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Original Studies |
Clinical Neuroscience Branch, National Institute of Neurological Disorders and Stroke (G.E., E.M., T.-T.H., B.H., T.E., S.D.) and Hypertension Endocrine Branch, National Heart Lung and Blood Institute (H.K.), National Institutes of Health, Bethesda, Maryland 20892-1424; Department of Clinical Physiology, University of Göteborg, Göteborg, Sweden (P.F.); and Departments of Rheumatology (A.E.) and Internal Medicine (J.W.M.L.), St. Radboud University Hospital, Nijmegen, The Netherlands
Address all correspondence and requests for reprints to: Graeme Eisenhofer, Building 10, Room 4D20, National Institutes of Health 10 Center Drive, Bethesda, Maryland 20892-1424. E-mail: ge{at}box-g.nih.gov
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Abstract |
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Introduction |
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A new test with more promise than other tests involves measurements of free metanephrines (normetanephrine and metanephrine) in plasma (7). These O-methylated metabolites of norepinephrine and epinephrine are produced by the enzyme, catechol-O-methyltransferase (COMT) and are rapidly metabolized to sulfate conjugates by the enzyme, monoamine-preferring phenolsulfotransferase. The resulting sulfate conjugates are present in plasma and urine in concentrations more than 25-fold higher than those of the free metanephrines (8). Assays of urine metanephrines typically employ a deconjugation step so that sulfate-conjugated metanephrines comprise the bulk of these measurements. Thus, these commonly used assays of urinary metanephrines are largely measurements of metabolites (i.e. sulfate-conjugated derivatives) that are different from those of the free metanephrines (7).
In a study of over 50 patients with pheochromocytoma, every patient had elevated plasma concentrations of free unconjugated metanephrines, showing that this test reliably excludes the presence of a tumor, whereas measurements of plasma catecholamines or urine metanephrines do not (9). A question arising from this study is why measurements of free metanephrines should provide a more superior method for diagnosis of pheochromocytoma than measurements of the parent amines or other metabolites? Our hypothesis for the superiority of plasma free metanephrines over other tests is that in patients with pheochromocytoma, free metanephrines are produced mainly from metabolism of catecholamines within the tumor, not from catecholamines metabolized after their secretion into the circulation. Because some tumors do not secrete catecholamines or secrete them episodically (10, 11, 12, 13), ongoing metabolism of catecholamines to free metanephrines within tumors would explain the better sensitivity of free metanephrines compared with the parent amines and other metabolites for diagnosis of the tumor.
In the present study, the hypothesis outlined above was examined by several procedures designed to assess the source of plasma metanephrines in patients with pheochromocytoma. The presence of COMT in tumor tissue was examined using Western blot analysis and immunohistochemical localization of the enzyme. The capacity of pheochromocytomas to produce free metanephrines was further examined from measurements of COMT enzyme activity and the presence of O-methylated amine metabolites in tumor tissue. Finally, comparisons of the production of metanephrines from the parent amines in patients with pheochromocytoma vs. production in other subjects receiving insulin tolerance tests or infusions of radiolabeled catecholamines enabled estimation of amounts of metanephrines derived from metabolism of circulating catecholamines, as distinct from those derived from metabolism of catecholamines within tumors.
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Materials and Methods |
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The patient database for this report includes 82 patients with histologically proven pheochromocytomas, half of whom were studied at the National Institutes of Health (Bethesda, MD), and the rest of whom were examined at several other centers within the United States or in The Netherlands (St. Radboud University Hospital, Nijmegen) or Sweden (Sahlgrenska Hospital, University of Göteborg, Göteborg). Other data from 52 of these patients have been reported previously in a study that examined the sensitivity and specificity of plasma metanephrines in the diagnosis of pheochromocytoma (9). The present study examines the source of the elevated plasma metanephrines in patients with pheochromocytoma. Blood samples were obtained from all patients, and samples of tumor tissue were obtained at surgery from 34 patients. Additional data includes the results of glucagon stimulation tests or multiple blood sampling in selected patients.
For comparison purposes, the data in this report are also derived from subject groups without pheochromocytoma (healthy normotensives and patients with angina pectoris or congestive heart failure) who received iv infusions of 3H-labeled catecholamines or bolus injections of insulin. Other subjects provided samples of normal liver or adrenal medulla during surgical procedures for conditions other than pheochromocytoma. Upper reference limits of normal (URLs) for plasma concentrations of metanephrines and catecholamines are the same as those established in our previous report (9).
All studies were approved by the appropriate institutional review boards, and all patients and subjects gave their informed consent to participate in studies.
