Dr. Ron’s Research Review – July 10, 2019

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This week’s research review focuses on urinary hormone testing

Hormone testing methods include first and second generation RIA (radioimmunoassay), LC-MS/MS (liquid chromatography tandem mass spectrometry) and GC-MS/MS (gas chromatography tandem mass spectrometry).
LC-MS/MS is considered the gold standard for accuracy. GC-MS/MS is less expensive and more widely used. Both have advantages, and are considered complimentary.
Measurement accuracy declines at the upper and lower limits. Testosterone and estradiol measurements are susceptible to accuracy issues at low levels.
Sample collections include serum, plasma, urine (24-hour, spot, dried), blood spot, saliva, etc. Each has advantages and disadvantages.
Many hormones are pulsatile (usually dependent on LH). 24-hour urine measures the total hormone output for the day. The larger amount may minimize accuracy issues.

Advantages of LC-MS/MS as an analytical technique include high specificity, possibility to simultaneously measure multiple analytes, and the ability to assess the specificity of the analysis in every sample. (Kushnir et al., 2010)
Mass spectrometry is considered the gold-standard method for testosterone measurement; however, due to its complexity and cost, it has not been widely adopted. (Kanakis et al., 2019)
GC/MS is excellent at providing an integrated picture of a person's steroid metabolome, or steroidome. However, in the end, both LC and GC will likely remain complementary and both should be available in advanced analytical laboratories. (Shackleton et al., 2018)
Highest specificity can be obtained using GC-MS, a sophisticated but most powerful tool for characterizing steroid metabolomes. LC-MS is a true high throughput technique and highly suited for detecting complex steroids. GC-MS and LC-MS are not competing but complementary techniques. (Wudy et al., 2018)
Urine, compared to other biofluids, is characterized by its ease of collection, richness in metabolites and its ability to reflect imbalances of all biochemical pathways within the body. (Khamis et al., 2017)
Urine often contains not only the original hormone but also key metabolites that may or may not have bio­ logic activity. The 24­ hour urine specimen is used for many endocrine tests. Such urine specimens represent a time average that integrates over the multiple pulsatile spikes of hormone secretion occurring throughout the day. The 24­ hour urine specimen also has the advantage of better analytic sensitivity for some hormones and metabolites. (Sluss and Hayes, 2016)
Liquid chromatography linked with tandem MS (LC-MS/MS) allows for rapid as well as highly specific and sensitive targeted steroid hormone analysis of multiple analytes from a single sample. Urinary steroid profile analysis by gas chromatography (GC)-MS is a non-invasive diagnostic approach and provides qualitative and quantitative data on the global excretion of steroid hormone metabolites. GC-MS remains the most powerful discovery tool for defining inborn errors of steroidogenesis, whereas LC-MS/MS represents a highly sensitive and specific method for targeted steroid hormone analysis. (Kamrath et al., 2014)
Liquid chromatography-tandem mass spectrometry (LC/MSMS) is considered the “gold standard” for measuring testosterone. (Demers, 2008)

 


Articles

 

Testosterone and estradiol assays: current and future trends.
            (Demers, 2008)  Download
Sex steroid measurements for the investigation of endocrine disorders have been fraught with accuracy and imprecision problems since the advent of high throughput, direct assays almost 10 years ago on automated analyzers. Results from testosterone and estradiol measurements at the low end of detectability have suffered the most and there are few automated systems that can accurately measure these steroids in women, children and hypogonadal males on a routine basis. With the advent of mass spectrometry coupled to either gas chromatography or liquid chromatography, an improved approach to the measurement of these steroids has developed that shows promise for accurately and precisely measuring testosterone and estradiol in all patient populations including women and children. These mass spectrometry based methods for the sex steroids have been established as higher order reference method procedures that will resolve the issues of low end sensitivity measurements for these steroids, provide for appropriate standardization and reference materials and align most laboratories in hospital and reference laboratories to generate results that are inter-changeable between laboratories and methods.

