1,720,985 research outputs found
Salting-Out Assisted Liquid-Liquid Extraction for UPLC-MS/MS Determination of Thyroxine and Steroid Hormones in Human Serum and Fish Plasma
Get PDFMeasuring the level of steroid and thyroxine hormones is key to understanding organism health conditions. Liquid chromatography coupled with tandem mass spectrometry has become the method of choice for such hormone analyses in clinical laboratories. Detection of hormones at low levels typically requires a time-consuming sample preparation, such as liquid-liquid extraction followed by solvent evaporation and re-solubilization of the sample extract. Instead, we applied salting-out assisted liquid-liquid extraction (SALLE) for the extraction of thyroxine, testosterone, cortisone, and cortisol from human serum and fish plasma samples. SALLE allowed direct injection of sample extracts. Sodium chloride and ammonium sulfate were evaluated as salting-out reagents together with four different organic solvents. High extraction recovery and reduced matrix interference were achieved by using ammonium sulfate together with 10% methanol in acetonitrile. Limits of quantification were in the range of 0.1–0.2 ng/mL and signal responses were linear (R2 > 0.997) up to at least 100 ng/mL for all hormones. The method was applied for hormone measurements in fish plasma. In conclusion, SALLE combines the simplicity of crude protein precipitation with the high analyte enrichment of a liquid-liquid extraction. Here we have presented it as a novel sample preparation method for clinical laboratories where mass spectrometry is utilized in the field of endocrinology
Utvikling og optimalisering av LC/MS-MS metode for kvantifisering av 11-ketotestosteron i plasma fra atlantisk laks.
Get PDFEn studie ble gjennomført for å optimalisere et derivatiseringsreagens for derivatisering av 11-ketotestosteron (11-KT), som ble analysert ved bruk av væskekromatografi – koblet til massespektrometri (LC/MS-MS). 11-KT er en viktig biomarkør for tidlig kjønnsmodning hos fisk, og en nøyaktig måling av denne biomarkøren er essensiell for fiskehelse og oppdrett.
Studien fokuserte på optimaliseringsprosessen for å indentifisere det mest effektive derivatiseringsreagenset som kunne tilsettes direkte i toppfasen under SALLE -metoden. SALLE – metoden ble valgt som prøveopparbeidelsestrinn på grunn av sin effektivitet i å isolere og oppkonsentrere 11-KT fra komplekse matrikser, noe som forbedrer påvisningsgrensen og kvantifiseringen av biomarkøren.
Den påvisbare grensen (LOD) for 11-KT ble bestemt til å være 0,14 ng/mL, mens kvantifiseringsgrensen (LOQ) ble funnet ved 0,31 ng/mL. Recovery av 11-KT ble målt ved spiket kalveserum og er 95,8 %. Repeterbarhet ligger på 1,9% og reproduserbarhet ble også evaluert og ligger på 5,9%
Exploring potential analytical methods for determination of glycerol-3-phosphate in human plasma
Get PDFChronic kidney disease (CKD) is a progressive condition where the kidneys gradually lose their ability to function properly. Biomarkers that indicate immediate renal tissue injury is needed for improved detection of early CKD. One potential biomarker is glycerol-3-phosphate (G3P), a small organic ion which recently has been inked to kidney injury through regulation of fibroblast growth factor 23 (FGF23). The aim of this study was to explore potential analytical methods for determination of G3P in human plasma. G3P was retained and analyzed on a hydrophilic interaction liquid chromatography (HILIC) column using liquid chromatography-mass spectrometry (LC-MS). Further, derivatization with alcohols through an esterification reaction was possible, which offered retention using reversed phase (RP) LC. However, the reaction conditions potentially lead to phospholipid hydrolysis, which increased the amount of G3P. Further research could explore the possibilities of phospholipid extraction through liquid-liquid extraction (LLE) or solid phase extraction (SPE)
Determination of eleven steroid hormones and bioavailable testosterone in human serum using salting-out assisted liquid-liquid extraction with UHPLC-MS/MS
Get PDFSteroid hormones are essential in various biological roles such as the regulation of sexual maturation, fertility, bone and muscle growth, stress-mediated responses, and inflammation, to name a few examples. Impaired steroidogenesis leads to anomalous steroid hormone concentrations and develops into endocrinological disorders, such as polycystic ovarian syndrome (PCOS) and hypogonadism, with physical and mental manifestations affecting life quality and possibly longevity. The detection of aberrant steroid concentrations is crucial for diagnosis and requires adequate biochemical assays to provide correct and reliable quantitation. Ultra-high-performance liquid chromatography (UHPLC-MS/MS) provides greater selectivity and sensitivity for endocrine analysis compared to modern direct immunoassays.