Insulin tolerance tests
Insulin tolerance tests were carried out in seven healthy volunteers (two males and five females; mean ± SD; age, 51.9 ± 8.5 yr) to examine plasma metanephrine responses to release of catecholamines from the adrenals. These studies were carried out between 0800 and 1100 h after an overnight fast with subjects supine. A catheter inserted into an antecubital vein was used for administration of insulin and sampling of blood. Thirty minutes later, insulin was administered as an iv bolus injection at a dose of 0.1 U/kg. Blood samples (10 mL) were obtained immediately before and at 30, 45, 120, and 180 min after the injection of insulin.
Infusions of 3H-labeled catecholamines
Infusions of 3H-labeled norepinephrine and epinephrine were carried out in 13 patients with angina pectoris, 25 patients with congestive heart failure, and 10 normal control subjects to examine production of metanephrines from circulating catecholamines. All subjects were supine during infusion of radiotracers. The radiotracers (L-2,5,6-[3H]norepinephrine, 40–60 Ci/mmol; L-N-methyl-[3H]epinephrine, 65–75 Ci/mmol; New England Nuclear Corp., Boston, MA) were infused simultaneously into a forearm vein at 1.0–1.5 µCi/min. Blood samples were taken at intervals between 15 and 146 min after the start of infusions.
Glucagon stimulation tests
Plasma concentrations of metanephrines and catecholamines were examined in six pheochromocytoma patients with a positive test result to glucagon (i.e. a more than 3-fold increase in plasma norepinephrine) and one patient with a negative test result but a large increase in plasma epinephrine. For this test, patients received a 1-mg iv bolus dose of glucagon. Venous blood was sampled in the supine position immediately before the bolus of glucagon and at 2 min after the bolus.
In the patient with the negative test result but the large increase in plasma epinephrine, blood was sampled at 5 min before and immediately before the glucagon and at intervals of 1, 2, 3, 4, 5, 10, and 20 min after glucagon. Two other blood samples were taken from this patient on 2 separate days preceding the day of the glucagon stimulation test.
Patient with paroxysmal hypertension
Blood samples were taken from an antecubital vein on three occasions in one 42-yr-old female patient with a pheochromocytoma and paroxysmal attacks of flushing, palpitations, and chest pain. On one occasion a blood sample was taken and measurements of blood pressure and heart rate were recorded when the patient was free of the above symptoms. On a second occasion blood samples and hemodynamic measurements were taken during a paroxysmal attack lasting 10 min. On a third occasion a blood sample was taken and hemodynamic measurements recorded within 10 min after the end of a paroxysmal attack.
Collection of blood and tissue samples
All blood samples were transferred into tubes containing an anticoagulant (heparin or EDTA) and immediately placed on ice until centrifuged (4 C) to separate the plasma. Plasma samples were stored at -80 C until assayed for concentrations of catecholamines and metanephrines.
Samples of pheochromocytoma tissue were obtained within 40 min of removal of tumors from 34 patients. For comparison purposes in assays of COMT enzyme activity, samples of normal liver were obtained from three patients who underwent partial hepatectomies. Samples of normal adrenal tissue were also obtained from another patient who underwent an adrenalectomy for a small adrenal cortical adenoma. Samples of excised tissue were placed on ice immediately after removal, extraneous tissue removed, and small 50- to 400-mg pieces were dissected apart and placed on dry ice before storage at -80 C.
Plasma and tissue catecholamines and metanephrines
Plasma and tissue concentrations of catecholamines and metanephrines were quantified by liquid chromatography with electrochemical detection. Tissue concentrations were determined in weighed samples of tissue that were homogenized in at least 5 vol 0.4 M perchloric acid with 0.5 mM EDTA. Homogenized samples were centrifuged (1500 x g for 15 min at 4 C), and supernatants were collected and stored at -80 C until assayed. Concentrations of catecholamines (norepinephrine and epinephrine) were determined after extraction from plasma or perchloric acid tissue supernatants using alumina absorption as described previously (14). Concentrations of metanephrines were determined using a different liquid chromatography procedure after extraction onto solid phase ion exchange columns (7). Concentrations of 3H-labeled norepinephrine, epinephrine, normetanephrine, and metanephrine were determined by scintillation counting of mobile phase eluants collected from the outlet of the electrochemical cell after separation by liquid chromatography.
Intraassay coefficients of variation (CVs) were 1.9% for norepinephrine, 3.0% for epinephrine, 4.2% for normetanephrine, and 3.3% for metanephrine. Interassay CVs were 6.5% for norepinephrine, 11.4% for epinephrine, 6.5% for normetanephrine, and 5.7% for metanephrine.
