Source of plasma adrenomedullin in patients with pheochromocytoma

AJH

2002; 15:994 –997

Source of Plasma Adrenomedullin in Patients With Pheochromocytoma Junichi Minami, Toshio Nishikimi, Masakatsu Todoroki, Chieko Kashiwakura, Hiroshi Yagi, Hidehiko Ono, Shigeo Horinaka, Toshihiko Ishimitsu, Kenji Kangawa, and Hiroaki Matsuoka Controversy exists as to the origin of plasma adrenomedullin (AM). To elucidate the source of plasma AM, we measured two molecular forms of AM, an active form of mature AM (AM-m) and an intermediate inactive form of glycine-extended AM (AM-Gly), by immunoradiometric assay using specific kits in two female patients with pheochromocytoma before and 3 weeks after surgery. We also measured plasma AM-m, AM-Gly, and AM-T (AM-m ⫹ AM-Gly) levels, in addition to plasma epinephrine (E) and norepinephrine (NE) levels, in bilateral adrenal veins of one patient. Although plasma E and NE levels decreased markedly after surgery in these patients, changes in plasma AM appeared to be confined to the normal range. There were no obvious differences in plasma AM-T, AM-m, or AM-Gly levels in adrenal veins between healthy tissue and tumor sides. Furthermore, plasma AM-T, AM-m, or AM-Gly levels in adrenal veins were comparable with those in the infrarenal inferior vena

A

cavae (IVC) or the suprarenal IVC. In contrast, plasma E and NE levels increased in the adrenal vein of the healthy side and increased further in the adrenal vein of the tumor side compared with those in the infrarenal IVC. These results suggest that the origin of plasma E and NE is the adrenal gland and that elevated plasma levels of E and NE in pheochromocytoma are due to excessive production of E and NE in the adrenal gland of the tumor side. In contrast, it is suggested that neither plasma AM levels in the adrenal vein of the healthy side nor those of the tumor side contribute to the systemic levels of plasma AM. The present results appear to be consistent with the hypothesis that the source of circulating AM is systemic vasculature. Am J Hypertens 2002;15:994 –997 © 2002 American Journal of Hypertension, Ltd. Key Words: Adrenomedullin, molecular form, cathecolamine, pheochromocytoma.

drenomedullin (AM) is a potent vasodilator peptide consisting of 52 amino acids.1 It was originally discovered from a human pheochromocytoma, and belongs to the calcitonin gene-related peptide (CGRP) superfamily. Adrenomedullin circulates in human blood and its levels are increased in proportion to the severity of cardiovascular disorders such as hypertension, chronic renal failure, or congestive heart failure.2,3 However, controversy appears to remain with regard to the origin of plasma AM. Moreover, inconsistent results have been reported regarding the behaviors of circulating AM in patients with pheochromocytoma.4,5 We previously reported that plasma AM levels did not increase at rest or even during a hypertensive attack in patients with pheochromocytoma, although epinephrine (E) and norepinephrine (NE) increased markedly.4 In contrast, Letizia et al5

recently reported that plasma AM levels increased in comparison with both normotensive subjects and those with essential hypertension. In addition, they have reported that plasma AM levels in these patients correlated with NE levels and were strongly related to the tumor, as demonstrated by a marked increase with tumor manipulation and subsequent reduction after surgical removal. These discrepancies may be due to the different methods of measuring plasma AM. Previous studies measured AM levels by radioimmunoassay using polyclonal antibodies after extraction of plasma. However, this method might not be able to detect minor changes in the plasma AM levels. Recent studies have revealed that two molecular forms of AM, that is, an active form of mature AM (AM-m) and an intermediate inactive form of glycineextended AM (AM- Gly), circulate in human plasma.6,7

Received April 24, 2002. First decision May 30, 2002. Accepted June 13, 2002.

Tochigi, and Research Institute, National Cardiovascular Center (KK), Suita, Osaka Japan. Address correspondence and reprint requests to Dr. Junichi Minami, Department of Hypertension and Cardiorenal Medicine, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan; e-mail: [email protected]

From the Department of Hypertension and Cardiorenal Medicine (JM, TN, MT, NY, HO, SH, JI, HM), and Department of Endocrinology and Metabolism (CK), Dokkyo University School of Medicine, Mibu, 0895-7061/02/$22.00 PII S0895-7061(02)02997-7

© 2002 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.

