Autoprzeciwciała skierowane przeciw karboksypeptydazie E w autoimmunologicznej chorobie Addisona

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1 / ORIGINAL PAPERS Endokrynologia Polska / Polish Journal of Endocrinology Tom/Volume 54; Numer/Number 6/2003 ISSN X Autoprzeciwciała skierowane przeciw karboksypeptydazie E w autoimmunologicznej chorobie Addisona Aleksandra Baumann-Antczak, Jerzy Kosowicz, Maria Gryczyńska, Cezary Mądrzak*, Jerzy Skrobisz** Klinika Endokrynologii, Akademia Medyczna im. Karola Marcinkowskiego w Poznaniu * Katedra Biochemii i Biotechnoilogii Akademii Rolniczej im. Augusta Cieszkowskiego w Poznaniu ** Oddział Chirurgii, Szpital Wojewódzki w Poznaniu Streszczenie Choroba Addisona często współistnieje z innymi chorobami o podłożu autoimmunizacyjnym (na przykład z chorobą Gravesa Basedowa, chorobą Hashimoto czy cukrzycą typu 1). Karboksypeptydaza E jest obecna w ludzkich przysadkach, nadnerczach, trzustce, a także tarczycy. Celem pracy było zbadanie obecności i częstości występowania przeciwciał przeciwko przysadkowej karboksypeptydazie E w surowicy pacjentów z chorobą Addisona. Materiał i metody. Badaniami objęto 46 pacjentów z chorobą Addisona oraz 41 osób z grupy kontrolnej. Karoksypeptydaza E była oczyszczana z przysadek ludzkich i bydlęcych według metody opisanej przez Parkinsona i użyta w radioimmunologicznej metodzie fazy stałej do badania obecności przeciwciał. Probówki polietylenowe opłaszczano 2 mikrogramami karboksypeptydazy, po blokowaniu inkubowano z surowicami badanych rozcieńczonymi 1:z100. Dla wykrycia związanych przeciwciał dodawano białko A znakowane izotopem 125 I. Badania wykazały dużą częstość występowania przeciwciał przeciwko ludzkiej (23,9%) i bydlęcej (32,6%) karboksypeptydazie przysadkowej w grupie pacjentów z chorobą Addisona. Natomiast w grupie kontrolnej przeciwciała przeciwko bydlęcej i ludzkiej przysadkowej karboksypeptydazie E występowały z tą sama częstością 2,4%. Wnioski. 1. W chorobie Addisona przeciwciała przeciwko przysadkowej karboksypeptydazie E występują z dużą częstością. 2. Radioimmunologiczna metoda fazy stałej z użyciem bydlecej karboksypeptydazy jako antygenu wykazywała się większa czułością metody. 3. Karboksypeptydaza E jako enzym obecny w wielu gruczołach dokrewnych może być antygenem odpowiedzialnym za wystąpienie autoimmunizacyjnej poliendokrynopatii w chorobie Addisona, lecz potwierdzenie tej tezy wymaga dalszych badań. Słowa kluczowe: karboksypeptydaza E, choroba Addisona, autoprzeciwciała 752

2 / ORIGINAL PAPERS Endokrynologia Polska / Polish Journal of Endocrinology Tom/Volume 54; Numer/Number 6/2003 ISSN X The prevalence of autoantibodies to pituitary carboxypeptidase E in autoimmune Addison s disease Aleksandra Baumann-Antczak, Jerzy Kosowicz, Maria Gryczyńska, Cezary Mądrzak*, Jerzy Skrobisz** Department of Endocrinology Karol Marcinkowski University of Medical Sciences, Poznań, Poland *Department of Biochemistry and Biotechnology August Cieszkowski Agricultural University of Poznań, Poland ** Department B of Surgery, Province Hospital, Poznań, Poland Summary Autoimmune Addison s disease is frequently associated with other autoimmune diseases (Graves disease, Hashimoto s thyroiditis, diabetes mellitus type 1). Carboxypeptidase E is found in human pituitary, adrenal glands, pancreas and thyroid. The aim of our studies was to evaluate the occurrence and prevalence of antibodies to pituitary carboxypeptidase E in patients with Addison s disease. Our studies involved 46 patients with autoimmune Addison s disease and 41 healthy subjects as a control group. Autoantibodies were evaluated by solid phase radioimmunoassay using polyethylene tubes coated with 2 g/tube of carboxy-peptidases isolated from human and bovine pituitaries and from porcine and bovine pancreas. After incubation with patients sera diluted 1:100 the contents of the tubes were decanted, washed and labeled. 125 Iodine Protein A was added to detect the retained antibodies. The studies revealed a high prevalence of autoantibodies to the human and bovine pituitary carboxypeptidase E as they were detected respectively in 23.9% and 32.6% patients with Addison s disease. While in a group of controls autoantibodies to human pituitary carboxypeptidase E and to bovine pituitary carboxypeptidase E were present with similar frequency in 2.4% subjects. From our studies we conclude that in autoimmune Addison s disease there is a high prevalence of autoantibodies to pituitary carboxypeptidase E. Furthermore, radioimmunoassay using bovine pituitary carboxypeptidase H as an antigen was more sensitive than these with human pituitary carboxypeptidase. Carboxypeptidase E is the enzyme present in many endocrine tissues and could be responsible for poliendocrine autoimmunity in addison s disease, but this must be investigate further. Key words: carboxypeptidase E, Addison s disease, autoantibodies A. Baumann Antczak, Department of Endocrinology, Karol Marcinkowski University of Medical Sciences, ul. Przybyszewskiego 49; Poznań, Poland Fax E- mail: a.baumann.antczak@wp.pl Introduction In serum of patients with adult onset autoimmune Addison s disease autoantibodies to steroid 21-hydroxylase are usually present [1]. Addison s disease is frequently associated with other autoimmune diseases such as Grave s disease, Hashimoto s thyroiditis or type 1 diabetes as the part of autoimmune polyendocrine syndrome. Therefore, we can presume that autoimmune process in this disease is more generalized involving not only adrenal specific antigen such as steroid 21-hydroxylase, but also affects other autoantigens (tubulin, adrenal, gastric, ovary and pituitary microsomal proteins) as reported in our previous studies [2, 3, 4]. Carboxypeptidase E named also the enkephalin convertase is present in many endocrine tissues, such as bovine pituitary, brain and adrenals as described by Fricker [5]. Carboxypeptidase E was also found in shark s neuron tissue, in rat heart, pituitary, brain and pancreatic islet cells [6] as well as in rat insulinoma [7]. Further studies revealed the presence of carboxypep- tidase E in human organs such as: brain [8], stomach [9], placenta [10] and pancreas [11]. The sequences of carboxypeptidases from human brain and other endocrine organs are known, and they show some similarities, for example the aminoacid sequence of carboxypeptidases from human thyroid and pancreas were identical, and differed from brain carboxypeptidase E in the absence of second ATG in the predicted 5 non-coding region [12]. Carboxypeptidase E is present in two forms: soluble and membrane bound [13]. Furthermore, they have two isoforms different in molecular weight 54 and 56 kda. Both forms of carboxypeptidase are glycoproteins. The presence of carboxypeptidase E in many neuroendocrine and endocrine tissues (pituitary, sympathetic ganglia, adrenal medulla, pancreas and thyroid) suggests that this enzyme is a common autoantigen in polyglandular autoimmune syndrome especially in patients in whom autoantibodies to different endocrine organs are present [14, 15, 16, 17]. 753

3 Autoprzeciwciała w chorobie Addisona Baumann-Antczak A. The aim of this study was to evaluate the autoantibodies to human and bovine pituitary carboxypeptidase E in patients with autoimmune Addison s disease. Materials and methods Patients Sera were obtained from 46 patients with autoimmune Addison s disease, 30 female and 16 male patients aged 19 to 47 years, mean age 28 yr, mean duration of disease 7 years (range from 1 to 15 yrs). Diagnosis was made on the basis of clinical and biochemical findings typical for chronic adrenal insufficiency. Serum ACTH was elevated above 200 pg/ml in all patients, cortisol concentration was low (0.5 6 g/dl), and there was no response in serum cortisol to rapid ACTH adrenal stimulation tests. Furthermore, all sera contained antibodies to steroid 21-hydroxylase. Healthy control aged matched group was chosen among blood donors and comprised of 41 subjects, 31 females and 10 males; in all of them the measurements of serum ACTH and cortisol were within the reference range. In control subjects 21-hydroxylase antibodies were not detected. Reagents Human pituitaries were obtained at autopsy and stored in -70 o C. Fresh bovine pituitaries were from slaughterhouse in Poznań. For affinity column we use p-aminobenzoylarginine kindly gifted by doctor Thomas H. Plummer Jr. from State of New York Department of Health. Protein A from Staphylococcus, CNBr Sepharose 4B, dextrose (Sephadex G25) were purchased from Pharmacia (Uppsala, Sweden). 125 Iodine for iodination of Protein A, manufactured by NEN was supplied by POLATOM Świerk, as well as polyethylene tubes. Fasting blood samples were collected and after separation sera were frozen at -20 o C until use. Methods Carboxypeptidase E was purified from human and bovine pituitaries according to Parkinson [18] by affinity chromatography method using p-aminobenzoyl-arginine coupled to CNBr-Sepharose 4 B as a sorbent. Frozen pituitaries were allowed to thaw for 10 minutes and after separation of adjoining connective tissue the pituitaries were homogenized in ice-cold 50 mm sodium acetate/200 mm sodium chloride ph 5.5 containing 1 mm phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 600g for 10 minutes to remove cell debris. The supernatant was then centrifuged at g for 30 minutes. The supernatant fraction from this step was made 50% saturated with solid ammonium sulfate and stirred for 30 minutes. Precipitated proteins were sedimented by centrifugation at g for 20 minutes, dissolved in 50 mm sodium acetate/200 mm sodium chloride ph 5.5 to 25% of the original volume and stirred for 30 minutes at room temperature. Any precipitates were removed by centrifugation and the supernatant was then loaded onto a column of affinity resin. For purification of human and bovine pituitary carboxypeptidase E affinity chromatography was performed using p-aminobenzoylarginine immobilized on CNBr activated agarose (Sepharose 4 B). The column was washed with 20 ml of 50 mm sodium acetate containing 800 mm sodium chloride ph 5.5. Carboxypeptidase E was eluted from the sorbent with 500 mm arginine in 50 mm sodium acetate/200 mm sodium chloride ph 5.5 and used for radioimmunoassay. The antibodies were assayed by radioimmunological solid phase method in polyethylene tubes coated with carboxypeptidase E as the antigen. Polyethylene tubes were coated with carboxypeptidases in 0.05 mol/l borate buffer ph 9.6 containing 0.1% sodium azide. To each tube 0.5 ml solution was added, antigen concentration was 2 5 g/ml. After hours of incubation in temperature 4 o C tubes were washed three times with PBS/Tween. The remaining active sites on the walls of the tubes were blocked by 3% skimmed milk in PBS/Tween. Patients sera diluted 1:100 in buffer were added to the coated tubes and incubated at 37 o C for 2 hours. Then the contents of the tubes were aspirated and the tubes were washed 3 times with 0.5 ml of PBS/Tween. Subsequently 125 I Protein A cpm per tube was added to detect the retained autoantibodies. After 18 hours of incubation in a cold room the tubes were decanted, washed three times with PBS/Tween and counted in gammacounter. The results were expressed as an antibody index the results of impulses counted per minute (cpm) of patients sera were divided by mean cpm of healthy controls. Results The t-student test was used for statistical analysis of the results. Additionally the Fisher s exact test was used to compare sensitivity and specificity of the method. In the serum of patients with autoimmune Addison s disease autoantibodies to carboxypeptidase E from bovine pituitary (Fig. 1) were detected in 32.6% (15 out of 46 patients). In these patients the antibody index was elevated above 1.46 and was statistically significant (p<0.0001). The antibodies to carboxypeptidase E from human pituitaries (Fig. 2) were less frequent as they were present in 11 out of 46 patients (23.9%) with autoimmune Addison s disease. In the group of control subjects the antibodies to human pituitary carboxypeptidase E as well as to bovine pituitary carboxypeptidase E (Fig. 1 and 2) were present only in 1 out of 41 controls (2.4%). 754

