madman
Super Moderator
Summary
The determination of steroid hormones and subsequent interpretation of results is accompanied by a range of difficulties. The amount of information that current technology can provide on the circulating concentrations of more than a hundred various steroid compounds can lead to problems with interpretation. The aim of this study is to help provide orientation in this maze of data on steroid hormones. First, we focus on specific aspects arising from the pre-analytical phase of steroid determination that needs to be considered when planning sampling, whether for diagnostics or research. Then, we provide a brief summary of the characteristics and diagnostic relevance of several steroid hormones and/or their metabolites: pregnenolone, 17α-hydroxypregnenolone, dehydroepiandrosterone, hydroxyderivatives of dehydroepiandrosterone, androstenedione, testosterone, estrone, estradiol, estriol, cortisol, cortisone, which in our institute are determined with validated LC-MS/MS methods. For these steroids, we also provide newly calculated reference values in fertile women according to the phase of their menstrual cycle.
Introduction
In the early 50s, Zimmermann’s reaction for the determination of 17-ketosteroids and the analysis of corticosteroids as 17-hydroxycorticosteroids or 17-ketogenic steroids in urine was the only way to obtain any information on steroid excretion. Since the 1970s, the discovery of immunologic analysis of hormones in the blood using Rosalyn Yellow opened the way for steroid analysis in fluids other than urine and allowed the determination of very small concentrations (pmol/l of analytes). Immunoanalysis of individual steroids still prevails for diagnostic uses in clinical practice; however, sophisticated methods of tandem chromatography and mass spectrometry have found their place not only in steroid analysis for scientific purposes but also for the clinical diagnosis of various pathologies.
Instrumental analysis combining gas or liquid chromatography with mass spectrometry allows the simultaneous measurement of a large number of steroid metabolites and has higher specificity than immunoanalytic methods. Measured values are therefore often somewhat lower than from immuneanalyses. The choice of method depends on the requirements for precision, economic factors, and the ability to incorporate automatization into routine analyses. Routine diagnostic examinations and other research use also have different requirements. The range of information that current technologies deliver can often lead to data on more than a hundred circulating steroids, resulting in difficulties in interpretation. We know little about the roles of many hormones in various pathogenic disorders, and while we have certain information from experimental physiology, particularly for neuroactive steroids that act in the brain, we know almost nothing about their effects on health and disease. The aim of this work is to provide an orientation into the maze of data on steroid hormones and their metabolites. We provide a brief summary of the characteristics and diagnostic relevance of several steroid hormones and/or their metabolites that in our institute are determined with validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. For these steroids and methods, we also provide newly calculated reference ranges in fertile women (Table 1), which are in accordance with another study presenting reference ranges measured by LC-MS/MS as well (Eisenhofer et al. 2017). We do not include those steroids that we currently determine using conventional immunoassays or the numerous steroids that can be determined using GC-MS/MS methods (Hill et al. 2019)
General comments
Either serum or plasma may be used to determine steroid hormones. It has been mentioned that serum is more advantageous than plasma in light of the possible interference from small precipitates occurring in plasma during freezing and thawing. This possible interference is important mainly during measurements in small plasma volumes (0.05 ml) (Stanczyk 2006). In clinical practice, the use of urine or saliva can also be useful, while determinations in other samples such as cerebrospinal fluid, seminal plasma, hair, or tissues are rather the subject of research.
Reference interval in the 2.5-97.5 percentile range is defined by concentration values of the given analyte in a defined population (i.e. a cohort of reference individuals defined as being healthy) (Racek et al. 2006). Each laboratory should determine reference values for the steroids they measure, as only such reference range enable a proper interpretation of results.
Steroid levels change physiologically with age (over years) as well as with reproductive function status (over months to years) and the menstrual cycle (over weeks), but also with seasonal and daily biorhythms. All these aspects cannot be captured in reference ranges, and therefore it is necessary to take the day of the menstrual cycle and time of day into account when determining reference norms. Physiological changes to steroid levels mean that different ranges are necessary for women and men, for different age groups, and for the menstrual cycle phase in women.
