Sviluppo e validazione di metodi di misurazione di ormoni mediante cromatografia liquida abbinata a spettrometria di massa tandem (LC-MS/MS): analisi di popolazioni composte da soggetti sani e affetti da malattie endocrino-metaboliche

Analytical chemistry has always had a pivotal role in determining major advancement in endocrine sciences. The development of the radioimmunoassay in the 50s allowed the measurement of hormone concentrations in biological fluids for the first time, leading to revolutionary improvements for the comprehension of endocrine physiology and disease. In the 80s, the development of high-throughput immunometric platforms allowed hormone measurement in routine clinic settings, clearly improving diagnosis accuracy and patient care. However, to achieve the impressive throughput required by large clinical laboratories, modern immunoassays eliminated sample pre-treatment steps, causing the worsening of the assay performance in terms of specificity and accuracy, thus giving rise to analytical as well as clinical uncertainties largely debated by the scientific community in the last decade. Liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) is an innovative technique displaying several key advantages such as the reliability of the measurement, the high throughput and the low cost required for the analysis. LC-MS/MS combines the high specificity and sensitivity of mass spectrometry with the high versatility and throughput of liquid chromatography, features conferring enormous potentialities that are successfully exploited in several fields, such as food, and environmental, pharmacology, toxicology and clinical sectors. LC-MS/MS has already been introduced in routine laboratories in critical fields that strongly rely on the reliability and rapidity of the measurement, such as newborn screening and immunosuppressant drug monitoring. The endocrinology laboratory is undergoing the same revolution, particularly in the field of the determination of steroid hormones, that are key regulators of the most important functions of the human body, from sexual development and reproduction to metabolism, stress recovery and mental health. A proper determination of the level of these hormones is mandatory in establishing the accurate diagnosis and treatment of important diseases such as hypogonadism, hyperandrogenism, infertility, genetic defects of sexual dimorphism, obesity, metabolic syndrome, salt imbalance and hypertension, Cushing’s Syndrome and osteoporosis, adrenal and gonadal tumors, overall affecting a significant part of the population at all ages. In this regard, several studies have reported the dramatic lack of accuracy and specificity of immunoassays for the measurement of critical steroids such as testosterone in females and children, 17OHprogesterone, aldosterone, 17βestradiol and vitamin D, and such a poor performance causes uncertainties in clinical practice and raises the indirect costs due to repeated tests and examinations. Thanks to its benefits in terms of analytical performance, cost saving and high-throughput, LC-MS/MS is replacing immunoassays for steroid determination in the major clinical laboratories all over the world; in Italy, however, no attempts have yet been made to introduce LC-MS/MS in the steroidology field. The aim of this study was to develop a quantitative method for a large serum steroid profile by using the innovative technology of LC-MS/MS, and to define new reference intervals of normal circulating values to be applied to the study of endocrine disease. A multi-analytical LC-MS/MS method for 9 serum steroids was developed, including major steroids and important intermediates of their biosynthesis, namely cortisol, 11deoxycortisol, corticosterone, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone. The 9 steroids were chosen not only for their scientific and clinic relevance, but also for their chemical and physical properties that allow them to be monitored by the same method. Important steroids not included in this first panel, such as estrogens, dihydrotestosterone, aldosterone, 17OHpregnenolone and pregnenolone, require different analytical strategies and will be developed hereafter. The method was carefully validated according to FDA guidelines and by using certified material: a satisfying level of sensitivity was obtained to determine the circulating levels of all the hormones included in the panel except DOC, which seems to circulate at levels below the sensitivity limit of 78pg/ml in most of the population. Nevertheless, the presence of this analyte in the profile proved very useful to indentify and characterize enzyme deficits of steroidogenesis causing its accumulation in the blood. The LC-MS/MS method was compared to the immunoassays currently used in the Central Laboratory of the S.Orsola-Malpighi Hospital in Bologna by measuring more than 300 samples of heterogeneous origin with both techniques. Immunoassays revealed a good agreement with LC-MS/MS for steroids circulating at high levels, such as cortisol, male testosterone and progesterone above 1ng/ml. A miscalibration problem was observed for immunoassays measuring androstenedione and DHEA compared to LC-MS/MS, and a dramatic inconsistency was observed between immunoassays and LC-MS/MS for testosterone in females, for progesterone below 1ng/ml and for 17OHprogesterone, caused by the lack of specificity of immunoassays and revealed by poor accuracy, proportional and systematic overestimation, respectively. No routine immunoassays were available for corticosterone, 11deoxycorticosterone and DOC, important intermediates that increase the informative power of LC-MS/MS for both research and clinical use. The LC-MS/MS 9 steroid profile was used to measure samples from an adult population consisting of 1244 subjects and from an adolescent population of 940 subjects. Healthy, normal weight and drug free subjects were selected for the estimation of reference intervals specific for gender, age ranges, fertility and menstrual phase. The estimation of reference intervals in narrow age ranges proved important for androgens, which showed a strong decrease with age both in males and females. By comparing steroid levels in different menstrual phases in young women, we observed a tendency for testosterone and androstenedione to increase during the ovulatory peak and in the luteal phase compared to the follicular phase: although statistical sensitivity has not been reached, these data support the importance of performing an evaluation of hyperandrogenism in the follicular phase. Moreover, we showed that also in healthy normal weight girls, conditions such as anovulation or a reduced efficiency of the luteal phase are characterized by increased testosterone levels. Our data collected on an obese population showed that female obesity is characterized by reduced glucocorticoid levels, apparently in contrast with previous reports stating the hyperactivation of the hypothalamus-pituitary-adrenal axis in obesity: in order to understand the importance of this observation, the evaluation of corticosterone, the inactive metabolite of cortisol, is mandatory to exclude the presence of mechanisms of compensation, as well as the evaluation of cortisol circadian fluctuation by measuring salivary fraction. In agreement with previous knowledge, we observed a decrease of testosterone levels in male obese subjects. The results obtained in this project clearly showed that LC-MS/MS has key advantages in terms of reliability and practicability, useful to conduct large epidemiological studies as well as the potential to be introduced in clinical routine. The impressive informative power of the LC-MS/MS technology proved useful to increase the comprehension of endocrine diseases. Our method, equipped with proper reference intervals generated in this study, specific for age, gender, fertility status and menstrual phase, represents a solid basis on which to conduct more reliable and detailed clinical and epidemiologic studies compared to what has been allowed so far by immunoassays.