4 Endokrynologia Polska / Polish Journal of Endocrinology 2003; 6 (54) Analysis by Fisher s Exact Test revealed that method with bovine pituitary CPE as an antigen was more sensitive and specific than method using human pituitary CPE. The two-sided p value < was considered extremely significant. Fig. 1. Binding index of antibodies to human pituitary carboxypeptidase E in serum of patients with autoimmune Addison s disease and control subjects. Shadowed area reflect the normal range. Fig. 2. Binding index of antibodies to bovine pituitary carboxypeptidase E in serum of patients with autoimmune Addison s disease and control subjects. Shadowed area reflect the normal range. Discussion Autoantibodies to carboxypeptidase E were only reported in autoimmune diabetes mellitus type 1, Castano et al. [11] revealed autoantibodies to rat CPE in serum of a prediabetic patient and his first degree relatives who were positive for islet cell antibodies. Other authors [19] found the presence of autoantibodies to CPE in 11% of IDDM patients at the onset of disease; the authors submitted the patients sera with in vitro translated rat CPE. Carboxypeptidase E was cloned [12] from human islet library but anti CPE assays using human pituitary carboxypeptidase E has not yet been reported. We have not found any publications on carboxypeptidase E autoantibodies in Addison s disease. In autoimmune Addison s disease antibodies to adrenal 21-hydroxylase were very frequent [17, 20]. There was a high association between adrenal cortex autoantibodies established by immunofluorescence studies and antibodies to steroid 21- hydroxylase measured by immunoprecipitation assays in patients with autoimmune Addison s disease [21, 22]. Steroid producing cells autoantibodies were found in 26% of patients with autoimmune Addison s disease, and antibodies to 17 alpha-hydroxylase and/or P-450 side chain cleavage enzyme in 36% of the patients [22]. These autoantibodies were also found in 85% of patients with Addison s disease associated with idiopathic premature ovarian failure. Autoantibodies to steroid 21-hydroxylase and 17 alpha hydroxylase or P-450 side chain cleavage (scc) enzyme were positive also in patients with autoimmune polyglandular syndromes 64% of APSI and 96% of APSII [23]. Autoantibodies to steroid 21-hydroxylase are sensitive markers for autoimmune Addison s disease [24] but the presence of antibodies to 17 alpha hydroxylase or scc may suggest the coexistence of or progression towards polyglandular autoimmune syndrome [25]. Further studies indicated that 21-hydroxylase autoantibodies bind to at least three different epitopes in the C-terminal part of 21 hydroxylase, and two of these epitopes appear to be human specific [26, 27, 28]. The autoimmunity in Addison s disease is often reported; in 47% of patients with Addison s disease at least one other autoimmune disorder was also present [29]. Of the highest prevalence in these patients was primary hypothyroidism (20.5%), then vitiligo (9.6%), premature ovarian failure (7.3% of 755

5 Autoprzeciwciała w chorobie Addisona Baumann-Antczak A. women patients), Graves disease (6%), pernicious anemia (4.8%), type 1 diabetes mellitus (1.2%) and celiac disease (1%) [29, 30, 31]. Furthermore, in Addison s disease patients the prevalence of Helicobacter pylori infection was markedly increased as it was found in 45.5% of cases [32]. Autoantibodies to 21 hydroxylase and scc or 17 alpha-hydroxylase are also reported in about 5% of patients with APS III (autoimmune thyroid disease + type 1 diabetes mellitus without Addison s disease) [33]. Addison s disease, autoimmune thyroid disease and autoimmune polyglandular syndrome type II were more frequent in patients with sarcoidosis compared with the general population [34]. Recently autoantibodies against aromatic L-amino acid decarboxylase (AADC) have been reported; they were present in 50% of patients with APS I, but also in some patients with autoimmune Addison s disease [35]. We found that in Addison s disease the antibodies to pituitary carboxypeptidase E are frequent and may be a marker or a target for pituitary, adrenal and pancreatic autoimmunity since carboxypeptidase E is present in all those endocrine glands. But this need to be investigate further. References 1. Bednarek J, Furmaniak J, Wedlock N, Kiso Y et al. : Steroid 21-hydroxylase is a major autoantigen involved in adult onset autoimmune Addison s disease. FEBS 1992; 309: Baumann Antczak A, Kosowicz J: Metoda wykrywania autoprzeciwciał antytubulinowych. Wyniki oznaczeń w autoimmunologicznych chorobach tarczycy. Endokrynol Pol 1992; 43, 4: Gryczyńska M, Kosowicz J: Radioimmunologiczne oznaczanie autoprzeciwciał przysadkowych u chorych z chorobą Addisona. Endokr Pol 1991; 43: Kosowicz J, Gryczyńska M, Botazzo G.F: A radioimmunoassay for the detection of adrenal autoantibodies. Clin Exp Immunol 1986; 63: Fricker LD, Snyder SH: Purification and characterization of enkephalin convertase an enkephalin synthesizing carboxypeptidase. The Journal of Biological Chemistry 1983; 258, 18: Rodriguez C, Brayton K A, Brownstein M: Rat preprocarboxypeptidase H. Cloning, characterization and for the cdna and regulation of mrna by corticotropin-releasing factor. J Biol Chem 1989; 264, Davidson H, Hutton J: The insulin secretory granule carboxypeptidase H. Biochem J 1987; 245: Manser E, Fernandez D, Loo L: Human carboxypeptidase E. Isolation and characterization of cdna, sequence conservation, expression and processing in vitro. Biochem J 1990; 267: Bunnet NW: Isolation of neuropeptide degrading carboxypeptidase from human stomach. Gastroenetrology 1992; 102 (1): Reznik SE, Salafia CM, Fricker LD: Immunohistochemical localization of carboxypeptidase E and D in human placenta and umbilical cord. J Histochem Cytochem 1998; 46: Castano E, Zhou L, Lipes MA, Eisenbarth GS: Identification and cloning of granule autoantigen (carboxypeptidase H) associated with type 1 diabetes. J Clin Endocrinol 1991; 73: Alcalde L, Tonacchera M, Costagliola S: Cloning of candidate autoantigen carboxypeptidase H from a human islet library: sequence identity with human brain CPH. J Autoimmunity 1996; 9: Suppattapone S, Fricker LD, Snyder SH: Purification and characterization of membrane bound enkephalin synthesizing carboxypeptidase, enkephalin convertase. J Neurochem 1984; 42: Botazzo GF, Florin-Christensen A, Doniach D: Islet cell antibodies in diabetes mellitus with autoimmune polyendocrine deficiency. Lancet 1974, Nov 30; 2 (7892): Gryczyńska M, Majkowska L, Czekalski S: Islet cell antibodies and complement fixing cell antibodies in blood serum of patients with chronic adrenal insufficiency (Addison s disease). Endokr Pol 1989; 40: Lowdell M, Botazzo GF: Autoimmnuity and insulin dependent diabetes. Lancet 1993, May 29; 341 (8857): Winquist O, Karlsson FA, Kampe O: 21-hydroxylase, a major autoantigen in idiopathic Addison s disease. Lancet 1992; 339: Parkinson D: Two soluble forms of bovine carboxypeptidase H have different NH 2 -terminal sequences. J Biol Chem 1990; 265: Genovese S, Bonifacio E, McNally JM, Dean BM et al..: Distinct cytoplasmic islet cell antibodies with different risks for Type 1 (insulindependent) diabetes mellitus. Diabetologia 1992; 35: Baumann-Antczak A, Wedlock N, Bednarek J, Kiso Y et al.: Autoimmune Addison s disease and 21-hydroxylase. Lancet 1992; 340, Colls J, Betterle C, Volpato M, Prentice L et al.: Immunoprecipitation assay for autoantibodies to steroid 21-hydroxylase in autoimmune adrenal diseases. Clin Chem 1995; 41: Betterle C, Volpato M, Pedini B, Chen S et al.: Adrenal-cortex autoantibodies and steroid-producing cells autoantibodies in patients with Addison s disease: comparison of immunofluorescence and immunoprecipitation assays. J Clin Endocrinol Metab 1999; 84, 2: Chen S, Sawicka J, Betterle C, Powell M et al.: Autoantibodies to steroidogenic enzymes in autoimmune polyglandular syndrome, Addison s disease, and premature ovarian failure. J Clin Endocrinol Metab 1996; 5, Falorini A, Laureti S, Nikoshkov A, Picchio ML et al. 21-hydroxylase autoantibodies in adult patients with endocrine autoimmune diseases are highly for Addison s disease. Clin Exp Immunol 1997; 107, 2: Seissler J, Schott M, Steinbrenner H, Peterson P et al.: Autoantibodies to adrenal cytochrome P450 antigen in isolated Addison s disease and autoimmune polyglandular syndrome type II. Exp Clin Endocrinol Diabetes 1999; 107, 3: Chen S, Sawicka J, Prentice L, Sanders J. F et al. Analysis of autoantibody epitopes on steroid 21-hydroxylase using a panel of monoclonal antibodies. J Clin Endocrinol Metab 1998; 83, 8: Tanaka H, Asawa T, Powell M, Chen S et al.: Autoantibody binding to steroid 21-hydroxylase effect of five mutations. Autoimmunity 1997; 26, 4: Volpato M, Prentice L, Chen S, Betterle C et al.: A study of epitopes on steroid 21-hydroxylase recognized by autoantibodies in patients with or without Addison s disease. Clin Exp Immunol 1998; 111, 2: Zellissen PM, Bast EJ, Croughs RJ: Associated autoimmunity in Addison s disease. J Autoimmun 1995; 8, 1: Sodebergh A, Winqvist O, Norheim I, Rorsman F et al. Adrenal autoantibodies and organ-specific autoimmunity in patients with Addison s disease. Clin Endocrinol 1996; 45, 4: Valentino R, Savastano S, Tommaselli AP, Dorato M et al. Unusual association of thyroiditis, Addison s disease, ovarian failure and celiac disease in a young woman. J Endocrinol Invest 1999; 22, 5: De Luis DA, Varela C, de La Calle H, Canton R et al. Helicobacter pylori infection is markedly increased in patients with autoimmune atrophic gastritis. J Clin Gastroenterology 1998; 26, 4: De Carmo Silva R, Kater CE, Dib SA, Lauretti S et al.: Autoantibodies against recombinant human steroidogenic enzymes 21-hydroxylase, side-chain cleavage and 17 alpha-hydroxylase in Addison s disease and autoimmune polyendocrine syndrome type III. Eur J Endocrinol 2000; 142, 2: Papadopoulos KI, Hornblad Y, Liljebladh H, Hallengren B: High frequency of endocrine autoimmunity in patients with sarcoidosis. Eur J Endocrinol 1996; 134, 3: Sodebergh A, Rorsman F, Halonen M, Ekwall O et al.: Autoantibodies against aromatic L-amino acid decarboxylase identifies a subgroup of patients with Addison s disease. J Clin Endocrinol Metab 2000; 85, 1: Work was supported by State Committee for Scientific Research Grant KBN 4PO5B

