scholarly journals The Potential of Steroid Profiling by Mass Spectrometry in the Management of Adrenocortical Carcinoma

Biomedicines ◽  
2020 ◽  
Vol 8 (9) ◽  
pp. 314 ◽  
Author(s):  
Claudia Rossi ◽  
Ilaria Cicalini ◽  
Sara Verrocchio ◽  
Giulia Di Dalmazi ◽  
Luca Federici ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Adrenocortical Carcinoma ◽  
Molecular Genetic ◽  
High Specificity ◽  
Adrenocortical Adenomas ◽  
Specificity And Sensitivity ◽  
Steroid Profiling ◽  
Clinical Dilemma ◽  
Work Up

Radiological and endocrinological work up of adrenal neoplasms is aimed at distinguishing between frequent non-functioning adenomas and rare but very aggressive adrenocortical carcinoma (ACC). Relevant research has addressed the identification of molecular, genetic and hormonal markers that could have clinical significance for malignancy, as well as a prognostic value. Regarding endocrine aspects, attention has been paid to the pattern of steroid secretion that can be affected by altered steroidogenic pathway in ACC. The advent of mass spectrometry techniques has overcome many limitations usually associated with immunoassays, allowing the determination of both common and rarely measured steroids in a single analysis with high specificity and sensitivity. Indeed, mass spectrometry strategies may be able to identify an individualized steroid profile of ACC, allowing a rapid diagnosis and a specific follow-up. In this review, insights, strengths and limitations of mass spectrometry-based approaches in steroid profiling, as well as of immunoassay in steroid measurements, will be specifically discussed. Moreover, the latest findings on steroid profiling by mass spectrometry-based techniques, the most promising analytical tool, will be summarized to evaluate if steroid profiling might be the clue for solving the clinical dilemma in differentiating ACC from non-functioning adrenocortical adenomas (ACA).

scholarly journals Comparison of Strategies for the Determination of Sterol Sulfates via GC-MS Leading to a Novel Deconjugation-Derivatization Protocol

Molecules ◽  
2019 ◽  
Vol 24 (13) ◽  
pp. 2353 ◽  
Author(s):  
Julia Junker ◽  
Isabelle Chong ◽  
Frits Kamp ◽  
Harald Steiner ◽  
Martin Giera ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography Mass Spectrometry ◽  
Liquid Chromatography Mass Spectrometry ◽  
Chemical Cleavage ◽  
Advantages And Disadvantages ◽  
Chromatography Mass Spectrometry ◽  
Steroid Profiling ◽  
Health And Disease ◽  
Indispensable Tool

Sulfoconjugates of sterols play important roles as neurosteroids, neurotransmitters, and ion channel ligands in health and disease. In most cases, sterol conjugate analysis is performed with liquid chromatography-mass spectrometry. This is a valuable tool for routine analytics with the advantage of direct sterol sulfates analysis without previous cleavage and/or derivatization. The complementary technique gas chromatography-mass spectrometry (GC-MS) is a preeminent discovery tool in the field of sterolomics, but the analysis of sterol sulfates is hampered by mandatory deconjugation and derivatization. Despite the difficulties in sample workup, GC-MS is an indispensable tool for untargeted analysis and steroid profiling. There are no general sample preparation protocols for sterol sulfate analysis using GC-MS. In this study we present a reinvestigation and evaluation of different deconjugation and derivatization procedures with a set of representative sterol sulfates. The advantages and disadvantages of trimethylsilyl (TMS), methyloxime-trimethylsilyl (MO-TMS), and trifluoroacetyl (TFA) derivatives were examined. Different published procedures of sterol sulfate deconjugation, including enzymatic and chemical cleavage, were reinvestigated and examined for diverse sterol sulfates. Finally, we present a new protocol for the chemical cleavage of sterol sulfates, allowing for simultaneous deconjugation and derivatization, simplifying GC-MS based sterol sulfate analysis.

scholarly journals Microalbuminuria and borderline-increased albumin excretion determined with a centrifugal analyzer and the Albumin Blue 580 fluorescence assay

