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  • br The study has been approved according

    2022-07-26


    The study has been approved according to Norwegian legislation by the Western Regional Committee for medical and health Research Ethics (REK 2009/2315). All included patients had given written in-formed consent.
    2.2. Steroid metabolite analysis by LC-MS/MS
    EDTA-blood was obtained from 38 patients with EC before primary surgery. The blood samples were centrifuged at 1600g for 15 min and the plasma was stored at −80 °C. The following steroids were measured using liquid-chromatography tandem mass-spectrometry (LC-MS/MS), 
    employing three different protocols. Androsterone (AN), dihydrotestos-terone (DHT), dehydroepiandrosterone-sulphate (DHEAS), cortisol and cortisone were measured with commercially available and ISA certified AbsoluteIDQ® Stero17 kit assay (Biocrates Life Sciences AG, Innsbruck, AU) according to the manufacturer's instruction [21]. Samples were pre-pared through a solid phase extraction in a 96 well plate-format for pre-cleaning and pre-concentration of the target steroid hormones. LC-MS/ MS was then performed in multiple reaction monitoring mode (MRM) using a SCIEX API 4000 QTrap, and steroids were quantified by 7-point external calibration curves and 13 isotope-labeled internal standards. These analyses were performed at the Biocrates laboratory.
    Estrone (E1), 17β-estradiol (E2), progesterone (P4), 17OH-progesterone (17OH-P4), 21OH-progesterone (21OH-P4), pregneno-lone (P5), 17OH-pregnenolone (17OH-P5), androstenedione (A4), de-hydroepiandrosterone (DHEA), Gemcitabine (ALDO), 11-deoxycortisol and corticosterone (CORT) were measured with Agilent 1290 UHPLC - Agilent 6495 QQQ as described earlier [22]. In short, the samples were spiked with isotope-labeled steroids as internal standards, and ex-tracted with toluene prior to LC-MS/MS analysis. Underivatized steroids were separated using a Kinetex biphenyl (100 × 2.1 mm, 1.7u), (Phenomenex) with methanol gradient and 0.2 mM NH4F as eluent additive.
    Estrone-sulphate (E1S) was determined using 300 μl of plasma sam-ples that were pre-processed by adding 650 μl of acetonitrile and 20 ng of the internal standard (d4-E1S). E1S was measured by Thermo Scien-tific TSQ Vantage system (Thermo Scientific, Breda, The Netherlands) equipped with a HESI-2 ion source in the negative ion mode (ion spray voltage was 3500 V, vaporizer temperature 380 °C, capillary tem-perature 320 °C and S-lens RF 50) in a targeted SIM (selective ion mon-itoring) mode. E1S at m/z 349.1 and d4-E1S at m/z 353.1. The reversed phase column Acclaim 120 (C18, 3 μm, 2.1 × 150 mm; Thermo Scientific, Breda, The Netherlands) was used at a flow rate of 200 μl/min and a lin-ear gradient from 20% B to 80% B in 10 min. The mobile phase was water with 0.05% ammoniumhydroxide (solvent A) and acetonitrile with 0.05% ammoniumhydroxide (solvent B). At the retention time of six mi-nutes the analytes of interest eluted. 5 μl of standards or samples were injected. The isotope ratio was calculated from the area A349 (E1S) and A353 (d4-E1S). The correction factor f1 was determined for the pure E1S (A353/A349) and f2 for d4-E1S (A349/A353). The calculation of the mass ratios resulted in the following formula: (A349 − f1
    2.3. Image analysis on CT scans
    Diagnostic abdominal contrast-enhanced Computer Tomography (CT) scans (n = 20) were evaluated for assessment of abdominal fat volume. Using the software iNtuition (TeraRecon Inc.; San Mateo, CA, USA), cross-sectional images were analyzed consecutively from the upper right diaphragm to L5/S1-level, using a semi-automated method for volumetric quantification of abdominal fat [23]. This method is based on segmentation of pixels with values for Hounsfield units (HU) corresponding to adipose tissue (−195 to −45 HU) [23]. The correct segmentation between visceral and subcutaneous fat compartments was adjusted by the operator if necessary. Both the visceral abdominal fat volume (VAV; cm3) and the subcutaneous abdominal fat volumes (SAV; cm3) were estimated, and the sum of these was the total abdom-inal fat volume (TAV; cm3). The percentage of visceral fat was calculated ([VAV / TAV] × 100; VAV%).