AJH–November 2002–VOL. 15, NO. 11

These studies have shown that AM measured by radioimmunoassay in earlier studies is equivalent to AM-total (AM-T) (ie, the sum of AM-m and AM-Gly) because the radioimmunoassay system used polyclonal antibodies, which could not distinguish between structures with or without a carboxy-terminal amide. Recently, we developed a one-step direct immunoradiometric assay system for AM-m and AM-T with different monoclonal antibodies.8,9 It easily enables measuring AM-m and AM-T specifically with a small sample without the prior extraction of plasma. In the present study, we measured the plasma AM-T and AM-m levels using this immunoradiometric assay system in two patients with pheochromocytoma before and after removal of the tumor. Furthermore, we measured plasma AM-T and AM-m levels in bilateral adrenal veins before surgery in one patient to address whether the adrenal gland was the origin of circulating AM.

Methods Patients and Case History Two patients with pheochromocytoma were studied. In patient 1, a 17-year-old woman, was admitted to our hospital with chief complaints of blurred vision and palpitation. The blood pressure (BP) was 164/116 mm Hg. A 4.0-cm mass in the right adrenal gland was seen on the computed tomographic scan. In patient 2, a 52-year-old woman, was admitted to our hospital with a 14-year history of hypertension, headache, and perspiration. The BP was 152/86 mm Hg. A 6.0-cm mass in the right adrenal gland was seen on the computed tomographic scan. In both patients, measurements of catecholamines and their metabolites confirmed the diagnosis of pheochromocytoma. In both patients, scintigraphy using metaiodobenzyl-guanidine, containing iodine 131 (131I-MIBG) showed the functioning tumor on the right side. After removal of the tumor, the BP of these patients decreased to about 120/70 mm Hg. In both patients, the histopathologic examination confirmed the diagnosis of pheochromocytoma. Each patient received a detailed explanation of the purpose of the study and gave written informed consent. Blood Sampling and Data Analysis In both patients, antecubital venous blood was taken after an overnight fast and 30 min of supine rest before and 3 weeks after surgery during the hospital stay. In patient 2, bilateral adrenal veins and the infrarenal and suprarenal inferior vena cavae (I-IVC, S-IVC) were cathetherized using a 5-F catheter before surgery. Selective venous samples for measurements of plasma E, NE, AM-m, and AM-T levels were obtained at each site. Plasma E and NE levels were measured using high-performance liquid chromatography.10 Both plasma AM-m and AM-T levels were measured by immunoradiometric assay using specific kits (AM ma-

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ture RIA SHIONOGI, AM RIA SHIONOGI) developed by the Diagnostic Science Department, Shionogi Pharmaceutical Co., Ltd., Osaka, Japan.8,9 These assay systems use two monoclonal antibodies against human AM, one recognizing a ring structure of human AM in both kits and the other recognizing the carboxy-terminal sequence in the AM-m kit or AM (25–36) in the AM-T kit. The assay measures human AM-m or AM-T by sandwiching it between the two antibodies without the extraction of plasma. The assay’s minimal detectable quantity of human AM-m or AM-T is 0.5 pmol/L in both kits. The coefficients of variation of the intra-assay and interassay in several blood samples were 4.4% to 8.2% and 5.5% to 8.3% in the AM-m kit and 3.4% to 7.3% and 5.3% to 9.0% in the AM-T kit, respectively. The recovery rate of 5 to 100 pmol/L of human AM added to several plasma samples was 91% to 118% in the AM-m kit and 91% to 118% in the AM-T kit. A reverse-phase high-performance liquid chromatography analysis revealed that the major peak of immunoreactive AM in the plasma detected by each immunoradiometric assay kit for AM-m and AM-T was identical to synthetic human AM (1-52).8,9 The AM-Gly was calculated using the following formula: AM-Gly ⫽ AM-T ⫺ AM-m. In the evaluation of plasma AM-T levels, 81 healthy subjects (normal group, 41 men and 40 women, mean age (⫾ SD) 48 ⫾ 9 years) and 28 patients with essential hypertension (hypertension group; 19 men and 9 women; mean age 53 ⫾ 18 years) served as reference.

