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    Can. La 1806; 2: 285-280 ORIGINAL ARTICLE y ENDOCRINOLOGY Glycoprotein hormone alpha-subunit release in sera and tumour cell cultures and the responses to thyrotropin-releasing hormone in the patients with clinically nonfunctioning pituitary adenomas Sekretion der Alpha-Untereinheit von Glycoproteinhormonen im Serum und in Tumorzellkulturen und die Reaktionen auf Thyroliberin bel Patienten mit klinisch funktionslosen Hypophysenadenomen Vesna Plavéie!, Mirko Korsié’, Neven Zarkovie*, Dunja Rogis!, Kameliia Zarkovie®, Zlatko Gifev', Simon Mikulandra’, Izet Aganovie', Josko Paladlino?, Blazenka Kozic! "ivan ot endceinelogy, Daparman femal Macs; ? Department af Haupathlogy:* Departmont of Nevesupery,Schest of Medic, Univer cokyot Zag, Crate," nethze Pugh Boskowe" Zags Cresta ‘Summary: Aipha-subunit (a-SU) release was studied in vivo and in vitro in 14 clinically nonfunctioning adeno- mas (NFA) using highly specific immunometric assays. ‘o-SU was secreted in vivo in 21% and in vitro in 71% of ‘the NFA. Significant correlations were found between LH release in vitro and its percent change (P<0.02) and between @-SU secretion in vivo and its area under the curve (AUG) after the TRH test (P<0.001), Two sub: groups of NFA were recognized according to gona dotropin and a-SU secretion in vitro, one with pronoun cad and the other with waak or absent secretory actvi- ty. Significant differences were found between these subgroups in gonadotropin and «-SU nAUC during the TRH test. Key words: Alpha-subunit, gonadotropins, thyrotropin- releasing hormone, nonfunctioning adenomas, cell cul- tur. Zusammentassung: Bel 14 funktionslosen Hypophy- Ssenadenomen (NFA) haben wir in vivo und in vitro die Alpha-Untereinheit (SU) mit einer hochspezifischen immunometrischen Methode bestimmt. In vivo fanden wir ohohte a-SU-Werto in 219% und in vitro bol 71% dor NFA. Eine signifxante Korrelation bestand zwischen der LH/-Sekretion in vitro und threr prozentualen Verdnde~ rung (P<0,02) sowie zwischen der a-SU-Sekretion in vivo und ihrer Oberflacha unter der Kurve (nAUC) nach dein TRH-Test (P<0,001), Gemas der Gonadotropin= und ‘-SU-Sekretion in vitro haben wir Zwei NFA-Subgrup- en erkannt: eine mit ausgepragter und die andere mit schwacher oder fehlender Sokretionsaktivitat. Signifi- ante Unterschiede zwischen diesen Subgruppen wur- den bs! den Gonadotropin und a-SU nAUC-Werten wahrend des TRH-Tests gefunden. SchllisselwOrter: Alpha-Uintereinhelt, Gonadatropine, ‘Thytolbern, funktionslose Adenome, Zellkutur Maryserip accaptd: December Tet, 1655 [Rants dos Mansi 18: Daze 1995 Introduction Alpha-subunits (e-SU) of glycoprotein hormones are identical, while the @-subunit is unique to each hormo- ne. A variable proportion of the a-SU remains unbound land Is secreted as the biologically Inactive free subunit ina given ratio together with the whole glycoprotein hormone molecule, while disproportionate amounts can be secreted in clinically nonfunctioning adenomas (NFA) and gonadotropinomas (1,2), or in GH, TSH or provactin (PAL) secreting adenomas @ 4). In the present study, a SU levels in serum and in culture of pitutary adenoma cells were measured and a TRH test was performed in Wo. The alm of the study was to evaluate whether the responsiveness to TRH can differentiate subclasses of ‘these tumors. Materials and Methods Clinical data ‘The amount of a-SU was measured in the sera of 14 patente wth NEA es well asin cel cutura media ofthe respective tumors. Eleven patients nad. nonsecreting tumors (No.t-12) and 2 had FSH-secreting tumors (No.13 and 14). One woman in the postmenopause (Nos) also had sloveted FSH socum levels (Tab 2) Seven patients were men aged 40-07 years, ahd seven were women aged 25-72 years. All men except one with an FSH-secretng adenoma (No.16) had decreased Scrum testosterone lovela. Elevated basal serum PRL values were found in 6 patients (62 5=188 pail, consi Stent with pituitary stalk compression. All patients re- sented with Visual disturbances, headaches and macro- adenoma 2s demonstrated by the preoperative CT ima- Gng scans. The histopathology ofthe tumors removed by transephenoidal surgery confirmed the clinical a gnosis of ptutary adenoma in al cases. Comespondence Ot vesna Pave, Dison of Eacccrinolgy, Depa ‘rent of emai Maciine Reto, Riépatoea 12, 41000 Zayed. Coa Glin, Lab, 4/96 285, Pvt etal: Aha-suburit lease Hormone assays a-SU was measured by immunoradiometric: method (RMA) using the Kit of Immunotech International (Fran- 9). The detection mit was 0.025 IU/L. Intra- and inter= assay coeficients of variation were 6.8 and 16.6%, res pectively. Cross-roactions with LH, FSH and TSH were loss than 0.19%. 