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The Jurassic relict Royal fern, Osmunda regalis L., is widely distributed across temperate zones in the Northern and Southern hemispheres. Even though this species has been utilised for centuries as a medicinal plant, its phytochemical composition mainly remains unknown. As part of our ongoing research to identify potential lead compounds for future anticancer drugs, 17 natural products were characterised from the aerial parts of Osmunda regalis L. Fifteen of these compounds were identified in this species for the first time, including the six previously undescribed compounds kaempferol 3- O -(2''- O -(2'''-α-rhamnopyranosyl)- β -glucopyranosyl)- β -glucopyranoside, quercetin 3- O -(2''- O -(2'''- α -rhamnopyranosyl)- β -glucopyranosyl)- β -glucopyranoside, kaempferol 3- O -(2''- O -(2'''-α-rhamnopyranosyl-6'''- O -( E )-caffeoyl-)- β -glucopyranosyl)- β -glucopyranoside, 3-methoxy-5-hydroxy-4-olide, 4-hydroxy-3-(3'-hydroxy-4'-(hydroxyethyl)-oxotetrafuranone-5-methyl tetrahydropyranone, and 4- O -(5-hydroxy-4-oxohexanoyl) osmundalactone. The molecular structures were determined by combining several 1D and 2D NMR experiments, circular dichroism spectroscopy, and HRMS. Determination of cytotoxicity against AML MOLM-13, H9c2, and NRK cell lines showed that two isolated lactones exhibited significant cytotoxic activity.

Background: The iron-regulatory hormone hepcidin is a promising biomarker to differentiate anaemia of inflammation from iron deficiency. Plasma hepcidin concentrations increase substantially during inflammation, and the amount of smaller, non-biologically active isoforms of hepcidin increase in inflammatory conditions. These smaller isoforms are measured in some, but not all analytical methods. Thus, we evaluated the comparability of two analytical methods with different isoform selectivity during and after acute-phase pneumonia as a highly inflammatory model disease., Methods: Blood samples from a cohort of 267 hospitalized community-acquired pneumonia patients collected at admission and a 6-week follow-up were analysed. Hepcidin was measured in plasma by an immunoassay, which recognizes all hepcidin isoforms, and a liquid chromatography tandem mass spectrometry (LC-MS/MS), which selectively measures the bioactive hepcidin-25. Additionally, a subset of serum samples was analysed by LC-MS/MS., Results: Hepcidin measurements by immunoassay were higher compared with LC-MS/MS. The relative mean difference of hepcidin plasma concentrations between the two analytical methods was larger in admission samples than in follow-up samples (admission samples <200 ng/mL: 37%, admission samples >200 ng/mL: 78%, follow-up samples >10 ng/mL: 22%). During acute-phase pneumonia, serum concentrations were on average 22% lower than plasma concentrations when measured by LC-MS/MS., Conclusions: Immunoassay measured higher hepcidin concentrations compared with LC-MS/MS, with more pronounced differences in high-concentration samples during acute-phase pneumonia. These findings should be considered in local method validations and in future harmonization and standardization optimization of hepcidin measurements., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Virtually all living organisms, including microbes and humans, depend on iron to survive and grow. During an infection, the plasma level of iron and several iron-related proteins change substantially. We hypothesized that iron and iron-related proteins could predict short- and long-term outcomes in community-acquired pneumonia., Methods: Blood samples from a prospective cohort of 267 in-patients with community-acquired pneumonia were analysed for hepcidin, ferritin, iron, transferrin, transferrin saturation, and soluble transferrin receptor at admission and 6-weeks post-discharge. Adverse short-term outcome was defined as admission to intensive care unit or death within 30 days, and long-term outcome was assessed as 5-year overall mortality. Logistic regression, Kaplan Meier survival curves, and Cox regression models with cut-offs at median for the potential biomarkers were used for statistical evaluation., Results: Low admission levels of hepcidin predicted 5-year overall mortality, independently of age, sex, comorbid conditions, and anaemia. Low levels of ferritin at admission as well as low levels of iron and transferrin saturation and high levels of soluble transferrin receptor at the 6-week follow-up were predictors of 5-year overall mortality in univariable, but not in multivariable analyses. Neither of these potential biomarkers predicted adverse short-term outcomes., Conclusions: In hospitalized patients with community-acquired pneumonia, low levels of hepcidin at admission predicted 5-year overall mortality, but not short-term adverse outcome.