Results In patient 1, the plasma E and NE levels decreased from 36 to 12 ng/L, and 10,090 to 466 ng/L, respectively, 3 weeks after surgery (Table 1). The ratio of the plasma E levels after to before surgery was 0.33, and that of plasma NE levels was 0.05. Plasma AM-T, AM-m, and AM-Gly levels were 14.2, 1.2, and 13.0 pmol/L, respectively, before surgery, whereas they were 12.2, 1.1, and 11.1 pmol/L, respectively, 3 weeks after surgery (Table 1). The ratio of the plasma AM-T levels after to before surgery was 0.86, that of plasma AM-m levels was 0.92, and that of plasma AM-Gly was 0.85. In patient 2, the plasma E and NE levels decreased from 1200 to 5 ng/L, and 5800 to 187 ng/L, respectively, 3 weeks after surgery (Table 1). The ratio of the plasma E levels after to before surgery was 0.004, and that of the plasma NE levels was 0.03. Plasma AM-T, AM-m, and AM-Gly levels were 7.0, 1.6, and 5.4 pmol/L, respectively, before surgery, whereas they were 13.3, 3.0, and 10.3 pmol/L, respectively, 3 weeks after surgery (Table 1). The ratio of the plasma AM-T levels after to before surgery was 1.90, that of the plasma AM-m levels was 1.88, and that of plasma AM-Gly was 1.91. In both patients, changes in plasma AM-T appeared to be confined to the normal range (Fig. 1), although plasma E and NE levels decreased markedly after surgery.

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Table 1. Plasma epinephrine, norepinephrine, total adrenomedullin, mature adrenomedullin, and glycineextended adrenomedullin levels before surgery and 3 weeks after surgery in two female patients with pheochromocytoma

Patient 1 Epinephrine (ng/L) Norepinephrine (ng/L) AM-T (pmol/L) AM-m (pmol/L) AM-Gly (pmol/L) Patient 2 Epinephrine (ng/L) Norepinephrine (ng/L) AM-T (pmol/L) AM-m (pmol/L) AM-Gly (pmol/L)

Before Surgery

After Surgery

Ratio (after/before)

36 10,090 14.2 1.2 13.0

12 466 12.2 1.1 11.1

0.33 0.05 0.86 0.92 0.85

1200 5800 7.0 1.6 5.4

5 187 13.3 3.0 10.3

0.004 0.03 1.90 1.88 1.91

AM-T ⫽ total adrenomedullin; AM-m ⫽ mature adrenomedullin; AM-Gly ⫽ glycine-extended adrenomedullin.

In patient 2, the plasma E and NE levels were 535 and 2350 ng/L in the I–IVC, 3200 and 3350 ng/L in the left adrenal vein, 5750 and 23,000 ng/L in the right adrenal vein, and 4200 and 12,500 ng/L in the S-IVC, respectively (Fig. 2). Plasma AM-T, AM-m, and Am-Gly levels were 7.0, 1.6, and 5.4 in the I-IVC, 7.9, 1.3, and 6.6 pmol/L in the left adrenal vein, 6.4, 1.8, and 4.6 pmol/L in the right adrenal vein, and 7.1, 2.3, and 4.8 pmol/L in the S-IVC, respectively (Fig. 2).

Discussion There have been several studies that assessed the plasma AM levels in patients with pheochromocytoma, although

the results are inconsistent.4,5 To the best of our knowledge, this is the first study to measure plasma AM levels using adrenal venous sampling. Moreover, we analyzed two molecular forms of plasma AM—AM-m and AMGly—in patients with pheochromocytoma. There were no obvious differences in plasma AM-T, AM-m, or AM-Gly levels in adrenal veins between the healthy and tumor sides. Furthermore, plasma AM-T, AM-m, or AM-Gly levels in adrenal veins were comparable with those in the I-IVC or S-IVC. In addition, the ratio of plasma AM-m to AM-T in the adrenal vein of the tumor side was also comparable with that in the I-IVC or S-IVC. In contrast, plasma E and NE levels increased in the adrenal vein of the healthy side and increased further

FIG. 1. Changes in the plasma total adrenomedullin levels before surgery and 3 weeks after surgery in two female patients with pheochromocytoma. In the evaluation of the plasma total adrenomedullin levels, 81 healthy subjects (normal group; 41 men and 40 women; mean age (⫾ SD) 48 ⫾ 1 years) and 28 patients with essential hypertension (hypertension group; 19 men and 9 women; mean age 53 ⫾ 3 years) served as reference.