Western blot analysis of COMT
Antibody to human COMT, raised in guinea pig against purified recombinant (Escherichia coli) soluble-COMT was kindly provided as a gift by Dr. Ismo Ulmanen (Orion Corp., Orion Pharma, Espoo, Finland). The antibody is highly selective for human COMT and does not cross-react with rat COMT (our unpublished data). Samples of human pheochromocytoma, adrenal medulla and liver tissue were homogenized in 2% SDS buffer (50 mM Tris, pH 6.8, 2.5% glycerol, 2% mercaptoethanol), boiled immediately for 10 min at 100 C, and then chilled for 5 min on ice. Samples of tissue preparations, with equivalent amounts of proteins (15 µg), were resolved on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes using a Bio-Rad transblot apparatus (Bio-Rad, Hercules, CA). After transfer, nitrocellulose membranes were blocked in Tris buffered saline (50 mM Tris, pH 7.4, 0.9% NaCl, 0.05% Tween-20 and 5% BSA) for 1 h. Nitrocellulose membranes were then incubated for 1 h with antihuman COMT primary antibody at a 1:1000 dilution. Membranes were then washed three times for 5 min with Tris buffered saline and incubated for 45 min with secondary antibody (antiguinea pig IgG) conjugated to horseradish peroxidase at 1:20,000 dilution. Detection of the two bands corresponding to the 30-kDa membrane-bound and 24-kDa soluble species of COMT was achieved by the enhanced chemiluminescence method.
COMT enzyme assay
Tissue samples (50–700 mg) were homogenized in at least 4 vol phosphate buffer (50 mM sodium phosphate and 0.5 mM dithiothreitol, pH 7.5). Homogenates were centrifuged at 100,000 x g for 30 min (4 C). Supernatant fractions were used for assays of soluble COMT activity, and the washed and resuspended pellets used for assays of membrane-bound COMT activity. The above method does not completely purify the two species of COMT (the supernatant fraction includes some of the larger membrane form of the enzyme), but does provide fractions enriched with either soluble or membrane-bound COMT.
COMT activity was assessed by incubation (37 C for 30 min) of 100 µL tissue preparation in a total volume of 500 µL containing 50 mM sodium phosphate buffer (pH 7.5), 5 mM MgCl2, 2 mM S-adenosylmethionine, and 0.4 mM 3,4-dihydroxybenzylamine as substrate (Sigma Chemical Co., St. Louis, MO). The reaction was terminated by addition of 100 µL 2 M perchloric acid. Samples were then centrifuged at 1500 x g for 15 min to separate the supernatant, which was used for measurements of the 3-methoxy-4-hydroxybenzylamine (MHBA) produced from 3,4-dihydroxybenzylamine. Amounts of MHBA in 10-µL samples of supernatant were determined by liquid chromatography with electrochemical detection. COMT activity was determined from the rate of production of MHBA and expressed as picomole of MHBA produced per minute per milligram of protein. These activities were determined under saturating conditions of substrate and therefore represent maximum velocity (Vmax) values. Division of these values by Michaelis-Menten affinity constant (Km) values previously determined by Lotta and colleagues (15) for human membrane-bound COMT (24.1 µM) and human soluble COMT (369 µM) provided additional estimates of COMT activity. The intra- and interassay CVs were 4.7% and 16.6%, respectively.
Immunohistochemistry
Localizations of COMT in different cells or the same cells where catecholamines are stored were examined by double staining of tissue sections with antibodies raised against COMT and against the vesicular monoamine transporters 1 and 2 (VMAT-1 and VMAT-2). Samples of pheochromocytoma tissue—frozen on dry ice soon after collection and stored at -80 C—were cut into 12-µm-thick sections using a Frigocut E 2800 cryostat (Reichert, Heidelberg, Germany). The frozen sections were mounted onto silanized slides and fixed in 4% paraformaldehyde fixative for 10 min, followed by multiple washes in PBS. To decrease nonspecific staining, the fixed sections were incubated at room temperature in PBS (pH 7.4) with 0.6% Triton X-100 and 1% BSA (diluent) for 30 min, and then washed in PBS. Primary antibody to COMT, raised in guinea pig (the same antibody used for Western blot analysis), was diluted 1:300 in the diluent described above. Primary antibodies to VMAT-1 and VMAT-2, raised in rabbits (Phoenix Pharmaceuticals, Mountain View, CA), were both diluted to 1:2000. The use of these antibodies in the immunohistochemical localization of VMAT-1 and VMAT-2 to endocrine cells and neurons has been documented previously (16). Tissue sections were first incubated overnight at 4 C in the guinea pig antibody to COMT and then incubated for 1 h at room temperature in the rabbit antibody to either VMAT-1 or VMAT-2. Sections were then incubated for consecutive 1-h periods at room temperature with a 1:2000 dilution of antiguinea pig IgG conjugated to CY3 fluorochrome followed by 1:100 dilution of antirabbit IgG conjugated to fluorescein isothiocyanate (Jackson Immunologicals, West Grove, PA). After application of secondary antibodies, sections were washed in distilled water, air dried, and coverslipped using Cytoseal 60 mounting medium (Stephens Scientific, Riverdale, NJ). Immunofluorescent labeling was viewed with a Leitz Dialux 20 fluorescence microscope (Leitz, Wetzlar, Germany) using appropriate filters. Negative controls included incubations in nonimmune mouse IgG or normal rabbit serum instead of the primary antibody or incubations without primary and/or the secondary antibodies.