6 / ORIGINAL PAPERS Endokrynologia Polska / Polish Journal of Endocrinology Tom/Volume 54; Numer/Number 6/2003 ISSN X Badanie kolonoskopowe u pacjentów z akromegalią. Nie ma dowodów na zwiększoną częstość występowania zmian złośliwych lub złośliwiejących śluzówki jelita grubego w akromegalii. Marek Bolanowski 1, Wojciech Kielan 2, Marta Rzeszutko 3, Jacek Daroszewski 1, Katarzyna Zatońska 1, Robert Tarnawa 2 1 Katedra i Klinika Endokrynologii i Diabetologii, A.M. we Wrocławiu 2 Katedra i Klinika Chirurgii Ogólnej i Onkologicznej A.M. we Wrocławiu 3 Zakład Anatomii Patologicznej A.M. we Wrocławiu Streszczenie Wstęp: U chorych na akromegalię sugerowane jest częstsze występowanie rozrostu śluzówki i polipów jelita grubego. Stwierdzano zależność zwiększonego wydzielania GH i IGF-I w akromegalii ze skłonnością do zmian nowotworowych. Celem pracy była ocena zagrożenia transformacją nowotworową w błonie śluzowej jelita grubego chorych na akromegalię zależnie od wieku, czasu trwania i aktywności choroby. Materiał i metoda: Kolonoskopię wykonano u 20 kolejnych chorych na akromegalię (15 kobiet, 5 mężczyzn) w wieku lat. W przypadku stwierdzenia patologicznego obrazu pobierano materiał do badania histologicznego. Wyniki: Obecność łagodnych polipów zapalnych wykazano u 2 mężczyzn z aktywną akromegalią, oporną na leczenie. U 3 kobiet występował niespecyficzny stan zapalny jelita grubego. Uchyłkowatość jelita grubego stwierdzono u mężczyzny z nawrotem choroby w 10 lat po skutecznym leczeniu operacyjnym. Miejscowy przerost komórek neuroendokrynnych obserwowano u pacjentki z akromegalią spowodowaną ektopowym wydzielaniem GHRH przez dużego rakowiaka oskrzela, przed jego operacyjnym leczeniem. U pozostałych 13 chorych nie stwierdzono patologicznych zmian w kolonoskopii. Wnioski: Obserwowane wyniki wskazują na częstą obecność zmian zapalnych jelita grubego u chorych na akromegalię. Nie możemy potwierdzić częstszego występowania gruczolakowych zmian nowotworowych ani przednowotworowych w błonie śluzowej jelita grubego chorych na akromegalię obserwowanego przez innych autorów. Słowa kluczowe: akromegalia, kolonoskopia, nowotwory, polipy jelita grubego Dr hab. n. med. M. Bolanowski, Katedra i Klinika Endokrynologii i Diabetologii, A.M. we Wrocławiu, Pasteura 4, Wrocław, tel/fax: (71) , bolan@endo.am.wroc.pl 757

7 / ORIGINAL PAPERS Endokrynologia Polska / Polish Journal of Endocrinology Tom/Volume 54; Numer/Number 6/2003 ISSN X Colonoscopic findings in patients with acromegaly. No evidence for increased prevalence of malignant or premalignant lesions in colon mucosa in acromegaly. Marek Bolanowski 1, Wojciech Kielan 2, Marta Rzeszutko 3, Jacek Daroszewski 1, Katarzyna Zatońska 1, Robert Tarnawa 2 Departments of 1 Endocrinology and Diabetology, 2 2nd General and Oncological Surgery, and 3 Pathological Anatomy, Wrocław Medical University, Wrocław, Poland Summary Introduction: Acromegaly seems to be associated with an increased mucosal hypertrophy and higher incidence of colonic polyps. There is a relation between GH and IGF-I overproduction in acromegaly and a tendency to malignancy. The aim of the study was to assess the risk of neoplastic colon mucosa transformation in patients with acromegaly according to age, duration and activity of the disease. Material and methods: Colonoscopy was performed in 20 consecutive patients with acromegaly (15 women and 5 men) in age years. If the pathological lesion was shown biopsy specimens were taken from it for the routine histological evaluation. B Benign, inflammatory polyps were found in 2 men with very active, treatment resistant disease. In 3 women nonspecific colitis was observed. Colonic diverticula were detected in man with recurrence of the disease 10 years after successful tumor surgery. A focal hyperplasia of neuroendocrine cells was shown in female patient presenting with acromegaly due to ectopic GHRH production in the course of large bronchial carcinoid prior to its surgical treatment. In the remaining 13 patients no pathological findings in colonoscopy were observed. Conclusions: Our results show frequent presence of inflammatory colonic changes in acromegaly. We can not support the evidence for increased prevalence of malignant or premalignant adenomatous lesions in colon mucosa in acromegaly shown in other studies. Key words: acromegaly, colonoscopy, neoplasia, colonic polyps M. Bolanowski, M.D., Ph.D., Department of Endocrinology and Diabetology, Wrocław Medical University, Pasteura 4, Wrocław, Poland, phone/fax: (71) , bolan@endo.am.wroc.pl Introduction Acromegaly, caused by a hypersecretion of growth hormone (GH) from anterior pituitary, is an uncommon disease associated with reduced life expectancy due to cardiovascular, cerebrovascular and respiratory diseases [1-3]. Other important cause of premature mortality seems to be increased incidence of malignancy including gastrointestinal tract, breast and prostate [4-9]. There is a relation between GH and IGF-I overproduction in acromegaly and a tendency to malignancy [10-11]. Most involved organ has been colon with an increased mucosal hypertrophy and higher incidence of adenomatous polyps [12-15]. This has prompted the suggestions of routinely performed repeated colonoscopy in patients with acromegaly to early detect premalignant changes [15-17]. On the contrary, increased prevalence of colonic polyps is not commonly accepted, other studies did not report their increased incidence in acromegalic patients in comparison to the general population [18, 19]. The influence of other factors i.e. dietary ones is also taken under the consideration [20]. Since the terminal part of colon is a localization with highest risk we performed current study in order to assess the incidence of neoplastic mucosa transformation within colon in acromegalic patients with respect to age, duration and activity of the disease. Materials and methods Colonoscopy was carried out in 20 consecutive patients with acromegaly (15 women, 5 men) in age years. The clinical data of the patients are shown in Table I. They had various status of the disease: 14 with active disease, out of them 4 before any treatment, 9 after failed surgery, and 1 presenting with the recurrence 10 years after successful surgery. Among 6 cured patients, 4 were cured by surgery, and 2 other by surgery followed by radiotherapy. Medical therapy was not given in patients studied. None had a history of previous neoplasm, no positive family history of colorectal cancer was known. 758

8 Endokrynologia Polska / Polish Journal of Endocrinology 2003; 6 (54) The diagnosis of acromegaly was established on the basis of typical clinical features, elevated serum GH level, not suppressed after oral glucose load, and elevated serum IGF-I. Cured patients had basal GH values less than 2.5 ng/ml. The duration of the disease was estimated by anamnesis when patients recalled certain signs and symptoms of the disease. Hormonal analyses were carried out using commercial kits, GH by RIA (Diagnostic Product Corporation, Los Angeles, USA; normal range ng/ml), IGF-I by extraction using RIA method (Nichols Institute Diagnostics, San Juan Capistrano, USA; normal range, ng/ml, dependent on age). Flexible colonoscopies were performed by 2 experienced endoscopists using the endoscope Pentax EC3800PL (120 cm in length) at the Endoscopy Unit of the 2 nd Dept. of General and Oncological Surgery. The endoscope was introduced under visual control through the anus and rectum, the sigmoid, transverse and ascendenting colon as far as to the caecum, when possible. The caecum was reached in all but 3 patients (No 2, 6, 7). If a pathological finding was shown, biopsy specimens were taken from it for the histological evaluation. The material from the mucosa excision was then examined by routine histopathological procedure at the Dept. of Pathological Anatomy. All patients gave informed consent for the endoscopic examination. Results No adenomatous polyp was shown in colonoscopy in our series of patients with acromegaly. Benign, inflammatory polyps were found in 2 men (No 4 and 11) with long-lasting, treatment resistant disease (Figure 1). In 3 women (No 2, 7, 9) nonspecific colitis Figure 1. Inflammatory colonic polyp in 35-yr. old male patient with active acromegaly. Mesenchymal lesions with an inflammatory infiltrate and vascular component dominating in mucosa (HE x40). was observed. Colonic diverticula were detected in 53-yr. old men with recurrence of the disease 10 yr. after previous successful tumor surgery (No 20). A focal hyperplasia of neuroendocrine cells was shown in 62-yr. old female patient (No 3) presenting with acromegaly due to ectopic GHRH production in the course of large bronchial carcinoid prior to its surgical treatment. In the remaining 13 patients no pathological findings in colonoscopy were observed. There were no endoscopy-associated complications. The results of colonoscopy and histological analysis are shown in Table I. Discussion At present, an increased prevalence of colonic polyps in patients with acromegaly is not commonly accepted [18, 19, 21]. Our results are in agreement with observation of above-mentioned authors. On the other hand, increased prevalence of malignant or premalignant changes in colon mucosa in acromegaly has been shown in other studies. The most common lesions are colon adenomas detected in 20-38%, hyperplastic polyps in 21-24% and carcinoma in % of patients [12-15, 17, 21, 22]. The neoplasms arose uniformly throughout the large bowel with the same number registered in proximal and distal colon [4]. The data from literature suggest coincidence of colon polyps with age over 50 yr., duration of the disease more than 10 yr., and the presence of more than 3 skin tags (acrochordons) [6, 14, 22]. It is of great importance, since malignancy attributed for about 15% of deaths in acromegaly [1, 2, 4, 21]. Nevertheless, circulating GH levels seem to be most important single determinant of the survival, regardless the cause of death, and the risk of colon polyps depends on the activity of poorly controlled disease [4, 21]. In our series colonic polyps were observed only in 2 male patients (35 and 50-yr.), both of them with long duration (13 and 9 yr., respectively) and therapy resistant disease (non-cured following 3 surgical attempts, and cured by 3 rd surgery, respectively). Both had high GH and IGF-I levels at the time of diagnosis of the disease. The lesions mentioned above were inflammatory polyps, without histological signs of adenomatous or hyperplastic structure. Inflammatory polyps (pseudopolyps) have no malignant potential, they account for 5% of colonic lesions, and they are reported mainly in association with ulcerative colitis [23]. Pseudopolyps and colitis found in 5 of our patients (25%) suggest relative high incidence of non-specific inflammatory colonic lesions. The incidence of inflammatory polyps in general population is difficult to estimate, since no epidemiological data existing. The prevalence of ulcerative colitis in USA is and of Crohn disease is per population, respectively [24]. 759