Steroidal compounds circulate in the blood or are excreted mainly in the urine as esters of sulfuric acid – sulfates, glucuronic – glucuronides, or bound to other acids and lipids as so-called conjugates, or non-bound, non-conjugated, sometimes called somewhat imprecisely as “free”. The term “free” is used in other contexts, and rather refers to steroid compounds circulating in the blood non-bound to proteins. These proteins have varying binding affinities, with specific globulins having the highest, for instance, sex hormone-binding globulin (SHBG) for C18 C19 steroids with 17β-hydroxy-groups such as estradiol or testosterone, and cortisol binding globulin (CBG) for corticoids. Albumin has a low affinity for testosterone, while other proteins such as orosomucoid or CBG have higher. In light of the complex dynamics of steroids binding to proteins, plasma methods for determining free or biologically-active steroids are the subject of some criticism (Goldman et al. 2017).
Instrumental methods at the Institute of Endocrinology in Prague
The Department of steroid hormones and proteofactors at the Institute of Endocrinology has long studied steroid hormones including those that are less common (Stárka and Brabencová 1959, Stárka and Brabencová 1960, Stárka et al. 1962). Because of their nanomolar concentrations in the blood, until the end of the 1960s, the main steroid hormones were determined in the urine. One of the first papers in the world describing the determination of testosterone using a protein binding method was published by scientists from our unit (Hampl et al. 1970). Radioimmunology methods, currently in wide use for routine diagnostics, were supplemented in our laboratory by measurements using gas chromatography with mass spectrometry (GC-MS) mainly for research purposes, and we currently provide determinations for almost 100 steroids (Hill et al. 2000, Kancheva et al. 2007, Hill et al. 2010, Hill et al. 2019). We have recently introduced new methods using LC-MS/MS (Sosvorova et al. 2015a, Sosvorova et al. 2015b, Vítků et al. 2015, Vítků et al. 2016, Vítků et al. 2018).
In the following section, we provide actual information on the steroids measured in our laboratory using these liquid chromatography-mass spectrometry methods. Our first LC-MS/MS method was validated for measuring dehydroepiandrosterone (DHEA), 7α-hydroxy-dehydroepiandrosterone (7α-OH–DHEA), 7β-hydroxy-dehydroepiandrosterone (7β-OH-DHEA), 7-oxo-dehydroepiandrosterone (7-oxo-DHEA), 16αhydroxy-dehydroepiandrosterone (16α-OH-DHEA), cortisol and cortisone (Sosvorova et al. 2015a), and was then widened to include aldosterone (Sosvorova et al. 2015b). In that same year, we validated a second method for estrone (E1), estradiol (E2), and estriol (E3) (Vítků et al. 2015). Then, the first method was expanded to include an additional steroid spectrum of pregnenolone, 17α-hydroxy-pregnenolone, androstenedione, testosterone (T) and dihydrotestosterone (DHT) (Vítků et al. 2016), plus 7β-hydroxy-epiandrosterone (7β-OH-EpiA) (Vítků et al. 2018). At this point only for research purposes, the method to determine cortisone, 11-deoxycortisol, corticosterone, and 11-deoxycorticosterone was developed (Šimková 2018, Šimková, unpublished data).
For these methods, LC-MS/MS was performed using an API 3200 (Sciex, Concord, Canada) triple quadrupole mass spectrometer with electrospray ionization (ESI) connected to an Eksigent ultra-high performance liquid chromatography (UPLC) UltraLC 110 system (Redwood City, CA, USA). Chromatographic separation was carried out on various Kinetex C18 (150 x 3.0 mm) columns (Phenomenex, Torrance, CA, USA) with a corresponding security guard. Measurements are done using 500 ml of serum or plasma. Due to the very low concentrations of steroids in biological materials, derivatization is performed, which significantly increases the analytical sensitivity. Detection limits of the methods can be found in the appropriate published papers (Sosvorova et al. 2015a, Vítků et al. 2015, Vítků et al. 2016).
The main drawback of these methods is the complex and time-consuming sample preparation, including extraction with organic solvents, chemical derivatization, and several evaporation and dilution steps. The main advantage is the higher precision and ability to determine multiple steroids at once
Individual steroids
*Pregnenolone
*17α-hydroxy-pregnenolone
*Dehydroepiandrosterone (DHEA)
*Hydroxy-derivatives of DHEA
*Androstenedione
*Testosterone
*Estrone
*Estradiol
*Estriol
*Cortisol
*Cortisone
Conclusions
Steroid hormones are determined in most laboratories using immunoanalytic methods. Recently, measurements using gas or liquid chromatography combined with mass spectrometry have become more common for routine analyses. Our laboratory has a long history of analyzing a wide spectrum of steroid hormones, for both routine clinical use and for research purposes. We focus not just on physiological relationships, but also the connections and associations with various diseases. The aim of this work is to help provide orientation in the labyrinth of data on steroid hormones and their metabolites.