Statistical analyses
The distributions of plasma and tumor tissue concentrations of catecholamines and metanephrines in patients with pheochromocytoma were highly skewed. Normal distributions were obtained after logarithmic transformation of the data. Mean values for these variables are therefore provided as geometric means. Corresponding confidence intervals and standard errors were established from the normalized data. All other results for normally distributed data are expressed as arithmetic means ± SEM. Where data showed nonnormal distributions, statistical tests of significance were carried out on normalized data. These tests included paired t tests and ANOVA for repeated measures with post hoc tests carried out using Scheffe’s F test. Relationships between tissue concentrations of catecholamines and metanephrines or between tumor mass and plasma catecholamines or metanephrines were examined by linear regression analysis, significance established using Pearson’s correlation coefficient.
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Results |
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Western blot analysis showed two forms of COMT corresponding to
the larger 30-kDa membrane-bound form and the smaller 25-kDa soluble
form of the enzyme (Fig. 1
). Both soluble
and membrane-bound COMT were detected consistently in pheochromocytoma
tissue, with the membrane-bound form of the enzyme showing a much
greater abundance than the soluble form. In a single sample of normal
adrenal medulla, COMT was almost totally present in the membrane-bound
form. Soluble and membrane-bound COMT were also detected in human liver
where the pattern was reversed with a considerably greater abundance of
the soluble over the membrane-bound form of the enzyme. The
membrane-bound form of COMT was considerably more abundant in both
adrenal medulla and pheochromocytoma tissue than in liver.
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The pattern of distribution of membrane-bound and soluble COMT
observed by Western blot analysis was mirrored by the results of COMT
enzyme activity assays (Table 1). These
assays showed higher (P < 0.05) Vmax
values for enzyme activity of COMT in the preparation of
pheochromocytoma tissue enriched with the membrane-bound form of the
enzyme (particulate fraction) than in that enriched with the soluble
form of the enzyme (supernatant fraction). In liver, the pattern was
reversed with considerably lower enzyme activity in the particulate
fraction than in the supernatant fraction. The difference in activities
of membrane-bound vs. soluble COMT in pheochromocytoma
tissue was considerably enhanced when activities were calculated by
dividing Vmax values with respective Km values
for membrane-bound and soluble forms of the enzyme. This additional
analysis also suggested relatively higher activities of membrane-bound
COMT in pheochromocytoma tissue than activities of both soluble and
membrane-bound COMT in liver.
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Sections of pheochromocytoma tissue showed clear
immunohistochemical staining with antibodies to COMT as well as
antibodies to both the VMATs (VMAT-1 and VMAT-2) involved in
translocation of biogenic amines into vesicular storage vesicles (Fig. 2). The morphology of the two tissue
sections taken from the benign adrenal tumors shown in Fig. 2
illustrates an arrangement of stained cells in clusters or
interconnecting cords, with a polygonal shape and large nuclei typical
of pheochromocytoma chromaffin cells. Chromaffin cells stained for COMT
in the cytoplasmic area surrounding the nuclei and all chromaffin cells
that stained for COMT also stained for VMAT-1 or VMAT-2, indicating
colocalization of COMT in the same cells that contained the VMAT
responsible for translocation of catecholamines into storage
vesicles.
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High concentrations of norepinephrine and epinephrine within the
micromole per gram of tissue range were present in pheochromocytoma
tissue (Table 2).
