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Catecholamine-synthesizing enzymes in carcinoid tumors and pheochromocytomas.

Carcinoids and pheochromocytomas are neuroendocrine tumors. Both have the ability to take up and decarboxylate amine precursors. This decarboxylation depends on aromatic-5-amino acid decarboxylase (AADC) [5] an enzyme involved in the synthesis of serotonin as well as catecholamines (Fig. 1). Because of these characteristics, Pearse (1) postulated the amine precursor uptake and decarboxylation (APUD) concept, according to which neuroendocrine tumors originate from a single neuroendocrine precursor cell. Because the embryologic origin of the precursor cells is not clarified, the APUD concept is not generally accepted (2).

In pheochromocytoma, grossly enhanced catecholamine synthesis leads to the classical presentation of palpitations, vasoconstriction, and increased blood pressure. The majority of pheochromocytomas are sporadic cases (3), but a familial predisposition has been found in, e.g., patients with the multiple endocrine neoplasia (MEN) syndrome type IIa/b (3). Catecholamine synthesis depends on three specific enzymes (Fig. 1): tyrosine hydroxylase (TH; EC; dopamine-[beta]-hydroxylase (DBH; EC; and phenylethanolamine-N-methyltransferase (PNMT; EC The rate of catecholamine synthesis is determined by TH (4). These enzymes are detectable in pheochromocytomas by immunohistochemical methods (5-8). Although catecholamines are the principal metabolic product of pheochromocytoma, these tumors also can lead to increased serotonin metabolism (9).


In midgut carcinoids derived from enterochromaffin cells located in the small intestines, cecal region, and ascending colon (10), serotonin (5-hydroxytryptamine) is the major metabolic product. Serotonin overproduction leads to the carcinoid syndrome, which consists of flushing, diarrhea, bronchial constriction, and carcinoid heart disease (11,12). In the rate-limiting step for serotonin synthesis, the essential amino acid tryptophan is converted to 5-hydroxytryptophan by tryptophan hydroxylase (Fig. 1). Serotonin is synthesized by decarboxylation of 5-hydroxytryptophan by AADC (13). In hepatic and pulmonary endothelial cells, serotonin is degraded to 5-hydroxyindoleacetic acid (5-HIAA) by monoamine oxidase (13). The metabolite 5-HIAA is excreted in urine and is widely used for the biochemical diagnosis of carcinoid tumors.

Although enhanced serotonin metabolism is a characteristic feature in carcinoid patients, the urinary excretion of catecholamines and their metabolites has been reported as increased as well (14,15). In one of these studies (14), the catecholamine content was higher in tissue homogenates of carcinoid tumors than in the surrounding healthy tissue. In another, immunohistochemical study (7), carcinoids appeared to be positive for norepinephrine but negative for epinephrine. Three hypothetical mechanisms can explain the increased excretion of urinary catecholamines in carcinoid patients. One mechanism is that carcinoids may contain the specific catecholamine-synthesizing enzymes and therefore have the ability to synthesize catecholamines autonomously. Alternatively, AADC in the carcinoid tumor may convert dihydroxyphenylalanine into dopamine, thus providing substrate for enhanced catecholamine synthesis outside the tumor. Finally, the enhanced production of serotonin might lead to physical stress, leading to increased urinary excretion of catecholamines.

Because it is unknown whether catecholamine-synthesizing enzymes are present in carcinoid tumors, our first aim was to detect these enzymes with an immunohistochemical method. In addition, we compared the immunohistochemical pattern found in carcinoid tumor specimens with that in pheochromocytoma specimens. Finally, we analyzed the relationship between the expression of catecholamine-synthesizing enzymes, the production of serotonin, and the urinary excretion of catecholamines and metabolites.

Materials and Methods


Paraffin-embedded tumor samples were obtained from carcinoid patients who had undergone surgery for tumor resection or debulking at the University Hospital Groningen between December 1987 and March 1999. Only patients with midgut tumors who had urinary catecholamines and metabolites determined within 1 year preceding resection were included in this immunohistologic study.

Paraffin-embedded tumor samples of sporadic as well as MEN type Ila-associated pheochromocytomas were obtained from adrenalectomies performed between January 1985 and December 1997 in the University Hospital Groningen. In both types of pheochromocytoma patients, urinary catecholamines and metabolites are routinely measured within 4 weeks before adrenalectomy.