The aquaculture industry has become a sustainable source of food for humans. Remaining challenges include disease issues and ethical concerns for the discomfort and stress of farmed fish. There is a need for reliable biomarkers to monitor welfare in fish, and the stress hormone cortisol has been suggested as a good candidate. This study presents a novel method for measurement of cortisol in fish feces based on enzymatic hydrolysis, liquid−liquid extraction, derivatization, and finally instrumental analysis by liquid chromatography coupled with tandem mass spectrometry. Hydrolysis and extraction conditions were optimized. Cortisol appeared to be mostly conjugated to sulfate and less conjugated to glucuronic acid in the studied samples of feces from farmed Atlantic salmon. The method was suitable for quantification of cortisol after enzymatic deconjugation by either combined glucuronidase and sulfatase activity, or by glucuronidase activity alone. The limit of detection was 0.15 ng/g, the limit of quantification was 0.34 ng/g, and the method was linear (R2 > 0.997) up to 380 ng/g, for measurement of cortisol in wet feces. Method repeatability and intermediate precision were acceptable, both with a coefficient of variation (CV) of 11%. Stress level was high in fish released into seawater, and significantly reduced after eight days.

The worldwide traditional Chinese medicine (TCM) herbs sales figures have increased considerably to 50 billion US$ (2018). Astragali Radix (AR) is amongst the most often sold TCM herbs; sales in the European Union (EU) need European Medicines Agency (EMA) approval. However, comparisons of characteristic bioactive molecules concentrations in AR from different EU vendors are lacking. This study uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) with standard addition to evaluate the influence of different sample and preparation types and ammonia treatment on bioactive molecules concentrations in AR. We also compare AR samples from different EU-vendors. Astragaloside IV (AG-IV), ononin and calycosin 7-O-β-D-glucoside concentrations were higher in root powder samples when extracted with boiled water than with ultrasonication using 70% methanol. AG-IV content was by far the highest in granulates from vendor 1 (202 ± 35 μg/g) but very low in hydrophilic concentrates from vendor 1 (32 ± 7 μg/g) and granulates from vendor 4 (36 ± 3 μg/g). Ammonia-treatment significantly increased AG-IV concentrations in all samples (e.g., to 536 ± 178 μg/g in vendor 1 granulates). Comparable effects were found for most other bioactive molecules. AG-IV and other bioactive molecules concentrations differed strongly depending on sample types, extraction processes, ammonia treatment-or-not and especially between different vendors samples. Ammonia-treatment is debatable, as it is supposed to convert other astragalosides, to AG-IV. The results indicate that routine quantitative analysis of major bioactive compounds present in AR, helps in quality control of AR and to guarantee the quality of commercial products., Competing Interests: Prof Jan P.A. Baak (JB) has a commercial affiliation with the company Dr med Jan Baak AS, Tananger, Norway. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction: Astragali radix (AR), the root of Astragalus, is an important medical herb widely used in traditional Chinese medicine. Bioactive components include isoflavones and a unique class of triterpenoid saponins (named astragalosides)., Objectives: Accurate measurement of bioactive components, especially astragaloside IV, is necessary for confirming AR authenticity, quality control and future medical research., Methodology: Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a suitable technique but suffers from ion suppression effects due to sample matrix. This can be corrected by using isotopic labelled internal standards, but these are not available for many phytochemicals. We explored the use of standard addition to circumvent this issue., Results: LC-MS/MS and liquid chromatography coupled with ultraviolet (LC-UV) detection provided linear calibration curves (R 2 > 0.99). LC-MS/MS provided superior selectivity and detection limits below 10 ng/mL, which was 2-3 magnitudes lower than LC-UV detection. Precision and accuracy were overall improved by using LC-MS/MS with diluted sample extracts, resulting in an inter series coefficient of variation (CV) of 12% or less and mean recovery estimates in the 85-115% range. LC-MS/MS quantification by standard addition resulted in significantly higher concentrations of astragaloside IV measured in the samples. Concentrations calculated by standard addition were unaffected by large variation in signal response caused by matrix effects, independent of variation in slope of the standard addition curves., Conclusion: Sample dilution was helpful but not sufficient for reducing effects of ion suppression. We have shown that LC-MS/MS quantification by standard addition can be a powerful approach for accurate measurement of phytochemicals in the absence of isotopic labelled internal standards., (© 2020 The Authors. Phytochemical Analysis published by John Wiley & Sons Ltd.)