Normetanephrine and metanephrine were also readily detected in samples
of pheochromocytoma tissue. Tumor tissue showed a considerable range in
concentrations of catecholamines, with those of epinephrine in
particular showing the widest range. Separate analysis of tumors with
either high and low concentrations of epinephrine (>1600 nmol/g and
<400 nmol/g) showed that tumors with high epinephrine concentrations
also had high metanephrine concentrations, and those with low
epinephrine concentrations had low metanephrine concentrations (Table 2). Corresponding with the above, there was a highly significant
positive relationship (r = 0.85, P < 0.0001)
between tumor tissue concentrations of epinephrine and metanephrine
(data not shown). There was also a positive relationship (r =
0.58, P < 0.003) between tumor tissue concentrations
of norepinephrine and normetanephrine (data not shown). Among the
tumors, there were four that were taken from extraadrenal locations.
These tumors had on average about 10-fold lower tissue concentrations
of epinephrine and metanephrine than tumors located in the adrenal
glands.
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Patients who had tumors with low epinephrine concentrations showed normal plasma concentrations of epinephrine (0.05–0.54 pmol/mL) and also tended to show normal plasma metanephrine levels (0.21–0.61 pmol/mL) before removal of tumors. Patients who had tumors with high concentrations of epinephrine all showed elevated plasma concentrations of metanephrine (1.13–16.52 pmol/mL) and also tended to show elevated plasma epinephrine levels (0.12–4.80 pmol/mL).
Although tumor tissue concentrations of metanephrines were much lower
than those of the parent amines, they were nevertheless several orders
of magnitude higher (P < 0.0001) than corresponding
concentrations of metanephrines in the plasma of the same patients
before removal of tumors (Fig. 3). Tumor
tissue concentrations of normetanephrine averaged 23,000 times higher
than the corresponding plasma concentrations and tissue concentrations
of metanephrine averaged 13,000 times higher than plasma
concentrations.
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). Similarly, there was a positive
relationship between tumor mass and plasma concentrations of
metanephrine (r = 0.46, P < 0.0005), but no
relationship with plasma epinephrine (r = 0.24) (data not shown).
The regression line for the relationship between plasma normetanephrine
and tumor size indicated that a patient with a small 10-g tumor might
be expected to show a 3-fold increase in plasma concentrations of
normetanephrine above the URL for normal compared with a 20-fold
increase for a large 200-g tumor.
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In normal subjects who received an iv bolus injection of insulin,
plasma glucose levels fell from baseline concentrations of 4.8 ±
0.1 mmol/L to 1.2 ± 0.1 mmol/L at 30 min after insulin. Plasma
concentrations of epinephrine increased (P < 0.0001)
by 27-fold, and concentrations of norepinephrine increased
(P < 0.0001) by 3-fold above baseline levels at 45 min
after insulin administration (Fig. 5). In
contrast to the large insulin-induced increases in plasma
catecholamines, plasma concentrations of normetanephrine remained
unchanged and plasma concentrations of metanephrine increased
(P < 0.0001) by only 2.8-fold above baseline
concentrations. The increase in absolute plasma concentrations of
metanephrine after insulin was only 7.8 ± 2.2% of the increase
in plasma concentrations of epinephrine.
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In normal control subjects and patients with angina or congestive
heart failure who received iv infusions of 3H-labeled
catecholamines, plasma concentrations of
[3H]norepinephrine and [3H]epinephrine
reached steady state levels close to 1000 dpm/mL within 30 min after
the start of radiotracer infusions (Fig. 6, A and C). In line with the rapid
circulatory clearance and short half-lives of plasma metanephrines
(17), steady state plasma levels of 3H-labeled
metanephrines were also reached rapidly. However, steady state plasma
concentrations of [3H]normetanephrine were only 3.3
± 0.1% those of plasma [3H]norepinephrine
concentrations, whereas concentrations of
[3H]metanephrine were only 7.7 ± 0.4% of
[3H]epinephrine concentrations. There were no differences
among subject groups in the formation of 3H-labeled
metanephrines from iv infused 3H-labeled catecholamines
as reflected by steady state ratios of plasma 3H-labeled
metanephrines to catecholamines.
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In six pheochromocytoma patients who showed a positive
glucagon-stimulation test result for the presence of a tumor (>3-fold
increase in plasma norepinephrine), plasma norepinephrine and
epinephrine concentrations both increased (P < 0.005)
by an average of 7-fold above baseline values (Fig. 7, A and C). Despite these large
increases in plasma catecholamines, plasma concentrations of
normetanephrine and metanephrine showed little change and were not
consistently elevated by glucagon (Fig. 7, B and D).
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, F and G).
The 10-fold increase in plasma epinephrine was associated with a
transient small (<20% above baseline) increase in plasma metanephrine
peaking at 2–3 min after glucagon. This nonsymptomatic patient, with
multiple endocrine neoplasia type II syndrome, was diagnosed by chance
with an adrenal pheochromocytoma following routine work-up for another
condition. The patient repeatedly had normal plasma concentrations
of norepinephrine on the 1st and 3rd days of blood sampling and normal
baseline concentrations of epinephrine on all 3 days of blood sampling.