Steroid biochemistry.
            (Kamrath et al., 2014) Download
Accurate analysis of steroid hormones represents an essential part in the evaluation of a patient with disorders or differences in sex development. Analytical methods based on mass spectrometry (MS) have become the state-of-the-art methodology allowing for the most specific qualitative and quantitative determination of steroid hormones and their metabolites. Liquid chromatography linked with tandem MS (LC-MS/MS) allows for rapid as well as highly specific and sensitive targeted steroid hormone analysis of multiple analytes from a single sample. Urinary steroid profile analysis by gas chromatography (GC)-MS is a non-invasive diagnostic approach and provides qualitative and quantitative data on the global excretion of steroid hormone metabolites. GC-MS remains the most powerful discovery tool for defining inborn errors of steroidogenesis, whereas LC-MS/MS represents a highly sensitive and specific method for targeted steroid hormone analysis.


 

Measuring testosterone in women and men.
            (Kanakis et al., 2019)  Download
Measurement of serum testosterone (T) level is of utmost importance for the evaluation of hypogonadism in men and androgen excess in women. Despite the advances in steroid hormone assessment, substantial variability exists regarding measurement of T concentrations. Several factors affect T measurement in men, including circadian rhythms, intra-individual daily variability and transient stressors, while T concentrations in women vary mainly according to the phase of the menstrual cycle. Most of the available immunoassays lack the required accuracy when dealing with T concentrations at the lower end of the normal range for men and across the entire range for females. Consequently, there is no universally accepted lower T threshold for healthy adult men and most immunoassays fail to detect states of mild androgen excess in women. Mass spectrometry is considered the gold-standard method for T measurement; however, due to its complexity and cost, it has not been widely adopted. To increase accuracy, T in men should be measured with a fasting morning sample and repeated if the level is found to be low; in women, measurement must be performed at the follicular phase of the cycle. In both cases, borderline results may be clarified by the assessment of free testosterone (fT). Since most fT assays are unreliable, calculated surrogates should be used instead. Collaborative efforts have been undertaken, with rigorous internal and external quality controls and the establishment of reference methods, to harmonise the commercial assays.

Mass spectrometric based approaches in urine metabolomics and biomarker discovery.
            (Khamis et al., 2017)  Download
Urine metabolomics has recently emerged as a prominent field for the discovery of non-invasive biomarkers that can detect subtle metabolic discrepancies in response to a specific disease or therapeutic intervention. Urine, compared to other biofluids, is characterized by its ease of collection, richness in metabolites and its ability to reflect imbalances of all biochemical pathways within the body. Following urine collection for metabolomic analysis, samples must be immediately frozen to quench any biogenic and/or non-biogenic chemical reactions. According to the aim of the experiment; sample preparation can vary from simple procedures such as filtration to more specific extraction protocols such as liquid-liquid extraction. Due to the lack of comprehensive studies on urine metabolome stability, higher storage temperatures (i.e. 4°C) and repetitive freeze-thaw cycles should be avoided. To date, among all analytical techniques, mass spectrometry (MS) provides the best sensitivity, selectivity and identification capabilities to analyze the majority of the metabolite composition in the urine. Combined with the qualitative and quantitative capabilities of MS, and due to the continuous improvements in its related technologies (i.e. ultra high-performance liquid chromatography [UPLC] and hydrophilic interaction liquid chromatography [HILIC]), liquid chromatography (LC)-MS is unequivocally the most utilized and the most informative analytical tool employed in urine metabolomics. Furthermore, differential isotope tagging techniques has provided a solution to ion suppression from urine matrix thus allowing for quantitative analysis. In addition to LC-MS, other MS-based technologies have been utilized in urine metabolomics. These include direct injection (infusion)-MS, capillary electrophoresis-MS and gas chromatography-MS. In this article, the current progresses of different MS-based techniques in exploring the urine metabolome as well as the recent findings in providing potentially diagnostic urinary biomarkers are discussed. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:115-134, 2017.

Liquid chromatography-tandem mass spectrometry applications in endocrinology.
            (Kushnir et al., 2010)  Download
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been recognized as a primary methodology for the accurate analysis of endogenous steroid hormones in biological samples. This review focuses on the use of LC-MS/MS in clinical laboratories to assist with the diagnosis of diverse groups of endocrine and metabolic diseases. Described analytical methods use on-line and off-line sample preparation and analytical derivatization to enhance analytical sensitivity, specificity, and clinical utility. Advantages of LC-MS/MS as an analytical technique include high specificity, possibility to simultaneously measure multiple analytes, and the ability to assess the specificity of the analysis in every sample. All described analytical methods were extensively validated, utilized in routine diagnostic practice, and were applied in a number of clinical and epidemiological studies, including a study of the steroidogenesis in ovarian follicles.