9 Kolonoskopia u pacjentów z akromegalią Bolanowski M. Table I. The data of patients with acromegaly and results of the colonoscopy. Patient sex age duration of therapy basal IGF-I result (yrs) of active GH (ng/ml) of disease (yrs) (ng/ml) endoscopy 1. IL f normal 2. GP f colitis 3. IS f NE cells 4. RZ m polyps 5. KC f normal 6. MA f normal 7. JR f colitis 8. JM f normal 9. ZB f colitis 10. WN f normal 11. JR m polyps 12. WK m normal 13. AK f normal 14. MC f normal 15. HR f normal 16. HA f normal 17. AW f normal 18. AK f normal 19. KU m normal 20. HD m diverticula f female, m male, therapy: 0 none (before any therapy), 1 surgery, 2 radiotherapy, NE cells neuroendocrine cells Ladas et al. observed low prevalence of colonic neoplasia, higher in elderly patients, but without correlation with activity and duration of the disease. They did not show an increased prevalence of gastric tumors in acromegaly [18]. Similarly, Renehan et al. failed to demonstrate an increased prevalence of neoplasia in patients with acromegaly compared with the expected prevalence in the general population [19]. Vasen at al. did not find any correlation between the duration of acromegaly, age of the patients and the presence of colonic adenomas. But, acromegalics with adenomas often had more active disease, although the differences were not statistically significant [13]. We observed one case of focal hyperplasia of neuroendocrine mucosa cells, but with negative staining for Neurone Specific Enolase and Chromogranin A. Further diagnostic procedure in this patient revealed bronchial carcinoid with ectopic GHRH secretion underlying signs and symptoms of acromegaly. Subsequent carcinoid surgery led to complete clinical and hormonal cure of this patient [25]. Ectopic GHRH secretion accounts for less than 1% of all cases of acromegaly, and metastasizing to other tissues, including gastrointestinal tract, is possible [26]. The incidence of lesions in our series of patients was greater among them over 50 yr. of age. Since the delay in the diagnosis of acromegaly is usually 10 years, this indicates the significant impact both of age of the patients and duration of the disease. We could not perform gender predisposition analysis, since small number of male patients in our series. Increased epithelial cell proliferation was shown in other study when biopsies from macroscopically normal-appearing mucosa in the midsigmoid from acromegalic patients with active disease were taken. It was absent in cases of patients with non-active disease [27]. This is, most probably, due to direct stimulatory effect of IGF-I, which contributes to the increased risk of colon neoplasms in acromegaly [4, 27]. Another recent study carried out in healthy volunteers indicates the importance of circulating IGF-II level in colorectal adenomas. The presence of adenoma was associated with elevated serum IGF-II, IGFBP-2 levels, obesity and hormonal replacement therapy. Additionally, adenomectomy was following in significant fall in mean IGF-II. Immunohistochemistry demonstrated IGF-II expression in almost all adenomas, in contrast to its absence in hyperplastic polyps and normal colon epithelium [28]. The lack of association between IGF-I and/ or IGFBP-3 and the occurrence of adenomas was another important finding of this study. The incidence of colon pathology in our, limited group of patients is less than in other studies reported [12-15, 17, 21]. This implicated us to the continuation of the study in more numerous group of patients. The influence of other factors, dietary, genetical or hormonal ones could be supposed also [20, 28, 29]. Nevertheless, the patients with 760

10 Endokrynologia Polska / Polish Journal of Endocrinology 2003; 6 (54) acromegaly require endoscopic examinations, since increased with age prevalence of lesions. Regular colonoscopy screening and polypectomy can reduce the incidence of subsequent carcinoma in these patients. The demonstration of a clear relation to serum GH and IGF-I levels is further indication for more aggressive treatment of the underlying acromegaly [22, 30]. Summary and conclusions We observed inflammatory polyps (pseudopolyps) in 10% and colitis in 15% of studied patients with acromegaly. The prevalnce of inflammatory polyps, as signs of colitis, is much less in general population. Since no adenomatous polyp was shown in our series, we can not support the evidence for increased prevalence of malignant or premalignant lesions in colon mucosa in acromegaly shown in other studies. References: 1. Bengtsson BA, Eden S, Ernest I et al. Epidemiology and longterm survival in acromegaly. A study of 166 cases diagnosed between 1955 and Acta Med Scand 1988; 223: Nabarro JDN. Acromegaly. Clin Endocrinol 1987; 26: Rajasoorya C, Holdaway IM, Wrightson P et al. Determinants of clinical outcome and survival in acromegaly. Clin Endocrinol 1994; 41: Orme SM, Mcnally RJQ, Cartwright RA, Belchetz PE. Mortality and cancer incidence in acromegaly: A retrospective cohort study. J Clin Endocrinol Metab 1998; 83: Colao A, Marzullo P, Spiezia S, Lombardi G. Acromegaly and prostate cancer. Growth Hormone & IGF Res 2000; Suppl A, S37-S Ezzat S, Melmed S. Are patients with acromegaly at increased risk for neoplasia? J Clin Endocrinol Metab 1991; 72: Cheung NW, Boyages SC. Increased incidence of neoplasia in females with acromegaly. Clin Endocrinol 1997; 47: Ron E, Gridley G, Hrubec Z, et al. Acromegaly and gastrointestinal cancer. Cancer 1991; 68: Bolanowski M, Zatońska K. Występowanie innych nowotworów u chorych na akromegalię. Adv Clin Exp Med 2002; 11: Holly JMP, Gunnell DJ, Davey Smith G. Growth hormone, IGF-I and cancer. Less intervention to avoid cancer? More intervention to prevent cancer? J Endocrinol 1999; 162: Khandwala HM, McCutcheon IE, Flyvbjerg A, Friend KE. The effects of insulin-like growth factors on tumorigenesis and neoplastic growth. Endocr Rev 2000; 21: Terzolo M, Tappero G, Borretta G et al. High prevalence of colonic polyps in patients with acromegaly. Influence of sex and age. Arch Intern Med 1994; 154: Vasen HFA, van Erpecum KJ, Roelfsema F et al. Increased prevalence of colonic adenomas in patients with acromegaly. Eur J Endocrinol 1994; 131: Colao A, Balzano A, Ferone D, et al. Increased prevalence of colonic polyps and altered subset pattern in the colonic lamina propria in acromegaly. Clin Endocrinol 1997; 47: Delhougne B, Deneux C, Abs R, et al. The prevalence of colonic polyps in acromegaly: A colonoscopic and pathological study in 103 patients. J Clin Endocrinol Metab 1995; 80: Freda PU. Advances in the diagnosis of acromegaly. The Endocrinologist 2000; 10: Jenkins PJ, Frajese V, Jones A-M et al. Insulin-like growth factor I and the development of colorectal neoplasia in acromegaly. J Clin Endocrinol Metab 2000; 85: Ladas SD, Thalassinos NC, Ioannides G, Raptis SA. Does acromegaly really predispose to an increased prevalence of gastrointestinal tumours? Clin Endocrinol 1994; 41: Renehan AG, Bhaskar P, Painter JE et al. The prevalence and characteristics of colorectal neoplasia in acromegaly. J Clin Endocrinol Metab 2000; 85: Giovannucci E, Willett WC. Dietary factors and risk of colon cancer. Ann Med 1994; 26: Melmed S. Acromegaly and cancer: Not a problem? J Clin Endocrinol Metab 2001; 86: Jenkins PJ, Besser M. Acromegaly and cancer: A problem. J Clin Endocrinol Metab 2001; 86: Welch CE, Hedberg SE. Other polypoid lesions of the colon and rectum. In: Polypoid lesions of the gastrointestinal tract. Second Edition, WB Saunders Co, 1975: Pascal RR, Fenoglio-Preiser CH, Noffsinger AE. Neoplastic diseases of the small and large intestine. In: Principles and practice of surgical pathology and cytopathology. Silverberg SG, Churchill Livingstone, New York, Bolanowski M, Schopohl J, Marciniak M et al. Acromegaly due to GHRH-secreting large bronchial carcinoid. Complete recovery following tumor surgery. Exp Clin Endocrinol Diabetes 2002; 110: Losa M, von Werder K. Pathophysiology and clinical aspects of the ectopic GH-releasing hormone secretion. Clin Endocrinol 1997; 47: Cats A, Dullaart R, Kleibeuker JH et al. Increased epithelial cell proliferation in the colon of patients with acromegaly. Cancer Res 1996; 56: Renehan AG, Painter JE, O Halloran D et al. Circulating insulin-like growth factor II and colorectal adenomas. J Clin Endocrinol Metab 2000; 85: Willett WC, Stampfer MJ, Colditz GA et al. Relation of meat, fat, and fiber intake to the risk of colon cancer in a prospective study among women. N Engl J Med 1990; 323: Bates AS, Van t Hoff W, Jones JM, Clayton RN. Does treatment of acromegaly affect life expectancy? Metabolism 1995; 44: (suppl.1)