Despite these normal plasma levels of catecholamines, plasma
concentrations of both normetanephrine and metanephrine were
consistently elevated well above the URLs of normal at all time points
and, un-like plasma catecholamines, showed little day-to-day
variation. Plasma metanephrines in a pheochromocytoma patient with a hypertensive paroxysm
In one pheochromocytoma patient who suffered from paroxysmal
attacks of flushing, palpitations, and chest pain, plasma
concentrations of catecholamines increased substantially during a
paroxysmal attack, whereas plasma concentrations of free metanephrines
increased by only a small amount in comparison (Table 3). In particular, plasma
concentrations of epinephrine increased by 83-fold, whereas plasma
concentrations of free metanephrine increased by only 3.6-fold. The
absolute increase in plasma concentrations of free metanephrine was
only 5% of the increase in plasma epinephrine, and the increase in
free normetanephrine was less than 1% of that of norepinephrine.
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In patients with pheochromocytoma, plasma concentrations of
normetanephrine were 53% those of plasma concentrations of
norepinephrine, and in patients with elevated epinephrine or
metanephrine concentrations, plasma concentrations of metanephrine were
3.38-fold higher than epinephrine concentrations (Fig. 6, B and
D).
Comparison of the relative plasma concentrations of normetanephrine to
norepinephrine in pheochromocytoma patients (0.53) with the relative
increase in plasma [3H]normetanephrine to
[3H]norepinephrine during iv infusion of radiolabeled
catecholamines (0.033) indicated that only 6.1% (0.033/0.53) of the
normetanephrine in plasma of pheochromocytoma patients was derived from
metabolism of norepinephrine after its release into the circulation
(Fig. 6, A and B). Similarly, comparison of the relative plasma
concentrations of metanephrine to epinephrine in pheochromocytoma
patients (3.38) with the relative increase in plasma
[3H]metanephrine to [3H]epinephrine during
iv infusion of radiolabeled catecholamines (0.077) indicated that only
2.3% (0.077/3.38) of the elevated metanephrine in plasma of
pheochromocytoma patients was derived from metabolism of epinephrine
after its release into the circulation (Fig. 6, C and D). Most of the
elevated plasma concentrations of normetanephrine (93.9%) and
metanephrine (97.7%) in patients with pheochromocytoma were derived
from metabolism of norepinephrine and epinephrine in tissue before
entry into the circulation, not from metabolism after entry of
precursor catecholamines into the circulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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COMT exists in two forms: one form is present as a soluble species in the cytoplasm, the other as a larger molecular weight species bound to membranes (18, 19). Both are coded for by the same gene, but differ in the presence or absence of a peptide tail that anchors the larger species to membranes. Variations in the presence of the two forms of COMT among tissues are dependent on transcriptional regulation by two promoters as well as posttranscriptional regulation at the translation step (20). Because the soluble form of COMT is by far the more abundant species in most tissues, particularly in the liver and kidneys (18), it was long considered the enzyme responsible for catecholamine metabolism. Other considerations now suggest that this notion may be incorrect (19). In particular, Km values for catecholamines of the membrane-bound form of COMT are much lower than those for the soluble form (15–25 vs. 200–370 µM), making the membrane-bound enzyme most likely responsible for metabolism of the low cytoplasmic concentrations of catecholamines found in vivo (15). Additionally, compared with the soluble form of COMT, the membrane-bound enzyme has a more than 10-fold higher selectivity for 3-O- than 4-O-methylation of catecholamines (15). Because the O-methylated derivatives of catecholamines are almost exclusively present in vivo as 3-methoxy metabolites, it seems likely that their formation reflects selective 3-O-methylation by the membrane-bound form of COMT.
The above suggestion that the membrane-bound form of COMT is the enzyme species largely responsible for metabolism of catecholamines in vivo is supported by the results of the present study in addition to those from some of our previous work (8, 21). This other work showed that over 90% of circulating metanephrine and between 23% and 40% of circulating normetanephrine are produced by metabolism of the parent catecholamines within the adrenal glands (8, 21). This makes the adrenals the single largest tissue source of circulating metanephrines in the body, with production surpassing by far that of metanephrines by the liver (21). The present findings that COMT in pheochromocytoma and adrenal medulla tissue exists predominantly in the membrane-bound form makes chromaffin tissue distinct from most other tissues, such as the liver, where the soluble form is the most predominant form of the enzyme (18, 19). The high rates of production of metanephrines by the adrenals together with the abundance of membrane-bound COMT within chromaffin tissue is consistent with the suggestion that it is the membrane-bound form of the enzyme that is responsible for in vivo metabolism of catecholamines (19).