1997 ◽  
Vol 43 (6) ◽  
pp. 996-1002 ◽  
Author(s):  
Manfred A Kessler ◽  
Andreas Meinitzer ◽  
Walter Petek ◽  
Otto S Wolfbeis
Keyword(s):  
Calibration Curve ◽  
Emission Intensity ◽  
Clinical Laboratory ◽  
Detection Limits ◽  
High Specificity ◽  
Fluorescence Assay ◽  
Urine Samples ◽  
Albumin Concentration ◽  
Specificity And Sensitivity

Abstract We report a new automated fluorescence assay for determination of albumin in urine. The dye Albumin Blue 580 specifically binds to albumin with exhibition of strong red fluorescence. The albumin concentration is calculated from emission intensity at 616 nm (excitation at 590 nm) and a calibration curve. Two Cobas Fara programs cover working ranges of 2–200 and 1–50 mg/L with detection limits of 1.4 and 0.4 mg/L, respectively. Within-run CVs (n = 10) ranged from 1.7% (189 mg/L) to 8.9% (7.2 mg/L) for 2–200 mg/L and from 2.9% (43.3 mg/L) to 5.7% (2.3 mg/L) for the 1–50 mg/L range. A test of urine samples (n = 100) submitted to routine analysis gave results that agreed well with those by the Behring nephelometric assay: AB 580 = 0.922 (± 0.010) BNA + 4.16 (± 0.78). No interference was detected from other urine components, including several proteins and 46 drugs. The high specificity and sensitivity make the method ideal for determination of microalbuminuria. In addition, the method is fast, inexpensive, and well-suited for clinical laboratory application and thus may be used instead of immunoassays.

scholarly journals MANAGEMENT OF ENDOCRINE DISEASE Hyperandrogenic states in women: pitfalls in laboratory diagnosis

2018 ◽  
Vol 178 (4) ◽  
pp. R141-R154 ◽  
Author(s):  
Michel Pugeat ◽  
Ingrid Plotton ◽  
Aude Brac de la Perrière ◽  
Gérald Raverot ◽  
Henri Déchaud ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Limit Of Detection ◽  
High Specificity ◽  
Free Testosterone ◽  
Laboratory Diagnosis ◽  
Sex Hormone Binding Globulin ◽  
Total Testosterone ◽  
Adrenal Androgen ◽  
Androgen Excess ◽  
Specificity And Sensitivity

Measuring total testosterone level is the first-line approach in assessing androgen excess in women. The main pitfalls in measuring testosterone relate to its low concentration and to the structural similarity between circulating androgens and testosterone, requiring accurate techniques with high specificity and sensitivity. These goals can be achieved by immunoassay using a specific anti-testosterone monoclonal antibody, ideally after an extraction step. Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) will be commonly used for measuring testosterone, providing optimal accuracy with a low limit of detection. Yet, the pitfalls of these two techniques are well identified and must be recognized and systematically addressed. In general, laboratories using direct testosterone immunoassay and mass spectrometry need to operate within a quality framework and be actively engaged in external quality control processes and standardization, so as to ensure appropriate interpretation irrespective of the particular laboratory. Circulating testosterone is strongly bound to sex-hormone-binding globulin (SHBG), and SHBG levels are typically low in overweight hyperandrogenic patients. Thus, low SHBG may decrease circulating testosterone to normal values, which will mask androgen excess status. One way to avoid this pitfall, awaiting direct free testosterone assays that are yet to be developed, is to measure SHBG and calculate free testosterone. A few other pitfalls will be discussed in this review, including those of adrenal androgen exploration, with the aim of helping clinicians to better handle laboratory investigation of androgen excess disorders in women.

scholarly journals Urine Steroid Metabolomics as a Novel Tool for Detection of Recurrent Adrenocortical Carcinoma

2019 ◽  
Vol 105 (3) ◽  
pp. e307-e318 ◽  
Author(s):  
Vasileios Chortis ◽  
Irina Bancos ◽  
Thomas Nijman ◽  
Lorna C Gilligan ◽  
Angela E Taylor ◽  
...  
Keyword(s):  
Machine Learning ◽  
Mass Spectrometry ◽  
Adrenocortical Carcinoma ◽  
Postoperative Recurrence ◽  
Urine Samples ◽  
Gas Chromatography Mass Spectrometry ◽  
R0 Resection ◽  
Steroid Profiling ◽  
Steroid Profiles ◽  
Urine Steroid