As controls for the immunohistochemical staining procedure, we used healthy human adrenal medulla tissue. The tissue samples used in this study were obtained from archival material.


The 24-h urine samples were collected into 2 L brown polypropylene bottles (Sarstedt) containing 250 mg each of NaZS,O, and EDTA as preservatives and were acidified to pH 4 with acetic acid before freezing. The samples were stored at -20 [degrees]C and analyzed within 1 week after collection. Urinary excretion of the free catecholamines dopamine [upper reference limit (URL), 300 [micro]mol/mol of creatinine], norepinephrine (URL, 30 [micro]mol/mol of creatinine), and epinephrine (URL,10 [micro]mol/mol of creatinine) was determined by HPLC with electrochemical detection (16). The sum of the free and conjugated catecholamine metabolites 3-methoxytyramine (URL, 167 [micro]mol/mol of creatinine), normetanephrine (URL, 260 [micro]mol/mol of creatinine), and metanephrine (URL, 69 [micro]mol/mol of creatinine) was determined in lyophilized urine samples by stable-isotope mass spectrometry (17).


We used urinary 5-HIAA and platelet serotonin content as markers of serotonin metabolism. Urinary 5-HIAA (mmol/mol of urinary creatinine) and the serotonin content of platelet-rich plasma (nmol serotonin/[10.sup.9] platelets) were determined by HPLC with fluorometric detection. The URL for urinary 5-HIAA was 3.8 mmol/mol of urinary creatinine. Platelet serotonin content was calculated by dividing the serotonin concentration in plateletrich plasma by the concentration of platelets. The URL for platelet serotonin was 5.4 nmol serotonin/[10.sup.9] platelets. An extended description of the sampling procedures and analytical methods has been published (18).


Paraffin-embedded sections (4 p,m), mounted on 3-aminopropyltriethoxysilane (Sigma-Aldrich Chemie) were deparaffinized according to standard procedures. The slides were incubated in an antigen retrieval solution [maleate buffer, pH 6.0, containing 20 g/L blocking reagent (Boehringer, Mannheim, Germany) and 2 g/L sodium dodecyl sulfate (BDH Laboratory Supplies)] and autoclaved (Certoclav Sterilizer GmbH) at 115 [degrees]C three times for 5 min. A 5-min cooling period at room temperature followed each autoclave cycle. After the antigen retrieval procedure, the sections were rinsed with phosphate-buffered saline (PBS; 8.75 g of NaCl, 1.37 g of [Na.sub.2]HP[0.sub.4], 0.215 g of K[H.sub.2]P[O.sub.4] in 1 L of distilled water, pH 7.4) three times for 5 min. This standard rinsing procedure was repeated between steps throughout the staining protocol. Endogenous peroxidase activity was blocked with 3 mL/L [H.sub.2][0.sub.2] in PBS for 30 min. Subsequently, the sections were preincubated with 20 g/L bovine serum albumin (BSA; Sigma Chemicals) in PBS for 30 min.

Immunohistochemistry for catecholamine-synthesizing enzymes was performed with anti-TH polyclonal antibodies (raised in rabbits with use of rat TH; Chemicon International) diluted 1:1000 in PBS containing 1 g/L BSA, anti-DBH polyclonal antibodies (raised in rabbits with use of bovine DBH; Eugene Tech International) diluted 1:100 in PBS containing 1 g/L BSA, and anti-PNMT polyclonal antibodies (raised in rabbits with use of bovine PNMT; Chemicon International) diluted 1:1000 in PBS containing 1 g/L BSA.

After overnight incubation at 4 [degrees]C, the sections were incubated with biotinylated goat anti-rabbit immunoglobulin (Amersham International), diluted 1:100 in PBS containing 1 g/L BSA, for 30 min at room temperature. The sections were then incubated with streptavidin-biotinylated horseradish peroxidase complex (Amersham), diluted 1:100 in PBS containing 1 g/L BSA, for 30 min at room temperature. Subsequently, a peroxidase staining reaction was performed with 20 mg of 9,9-diaminobenzidine (Sigma Chemicals) and 50 /,L of 300 mL/L [H.sub.2][0.sub.2] in 50 mL of Tris-buffered saline (0.05 mol/L Tris-HCI in a solution of 9 g/L NaCl, pH 7.4). The staining reaction was stopped after 10 min by rinsing three times for 5 min with Tris-buffered saline. Nuclear staining was performed with hematoxylin for 1 min. After dehydration with ethanol (960 mL/L ethanol followed by absolute ethanol), the sections were embedded with DePeX mounting medium "Gurr" (BDH Laboratory Supplies).