1 IU/L a-SU corresponds to 1 ng/ml. LH and FSH were measured by immunoenzymometric method with luminescent substrate using Amerite kits (ohnson&Johnson Clinical Diagnostics, UK). The detection limits wore 0.12 and 0.5 IU/L for LH and FSH, respectively, The standards used were 1®IRP 68/40 for LH and 2° IRP 78/549 for FSH. PRL was determined by radicimmuncassay (RIA) using the kits of Sorin-Blome- dica (aly), ‘TRH test. Blood was sampled -30 and 0 minutes before and 10,20,30 and 90 minutes after iv. administration of 200 41g TRH (Relefact, Hoechst). The sera were stored at -20°C for subsequent assays, Cell cultures Pituitary adenoma tissue was collected in iced saline at the time of surgery and centrifuged 10 minutes at 150 9. All suspensions were prepared under sterile conditions from mechanically and enzymatically cisaggregated tis- sue. A number of live oes per mi of medium was adju- Sted to 2x10; the cols were incubated in DMEM F-12 Ham's medium supplemented with 5% fetal calf serum, ‘and cultured for 72 hours at 37°C in humidified air ‘atmosphere with 8% CO. The same amount of medium was incubated without pituitary tissue as the control sample, Multivell plates were centrifuged at 4° C for 10 Table 1. Secretion of e-subunitin serum and release of e-eubunit, LH a minutes at 600g and the supernatants were collected, ‘ilered and frozen for hormone analysis. Statistical analysis, The maximal response to TRH was calculated as follows: The maximal response = (maximal response-mean basal value) x 100/mean besal value For statistical purposos, the responses to TRH were assessed by calculating of the net incremental area Under the curve (nAUC) over 90 minutes (trapezoidal ‘mathod), Statistical comparisons were made using the Mann-Whitney U test. Correlations were made using linear regression analysis. Results Elevated serum a-SU levels were found in 9 of 14 pati- ‘ents (219%) and a-SU was secratod in the culture media in 10 of 14 adenomas (71%). The same adenomas secreted LH and/or FSH. After the administration of TRH to 9 patients who con- sented to the test, the @-SU responses varied from 18.7-175% (Table 2). A significant correlation was demonstrated between the a-SU serum level and nAUC following the TRH test ('=0.99, p<0,001, Fig.1) and bat- ween LH secretion in vitro and its percent change aftr TRH (0.87, p<0.02, Fig.2). ‘The patients were divided into two groups according to the cell culture findings regardless of the secretion of hormones in vivo (Table 2). Group 1 (n=4) consisted of patients whose tumors secreted abundant quantities of 3nd FSH in vito in 14 patients with cnically nonfunctioning pu tary adenomas. Secretion Into Call culture Is expresses pr 10" col/72 hours. Controls: In vivo o-SU m.0-0.8, 10-0.9 ILM (prams ‘epausell 01.6 (posimenopauealy invita «eSU0.20 MIL: LH 1.19 IL: FSH 1.72 IU/L: n.p not performed, Patient Invivo Invitro | Sex Age brs) eS w8U uH FoH | au uy quay uy La M 0 020 020 119 =172 2 Mt 6 010 =020 52 35 3 F 28 0.30 020 118 192 4 F % 50 5.99 <1.19 <2 3 F 3s 050 134 357 134 6 ae | EEE re oa7 1.42, 487 190.41 7 M a =0.025 020 = 8 _M 58 024 359 33 ° F 6 02 De 33. 10 F 25 04 Bar 1 M 40 7a =u 72 F 50 87 766 13 M oa 72 3000 286 Glin. Lab. 4/96 Pav etal: Ale subunit lease Table 2. LH, FSH and «-SU release in vitro, basal values in sera and responses to TRH (% maximal increase over basal value and nAUG over 90 min) in 4 patients with ebundant release (Group 1) and in 8 patients with negligible or no relea- ‘8¢ (Group 2) of gonadotropins and a-SU in vitro. | Patient a Fst SU | Wo.sex, | invitro [Becatieve] max(%) | nAUC | Invitro |Bacaliovel] max (%} | nAUC | Invitro [Rasalieve] maxis6) | nAUC ae | au | aun) outed | qu) | AU aunomin| cua | quay puso Group | 6n72 | 47 | o70 [tix | 77a | oon | 102 | oo | cose | 14 | 020 | 50° | a3 verso | 76 | 050 | 1900] s08 | 766 | 51 | 240 | soa | aor | 021 | a8 | 126 ramsa| sts | 98 [oss | arse | 13 | 15 | aos | ans | 72 | a5 | aor | ovo sams7| ana | 22 | 1044 | 1401 | sooo | sso | 124 | sor | 131 | 17 | aaa | 102 ase | «a0 7796 | 1602s | st7= aise | 27ase | aaae 9232 | 605: 40 29 | 70 | 728 yes | 661 | 200 sa7 | aa Group 2 sma]

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