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was discovered from pheochromocytoma and termed adrenomedullin. Therefore, the source of plasma AM needs to be clarified. Sugo et al8 previously demonstrated that cultured vascular endothelial cells and smooth muscle cells synthesized and secreted AM more actively than adrenal glands, suggesting that the systemic vessel wall is a potential site of AM production. Furthermore, we previously reported that plasma AM levels did not increase in the veins across the organs in which AM mRNA was highly expressed.4 These results are consistent with the hypothesis11 that the systemic vascular wall is a source of plasma AM. This hypothesis is also in agreement with the present findings that plasma AM levels were comparable at all the sampling sites.

References 1.

FIG. 2. Plasma epinephrine, norepinephrine, and total adrenomedullin, mature adrenomedullin (AM-m), and glycine-extended adrenomedullin (AM-Gly) levels in the infrarenal inferior vena cavae (I-IVC), in the left adrenal vein (L-Adr V), in the right adrenal vein (R-Ad V), and in the suprarenal inferior vena cavae (S-IVC) before surgery in patient 2.

in the adrenal vein of the tumor side compared with those in the I-IVC. These results suggest that the origin of plasma E and NE is the adrenal gland and that elevated plasma levels of E and NE in pheochromocytoma are due to excessive production of E and NE in the adrenal gland of the tumor side. In contrast, it is suggested that neither plasma AM levels in the adrenal vein of the healthy side nor those in the tumor side contribute to systemic levels of plasma AM. These findings are in agreement with our previous observations that plasma AM levels did not increase at rest or even during the hypertensive attack in patients with pheochromocytoma.4 In the present study, the investigation was extended to the measurement of two molecular forms of AM, and it was found that plasma AMm or AM-Gly levels did not increase in pheochromocytoma. Thus, it is suggested that the adrenal gland is not a major site of production of circulating AM, although AM

Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, Matsuo H, Eto T: Adrenomedullin: A novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun 1993;192:553–560. 2. Ishimitsu T, Nishikimi T, Saito Y, Kitamura K, Eto T, Kangawa K, Matsuo H, Omae T, Matsuoka H: Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure. J Clin Invest 1994;94:2158 –2161. 3. Nishikimi T, Saito Y, Kitamura K, Ishimitsu T, Eto T, Kangawa K, Matsuo H, Omae T, Matsuoka H: Increased plasma levels of adrenomedullin in patients with heart failure. J Am Coll Cardiol 1995;26:1424 –1431. 4. Nishikimi T, Kitamura K, Saito Y, Shimada K, Ishimitsu T, Takamiya M, Kangawa K, Matsuo H, Eto T, Omae T, Matsuoka H: Clinical studies on the sites of production and clearance of circulating adrenomedullin in human subjects. Hypertension 1994;24: 600 –604. 5. Letizia C, De Toma G, Caliumi C, Cerci S, Massa R, Loria RD, Alo P, Marinoni EM, Diacinti D, D’Erasmo E: Plasma adrenomedullin concentrations in patients with adrenal pheochromocytoma. Horm Metab Res 2001;33:290 –294. 6. Kitamura K, Kato J, Kawamoto M, Tanaka M, Chino N, Kangawa K, Eto T: The intermediate form of glycine-extended adrenomedullin is the major circulating molecular form in human plasma. Biochem Biophys Res Commun 1998;244:551–555. 7. Nishikimi T, Matsuoka H, Shimada K, Matsuo H, Kangawa K: Production and clearance sites of two molecular forms of adrenomedullin in human plasma. Am J Hypertens 2000;13:1032– 1034. 8. Sugo S, Minamino N, Kangawa K, Miyamoto K, Kitamura K, Sakata J, Eto T, Matsuo H: Endothelial cells actively synthesize and secrete adrenomedullin. Biochem Biophys Res Commun 1994;201: 1160 –1166. 9. Sugo S, Minamino N, Shoji H, Kangawa K, Kitamura K, Eto T, Matsuo H: Production and secretion of adrenomedullin from vascular smooth muscle cells: augmented production by tumor necrosis factor-alpha. Biochem Biophys Res Commun 1994;203:719 –726. 10. Speek AJ, Odink J, Schrijver J, Schreurs WH: High-performance liquid chromatographic determination of urinary free catecholamines with electrochemical detection after prepurification on immobilized boric acid. Clin Chim Acta 1983;128:103–113. 11. Charles CJ, Lainchbury JG, Lewis LK, Rademaker MT, Richards AM, Yandle TG, Nicholls MG: The role of adrenomedullin. Am J Hypertens 1999;12:166 –173.