As controls we used four slides of healthy human adrenal medulla for each of the respective staining procedures for TH, DBH, and PNMT. Two of the four slides were used as positive controls and underwent the immunochemical staining protocol as described above. For the other two slides, used as negative controls, the first step was substituted by incubation with medium (PBS containing 1 g/L BSA) without antibodies against TH, DBH, or PNMT, respectively.


The immunohistochemical sections were evaluated by one pathologist (H.H.) with wide experience in the field of neuroendocrine tumors. In addition to determining the site of intracellular staining within the tumor, the pathologist estimated the proportion of immunoreactive cells. The intensity of staining was classified using a gradual scale, from negative, to weakly positive, to moderately positive, to strongly positive. The assessments were made without knowledge of the catecholamine secretory characteristics. For all samples, the diagnosis (pheochromocytoma or carcinoid tumor) was evident from the section.


The results for the markers of serotonin metabolism and for urinary catecholamines are reported as medians with lower and upper ranges. For comparison of catecholamines and metabolites within midgut carcinoids and sporadic and MEN type IIa-associated pheochromocytomas, respectively, we used the Mann-Whitney U-test. We used the [[chi].sup.2] test or Fisher exact test to evaluate differences in catecholamine-synthesizing enzymes among the respective groups of patients and to evaluate the relationship between catecholamine-synthesizing enzymes and urinary catecholamine and metabolite excretion. P <0.05 was considered statistically significant.



The characteristics of the 21 carcinoid and 20 pheochromocytoma patients are shown in Table 1. All carcinoid patients had evidence of serotonin overproduction: median (range) urinary 5-HIAA excretion was 42.8 (1.0-194.0) mmol/mol urinary of creatinine. Median (range) platelet serotonin content was 24.7 (8.2-53.6) nmol serotonin/[10.sup.9] platelets. Markers of serotonin metabolism were not available for the pheochromocytoma patients.


The urinary excretion rates for catecholamines and metabolites in the three groups of patients are shown in Fig. 2. In carcinoid patients, urinary catecholamines (dopamine, norepinephrine, and epinephrine) were available in 17 patients and urinary catecholamine metabolites (3-methoxytyramine, normetanephrine, and metanephrine) in 14 carcinoid patients. Among the carcinoid patients, eight had increased urinary excretion of dopamine or 3-methoxytyramine, indicating increased dopamine synthesis. Indications of increased synthesis of norepinephrine or epinephrine were found in six patients each. Eleven carcinoid patients had urinary excretion rates for catecholamines or metabolites that were within the relevant reference intervals. We found no quantitative correlation between the markers of serotonin metabolism (urinary 5-HIAA and platelet serotonin) and urinary excretion of catecholamines or their metabolites.


Urinary catecholamine or metabolite excretion was increased in all pheochromocytoma patients. Urinary excretion of the catecholamine metabolites differed between carcinoid and pheochromocytoma patients. For 3-methoxytyramine, pheochromocytoma patients had a median urinary excretion of 300 (97-5398) [micro]mol/mol of creatinine, compared with 211 (39-865) [micro]mol/mol of creatinine in carcinoid patients (P = 0.04). Urinary normetanephrine excretion was higher (P = 0.0001) in pheochromocytoma patients (median, 1703 [micro]mol/mol of creatinine; range, 132-15 400 [micro]mol/mol of creatinine) than in carcinoid patients (median, 209 mol/mol of creatinine; range, 50-500 [micro]mol/mol of creatinine); the urinary excretion of metanephrine was also higher (P = 0.0001) in pheochromocytoma (median, 665 [micro]mol/mol of creatinine; range, 132-10 070 [micro]mol/mol of creatinine) than in carcinoid patients (median, 55 [micro]mol/mol creatinine; range, 37-120 [micro]mol/mol of creatinine). We found no differences in the urinary excretion of the other catecholamine metabolites.