The determination of steroid hormones and subsequent interpretation of results is accompanied by a range of difficulties. The amount of information that current technology can provide on the circulating concentrations of more than a hundred various steroid compounds can lead to problems with interpretation. The aim of this study is to help provide orientation in this maze of data on steroid hormones. First, we focus on specific aspects arising from the pre-analytical phase of steroid determination that needs to be considered when planning sampling, whether for diagnostics or research. Then, we provide a brief summary of the characteristics and diagnostic relevance of several steroid hormones and/or their metabolites: pregnenolone, 17α-hydroxypregnenolone, dehydroepiandrosterone, hydroxyderivatives of dehydroepiandrosterone, androstenedione, testosterone, estrone, estradiol, estriol, cortisol, cortisone, which in our institute are determined with validated LC-MS/MS methods. For these steroids, we also provide newly calculated reference values in fertile women according to the phase of their menstrual cycle.
Introduction
In the early 50s, Zimmermann’s reaction for the determination of 17-ketosteroids and the analysis of corticosteroids as 17-hydroxycorticosteroids or 17-ketogenic steroids in urine was the only way to obtain any information on steroid excretion. Since the 1970s, the discovery of immunologic analysis of hormones in the blood using Rosalyn Yellow opened the way for steroid analysis in fluids other than urine and allowed the determination of very small concentrations (pmol/l of analytes). Immunoanalysis of individual steroids still prevails for diagnostic uses in clinical practice; however, sophisticated methods of tandem chromatography and mass spectrometry have found their place not only in steroid analysis for scientific purposes but also for the clinical diagnosis of various pathologies.
Instrumental analysis combining gas or liquid chromatography with mass spectrometry allows the simultaneous measurement of a large number of steroid metabolites and has higher specificity than immunoanalytic methods. Measured values are therefore often somewhat lower than from immuneanalyses. The choice of method depends on the requirements for precision, economic factors, and the ability to incorporate automatization into routine analyses. Routine diagnostic examinations and other research use also have different requirements. The range of information that current technologies deliver can often lead to data on more than a hundred circulating steroids, resulting in difficulties in interpretation. We know little about the roles of many hormones in various pathogenic disorders, and while we have certain information from experimental physiology, particularly for neuroactive steroids that act in the brain, we know almost nothing about their effects on health and disease. The aim of this work is to provide an orientation into the maze of data on steroid hormones and their metabolites. We provide a brief summary of the characteristics and diagnostic relevance of several steroid hormones and/or their metabolites that in our institute are determined with validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. For these steroids and methods, we also provide newly calculated reference ranges in fertile women (Table 1), which are in accordance with another study presenting reference ranges measured by LC-MS/MS as well (Eisenhofer et al. 2017). We do not include those steroids that we currently determine using conventional immunoassays or the numerous steroids that can be determined using GC-MS/MS methods (Hill et al. 2019)
General comments
Either serum or plasma may be used to determine steroid hormones. It has been mentioned that serum is more advantageous than plasma in light of the possible interference from small precipitates occurring in plasma during freezing and thawing. This possible interference is important mainly during measurements in small plasma volumes (0.05 ml) (Stanczyk 2006). In clinical practice, the use of urine or saliva can also be useful, while determinations in other samples such as cerebrospinal fluid, seminal plasma, hair, or tissues are rather the subject of research.
Reference interval in the 2.5-97.5 percentile range is defined by concentration values of the given analyte in a defined population (i.e. a cohort of reference individuals defined as being healthy) (Racek et al. 2006). Each laboratory should determine reference values for the steroids they measure, as only such reference range enable a proper interpretation of results.
Steroid levels change physiologically with age (over years) as well as with reproductive function status (over months to years) and the menstrual cycle (over weeks), but also with seasonal and daily biorhythms. All these aspects cannot be captured in reference ranges, and therefore it is necessary to take the day of the menstrual cycle and time of day into account when determining reference norms. Physiological changes to steroid levels mean that different ranges are necessary for women and men, for different age groups, and for the menstrual cycle phase in women.
Steroidal compounds circulate in the blood or are excreted mainly in the urine as esters of sulfuric acid – sulfates, glucuronic – glucuronides, or bound to other acids and lipids as so-called conjugates, or non-bound, non-conjugated, sometimes called somewhat imprecisely as “free”. The term “free” is used in other contexts, and rather refers to steroid compounds circulating in the blood non-bound to proteins. These proteins have varying binding affinities, with specific globulins having the highest, for instance, sex hormone-binding globulin (SHBG) for C18 C19 steroids with 17β-hydroxy-groups such as estradiol or testosterone, and cortisol binding globulin (CBG) for corticoids. Albumin has a low affinity for testosterone, while other proteins such as orosomucoid or CBG have higher. In light of the complex dynamics of steroids binding to proteins, plasma methods for determining free or biologically-active steroids are the subject of some criticism (Goldman et al. 2017).
