma glucose decrement for the third hour. Therefore, dumping symptoms clearly are accompanied by postprandial sympathoadrenal activation, but sympathoadrenal activation cannot account completely for development of dumping symptoms.

Comparison of blood pressure response to exogenous epinephrine in hypertensive men and women.

This study investigated possible differences between hypertensive men and hypertensive women concerning the hemodynamic effects of incremental doses of exogenous epinephrine. The study population comprised 38 men (37 +/- 10 years) (standard deviation) and 25 women (33 +/- 9 years) with mild essential hypertension (mean blood pressure 147/90 and 147/93 mm Hg, respectively). Body mass index was slightly higher in men (25 +/- 3 kg/m2) than in women (23 +/- 2 kg/m2). Both groups received an intravenous infusion with epinephrine of 15 and 30 ng/kg/min for 8 minutes each. Despite the similar doses of epinephrine infused in both groups, the increase of venous plasma epinephrine in men was nearly twice that in women (1.04 +/- 0.09 vs 0.67 +/- 0.09 nmol/liter, p less than 0.01), suggesting that women cleared the infused epinephrine more efficiently than men. At the highest infusion dose, the increase of systolic blood pressure was larger in men than in women (5.3 +/- 1.2 vs 1.7 +/- 1.1 mm Hg, p less than 0.05). Conversely, the decrease of diastolic blood pressure was also larger in men than in women (-8.8 +/- 1.0 vs -5.8 +/- 1.0 mm Hg, p less than 0.05). The heart rate increased to the same extent in both groups (11.5 +/- 0.8 and 13.7 +/- 1.2 beats/min). If the blood pressure responses were corrected for the increase of plasma epinephrine, the difference between men and women disappeared.(ABSTRACT TRUNCATED AT 250 WORDS)