11 / ORIGINAL PAPERS Endokrynologia Polska / Polish Journal of Endocrinology Tom/Volume 54; Numer/Number 6/2003 ISSN X Leptyna u mężczyzn z chorobą niedokrwienną serca. Lucyna Siemińska, Celina Wojciechowska 1, Ewa Nowalany-Kozielska 1, Bogdan Marek, Beata Kos-Kudła, Mariusz Nowak, Wanda Foltyn, Dariusz Kajdaniuk, Anna Borowska-Kozler 2 Katedra Patofizjologii i Endokrynologii Śląskiej Akademii Medycznej 1 II Katedra i Klinika Kardiologii Śląskiej Akademii Medycznej 2 Oddział Chorób Wewnętrznycyh, Szpital im. Dr J. Rostka, Chorzów Streszczenie W badaniach eksperymentalnych wykazano, że leptyna produkowana przez adipocyty odgrywa rolę w rozwoju miażdżycy. W obecnej pracy oceniano powiązania pomiędzy leptyną a rozwojem zmian miażdżycowych w tętnicach wieńcowych. W tym celu przebadano 132 mężczyzn z udokumentowaną koronarograficznie chorobą wieńcową. 23 mężczyzn z prawidłowym angiogramem wieńcowym stanowiło grupę kontrolną. Analizowano powiązania między stężeniem leptyny w surowicy a czynnikami ryzyka choroby niedokrwiennej serca. Stężenia leptyny były istotnie wyższe u mężczyzn chorobą niedokrwienną serca niż u mężczyzn z grupy kontrolnej. Jednakże mężczyźni z chorobą wieńcową mieli wyższy wskaźnik masy ciała i po uwzględnieniu tego czynnika różnice nie były już istotne. Stężenie leptyny korelowało dodatnio z 3 czynnikami ryzyka choroby wieńcowej: wskaźnikiem masy ciała, nadciśnieniem tętniczym i stężeniem cholesterolu. Po przeprowadzeniu analizy wieloczynnikowej okazało się, że jedynie wskaźnik masy ciała i obecność nadciśnienia wiążą się istotnie ze stężeniem leptyny. Pomimo wykazania związków pomiędzy podwyższonymi stężeniami leptyny i czynnikami ryzyka choroby wieńcowej rezultaty obecnej pracy nie potwierdzają bezpośredniego wpływu leptyny na rozwój miażdżycy w tętnicach wieńcowych. Słowa kluczowe: leptyna, miażdżyca, choroba wieńcowa Leptin in men with coronary artery disease Lucyna Siemińska, Celina Wojciechowska 1, Ewa Nowalany-Kozielska 1, Bogdan Marek, Beata Kos-Kudła, Mariusz Nowak, Wanda Foltyn, Dariusz Kajdaniuk, Anna Borowska-Kozler 2 Department of Pathophysiology and Endocrinology 1 II Department of Cardiology Silesian Medical University, Zabrze, Poland 2 Internal Diseases Department, The Rostka Hospital, Chorzów Abstract Experimental studies suggest that leptin produced by adipocytes plays a role in the etiology of atherosclerosis. In this study we examined whether serum level of leptin is associated with coronary atherosclerosis assessed by coronary angiography. We studied a total of 132 male patients with angiographically documented coronary atherosclerosis. As a control group, 23 men with normal coronary angiogram were included. We also explored the associations between circulating serum leptin and risk factors of coronary artery disease. Leptin levels were higher in men with coronary artery disease than in control men. However, subjects with coronary heart disease had a larger body mass index, and when subjects were matched for body mass index, the differences were not significant. Leptin correlated positively with three independent coronary artery heart disease risk factors: body mass index, hypertension and cholesterol. In multivariate analysis only body mass index and hypertension significantly related to serum leptin concentration. Although the elevated levels of leptin are associated with the factors of increased risk of atherosclerosis the results of this study do not support the direct role of leptin in coronary artery disease. Key words: leptin, atherosclerosis, coronary artery disease. Department of Pathophysiology and Endocrinology Silesian Medical University Zabrze, pl. Traugutta 2, Poland siem@pik-net.pl 762

12 Endokrynologia Polska / Polish Journal of Endocrinology 2003; 6 (54) Introduction In recent years it has become evident that adipose tissue is a secretory organ, producing a variety of bioactive substances such as: plasminogen activator inhibitor type 1 (PAI-1), tumor necrosis factor- (TNF- ), interleukin-6 (IL-6), adiponectin, angiotensinogen and leptin. These molecules, called adipocytokines, have both paracrine and endocrine effects. It has been shown that all adipocytokines are involved in the pathogenesis of complications of obesity such as atherosclerosis, hypertension and insulin resistance [1]. Among them PAI-1 is a regulator of the fibrinolytic system and plays some role in the thrombosis, acute occlusion and acceleration of atherosclerosis. Increased serum PAI-1 level is an independent risk factor for cardiovascular events [2]. TNF- is produced in adipose tissue and increased levels in obese people may injure endothelial cells contributing to the development of atherosclerosis. Ridker et al. have shown that elevations of TNF- in the stable phase after myocardial infarction were associated with an increased risk of recurrent coronary events [3]. Adiponectin is secreted only from adipose tissue and has anti-inflammatory effects on the vascular wall. It inhibits monocyte adhesion to endothelial cells, suppresses macrophage-to-foam-cell transformation and attenuates proliferation of smooth muscle cells. Its serum level is significantly lower in subjects with coronary artery disease so adiponectin has antiatherogenic properties [4]. Adipose tissue is an important source of angiotensinogen, which is contributing to hypertension, commonly associated with obesity. Leptin is one of a number of proteins secreted from adipocytes. High concentrations of leptin are associated with such risk factors for cardiovascular disease as insulin resistance and obesity. Leptin is also proposed to be associated with the development of essential hypertension [5]. Nevertheless, there is little evidence about the potential role of leptin in the development of atherosclerosis. Leptin deficient mice (ob/ob) on atherogenic diet for 4 months failed to develop atherosclerosis despite occurrence of obesity and hypercholesterolemia [6]. These experimental studies suggest that leptin may be one of the mediators promoting atherosclerosis but exact mechanism is not fully elucidated. In the present study we hypothesize that leptinemia may be related to atherosclerosis in coronary arteries. The objectives of the work were to estimate the concentrations of serum leptin in men with angiographically proven ischemic heart disease and compare them with the concentrations in control group. Furthermore, the relationships of serum leptin and such of cardiovascular risk factors as: hypertension, body mass, age and lipid profile were investigated. Material and methods The tests comprised 132 men with angiographically diagnosed coronary artery disease (CAD). All of the patients underwent coronarography because of chest pain syndromes or abnormal stress test. Eighty five men had recognized arterial hypertension. Hypertension was defined as systolic blood pressure 140 mmhg and/or diastolic blood pressure 90 mmhg or the recognition was based on reported use of antihypertensive medication before the admission to the hospital. The control group consisted of 23 healthy men without hypertension in whom coronarography excluded atherosclerotic changes in coronary arteries. In these cases the coronarography was performed to search for the cause of atypical chest pain or unexplained arrhythmias or was carried out before cardiosurgical operations (for congenital anomalies or valvular heart disease). All subjects had blood samples taken between 7.00 AM and 8.00 AM and the body mass index (BMI) was calculated for each patient. Samples of serum were frozen at 80 o C until assay. Leptin concentrations were measured with a commercial RIA kit (Linco Research, Inc.). Total cholesterol (chol), HDL-cholesterol (chdl) and triglycerides (TG) were measured according to standard enzymatic methods. LDL-cholesterol (cldl) was calculated using the Friedwald formula. The results are presented as means ±SD. The significance of differences between mean values was estimated by the Student s test or by the U Mann-Whitney test. It was assumed that the difference is essential at p<0.05. The relations between particular parameters were estimated by calculating the correlation coefficient r by Pearson s or Spearman s methods. Results Table 1 shows the baseline characteristics and laboratory findings for men with CAD and control group. The cases had higher levels of chol, TG, cldl and lower levels of chdl. The mean leptin concentration in men with CAD was 8.2 ng/ml (SD=5.3) and ranged from 1 41 ng/ml. The leptin serum levels were higher and statistically significant in men with CAD than they were in control men (5.2 ng/ml, SD=1.7). However, subjects with CAD had a slightly larger BMI, and when subjects were matched for BMI, the differences were not significant. When the male subjects with CAD were subdivided into 2 groups, those with hypertension (n=85) and without hypertension (n=47), the serum leptin was higher in CAD men with hypertension 763

13 Leptyna u mężczyzn z chorobą niedokrwienną serca Siemińska L. compared with the CAD normotensive men (8.9±5.6 vs. 7.1±4.2 ng/ml) and higher than the control group (5.2±1.7 ng/ml), Fig. 1. The differences were significant (p<0.05 and p<0.01). Relations of serum leptin with BMI and cardiovascular risk factors. Next we analyzed separately the linear correlations between serum levels of leptin and other parameters: BMI, lipids, age in the overall sample and in the CAD group. Correlations results are given in Table 2. Leptin was significantly correlated with BMI (r=0.65, p<0.001) and chol (r=0.28, p<0.01) in the overall sample. We found no evidence of association between leptin levels and chdl, although weak correlation was observed between leptin and cldl (r=0.10, p=0.05). The relationships between serum leptin and BMI and serum leptin and chol were reproducible in men with CAD and in the subgroup of men with CAD and hypertension. No significant correlation was found between the serum levels of leptin and age. The population of men with CAD was then categorized in quartiles based on the distribution of serum leptin levels. We compared the group of men with the highest quartiles of serum leptin ( 10.6 ng/ml, n=39) with group with the lowest quartiles of serum leptin ( 4.7 ng/ml, n=35). We observed a greater prevalence of hypertension in men with the highest levels of leptin compared with men within the lowest quartile (65% vs. 25%). Variables that were found to be correlated with serum leptin levels on univariate analyses were used in multiple linear regression analysis. After the analysis, where leptin was a dependent variable it was found that BMI remained significantly correlated with leptin. The presence of hypertension was also significantly associated with the increased serum level of leptin, independent of BMI (see Table 3). Table 1. Clinical and metabolic characteristics of the CAD and the control subjects parameter CAD control p number Age (years) 56 ± 7 51 ± 11 ns BMI 27 ± 3 26 ± Leptin (ng/ml) 8.2 ± ± Cholesterol (mg%) 225 ± ± cldl (mg%) 155 ± ± chdl (mg%) 34 ± 8 42 ± Triglyceride (mg%) 184 ± ± Table 2. Correlation of serum leptin with selected anthropometric and metabolic variables for all men and for CAD men parameter All subjects n=155 CAD men n=132 r p r p BMI 0.65 < <0.001 chol 0.28 < <0.01 chdl 0.01 ns 0.02 ns cldl ns TG 0.09 ns 0.10 ns age 0.03 ns 0.06 ns Table 3. Multiple linear regression analysis for serum leptin. Dependent variable: leptin Independent Variable Regression Coefficient SE of Regression Coefficient BMI kg/m <0.001 chol mg% CAD, yes/no ns hypertension, yes/no <0.01 p Fig. 1 7KHFRPSDULVRQRIVHUXPOHSWLQOHYHOEHWZHHQ&$'PHQZLWKK\SHUWHQVLRQDQG &$'PHQZLWKRXWK\SHUWHQVLRQDQGFRQWUROJURXS VHUXPOHSWLQQJPO S S &$'ZLWKK\SHUWHQVLRQ &$'ZLWKRXWK\SHUWHQVLRQ FRQWUROJURXS 764