Abstract Context Urine steroid metabolomics, combining mass spectrometry-based steroid profiling and machine learning, has been described as a novel diagnostic tool for detection of adrenocortical carcinoma (ACC). Objective, Design, Setting This proof-of-concept study evaluated the performance of urine steroid metabolomics as a tool for postoperative recurrence detection after microscopically complete (R0) resection of ACC. Patients and Methods 135 patients from 14 clinical centers provided postoperative urine samples, which were analyzed by gas chromatography–mass spectrometry. We assessed the utility of these urine steroid profiles in detecting ACC recurrence, either when interpreted by expert clinicians or when analyzed by random forest, a machine learning-based classifier. Radiological recurrence detection served as the reference standard. Results Imaging detected recurrent disease in 42 of 135 patients; 32 had provided pre- and post-recurrence urine samples. 39 patients remained disease-free for ≥3 years. The urine “steroid fingerprint” at recurrence resembled that observed before R0 resection in the majority of cases. Review of longitudinally collected urine steroid profiles by 3 blinded experts detected recurrence by the time of radiological diagnosis in 50% to 72% of cases, improving to 69% to 92%, if a preoperative urine steroid result was available. Recurrence detection by steroid profiling preceded detection by imaging by more than 2 months in 22% to 39% of patients. Specificities varied considerably, ranging from 61% to 97%. The computational classifier detected ACC recurrence with superior accuracy (sensitivity = specificity = 81%). Conclusion Urine steroid metabolomics is a promising tool for postoperative recurrence detection in ACC; availability of a preoperative urine considerably improves the ability to detect ACC recurrence.

scholarly journals Optimizing the fragment complementation of APEX2 for detection of specific protein-protein interactions in live cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Miaomiao Xue ◽  
Junjie Hou ◽  
Linlin Wang ◽  
Dongwan Cheng ◽  
Jingze Lu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
High Specificity ◽  
Mass Spectrometry Analysis ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Hek 293 ◽  
Spectrometry Analysis ◽  
Specificity And Sensitivity ◽  
Fragment Complementation

Abstract Dynamic protein-protein interactions (PPIs) play crucial roles in cell physiological processes. The protein-fragment complementation (PFC) assay has been developed as a powerful approach for the detection of PPIs, but its potential for identifying protein interacting regions is not optimized. Recently, an ascorbate peroxidase (APEX2)-based proximity-tagging method combined with mass spectrometry was developed to identify potential protein interactions in live cells. In this study, we tested whether APEX2 could be employed for PFC. By screening split APEX2 pairs attached to FK506-binding protein 12 (FKBP) and the FKBP12-rapamycin binding (FRB) domain, which interact with each other only in the presence of rapamycin, we successfully obtained an optimized pair for visualizing the interaction between FRB and FKBP12 with high specificity and sensitivity in live cells. The robustness of this APEX2 pair was confirmed by its application toward detecting the STIM1 and Orial1 homodimers in HEK-293 cells. With a subsequent mass spectrometry analysis, we obtained five different biotinylated sites that were localized to the known interaction region on STIM1 and were only detected when the homodimer formed. These results suggest that our PFC pair of APEX2 provides a potential tool for detecting PPIs and identifying binding regions with high specificity in live cells.

scholarly journals Discovery of protein modifications using high resolution differential mass spectrometry proteomics

2020 ◽  
Author(s):  
Paolo Cifani ◽  
Zhi Li ◽  
Danmeng Luo ◽  
Mark Grivainis ◽  
Andrew M. Intlekofer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Open Source ◽  
High Resolution Mass Spectrometry ◽  
Peptide Identification ◽  
High Specificity ◽  
Functional Protein ◽  
Protein Modifications ◽  
Specificity And Sensitivity ◽  
Spectral Alignment

SummaryRecent studies have revealed diverse amino acid, post-translational and non-canonical modifications of proteins in diverse organisms and tissues. However, their unbiased detection and analysis remain hindered by technical limitations. Here, we present a spectral alignment method for the identification of protein modifications using high-resolution mass spectrometry proteomics. Termed SAMPEI for Spectral Alignment-based Modified PEptide Identification, this open-source algorithm is designed for the discovery of functional protein and peptide signaling modifications, without prior knowledge of their identities. Using synthetic standards and controlled chemical labeling experiments, we demonstrate its high specificity and sensitivity for the discovery of sub-stoichiometric protein modifications in complex cellular extracts. SAMPEI mapping of mouse macrophage differentiation revealed diverse post-translational protein modifications, including distinct forms of cysteine itaconatylation. SAMPEI’s robust parameterization and versatility are expected to facilitate the discovery of biological modifications of diverse macromolecules. SAMPEI is implemented as a Python package, and is available open-source from BioConda and GitHub (https://github.com/FenyoLab/SAMPEI).