Although Western blot and Vmax values from COMT enzyme assays both indicated higher amounts of membrane-bound than soluble COMT in pheochromocytoma tissue, this difference appeared more pronounced by Western blot analysis. This likely reflects two factors: first, in enzyme assays, although the supernatant fraction is enriched with the smaller soluble form of COMT, it also contains some of the larger species of COMT in unbound form, and, second, enzyme activities are determined under saturating conditions of substrate favoring the soluble species of COMT, which has a higher Vmax than the membrane-bound form (15, 19).
As described by Roth (19), a more realistic method to compare likely relative activities in vivo of membrane-bound and soluble forms of COMT can be achieved by dividing Vmax values by the corresponding Km values for each form of the enzyme. Doing this by using the Km values of recombinant human soluble and membrane-bound COMT determined by Lotta et al. (15) yields activities of the membrane-bound form of COMT in pheochromocytoma tissue many fold higher than activities of the soluble form. This additional analysis also revealed higher activity of membrane-bound COMT in pheochromocytoma tissue than both soluble and membrane-bound COMT activity in liver.
VMAT-1 is preferentially localized to large dense core vesicles where it is involved in the translocation and packaging of biogenic amines into secretory granules (22). Although VMAT-1 is predominantly located within endocrine cells, such as chromaffin cells of the adrenal medulla, these cells also often contain the neuronal VMAT-2 (16). Immunohistochemical colocalization of COMT with both VMAT-1 and VMAT-2 indicates the presence of the metabolizing enzyme within the same chromaffin cells where catecholamines are stored. This colocalization may seem surprising, but is not without precedence. In sympathetic neurons, the catecholamine- metabolizing and mitochondrial-bound enzyme, monoamine oxidase, is responsible for most norepinephrine turnover (23). The majority of this is secondary to norepinephrine leaking from vesicular stores into the axoplasm where the transmitter is available for metabolism by monoamine oxidase (23, 24). Presumably this high rate of intraneuronal metabolism serves to safeguard against high toxic axoplasmic concentrations of catecholamines. The considerable intraneuronal metabolism of catecholamines leaking from storage vesicles also serves as a gearing mechanism to minimize the requirement for increases in the activity of the rate-limiting enzyme in catecholamine synthesis, tyrosine hydroxylase, to match increases in exocytotic transmitter release (24). Perhaps similar reasons apply in the adrenal medulla where metabolism to metanephrine almost equals epinephrine release (21).
Immunohistochemical staining of COMT in the cytoplasm of pheochromocytoma chromaffin cells is in agreement with recent findings on the intracellular localization of membrane-bound COMT to the rough endoplasmic reticulum (25). Perhaps the presence of COMT bound to these structures in chromaffin cells may provide distinct locations within the cytoplasm where catecholamines are O-methylated. This may serve to compartmentalize intracellular sites of catecholamine metabolism away from those of synthesis and storage.
Apart from the presence in tumor tissue of membrane-bound COMT, the capacity of COMT in pheochromocytomas to metabolize catecholamines is also reflected by the high concentrations of metanephrines in tumor tissue. Although these concentrations are less than 2% those of the parent amines, it must be recognized that catecholamines are sequestered in chromaffin granules at concentrations 104–105 times higher than those in the cytoplasm (26), where they are available for metabolism by COMT. The high tissue levels of metanephrines in pheochromocytomas are in agreement with previous findings (27, 28, 29). However, the additional findings of the present study that these levels are over 10,000 times higher than plasma concentrations of metanephrines illustrate how these high tissue levels may easily make a major contribution to the elevated levels of plasma metanephrines found in patients with pheochromocytoma.
Estimation of the proportion of circulating metanephrines produced from metabolism of catecholamines released into the circulation can be achieved by examination of the production of metanephrines from iv infused catecholamines or from catecholamines released directly into the circulation by the adrenals (30). The results of the insulin tolerance tests showed that plasma metanephrines are relatively insensitive to large increases in adrenal release of catecholamines, with the increase in plasma metanephrine only 7.8% that of the increase in plasma epinephrine. This result is remarkably close to the findings during iv infusions of 3H-labeled catecholamines, where the increase in plasma [3H]metanephrine was 7.7% of the increase in [3H]epinephrine. Assuming similar metabolism of circulating catecholamines in patients with pheochromocytoma (an assumption dealt with in the paragraph below), if all the metanephrine in plasma was derived from epinephrine released into the circulation, then plasma concentrations of metanephrine would be expected to be close to 8% of those of epinephrine. Instead, the elevated plasma concentrations of metanephrine in patients with pheochromocytoma were over 200% higher than concentrations of epinephrine. A similar situation was observed for normetanephrine indicating that only small proportions of both plasma-free normetanephrine (<7%) and metanephrine (<3%) are derived from metabolism of catecholamines after their release into the circulation from pheochromocytomas.