14 Endokrynologia Polska / Polish Journal of Endocrinology 2003; 6 (54) Discussion Leptin is one of the adipocytokines produced by adipose tissue. It regulates body weight, food intake and energy expenditure, activity of the hypothalamic-pituitary-gonadal axis, sexual maturation and fertility, haematopoiesis, immune response. Leptin also affects glucose metabolism and insulin sensitivity and is relevant to cardiovascular function [7]. Recently, new peripheral roles for leptin have been identified and its receptors are widely expressed throughout the body. Leptin increases the accumulation of oxidant radicals in endothelial cells and thereby might play a role in the inflammation process and genesis of atherosclerosis [8]. Leptin also potentates the aggregation of platelets [9]. The prothrombotic effect of leptin is mediated through receptors which have been identified on the surface of endothelial cells and platelets [10]. O Rourke et al. have recently found that leptin effects the cholesterol accumulation in macrophages and regulates the formation of the foam cells [11]. Leptin enhances calcification of vascular cells [12] and, in concentrations observed in obese patients, induces proliferations and migration of smooth muscle cell [13]. Receptors are expressed in vascular endothelial [14] and smooth muscle cells [13]. There are differing opinions regarding the direct association between leptin and coronary artery disease. Recently, results from the West of Scotland Coronary Prevention Study (WOSCOPS) showed that high leptin level may identify men with increased risk of coronary event [15]. In the study of Stungl et al. [16] patients with documented coronary artery disease exhibit higher serum leptin levels than controls, indicating that this protein could contribute to the development of atherosclerotic changes in coronary arteries. However, Couillard et al. observed the lack of association between leptinemia and development of ischaemic heart disease in men during the 5-year observation [17]. In this study we do not confirm that leptin directly contributes to the coronary artery disease. However, a single hormone assay doesn t reflect the connections with endothelial dysfunction and cannot exclude the promoting role of leptin in atherogenesis. We have found that serum leptin concentrations are related to such risk factors of coronary artery disease as hypercholesterolemia and overweight. Furthermore, the subgroup of patients with CAD and hypertension had higher concentrations of serum leptin in comparison to the CAD group without hypertension. Our results are consistent with earlier studies. Leyva et al. [18] have shown the relationships between hyperleptinemia and these components of the metabolic syndrome which directly enhance the progression of atherosclerosis: obesity, increased blood pressure, insulin resistance, hyperinsulinemia and hipertriglyceridemia. Leptin plays a role in the development of hypertension via activation of the sympathetic nervous system and renin angiotensin system [19]. Besides, high levels of leptin contribute to insulin resistance via interaction with the insulin-signaling pathway [20]. Recently, it has been reported that high levels of leptin decreases the reactivity of arteries [21]. In our study a greater prevalence of hypertension was observed in CAD men with the highest concentration of leptin. The associations between leptin and lipids are controversial. A number of studies have reported that leptin has direct effects on lipids metabolism [22, 23] and other studies have found that leptin has no direct effects [24, 25]. In our study a positive correlation emerged between serum levels of leptin and total cholesterol. No other significant relationships between leptin and serum lipids were found. Conclusions Although the elevated serum leptin concentrations are associated with the occurrence of cardiovascular risk factors, leptin seems not to affect directly development of coronary artery disease in men. It is probable that leptin is only one of the many factors which have an impact on pathogenesis of atherosclerosis and hypertension. References: 1. Poirier P, Eckel RH. Obesity and cardiovascular disease. Curr Atheroscler Rep 2002, 4: Alessi MC, Peiretti F, Morange P, Henry M, Nalbone G, Juhan-Vague I. Production of plasminogen activator inhibitor-1 by human adipose tissue. Possible link between visceral fat accumulation and vascular disease. Diabetes 1997, 46: Ridker PM, Rifai N, Pfeffer M, Sacks F, Lepage S, Braunwald E. Elevation of tumor necrosis factor-??and increased risk of recurrent coronary events after myocardial infarction. Circulation 2000, 101: Ouchi N, Kihara S, Arita Y, Maeda K, Kuriyama H, Okamoto Y, Hotta K, Nishida M, Takahashi M, Nakamura T, Yamashita S, Funahashi T, Matsuzawa Y. Novel modulator for endothelial adhesion molecules: adipocyte-derived plasma protein adiponectin. Circulation 1999, 100: Agata J, Masuda A, Takada M, Higashiura K, Murakami H, Miyazaki Y, Shimamoto K. High plasma immunoreactive leptin level in essential hypertension. Am J Hypertens 1997, 10: Yen TT, Allan JA, Pearson DV, Schinitsky MR. Dissociation of obesity, hypercholesterolemia and diabetes from atherosclerosis in ob/ob mice. Experientia 1977, 33: Wauters M, Considine RV, Van Gaal LF. Human leptin: from an adipocyte hormone to an endocrine mediator. Eur J Endocrinol 2000, 143: Bouloumie A, Marumo T, Lafontan M, Busse R. Leptin induces oxidative stress in human endothelial stress. FASEB J 1999, 13: Maruyama I, Nakata M, Yamaji K. Effect of leptin in platelet and endothelial cells. Obesity and arterial thrombosis. Ann N Y Ac Sci 2000, 902: Konstantinides S, Schafer K, Loskutoff DJ. The prothrombotic effects of leptin possible implications for the risk of 765

15 Leptyna u mężczyzn z chorobą niedokrwienną serca Siemińska L. cardiovascular disease in obesity. Ann N Y Acad Sci 2001, 947: O`Rourke, Yeaman SJ, Shepherd PR. Insulin and leptin acutely regulate cholesterol ester metabolism in macrophages by novel signaling pathways. Diabetes 2001, 50: Parhami F, Tintut Y, Ballard A, Fogelman AM, Demer LL. Leptin enhances the calcification of vascular cells: artery wall as a target of leptin. Cir Res 2001, 88: Oda A, Taniguchi T, Yokoyama M. Leptin stimulates rat aortic smooth muscle cell proliferation and migration. Kobe J Med Sci. 2001, 47: Bouloumie A, Drexler HC, Lafontan M, Busse R. Leptin, the product of Ob gene, promotes angiogenesis. Circ Res 1998, 83: Wallace AM, McMahon AD, Packard CJ, Kelly A, Shepherd J, Gaw A, Sattar N. Plasma leptin and the risk pf cardiovascular disease in the west of scotland coronary prevention study (WOSCOPS). Circulation 2001, 104: Stangl K, Cascorbi I, Laule M, Stangl V, Vogt M, Ziemer S, Roots I, Wernecke K, Baumann G, Hauner H. Elevated serum leptin in patients with coronary artery disease: no association with the Trp64Arg polymorphism of the beta3-adrenergic receptor. Int J Obes Relat Metab Disord 2000, 24: Couillard C, Lamarche B, Mauriege P, Cantin B, Dagenais GR, Moorjani S, Lupien PJ, Despres JP. Leptinemia is not a risk factor for ischemic heart disease in men. Prospective results from the Quebec Cardiovascular Study. Diabetes Care 1998, 21: Leyva F, Godsland IF, Ghatei M, Proudler AJ, Aldis S, Walton C, Bloom S, Stevenson JC. Hyperleptinemia as a component of a metabolic syndrome of cardiovascular risk. Arterioscler Thromb Vasc Biol 1998, 18: Montani JP, Antic V, Yang Z, Dulloo A. Pathways from obesity to hypertension: from the perspective of a vicious triangle. Int J Obes Relat Metab Disord 2002 Suppl 2: S Kellerer M, Lammers R, Fritsche A, Strack V, Machicao F, Borboni P, Ullrich A, Haring HU. Insulin inhibits leptin receptor signalling in HEK293 cells at the level of janus kinase-2: a potential mechanism for hyperinsulinaemiaassociated leptin resistance. Diabetologia 2001, 44: Singhal A, Farooqi IS, Cole TJ, O Rahilly S, Fewtrell M, Kattenhorn M, Lucas A, Deanfield J. Influence of leptin on arterial distensibility: a novel link between obesity and cardiovascular disease? Circulation 2002, 106: Hynes GR, Jones PJ. Leptin and its role in lipid metabolism. Curr Opin Lipidol 2001, 12: Hasty AH, Shimano H, Osuga J, Namatame I, Takahashi A, Yahagi N, Perrey S, Iizuka Y, Tamura Y, Amemiya-Kudo M, Yoshikawa T, Okazaki H, Ohashi K, Harada K, Matsuzaka T, Sone H, Gotoda T, Nagai R, Ishibashi S, Yamada N. Severe hypercholesterolemia, hypertriglyceridemia, and atherosclerosis in mice lacking both leptin and the low density lipoprotein receptor. J Biol Chem 2001, 276: Haluzik M, Fiedler J, Nedvidkova J, Ceska R. Serum leptin levels in patients with hyperlipidemias. Nutrition 2000, 16: Marinari GM, Scopinaro N, Adami GF. Leptin and HDLcholesterol in non-diabetic normotensive subjects. Obes Surg 2001, 11:

16 / ORIGINAL PAPERS Endokrynologia Polska / Polish Journal of Endocrinology Tom/Volume 54; Numer/Number 6/2003 ISSN X Elevated concentrations of Insulin-like growth factor binding protein-2 in the serum of the adrenal tumours patients. Tomasz Stępień, Krzysztof Kołomecki, Krzysztof Kuzdak Dept of Endocrinological and General Surgery, Medical University, Lodz, Poland Abstract The IGFs system plays a key role in the hormonal regulation of growth, development and hormonal activity of adrenal gland. Various abnormalities of the IGF axis have been recently described in the pathomechanism of malignant transformation and tumourigenesis adrenal cortex and adrenal medulla. The aim of this study, was assessement the value of plasma IGF-II, IGFBP-2 and IGFBP-3 concentrations as a markers of malignancy in preoperative adrenal tumours and in the follow-up of patients, one month after surgical operation. Forthy nine patients (28 female, 21 male; mean age, 45.6 ±7.3 years) treated in our institutions were included in this study. Tumours were divided according to histopathological criteria into 4 groups : cortex carcinoma (n=14), benign epithelial adenoma (n=15), nonepithelial tumours (n=11) and pheochromocytoma (n=9). The control group was consisted of 10 healthy persons (6 male, 4 female: mean age 39.5 ± 2.3 years). Plasma IGF-2, IGFBP-2 and IGFBP-3 were determined by RIA or IRMA methods. The IGFBP-2 level before operation in groups of patients with cortex carcinoma ( ng/ml ± ; p<0.001), benign adenoma ( ng/ml ± ; p< 0.05) and pheochromocytoma (532.44ng/ml ± 86.90) was significantly higher in comparison to the control ( ng/ml ± 30.34). Surgical treatment patients with adrenocortical cancer decreased significantly the IGFBP-2 plasma level (p <0.05) but did not modyfied the peptide concentration in patients with adrenal adenoma, nonepithelial tumours and pheochromocytoma.there was no significant difference in plasma IGF-2 and IGFBP- 3 concentrations between healthy controls and all groups of patients with adrenal tumours before and one month after surgery. These results suggest that determination of IGFBP-2 plasma concentration can be used as an additional marker in the diagnosis and follow-up observation of patients with adrenocortical malignancy. Key words: adrenal tumours, IGF-2, IGFBP-2, IGFBP-3 Tomasz Stępień Dept. of Endocrinological and General Surgery, Medical University of Lodz Łódź ul.pabianicka