This study optimized and validated an UHPLC-MS/MS method using salting-out assisted liquid-liquid extraction (SALLE) for sample clean-up and analyte extraction of 11 steroid hormones from human serum. The analyte panel includes glucocorticoids (cortisol, cortisone, corticosterone, deoxycorticosterone, 11-deoxycortisol), progestogens (progesterone, 17α-hydroxyprogesterone) and androgens (total testosterone, bioavailable testosterone, androstenedione, 11-ketotestosterone, 11β-hydroxyandrostenedione). Additionally, the study involved the analysis of 38 sera samples from healthy individuals through the project “Blood Reference in Stavanger (BRIS)”. The method exhibited its ability as a diagnostic tool by identifying three individuals with anomalous androgen concentrations, including the 11-oxygenated androgens, from the small population cohort studied.
The proposed multiplex method demonstrated high-throughput performance with a 2.67-minute cycle time per injection and a fully automated sample preparation with large sample capacity and high time and cost efficiency. Sensitive detection for all analytes was achieved with detection limits (LOD) and quantification limits (LOQ) at 0.01 – 0.12 nmol/L and 0.03 – 0.29 nmol/L, respectively. Satisfactory intermediate precision was found for total testosterone (6%), cortisol (11%), cortisone (4%), progesterone (5%), 17α-hydroxyprogesterone (7%), androstenedione (11%), and corticosterone (6%) in the upper range of endogenous hormone concentrations in real human serum. Additionally, the imprecision of total testosterone in the lower concentration range, corresponding to female endogenous levels, was 8%. Higher imprecisions were observed for deoxycorticosterone (23%), 11-deoxycortisol (16%), 11-ketotestosterone (17%), 11β-hydroxyandrostenedione (11%) due to limitations in the endogenous levels of these steroid hormones, and bioavailable testosterone (21%).
The method can simultaneously determine 11 steroid hormones from one single analysis, enabling the exploration of hormone patterns and the intracrine relationship in endocrinological conditions. Multiplex steroid analysis by UHPLC-MS/MS is advantageous in the exclusion of differential diagnoses, replacing multiple single-analyte assays, and expanding the analyte panel to include other steroid hormones such as 21-deoxycortisol and oestrogens
Develop an LC-MS/MS method for the determination of vitamin D and 25-hydroxyvitamin D in fish fillets
Get PDFInterleukin-1β, heat shock protein 90α, and hypocretin-1 in chronic fatigue
Get PDFBackground: Fatigue, defined as an overwhelming sense of tiredness, lack of energy, and feeling of exhaustion, is a phenomenon many people have experienced in connection with infections such as influenza, Epstein-Barr virus, etc. Fatigue is also common in cancer, neurological conditions like multiple sclerosis, Parkinson’s disease, and in chronic inflammatory and autoimmune diseases such as rheumatoid arthritis, psoriasis, and others.
“Sickness behavior” observed in animals is a conceptual model for understanding fatigue. In this model, infection or tissue damage is followed by behavioral changes like social withdrawal, inactivity, sleepiness, fatigue, and reduced food and water intake. The proinflammatory cytokine interleukin (IL)-1β produced during activation of innate immune cells has a prominent role in mediating this behavior. IL-1β crosses the blood-brain barrier and in the brain IL-1β amplifies its own signaling by inducing microglia to produce IL-1β. In cerebral neurons IL-1β signals through a receptor complex including interleukin-1 receptor I (IL-1RI) and an alternative IL-1 receptor accessory protein that does not mediate inflammation but induce neuronal activation and sickness behavior.