Sickness behavior and fatigue are induced by cerebral mechanisms involving inflammatory cytokines. High mobility group box 1 (HMGB1) is an alarmin, and a potential key player in this process. Reliable quantification methods for total HMGB1 and its redox variants must be established in order to clearly understand how it functions. Current methods pose significant challenges due to interference from other plasma proteins and autoantibodies. We aimed to develop an antibody-free sample preparation method followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to measure HMGB1 in human plasma. Different methods were applied for the removal of interfering proteins and the enrichment of HMGB1 from spiked human plasma samples. A comparison of methods showed an overall low extraction recovery (<40%), probably due to the stickiness of HMGB1. Reversed-phase liquid chromatography separation of intact proteins in diluted plasma yielded the most promising results. The method produced an even higher degree of HMGB1 purification than that observed with immunoaffinity extraction. Detection sensitivity needs to be further improved for the measurement of HMGB1 in patient samples. Nevertheless, it has been demonstrated that a versatile and fully antibody-free sample preparation method is possible, which could be of great use in further investigations.

Competing Interests: The authors declare no conflict of interest.

Background: Iron is crucial for survival and growth of microbes. Consequently, limiting iron availability is a human antimicrobial defense mechanism. We explored iron and iron-related proteins as potential biomarkers in community-acquired pneumonia and hypothesized that infection-induced changes in these potential biomarkers differ between groups of pathogens and could predict microbial etiology., Methods: Blood samples from a prospective cohort of 267 patients with community-acquired pneumonia were analyzed for hepcidin, ferritin, iron, transferrin, and soluble transferrin receptor at admission, clinical stabilization, and a 6-week follow-up. A total of 111 patients with an established microbiological diagnosis confined to 1 microbial group (atypical bacterial, typical bacterial, or viral) were included in predictive analyses., Results: High admission levels of ferritin predicted atypical bacterial versus typical bacterial etiology (odds ratio [OR], 2.26; 95% confidence interval [CI], 1.18-4.32; P = .014). Furthermore, hepcidin and ferritin predicted atypical bacterial versus viral etiology (hepcidin: OR = 3.12, 95% CI = 1.34-7.28, P = .008; ferritin: OR = 2.38, 95% CI = 1.28-4.45, P = .006). The findings were independent of C-reactive protein and procalcitonin., Conclusions: Hepcidin and ferritin are potential biomarkers of microbial etiology in community-acquired pneumonia., (© The Author(s) 2021. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: Therapeutic drug monitoring of the immunosuppressants tacrolimus, sirolimus, everolimus, and cyclosporine A is effectively performed by analyzing whole-blood samples using liquid chromatography coupled with tandem mass spectrometry. Samples are usually prepared using simple protein precipitation (PPT) with methanol and zinc sulfate (ZnSO4). Significant sample dilution is necessary to obtain clean extracts but may increase the limit of quantification of the method. Salting out-assisted liquid-liquid extraction (SALLE) was explored as a novel sample preparation method for measuring these drugs in blood., Method: SALLE, which simply consists of LLE with a water-miscible solvent where phase separation is achieved by adding salt, was used to analyze treated blood samples., Results: SALLE allowed direct injection of a 5-µL extract from the upper solvent phase into a reversed phase LC column, which would not be feasible using standard LLE. Compared with PPT, SALLE provided better extraction efficiencies and more ion enhancement, resulting in limit of quantification of 0.4, 1.4, 0.06, and 0.4 ng/mL for tacrolimus, sirolimus, everolimus, and cyclosporine A, respectively. Full-method validation was performed, including a comparison of results with those of another laboratory. A ≤10% bias was observed for tacrolimus and cyclosporine A, whereas further investigation of that for sirolimus (-12%) and everolimus (-18%) revealed that it was caused by the different calibrators used., Conclusions: This is the first report of the use of SALLE for the measurement of tacrolimus, sirolimus, everolimus, and cyclosporine A in whole blood. The advantages of SALLE over PPT and conventional LLE would make it an attractive sample preparation method for clinical laboratories.