17 / ORIGINAL PAPERS Endokrynologia Polska / Polish Journal of Endocrinology Tom/Volume 54; Numer/Number 6/2003 ISSN X Podwyższone stężenia białka wiążącego insulinopodobne czynniki wzrostowe ( IGFBP-2 ) w surowicy krwi chorych z guzami nadnerczy. Tomasz Stępień, Krzysztof Kołomecki, Krzysztof Kuzdak Klinika Chirurgii Ogólnej i Endokrynologicznej Instytutu Endokrynologii Uniwersytetu Medycznego w Łodzi Streszczenie Układ insulinopodobnych czynników wzrostowych (IGF) odgrywa istotną rolę w regulacji wzrostu, różnicowania i czynności hormonalnej nadnerczy. Wykazano ostatnio, że zaburzenia w obrębie tego układu odgrywają istotną rolę w procesie transformacji nowotworowej i powstawaniu guzów wywodzących się z komórek warstwy korowej i rdzenia nadnerczy. Celem pracy była ocena pomiarów stężeń insulinopodobnego czynnika wzrostowego-2 (IGF-2) oraz białek wiążących insulinopodobne czynniki wzrostowe (IGFBP- 2 i IGFBP-3) jako potencjalnych markerów złośliwości, w surowicy krwi chorych przed i w 30 dni po chirurgicznym usunięciu guza nadnercza. Zbadano 49 chorych (21M/28K: w wieku 45,6±7,3 lata) z guzami nadnerczy leczonych chirurgicznie w Klinice Chirurgii Ogólnej i Endokrynologicznej Instytutu Endokrynologii A.M. w Łodzi, podzielonymi zgodnie z klasyfikacją histopatologiczną na 4 grupy: raki (n=14), gruczolaki (n=15), guzy pochodzenia nienabłonkowego (n=11) oraz barwiaki rdzenia nadnerczy (n=9). Grupę kontrolną stanowiło 10 zdrowych osób (6K/4M: w wieku 39,5±6,3 lata). Pomiary stężeń IGF-2, IGFBP- 2, IGFBP-3 w surowicy krwi przeprowadzono posługując się techniką IRMA i RIA. Stężenia IGFBP-2 w grupie kontrolnej wynosiły 292,23±30,34 ng/ml. Przed zabiegiem operacyjnym w grupie chorych z rakami kory osiągały wartości najwyższe (1246,95±256,31 ng/ml; p<0,01); u chorych z gruczolakami (759,90±189,70 ng/ml; p<0,05); z barwiakami (532,44±86,90 ng/ml) były istotnie wyższe w porównaniu z kontrolą. Stężenia IGFBP-2 w grupie chorych z guzami nienabłonkowymi wynosiły 420,75±74,72 ng/ml i nie różniły się istotnie od wartości w grupie kontrolnej (p>0,05). Zabieg operacyjny spowodował istotne obniżenie stężenia IGFBP-2 u chorych z rakami kory (p<0,05), natomiast u chorych z gruczolakami kory nadnerczy oraz barwiakami rdzenia nadnerczy zabieg operacyjny nie wpłynął istotnie na stężenie tego czynnika. Stężenia IGF-2 oraz IGFBP-3 przed operacją oraz po zabiegu chirurgicznym nie różniły się znamiennie od wartości stężeń tych czynników w grupie kontrolnej. Wnioskujemy, że oznaczanie stężeń IGFBP-2 w surowicy krwi może mieć istotne znaczenie w diagnostyce różnicowej, monitorowaniu leczenia chirurgicznego i rokowaniu w chorobach rozrostowych nadnerczy. Slowa kluczowe: guzy nadnerczy, IGF-2, IGFBP-2, IGFBP-3 Tomasz Stępień Klinika Chirurgii Ogólnej i Endokrynologicznej Łódź ul.pabianicka 62 tomsamaz@wp.pl Wstęp Insulinopodobne czynniki wzrostowe (IGF-1 i IGF- 2) są polipeptydowymi hormonami tkankowymi wykazującymi duże podobieństwo strukturalne do proinsuliny [1]. Produkowane są one głównie w wątrobie, ale także inne tkanki mają zdolność wytwarzania tych czynników. Synteza IGF-1 regulowana jest przez hormon wzrostu (GH), podczas gdy wytwarzanie IGF-2 jest niezależne od tego hormonu. Przyjmuje się, że układ GH/IGF-1 odgrywa fundamentalną rolę w pobudzaniu procesów wzrostu i różnicowania komórkowego w okresie postnatalnym, podczas gdy IGF-2 uznawany jest za główny czynnik mitogenny w okresie życia płodowego. IGF-1, IGF-2 i ich receptory zostały wykryte w obrębie komórek kory i rdzenia nadnerczy, gdzie zdają się odgrywać istotną rolę w regulacji ich proliferacji, dojrzewania i procesu apoptozy [2, 3]. IGF-1 i IGF-2 wywierają także pobudzający wpływ na wytwarzanie i sekrecję sterydów nadnerczowych [4]. Efekty biologiczne tych peptydów wywierane są za pośrednictwem specyficznych receptorów błonowych. Receptor typu 1 zbudowany jest z dwóch podjednostek α i dwóch podjednostek β o strukturze homologicznej w 60% z receptorem insulinowym, wykazującym aktywność kinazy tyrozynowej. Receptor typu 2 jest monomerem, który funkcjonuje także jako receptor dla mannozo- 6-fosforanu. W większości efektów biologicznych IGF-2 pośredniczy receptor typu 1. Funkcja receptora typu 2 pozostaje niejasna. Sugerowany jest ostatnio udział tego receptora w procesie internalizacji i degradacji IGF-2 [5]. W płynach ustrojowych insulinopodobne 768

18 Endokrynologia Polska / Polish Journal of Endocrinology 2003; 6 (54) czynniki wzrostowe wiązane są przez specyficzne białka wiążące (IGF-BP). Do chwili obecnej wykryto i scharakteryzowano sześć białek wiążących, które wykazują między sobą podobieństwo strukturalne w 35% [5]. Obecność IGF-BP wykazano w wielu ludzkich komórkach i tkankach, w tym także w nadnerczach [2, 7]. Sugeruje się, że zasadniczą rolą białek wiążących jest modulowanie działania IGF-1 i IGF-2, a stosunek IGF-1,2/IGF-BP w tkankach obwodowych ma znaczenie fundamentalne dla ich efektów fizjologicznych. W ostatnim dziesięcioleciu przeprowadzono szereg badań doświadczalnych i klinicznych wskazujących na istotną rolę układu insulinopodobnych czynników wzrostowych, ich receptorów oraz białek wiążących te czynniki, w powstawaniu i progresji różnych nowotworów. Wzmożoną ekspresję tkankową mrna dla IGF-1 i IGF-2 wykazano w glejakach i oponiakach ośrodkowego układu nerwowego, w nowotworach trzustki, żołądka i jelit, rakach płuca, piersi, jajnika, gruczołu krokowego oraz pęcherza moczowego [5]. Także w warunkach in vitro peptydy te pobudzają proliferację szeregu stransformowanych linii komórkowych. Sugeruje się ostatnio, że dysfunkcja układu insulinopodobnych czynników wzrostowych odgrywać może istotną rolę w patogenezie raków kory i rdzenia nadnerczy. Wykazano silne mitogenne efekty IGF-1 i IGF-2 na ludzką linię komórek raka nadnerczy NCI H295R w warunkach in vitro [8]. W oparciu o badania immunohistochemiczne oraz hybrydyzację in situ wykazano wzmożoną ekspresję tkankowej zawartości mrna dla IGF-2 w ponad 60% raków kory nadnerczy [2, 9]. Zaobserwowano także zwiększoną ekspresję receptora IGF-1R (ale nie IGF-2R) w rakach kory wywołujących zespół Cushinga [10]. Także tkankowa ekspresja niektórych białek wiążących IGF-1 i IGF-2 była wyższa w przypadku raków w porównaniu z gruczolakami i prawidłową tkanką nadnerczową [9, 11]. W przeprowadzonych ostatnio badaniach przez Boulle i wsp. [12] wykryto podwyższone stężenia IGFBP-2 w surowicy krwi chorych z rakami nadnerczy, które dodatnio korelowały ze stopniem zaawansowania klinicznego. Wypada w tym miejscu dodać, że wzmożoną ekspresję mrna dla IGF-2 oraz IGFBP-2 i IGFBP-4 zaobserwowano również w komórkach chromochłonnych barwiaka [11, 13]. Danych dotyczących pomiarów zawartości IGF-2 oraz białek wiążących insulinopodobne czynniki wzrostowe (IGFBP1-6) w płynach ustrojowych w zależności od rozpoznania histopatologicznego nowotworu nadnerczy dotychczas nie publikowano. Cel pracy Celem podjętych badań była ocena pomiarów stężeń insulinopodobnego czynnika wzrostowego-2 (IGF-2) oraz białek wiążących insulinopodobne czynniki wzrostowe (IGFBP-2 i IGFBP-3) w surowicy krwi chorych z łagodnymi i złośliwymi guzami nadnerczy przed i w 30 dni po chirurgicznym usunięciu guza nadnercza. Materiał i metody Badaniem objęto 49 chorych leczonych z powodu guzów nadnercza w Klinice Chirurgii Ogólnej i Endokrynologicznej Instytutu Endokrynologii Uniwersytetu Medycznego w Łodzi, w latach Grupę badaną stanowiło 28 kobiet i 21 mężczyzn w wieku od 22 do 69 lat (45,6±7,3 lat; wartość średnia arytmetyczna ± błąd standardowy wartości średniej arytmetycznej ±SEM). Wszystkich chorych zakwalifikowano do zabiegu, a następnie operowano techniką klasyczną z dostępu bocznego. Czas pobytu chorych w szpitalu po operacji wynosił od 6 do 9 dni. U chorych stosowano rutynową, okołooperacyjną profilaktykę antybakteryjną i przeciwzakrzepową. Kobiet nie operowano, ani nie pobierano krwi w trakcie miesiączki. Krew do oznaczeń biochemicznych pobierano w godzinach rannych, na czczo przed śniadaniem, 1 dzień przed zabiegiem operacyjnym i w 30 dniu po operacji. Próbki krwi żylnej pobrane z żyły łokciowej odwirowywano, a uzyskaną surowicę przechowywano w temperaturze -25 stopni C do momentu wykonania oznaczeń: IGF-2, IGFBP-2 oraz IGFBP-3. Rozpoznanie określano na podstawie objawów klinicznych, rutynowych badań hormonalnych oraz badania histopatologicznego usuniętej zmiany. Czynność glikokortykoidową oceniano na podstawie pomiaru porannego stężenia ACTH i kortyzolu w surowicy krwi oraz skróconego testu hamowania deksametazonem (po 1 i 8 mg). Czynność mineralokortykoidową oceniano na podstawie pomiarów stężeń elektrolitów i aldosteronu w surowicy krwi. U wszystkich chorych wykonano ponadto pomiary stężeń androstendionu, siarczanu dehydroepiandrosteronu, testosteronu i estradiolu. Czynność rdzenia nadnerczy oceniano na podstawie pomiaru stężenia kwasu wanilinomigdałowego w 24- godzinnej zbiórce moczu oraz chromograniny A w surowicy krwi. U każdego chorego przed zabiegiem operacyjnym, celem dokładnej wizualizacji guza wykonywano badania USG i CT jamy brzusznej (warstwowe lub spiralne). W oparciu o uzyskany wynik rozpoznania histopatologicznego badaną grupę chorych z guzami nadnerczy podzielono zgodnie z klasyfikacją Seagera [14]. Grupę kontrolną stanowiło 10 zdrowych ochotników, 6 kobiet i 4 mężczyzn, w wieku 39,5±6,3 lat, u których pobrano krew celem dokonania oznaczeń stężeń badanych czynników wzrostowych oraz białek wiążących te czynniki. Stężenia IGF-2 oznaczano metodą immunoradiometryczną (IRMA) posługując się zestawami firmy DIAGNOSTIC SYSTEM LABORATORIES 769