Immunostaining for the catecholamine-synthesizing enzymes TH, DBH, and PNMT showed a pattern of diffuse cytoplasmic staining in carcinoids as well as in pheochromocytomas. In tumors staining for two or three catecholamine-synthesizing enzymes, the immunoreactive cells showed an overlapping distribution within the tumor. Especially in carcinoids, groups of cells staining positive for catecholamine-synthesizing enzymes could be recognized. Representative examples of immunohistochemical staining for the three catecholamine-synthesizing enzymes in carcinoid tumors and pheochromocytomas are shown in Fig. 3, and the results of the semiquantitative assessment of the immunohistochemical staining in the 21 carcinoid tumors and 20 pheochromocytomas are presented in Table 2.

Comparison of carcinoid tumors with pheochromocytomas revealed differences in the immunohistochemistry for the three catecholamine-synthesizing enzymes. Staining for TH was positive in nine carcinoid tumors, whereas all pheochromocytomas stained positive (P = 0.003). Staining for DBH was positive in 8 carcinoids and 15 pheochromocytomas (P = 0.04). Staining for PNMT was positive in 7 carcinoids and 13 pheochromocytomas (not significant, P = 0.09). Although we observed a trend toward higher intensity of staining and a higher proportion of immunoreactive cells in pheochromocytoma (Table 2), the numbers were too small to reveal significant differences with carcinoid tumors. We found several distribution patterns for the catecholamine-synthesizing enzymes in both carcinoid and pheochromocytoma specimens (Table 3). The concurrent presence of all three enzymes was the most frequent. TH was detectable in all tissue samples from pheochromocytoma patients.


Of 12 primary carcinoid tumors, 8 (67%) were positive for at least one catecholamine-synthesizing enzyme. In nine samples obtained from metastatic sites, immunohistochemistry was positive in five (55%) specimens.


Urinary catecholamine excretion was increased in 8 of 13 carcinoid tumor samples that stained positive for at least one enzyme. In contrast, urinary catecholamine excretion was increased in two of eight carcinoid samples that did not stain positive (not significant). Excretion of catecholamines and metabolites was higher in patients with carcinoid tumors that stained positive for PNMT than in those with carcinoids with negative staining (Fig. 4). The urinary excretion of dopamine [median, 177 (123-263) vs 117 (80-183) Amol/mol of creatinine; P = 0.03], normetanephrine [median, 352 (249-500) vs 114 (50-308) [micro]mol/ mol of creatinine; P = 0.009], and metanephrine [median, 100 (60-120) vs 52 (37-98) Amol/mol of creatinine; P = 0.04] was higher in carcinoid patients with tumors that stained positive for PNMT than in patients with negatively staining tumors. In contrast, we observed no differences in urinary excretion of catecholamine metabolites between patients with tumor specimens positive for TH or DBH and those with tumors negative for TH or DBH. Furthermore, we found no correlation between increased urinary dopamine (metabolite) excretion and immunoreactivity for TH, between increased urinary norepinephrine (metabolite) excretion and DBH, or between increased urinary epinephrine (metabolite) excretion and PNMT.


In the 20 patients with pheochromocytomas, the presence or absence of one specific catecholamine-synthesizing enzyme was not predictive of higher urinary excretion of the catecholamine produced by that enzyme.


In this study we demonstrate the presence of catecholamine-synthesizing enzymes in midgut carcinoid tumors. At least one of three investigated catecholamine-synthesizing enzymes was detectable in 13 (62%) of 21 carcinoid tumors and in all pheochromocytomas (P = 0.007; Table 2). The presence of these enzymes indicates that carcinoid tumors can synthesize catecholamines autonomously. Moreover, our study shows the presence of a pathway for catecholamine synthesis in carcinoid tumors that is similar to the pathway in pheochromocytoma.

Previous studies were performed in cultured carcinoid tumor cells derived from a limited number of patients. In these studies, cultured carcinoid cells from three midgut carcinoids showed positive staining for TH and DBH (19, 20). hnmunohistochemistry performed on a specimen of one midgut carcinoid tumor showed positive staining for TH, whereas staining for DBH and PNMT was negative (7). Other studies addressing AADC, the enzyme implicated in both the catecholamine and the serotonin metabolic pathways, showed expression in both carcinoids (21-23) and pheochromocytomas (8,22). Our study reveals a similar pattern of diffuse cytoplasmic staining in both carcinoids and pheochromocytomas. Both TH and DBH have been described as staining uniformly throughout the healthy adrenal medulla, pheochromocytomas, and one midgut carcinoid tumor (7), but PNMT showed focal staining in a small subset of cells adjacent to the adrenal cortex (7). This finding is probably explained by the induction of PNMT in an environment rich in glucocorticoids (3). This was illustrated in a study comparing adrenal with extra-adrenal pheochromocytomas (5). The carcinoid tumors evaluated in the present study showed a focal staining pattern for PNMT as well. However, because the tumors were located in the intestines or in the liver, a stimulating role of glucocorticoids is unlikely. The focal distribution within the tumor of immunoreactivity for the three catecholamine-synthesizing enzymes suggests that a subpopulation of carcinoid cells is responsible for the observed catecholamine synthesis.