The independence of metanephrine production on metabolism of catecholamines released into the circulation from tumors was further illustrated by the relative lack of increase in plasma metanephrines after glucagon in patients with a positive glucagon stimulation test characterized by large increases in plasma catecholamines. Similarly, in one patient with an episodically active tumor, plasma concentrations of norepinephrine increased by nearly 5-fold and epinephrine by more than 80-fold during a paroxysmal hypertensive attack. Despite these large increases, plasma concentrations of free metanephrine increased by only 6% of the increase in epinephrine, and plasma concentrations of free normetanephrine remained stable. The relative increases in plasma free metanephrines after increased catecholamine release into the circulation in patients with pheochromocytoma were slight and thus similar to those in normal subjects during insulin tolerance tests and iv infusions of catecholamines. This indicates little difference in metabolism of circulating catecholamines that could account for the high plasma concentrations of free metanephrines relative to catecholamines in patients with pheochromocytoma. Thus, in patients with pheochromocytoma, more than 93% of the free normetanephrine and 97% of the free metanephrine in plasma are derived from metabolism of catecholamines before release into the circulation, almost all of this representing metabolism within tumors.
A similar conclusion to the above was reached by Crout and Sjoerdsma (29) many years ago when they observed that the urinary excretion of catecholamines and their metabolites in patients with pheochromocytoma showed a completely different profile from that in subjects receiving iv infusions of catecholamines. These investigators also showed that tumor size was not a determinant of catecholamine excretion, but did influence the excretion of catecholamine metabolites. Similarly, in the present study highly significant positive relationships between tumor size and plasma concentrations of free metanephrines contrasted with complete lack of relationships between tumor size and plasma concentrations of catecholamines. These findings provide further evidence for the independence of elevations in plasma levels of metanephrines on catecholamine release from pheochromocytomas. More importantly, the findings also show how the magnitude of increases in plasma free metanephrines above normal can provide information about tumor size, whereas elevations in catecholamines do not.
Although the above analysis establishes that elevated circulating
levels of free metanephrines in patients with pheochromocytoma are
largely produced independently of catecholamine release from tumors,
this does not in itself explain why plasma free metanephrines should
provide a better diagnostic test for a tumor than the parent amines
themselves. A plausible explanation, however, follows from other
findings that a proportion of patients with pheochromocytoma harbor
tumors that are nonfunctional or quiescent and do not secrete
catecholamines or secrete catecholamines episodically (10, 11, 12, 13). In
these patients, plasma and urinary catecholamines typically yield false
negative tests. In contrast, plasma free metanephrines (normetanephrine
or metanephrine) appear to be much more consistently elevated (9),
indicating that even when tumors are not secreting catecholamines, they
are actively metabolizing catecholamines to the free metanephrines. An
example of a catecholamine producing and metabolizing, yet nonsecreting
tumor, is illustrated by the present results for the patient shown in
Fig. 7, F and G. This patient, with multiple endocrine neoplasia type
II syndrome, was only diagnosed with a tumor after a routine computed
tomography (CT) scan revealed an adrenal lesion. Plasma catecholamines
were normal or near normal in every sample, whereas plasma
concentrations of normetanephrine and metanephrine were both
consistently elevated well above the URLs of normal in each of 11
separate samples taken over the course of 3 separate days. Clearly this
patient had a tumor that produced and metabolized catecholamines to
metanephrines, but did not secrete catecholamines into the
bloodstream in amounts sufficient to cause a positive test result.
In conclusion, the present study shows that pheochromocytomas have an unusually higher content of the membrane-bound than the soluble form of COMT. However, it appears that the membrane-bound and not the soluble form of COMT is the enzyme species responsible for metabolism of catecholamines in vivo. The high content of membrane-bound COMT in the same tumor cells where catecholamines are synthesized and stored leads to considerable production of free metanephrines within pheochromocytomas. Thus, the elevated plasma levels of free metanephrines in patients with pheochromocytoma are produced independently of catecholamine release by tumors. Because some tumors are quiescent or nonfunctional and do not readily or continually secrete catecholamines, measurements of free metanephrines provide a more reliable marker for the presence of a pheochromocytoma than do the parent amines.
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Acknowledgments |
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Received January 22, 1998.
Revised February 23, 1998.
Accepted March 2, 1998.
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