Evaluation of Age and Metabolic Status Impact on Serum Steroid Hormones and Product/Precursor Ratios for the Generation of LC-MS/MS Reference Interval in Men Marco Mezzullo (Presenter) University of Bologna Presenter Bio: Marco Mezzullo’s achieved his MSc in Medical Biotechnology in 2011 by Alma Mater Studiorum, University of Bologna, Italy, after internship in the research group of Prof. Pasquali. He continued his career in the same lab as a research fellow in the mass spec team of Dr. Flamina Fanelli. He improved his expertise in LC-MS/MS method development and validation, he tutored student activity and managed several research activities. His main research topic is represented by salivary steroid hormones evaluation in polycystic ovary syndrome. He is co-author of 23 papers, and is first author three papers on salivary steroids measurement by LC-MS/MS, providing data on assays’ analytical and biological validation as well as on clinical application [Mezzullo et al. 2016, JSBMB; Mezzullo et al. 2017, CCLM; Mezzullo et al. 2018, Psychoneuroendocrinology]. In 2016 he started PhD program.

Authors: Marco Mezzullo, Alessia Fazzini, Uberto Pagotto and Flaminia Fanelli
Endocrinology Unit, Department of Medical and Surgical Sciences (DIMEC), Centre for Applied Biomedical Research (CRBA), S. Orsola-Malpighi University Hospital - University of Bologna, Bologna, Italy

Aiming at generating the reference intervals (RIs) for steroid levels and product/precursor ratios, we selected 321 adult drug- and disease-free Italian males, and defined a sub-cohort of 137 metabolically healthy individuals. Twelve serum steroids were measured by two validated LC-MS/MS assays. The independent impact of age, adiposity and metabolic parameters on steroid values was estimated, then, RIs at age 20, 30, 40, 50, 60, 70 and 80 were calculated in the overall and in the metabolically healthy cohort. We provided age- and metabolic status-specific RIs for a broad steroid profile, useful to implement LC-MS/MS in the management of endocrine diseases.

Introduction

Steroid hormone determination in clinical and research settings is undergoing a profound technical upgrade as represented by the switching from direct antibody-based assays to mass spectrometric multi-analyte quantitation. The current knowledge about circulating steroid normative values has mainly been generated by immunoassays’ determinations on reference cohorts heterogeneously defined as to the health criteria and by unstandardized sampling conditions. In the last decade, a profound disagreement was documented between immunoassays and LC-MS/MS for serum steroid measurements. Furthermore, LC-MS/MS opened to the evaluation of steroid metabolites previously not available by immunoassays. Very few data exist about the circulating values of these molecules as well as about the steroid product-over-precursor ratios often used for estimating the steroidogenic activity. Given these considerations, a careful update and implementation of circulating steroid normative values is required in order to prompt the introduction of LC-MS/MS profiling potential into the clinical practice. In such a frame, taking into account physiological aspects, such as age, gender, obesity and metabolic status, wake-sleep cycle, as well as the sampling conditions, such as the fasting state, the time of the day and the venipuncture stress, known to influence steroid values, is mandatory for establishing robust RIs [1].

Methods

Male volunteers aged 18-90y were recruited from the general population of the Emilia Romagna Region, Italy. Among 590 subjects examined, 321 reported not to be affected by any disease, to be drug-free and to have a normal wake-sleep cycle, and were therefore invited to give their blood between 8:00 – 10:00 am, in overnight fasting condition and after 10 minutes saline infusion to avoid venipuncture stress influence. Main routine biochemical parameters were measured. In addition, the steroid panel including cortisol (F), 11-deoxycortisol (11S), 17-hydroxyprogesterone (17OHP4), 17-hydroxypregnenolone (17OHP5), testosterone (T), androstenedione (A4), dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), progesterone (P4), corticosterone (B), estrone (E1), estradiol (E2) was determined by two in-house LC-MS/MS methods previously validated by Certified Reference Materials and multicenter comparison trials [2-4]. Molar product/precursor ratios were computed. We evaluated the independent impact of age, BMI, waist circumference, systolic (SBP) and diastolic blood pressures (DBP), homeostatic model assessment – insulin resistance (HOMA-IR), total/HDL-cholesterol ratio and triglyceride levels on each steroid value by multiple stepwise regression. Age-related lower and upper reference limits, corresponding to the 2.5th and 97.5th percentiles of the distribution, respectively, were calculated by weighted polynomial regression for each steroid value in the overall cohort [5, 6]. In addition, for those steroid values who were significantly impacted by metabolic parameters, age-related reference limits were calculated in a sub-cohort of 137 normal weight (body mass index, BMI: 18.5 – 25.0 kg/m2) subjects displaying waist circumference<102 cm, normal blood pressure (SBP <140 and DBP <90 mmHg), no insulin resistance (HOMA-IR <2.5) and normal lipid profile (total/HDL-cholesterol <5; triglycerides <150 mg/dL).

Results

Age was found not to influence T, DHT, E1and E2 levels, and to negatively influence 17OHP4, 17OHP5, A4, DHEA, P4and B (all P<0.001), F (P=0.016) and 11S (P=0.006). In addition, aging was negatively associated with DHEA/17OHP5 (P=0.005) and E2/E1 (P=0.027), and positively associated with A4/DHEA, T/A4, 17OHP4/17OHP5 and E1/A4 ratios (P<0.0001). E2 (P=0.031), E2/T (P<0.0001) and E1/A4 (P=0.001) increased, while 17OHP4 (P=0.002), DHT (P=0.003) and DHT/T (P=0.001) decreased with BMI. E1 (P=0.012) and DHT/T (P=0.009) increased, while F decreased (P=0.004) with waist circumference. T/A4 increased (P=0.035), while B (P=0.001), 17OHP5 (P=0.024), and A4/17OHP4 (P=0.029) decreased with increasing total/HDL-cholesterol ratio. HOMA-IR negatively influenced T (P=0.002) and A4/DHEA (P=0.025). A4/17OHP4 (P=0.001) and DHEA/17OHP5 (P=0.036) increased, while T/A4 (P=0.003) and 17OHP4/P4 (P=0.009) decreased with triglyceride levels. Finally, a positive association was found between SBP and E1 (P=0.034). According to the independent effects of the metabolic parameters, 2.5th and 97.5th centiles of distribution at age 20, 30, 40, 50, 60, 70 and 80 were estimated in the overall cohort for A4, DHEA, P4, 11S, 17OHP4/17OHP5, A4/DHEA and E2/E1 ratios, and in the metabolically healthy sub-cohort for F, 17OHP4, 17OHP5, T, DHT, B, E1, E2 and 17OHP4/P4, DHEA/17OHP5, A4/17OHP4, T/A4, DHT/T, E2/T and E1/A4.

Conclusions & Discussion

This study provided a comprehensive panel of LC-MS/MS-based, age and metabolic status-specific RIs for circulating steroid levels and ratios in a properly selected cohort of adult men. These data represent a valuable tool in diagnostic work-up of diseases subtended by the steroidogenesis imbalance.

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