A further aim of our study was to correlate the presence of catecholamine-synthesizing enzymes with the urinary excretion of catecholamine (metabolites). Carcinoid patients with tumors staining for PNMT had higher urinary excretion of dopamine, normetanephrine, and metanephrine compared with those with PNMT-negative tumors (Fig. 4). In contrast, immunoreactivity for either TH or DBH was not associated with increased urinary excretion of catecholamines or metabolites. Remarkably, the presence of immunoreactivity for TH, DBH, or PNMT was not predictive of increased urinary excretion of (metabolites of) dopamine, norepinephrine, or epinephrine, respectively. The poor correlation between the presence of TH or DBH and catecholamines and metabolites excreted in urine might result from the complementary action of the catecholamine-synthesizing capacity at other sites. In this way, a TH-deficient tumor could take up a precursor (e.g., t-dihydroxyphenylalanine), with subsequent synthesis of dopamine, norepinephrine, or epinephrine.

Conversely, intermediate tumor products are possibly further synthesized at sites located outside the tumor. Additional studies on enzyme activity and catecholamine content of tumor specimens are needed to elucidate this complex interaction between the respective sites of catecholamine synthesis in the body.

The data in Table 3 show that we found no histochemical evidence of PNMT expression in 7 of 20 pheochromocytoma tissue samples, whereas all of the corresponding patients presented with increased urinary excretion of metanephrine (Fig. 2). Thus, there is a discrepancy between the (in)ability of the tumor to synthesize epinephrine and the increased urinary excretion of its metabolite in urine of the corresponding patients. Using enzyme activity assays and gene expression analyses, Eisenhofer et al. (24) showed consistent tumor PNMT expression in epinephrine-producing pheochromocytoma patients. These data indicate that false-negative results can be obtained with the immunohistochemical procedures used in this study on paraffin-embedded tissue samples. Measurements of enzyme activities, Western blotting, and gene expression analyses give more sensitive and specific information and thus could clarify the discrepancies found.

The present study illustrates that carcinoids and pheochromocytomas share biosynthetic characteristics, as was postulated in the APUD concept (1). Corresponding to the capacity of carcinoids to synthesize catecholamines, pheochromocytomas probably can synthesize serotonin, as was described for human pheochromocytoma cells transplanted in the anterior eye chamber of the rat (25). An interesting theory elaborating the APUD concept was presented by Ahlman et al. (19). Both pheochromocytomas and carcinoids are possibly derived from a multipotent progenitor cell able to synthesize catecholamines. In the case of gastrointestinal chromaffin cells, the catecholamine-synthesizing capacity becomes suppressed and serotonin synthesis supervenes. In carcinoid tumors, dedifferentiation takes place, leading to a more progenitor-like cell type, with loss of suppression of catecholamine synthesis. Further loss of suppression of catecholamine synthesis in cell culture possibly explains the active catecholamine synthesis of carcinoid cells in vitro.

The clinical consequences of catecholamine synthesis in carcinoid patients remain to be determined. Depending on the amounts and the type of catecholamines synthesized, catecholamines contribute to the clinical presentation of carcinoid tumors. Catecholamines play a role in the pathophysiology of the carcinoid flush (26) and in abnormal cardiovascular function (27,28). Furthermore, catecholamines can stimulate serotonin secretion by carcinoids via a [beta]-adrenoreceptor located on the carcinoid cell (20). This mechanism can produce positive feedback, eventually leading to a carcinoid crisis. Appreciation of the catecholamine-synthesizing capacity of carcinoid tumors can lead to optimization of the perioperative management of carcinoid patients. In addition to octreotide, selective blockade of catecholamine receptors might prevent carcinoid crisis in stressful situations.