19 IGFBP-2 w surowicy krwi chorych z guzami nadnerczy Stępień T. Tab.1. Podział chorych z guzami nadnerczy leczonych w Klinice Chirurgii Ogólnej i Endokrynologicznej Instytutu Endokrynologii Uniwersytetu Medycznego w Łodzi w latach w zależności od rozpoznania histopatologicznego [14]. Grupa Liczba chorych Rozpoznanie I 14 Raki kory II 15 Gruczolaki kory III 11 Guzy nienabłonkowe IV 9 Barwiaki rdzenia Kontrola 10 INC. (DSL Inc., Webster, Texas, USA). Według opisu metody stężenia IGF-2 w surowicy krwi ludzi zdrowych wahały się od 133 do 1077 ng/ml. Dolna granica czułości metody wynosiła 12 ng/ml. Współczynnik zmienności wewnątrzseryjnej wynosił 6,3%, a współczynnik zmienności międzyseryjnej 6,5%. Stężenia IGFBP-2 oznaczano metodą radioimmunologiczną (RIA) posługując się zestawami firmy DIAGNOSTIC SYSTEM LABORATORIES, INC., (DSL Inc. 445 Medical Center Blvd., Webster, Texas USA). Dolna granica czułości metody wynosiła 0,5 ng/ml. Współczynnik zmienności wewnątrzseryjnej wynosił 7,4%, a współczynnik zmienności międzyseryjnej 8,5%. Stężenia IGFBP-3 oznaczano metodą immunoradiometryczną (IRMA) posługując się zestawami firmy IMMUNOTECH AND BECKMAN COULTER COMPANy (Immunotech and Beckman Coulter Company 130, Marseille, Cedex, 9 France). Według opisu metody stężenia IGFBP-3 w surowicy krwi ludzi zdrowych wahały się od 1531 do 4277 ng/ml, średnio 2904 ng/ml. Dolna granica czułości metody wynosiła 50 ng/ml. Współczynnik zmienności wewnątrzseryjnej wynosił 4,8 %, a współczynnik zmienności międzyseryjnej 6,4%. Uzyskane wyniki poddano analizie statystycznej z wykorzystaniem komputera typu PC przy zastosowaniu programów Excel i Statgraphics Plus v.5.1. Przy opracowaniu zebranego materiału zastosowano następujące parametry opisowe w badanych grupach: wartość średnia arytmetyczna ( ) oraz błąd standardowy wartości średniej arytmetycznej (SEM).Analizowano istotność różnicy średnich między grupą kontrolną i grupami badanymi, jak również istotność różnicy średnich między poszczególnymi grupami chorych. Stosowano test t-studenta dla dwóch średnich oraz analizę wariancji ANOVA. Za istotne statystycznie uznano różnice pomiędzy średnimi arytmetycznymi, dla których obliczona wartość testu była równa lub większa od wartości krytycznej odczytanej z tablic rozkładu t-studenta przy odpowiedniej liczbie stopni swobody i prawdopodobieństwie błędu p<0,05. W przypadku niejednorodnych wariancji stosowano poprawkę do testu t-studenta. Wyniki Stężenia IGF-2 przed operacją oraz po zabiegu chirurgicznym we wszystkich badanych grupach chorych nie różniły się znamiennie od wartości stężeń tego czynnika w grupie kontrolnej (Ryc. 1) Stężenia IGFBP-2 w grupie kontrolnej wynosiły 292,23±30,34 ng/ml. Przed zabiegiem operacyjnym w grupie chorych z rakami kory osiągały wartości najwyższe (1246,95±256,31 ng/ml; p<0,01); u chorych z gruczolakami (759,90±189,70 ng/ml; p<0,05); z barwiakami (532,44±86,90 ng/ml) były istotnie wyższe w porównaniu z kontrolą. Stężenia IGFBP-2 w grupie chorych z guzami nienabłonkowymi wynosiły 420,75±74,72 ng/ml i nie różniły się istotnie od wartości w grupie kontrolnej (p>0,05). Zabieg operacyjny spowodował istotne obniżenie stężenia IGFBP-2 u chorych z rakami kory (p<0,05), natomiast u chorych z gruczolakami kory nadnerczy, guzami nienabłonkowymi oraz barwiakami rdzenia nadnerczy zabieg operacyjny nie wpłynął istotnie na stężenie tego czynnika (Ryc. 2). Stężenia IGFBP-3 przed operacją oraz po zabiegu chirurgicznym nie różniły się znamiennie od wartości stężeń tych czynników w grupie kontrolnej (Ryc. 3) Dyskusja W diagnostyce guzów kory i rdzenia nadnerczy problemem fundamentalnym z punktu widzenia postępowania terapeutycznego, poza określeniem aktywności hormonalnej, jest udzielenie Ryc.1. Stężenie IGF-II I w surowicy krwi chorych z guzami nadnerczy przed i w 30 dniu po zabiegu operacyjnym oraz w grupie kontrolnej. Histogram przedstawia wartości średnie ±SEM. 770

20 Endokrynologia Polska / Polish Journal of Endocrinology 2003; 6 (54) Ryc. 2. Stężenie IGFBP-2 w surowicy krwi chorych z guzami nadnerczy przed i w 30 dniu po zabiegu operacyjnym oraz w grupie kontrolnej. Histogram przedstawia wartości średnie ±SEM Ryc. 3. Stężenie IGFBP-3 w surowicy krwi chorych z guzami nadnerczy przed i w 30 dniu po zabiegu operacyjnym oraz w grupie kontrolnej. Histogram przedstawia wartości średnie ±SEM odpowiedzi na pytanie czy stwierdzona w oparciu o badania obrazowe zmiana ma charakter złośliwy czy łagodny. Mimo stosowania coraz bardziej dokładnych i czułych technik badawczych w wielu przypadkach odpowiedź na powyżej postawione pytanie jest niejednoznaczna. Sugestie wynikające z analizy obrazów USG, TK, MRJ są na tyle niepewne, że nie dają podstaw do odstąpienia od zabiegu chirurgicznego bez ewentualnego narażenia chorego na przeoczenie u niego nowotworu złośliwego. Z tych też powodów w wielu laboratoriach podejmowane są próby poszukiwania nowych metod diagnostycznych, na podstawie których można by z większym prawdopodobieństwem rozróżniać te guzy, a także różnicować je z przerzutami do nadnerczy nowotworów wywodzących się z innych narządów i tkanek. W ostatnich latach dokonano szeregu bardzo ważnych odkryć wskazujących na istotną rolę układu insulinopodobnych czynników wzrostowych, białek wiążących te czynniki oraz ich receptorów w procesie powstawania nowotworów [15, 16, 17, 18]. Badania te oparte były na ocenie wpływu tych czynników na proliferację licznych linii komórek prawidłowych i nowotworowych w warunkach in vitro i in vivo oraz ekspresji tkankowej tych czynników w procesie transformacji nowotworowej. Opracowano również coraz czulsze metody RIA i ELISA, pozwalające na precyzyjne określenie stężeń tych czynników w surowicy krwi i innych płynach ustrojowych. W wielu ośrodkach podjęto także badania nad oceną pomiarów stężeń IGF-1, IGF-2, białek wiążących te czynniki (IGFBP 1-6) i ich receptorów w diagnostyce wielu chorób, zwłaszcza o podłożu nowotworowym. Coraz powszechniej wyrażana jest opinia, że pomiary stężeń tych czynników we krwi lub w moczu mogą być bardzo przydatne w diagnostyce, monitorowaniu leczenia oraz rokowaniu u chorych z nowotworami nadnerczy. W naszym materiale stężenia badanych peptydów ocenialiśmy dwukrotnie: przed operacją i w 30 dniu od momentu chirurgicznego usunięcia guza. W tym czasie gojenie się rany pooperacyjnej było praktycznie zakończone i nie powinno rzutować istotnie na wyniki przeprowadzonych pomiarów. W przedstawionych wynikach badań nie wykazaliśmy uchwytnych różnic w stężeniach IGF-2. Stężenia tego peptydu przed operacją oraz po zabiegu chirurgicznym we wszystkich badanych grupach nie odbiegały znamiennie od wartości stężeń tego czynnika w grupie kontrolnej. Prezentowane w światowej literaturze wyniki badań wskazują, że w przypadku nowotworów nadnerczy u ludzi wzmożona ekspresja tkankowa IGF-2 oraz receptorów IGF-2 jest ściśle, dodatnio skorelowana ze stopniem złośliwości guza [2, 9, 10, 19]. Brak wzrostu stężenia IGF-2 w surowicy krwi chorych przy niewątpliwie wzmożonej syntezie tego peptydu w złośliwych nowotworach nadnerczy spowodowany jest najprawdopodobniej zaburzeniami na poziomie translacji. Oznaczany przeze mnie techniką IRMA czynnik jest 67-aminokwasowym peptydem o ciężarze molekularnym 7,5 771