Instrumental methods at the Institute of Endocrinology in Prague
The Department of steroid hormones and proteofactors at the Institute of Endocrinology has long studied steroid hormones including those that are less common (Stárka and Brabencová 1959, Stárka and Brabencová 1960, Stárka et al. 1962). Because of their nanomolar concentrations in the blood, until the end of the 1960s, the main steroid hormones were determined in the urine. One of the first papers in the world describing the determination of testosterone using a protein binding method was published by scientists from our unit (Hampl et al. 1970). Radioimmunology methods, currently in wide use for routine diagnostics, were supplemented in our laboratory by measurements using gas chromatography with mass spectrometry (GC-MS) mainly for research purposes, and we currently provide determinations for almost 100 steroids (Hill et al. 2000, Kancheva et al. 2007, Hill et al. 2010, Hill et al. 2019). We have recently introduced new methods using LC-MS/MS (Sosvorova et al. 2015a, Sosvorova et al. 2015b, Vítků et al. 2015, Vítků et al. 2016, Vítků et al. 2018).
In the following section, we provide actual information on the steroids measured in our laboratory using these liquid chromatography-mass spectrometry methods. Our first LC-MS/MS method was validated for measuring dehydroepiandrosterone (DHEA), 7α-hydroxy-dehydroepiandrosterone (7α-OH–DHEA), 7β-hydroxy-dehydroepiandrosterone (7β-OH-DHEA), 7-oxo-dehydroepiandrosterone (7-oxo-DHEA), 16αhydroxy-dehydroepiandrosterone (16α-OH-DHEA), cortisol and cortisone (Sosvorova et al. 2015a), and was then widened to include aldosterone (Sosvorova et al. 2015b). In that same year, we validated a second method for estrone (E1), estradiol (E2), and estriol (E3) (Vítků et al. 2015). Then, the first method was expanded to include an additional steroid spectrum of pregnenolone, 17α-hydroxy-pregnenolone, androstenedione, testosterone (T) and dihydrotestosterone (DHT) (Vítků et al. 2016), plus 7β-hydroxy-epiandrosterone (7β-OH-EpiA) (Vítků et al. 2018). At this point only for research purposes, the method to determine cortisone, 11-deoxycortisol, corticosterone, and 11-deoxycorticosterone was developed (Šimková 2018, Šimková, unpublished data).
For these methods, LC-MS/MS was performed using an API 3200 (Sciex, Concord, Canada) triple quadrupole mass spectrometer with electrospray ionization (ESI) connected to an Eksigent ultra-high performance liquid chromatography (UPLC) UltraLC 110 system (Redwood City, CA, USA). Chromatographic separation was carried out on various Kinetex C18 (150 x 3.0 mm) columns (Phenomenex, Torrance, CA, USA) with a corresponding security guard. Measurements are done using 500 ml of serum or plasma. Due to the very low concentrations of steroids in biological materials, derivatization is performed, which significantly increases the analytical sensitivity. Detection limits of the methods can be found in the appropriate published papers (Sosvorova et al. 2015a, Vítků et al. 2015, Vítků et al. 2016).
The main drawback of these methods is the complex and time-consuming sample preparation, including extraction with organic solvents, chemical derivatization, and several evaporation and dilution steps. The main advantage is the higher precision and ability to determine multiple steroids at once
Individual steroids
*Pregnenolone
*17α-hydroxy-pregnenolone
*Dehydroepiandrosterone (DHEA)
*Hydroxy-derivatives of DHEA
*Androstenedione
*Testosterone
*Estrone
*Estradiol
*Estriol
*Cortisol
*Cortisone
Conclusions
Steroid hormones are determined in most laboratories using immunoanalytic methods. Recently, measurements using gas or liquid chromatography combined with mass spectrometry have become more common for routine analyses. Our laboratory has a long history of analyzing a wide spectrum of steroid hormones, for both routine clinical use and for research purposes. We focus not just on physiological relationships, but also the connections and associations with various diseases. The aim of this work is to help provide orientation in the labyrinth of data on steroid hormones and their metabolites.