In conclusion, the catecholamine-synthesizing enzymes TH, DBH, and PNMT are present in 42%, 38%, and 33%, respectively, of tumors in midgut carcinoid patients. Immunoreactivity for these enzymes showed a similar pattern for carcinoid tumors, sporadic pheochromocytoma, and MEN-associated pheochromocytoma. Increased urinary excretion of catecholamines and metabolites was observed in 48% of carcinoid patients. No clinically relevant association between the presence of catecholamine-synthesizing enzymes and urinary catecholamine (metabolite) excretion was found. This, however, does not exclude a possible relationship because urinary catecholamine (metabolite) excretion is an indirect measurement that is not sensitive enough to detect catecholamine overproduction from tumor sites. The presence of these catecholamine-synthesizing enzymes in carcinoid tumors demonstrates their ability to synthesize catecholamines. These catecholamines can cause symptoms, and knowledge of the presence of catecholamine synthesis can therefore be useful in clinical management of the carcinoid syndrome.

Received November 18, 2002; accepted January 16, 2003.


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Departments of [1] Medical Oncology, [2] Medical Physiology, [3] Pathology and Laboratory Medicine, and [4] Endocrinology, University Hospital Groningen, 9700 RB Groningen, The Netherlands.

* Address correspondence to this author at: Department of Medical Oncology, University Hospital, PO Box 30.001, 9700 RB Groningen, The Netherlands. Fax 31-503614862; e-mail

[5] Nonstandard abbreviations: AADC, aromatic-L-amino acid decarboxylase; APUD, amine precursor uptake and decarboxylafion; MEN, multiple endocrine neoplasia; TH, tyrosine hydroxylase; DBH, dopamine-0-hydroxylase; PNMT, phenylethanolamine-N-methyltransferase; 5-HIAA, 5-hydroxyindoleacetic acid; URL, upper reference limit; PBS, phosphate-buffered saline; and BSA, bovine serum albumin.
Table 1. Characteristics of carcinoid and
pheochromocytoma patients and sources of tumor

 carcinoids Sporadic MEN

Number of patients 21 10 10
Median (range) age, years 61 (44-75) 39 (31-49) 33 (26-41)
M/F 8/13 4/6 4/6
Source of tumor specimen
 Adrenal tumor 10 10
 Primary gut tumor 12
 Mesenterial lymph node 6
 Liver metastasis 3

Table 2. Immunohistochemical staining for catecholamine-synthesizing
enzymes in carcinoid and pheochromocytoma patients.

Enzyme carcinoid Pheochromocytoma P

 All patients, n 21 20
 Overall classification, n 0.003
 Negative 12 0
 Positive 9 20
 Median (range) % 90 (10-100) 100 (5-100)
 positive cells
 Intensity of staining, n
 Weak 7 7
 Moderate 2 10
 Strong 0 3
 Overall classification, n 0.04
 Negative 13 5
 Positive 8 15
 Median (range) % 20 (5-100) 100 (5-100)
 positive cells
 Intensity of staining, n
 Weak 3 9
 Moderate 5 4
 Strong 0 2
 Overall classification, n 0.09
 Negative 14 7
 Positive 7 13
 Median (range) % 80 (5-100) 60 (5-100)
 positive cells
 Intensity of staining, n
 Weak 3 6
 Moderate 2 2
 Strong 2 5
At least one enzyme
 Negative 8 0 0.007
 Positive 13 20

Table 3. Enzyme expression profiles in tumor specimens from carcinoid
and pheochromocytoma patients. (a)

 No. of patients expressing
 the enzyme profile

 TH + + + + - - - -
 DBH + + - - + + - -
 PNMT + - + - + - + -
Carcinoid (n = 21) 4 (b) 2 0 3 1 1 2 8
 Sporadic (n = 10) 5 4 1 0 0 0 0 0
 MEN (n = 10) 5 1 2 2 0 0 0 0

(a) +, enzyme detectable; -, enzyme not detectable.

(b) Number of patients.
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Title Annotation:Proteomics and Protein Markers
Author:Meijer, Wim G.; Copray, Sjef C.V.M.; Hollema, Harry; Kema, Ido P.; Zwart, Nynke; Mantingh-Otter, Iet
Publication:Clinical Chemistry
Date:Apr 1, 2003
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