The inflammatory response needs to be controlled and is therefore downregulated in a timely manner not to run rampant. In addition, cellular protection mechanisms are activated during inflammation and tissue damage to preserve cellular life from reactive molecules that kill pathogens. Some variants of heat shock proteins (HSPs) released into the extracellular space could represent a defense mechanism of cellular life that also influence fatigue mechanisms.
In addition, sleepiness and weariness are closely related to fatigue and part of the sickness behavior response. Inflammation can alter sleep patterns. The master regulator of sleep- and wakefulness, neuropeptide hypocretin-1 (Hcrt1), could therefore have a role in fatigue generation.
Objectives:
I) Investigate if mechanisms that protect cellular life and homeostasis are involved in the generation of fatigue.
II) Develop a non-radioactive, sensitive and selective method for measurement of Hcrt1 in cerebrospinal fluid (CSF).
III) Explore how IL-1β and other selected molecules interact in generation of fatigue, and to investigate a possible link between the neuropeptide Hcrt1 and fatigue.
Methods: To explore mechanism of fatigue, a cohort of 71 patients with primary Sjögren’s syndrome were investigated. CSF samples where available from 49 patients. A method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for measurements of Hcrt1. Hcrt1 was measured in CSF samples from 22 healthy subjects and 9 patients with narcolepsy type 1.
The clinical variables fatigue, depression and pain were scored using the fatigue Visual Analogue Scale (fVAS), Beck Depression Inventory, and the pain item of the Medical Outcome Survey short form 36, respectively. ELISAs were used to measure HSP32, -60, -72, and -90α in plasma, and in CSF to measure concentrations of IL-1Ra, IL-1RII, IL-6, and the calcium binding protein S100B. Hcrt1 in CSF was measured using a radioimmunoassay (RIA) method, in addition to a non-radioactive method based on liquid chromatography coupled with tandem mass spectrometry. Results were analyzed by non-parametric group comparisons, logistic regression, univariate- and multiple regression, and principal component analysis (PCA).
Results: Measures of HSP32, -60, -72, and -90α in plasma revealed that the concentrations of HSP90α were significantly higher in pSS patients with high fatigue versus low fatigue. A tendency toward higher concentrations of HSP72 was observed in patients with high fatigue compared to patients with low fatigue.
The LC-MS/MS method for Hcrt1 in CSF revealed much lower
concentrations in healthy subjects than what has previously been published. Patients with narcolepsy type 1, a sleep disorder characterized by low levels of Hcrt1 in CSF, also had lower levels of Hcrt1 in CSF compared to previous published studies. The LC-MS/MS method was compared to the commonly used RIA method. A Bland-Altman plot showed agreement between the two methods.
Analysis of IL-1β related proteins (IL-1Ra, IL-1RII, and S100B), IL-6, and Hcrt1 in CSF demonstrated that IL-1Ra showed significant association with fVAS scores together with the clinical variables BDI scores and pain scores. The relationship of the biochemical variables was explored in PCA, and two significant components appeared: Variables related to IL-1β activity dominated the first component while in the second component there was a negative association between IL-6 and Hcrt1. Fatigue was introduced as an additional variable in a second model. In this PCA, fVAS scores were associated with the first component as was the IL-1β related variables. In addition, the second PCA model revealed a third component that showed a negative relationship between Hcrt1 and fatigue.
Conclusions:
I) HSP90α and to a lesser degree HSP72 in blood may possibly be parts of a fatigue inducing mechanism.
II) The LC-MS/MS method with high selectivity and accuracy revealed considerably lower levels of Hcrt1 in CSF than previously reported.
III) IL-1β signaling is a primary driver in fatigue. Several other proteins and molecules interact with IL-1β in a complex network, in which several cell types (neurons, microglia, and astrocytes) probably participate.
IV) Hcrt1 also influences fatigue, but probably through another pathway than the IL-1 route
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
- …