Diagnostic value of urine organic acids analysis among patients with primary mitochondrial disorders

2021 ◽  
pp. 13-24
Author(s):  
Т.Д. Крылова ◽  
М.В. Куркина ◽  
П.В. Баранова ◽  
Е.Ю. Пыркова ◽  
П.Г. Цыганкова ◽  
...  
Keyword(s):  
Organic Acids ◽  
Molecular Genetic ◽  
High Specificity ◽  
Diagnostic Value ◽  
Mitochondrial Disorders ◽  
Test Methods ◽  
Molecular Genetic Analysis ◽  
Defective Structure ◽  
Specificity And Sensitivity ◽  
Urine Organic Acids

Первичные митохондриальные заболевания (ПМЗ) - генетически и клинически гетерогенные заболевания, характеризующиеся нарушением структуры или функций системы окислительного фосфорилирования (OXPHOS), включая электрон-транспортную цепь. Несмотря на успешное применение методов секвенирования нового поколения в диагностике наследственных заболеваний в последнее десятилетие, существует ряд объективных трудностей в интерпретации результатов, особенно при обнаружении новых генов или новых вариантов нуклеотидной последовательности. Анализ биомаркеров, которые являются индикаторами нарушения функций митохондрий, является важным этапом в диагностике многих ПМЗ. Целью данной работы было проведение анализа спектра и концентраций 72 органических кислот в моче методом газовой хроматографии с масс-спектрометнией (ГХ-МС,7890А/5975С, Agilent Technologies, США) в выборке из 84 пациентов с подтвержденным молекулярно-генетическими методами диагнозом ПМЗ и оценка их диагностической значимости. Среди 84 пациентов с ПМЗ, отклонения в спектре органических кислот были выявлены в 78% (66/84) случаев. Уникальный спектр органических кислот наблюдался при митохондриальных гепатопатиях, связанных с мутациями в гене DGUOK: наравне с повышением уровня лактата, пирувата, 3-гидроксибутирата было выявлено повышение концентрации 4-гидроксифениллактата, 4-гидроксифенилпирувата. При анализе ROC-кривых было показано, что диагностическая значимость маркеров убывает в ряду: 3-гидроксибутират, лактат, пируват. При проведении оценки достоверности теста показано, что повышение концентраций пирувата и 4-гидроксифениллактата может быть принято во внимание при предположении ПМЗ у пациента. Introduction. Primary mitochondrial disorders (PMD) are a group of clinically and genetically heterogeneous group of diseases characterized by a defective structure and functions of the Oxidative Phosphorylation System (OXPHOS). Despite the advantages of the next generation sequencing, diagnosis of PMD is still challenging. There is no currently available biomarker with high specificity and sensitivity. But the level of metabolites reflecting the defective OXPHOS is needed for making of a diagnosis of PMD. Aim: to reveal the level and spectrum of urine organic acids among patients with confirmed diagnosis (by molecular-genetic analysis) of PMD and to estimate the diagnostic value of the test. Methods. We measured 72 different metabolites in 84 urine samples from patients with PMD by GC-MS (7890А/5975С, Agilent Technologies, USA). Results. In 66/84 cases among the patients, we detected the abnormal level of urine organic acids. We observed a unique spectrum of metabolites in the patients with DGUOK-associated hepatopathy (abnormal levels of lactate, pyruvate, 3-hydroxybutyrate, and at the same time 4-hydroxyphenyllactate and 4-hydroxyphenylpyruvate). Using ROC-analysis one of the most informative biomarkers was 3-hydroxybutyrate. But due to the lack of specificity, it could not be classified as a valuable biomarker for PMD. The high level of pyruvate and 4-hydroxyphenyllactate could be taken into account to make a diagnosis of PMD

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