GC/MS in Recent Years Has Defined the Normal and Clinically Disordered Steroidome: Will It Soon Be Surpassed by LC/Tandem MS in This Role
            (Shackleton et al., 2018)  Download
Gas chromatography/mass spectrometry (GC/MS) has been used for steroid analysis since the 1960s. The advent of protective derivatization, capillary columns, and inexpensive electron ionization bench-top single quadrupole soon made it the method of choice for studying disorders of steroid synthesis and metabolism. However, the lengthy sample workup prevented GC/MS from becoming routine for steroid hormone measurement, which was dominated by radioimmunoassay. It was the emergence of liquid chromatography/tandem MS (LC/MS/MS) that sparked a renewed interest in GC/MS for the multicomponent analysis of steroids. GC/MS is excellent at providing an integrated picture of a person's steroid metabolome, or steroidome, as we term it. We review the recent work on newly described disorders and discuss the technical advances such as GC coupling to triple quadrupole and ion trap analyzers, two-dimensional GC/MS, and alternative ionization and detection systems such as atmospheric pressure chemical ionization (APCI) and time of flight. We believe that no novel GC/MS-based technique has the power of GC(electron ionization)/MS/MS as a "discovery tool," although APCI might provide ultimate sensitivity, which might be required in tissue steroidomics. Finally, we discuss the role of LC/MS/MS in steroidomics. This remains a challenge but offers shorter analysis times and advantages in the detection and discovery of steroids with a known structure. We describe recent advances in LC/MS steroidomics of hydrolyzed and intact steroid conjugates and suggest the technique is catching up with GC/MS in this area. However, in the end, both techniques will likely remain complementary and both should be available in advanced analytical laboratories.

Laboratory Techniques for Recognition of Endocrine Disorders
            (Sluss and Hayes, 2016) Download
The practice of endocrinology relies heavily on accurate laboratory measurements. Small changes in hormone levels, biomarkers, or molecular markers are often more specific and earlier indicators of disease than the appearance of physical symptoms. Urine often contains not only the original hormone but also key metabolites that may or may not have bio­ logic activity. The 24­ hour urine specimen is used for many endocrine tests. Such urine specimens represent a time average that integrates over the multiple pulsatile spikes of hormone secretion occurring throughout the day. The 24­ hour urine specimen also has the advantage of better analytic sensitivity for some hormones and metabolites.

 


References

Demers, LM (2008), ‘Testosterone and estradiol assays: current and future trends.’, Steroids, 73 (13), 1333-38. PubMed: 18565562
Kamrath, C, SA Wudy, and N Krone (2014), ‘Steroid biochemistry.’, Endocr Dev, 27 41-52. PubMed: 25247643
Kanakis, GA, CP Tsametis, and DG Goulis (2019), ‘Measuring testosterone in women and men.’, Maturitas, 125 41-44. PubMed: 31133215
Khamis, MM, DJ Adamko, and A El-Aneed (2017), ‘Mass spectrometric based approaches in urine metabolomics and biomarker discovery.’, Mass Spectrom Rev, 36 (2), 115-34. PubMed: 25881008
Kushnir, MM, AL Rockwood, and J Bergquist (2010), ‘Liquid chromatography-tandem mass spectrometry applications in endocrinology.’, Mass Spectrom Rev, 29 (3), 480-502. PubMed: 19708015
Shackleton, C, OJ Pozo, and J Marcos (2018), ‘GC/MS in Recent Years Has Defined the Normal and Clinically Disordered Steroidome: Will It Soon Be Surpassed by LC/Tandem MS in This Role’, J Endocr Soc, 2 (8), 974-96. PubMed: 30094411
Sluss, PM and FJ Hayes (2016), ‘Laboratory Techniques for Recognition of Endocrine Disorders’, Williams Textbook of Endocrinology (77-107.
Wudy, SA, et al. (2018), ‘The art of measuring steroids: Principles and practice of current hormonal steroid analysis.’, J Steroid Biochem Mol Biol, 179 88-103. PubMed: 28962971