Significant quantities of several important herbs are processed and consumed from Norwegian commercial kitchens annually although surprisingly the contents of polyphenols have been scarcely characterized. We here report on the qualitative and quantitative content of polyphenolic compounds from ten of the most utilized herbs. From parsley (Petroselinum crispum) var. Darki, isorhamnetin 3-(6″-malonylglucoside)-7-glucoside (2) and diosmetin 7-(2″-apiosyl-6″-malonylglucoside) (8) are reported for the first time, in addition to seven known flavonoids, some of which are reported for the first time from this plant species. Oregano, rosemary and thyme contained the highest amounts of total phenolics with maximum levels of 23.8, 24.2 and 23.4 mg GAE g -1 dry matter, respectively. Fresh herbs contained significantly higher quantities of phenolics than processed, dried herbs. Parsley, coriander, dill and thyme were the richest sources of flavonoids among the investigated herbs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Low levels of hypocretin-1 (Hcrt1) in cerebrospinal fluid (CSF) are associated with narcolepsy type 1 (NT1). Although immunoassays are prone to antibody batch differences, detection methods and variation between laboratories, the standard method for Hcrt1 measurement is a radioimmunoassay (RIA). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is an antibody- and radioactive free alternative for precise measurement of Hcrt1. We developed an LC-MS/MS method for measurement of Hcrt1 in CSF with automated sample preparation by solid-phase extraction (SPE). The LC-MS/MS method was compared with the RIA method for Hcrt1 detection. CSF samples from healthy subjects and NT1 patients was obtained by lumbar puncture. NT1 patients were diagnosed according to the minimal criteria by the International Classification of Sleep Disorders (ICSD). The LC-MS/MS method showed linearity across the range of calibrators and had a limit of detection (LOD) of 2.5 pg/mL and a limit of quantitation (LOQ) of 3.6 pg/mL. Comparison of the LC-MS/MS method with RIA revealed a 19 times lower level in healthy controls and 22 times lower level in NT1 patients with the LC-MS/MS method than with RIA. Bland-Altman analysis demonstrated agreement between the methods. These results question what is detected by RIA and strongly suggest that the physiological concentrations of the peptide are much lower than previously believed. LC-MS/MS proves to be an alternative for detection of Hcrt1 for diagnosis of narcolepsy.

Background: Fatigue is a common and sometimes debilitating phenomenon in primary Sjögren's syndrome (pSS) and other chronic inflammatory diseases. We aimed to investigate how IL-1 β-related molecules and the neuropeptide hypocretin-1 (Hcrt1), a regulator of wakefulness, influence fatigue., Methods: Hcrt1 was measured by radioimmunoassay (RIA) in cerebrospinal fluid (CSF) from 49 patients with pSS. Interleukin-1 receptor antagonist (IL-1Ra), IL-1 receptor type 2 (IL-1RII), IL-6, and S100B protein were measured by enzyme-linked immunosorbent assay (ELISA). Fatigue was rated by the fatigue visual analog scale (fVAS)., Results: Simple univariate regression and multiple regression analyses with fatigue as a dependent variable revealed that depression, pain, and the biochemical variable IL-1Ra had a significant association with fatigue. In PCA, two significant components were revealed. The first component (PC1) was dominated by variables related to IL-1β activity (IL-1Ra, IL-1RII, and S100B). PC2 showed a negative association between IL-6 and Hcrt1. fVAS was then introduced as an additional variable. This new model demonstrated that fatigue had a higher association with the IL-1β-related PC1 than to PC2. Additionally, a third component (PC3) became significant between low Hcrt1 concentrations and fVAS scores., Conclusions: The main findings of this study indicate a functional network in which several IL-1β-related molecules in CSF influence fatigue in addition to the classical clinical factors of depression and pain. The neuropeptide Hcrt1 seems to participate in fatigue generation, but likely not through the IL-1 pathway.

Objectives: Fatigue is a frequent and often disabling phenomenon that occurs in patients with chronic inflammatory and immunological diseases, and the underlying biological mechanisms are largely unknown. Because fatigue is generated in the brain, we aimed to investigate cerebrospinal fluid and search for molecules that participate in the pathophysiology of fatigue processes., Methods: A label-free shotgun proteomics approach was applied to analyze the cerebrospinal fluid proteome of 20 patients with primary Sjögren's syndrome. Fatigue was measured with the fatigue visual analog scale., Results: A total of 828 proteins were identified and the 15 top discriminatory proteins between patients with high and low fatigue were selected. Among these were apolipoprotein A4, hemopexin, pigment epithelium-derived factor, secretogranin-1, secretogranin-3, selenium-binding protein 1, and complement factor B., Conclusion: Most of the discriminatory proteins have important roles in regulation of innate immunity, cellular stress defense, and/or functions in the central nervous system. These proteins and their interacting protein networks may therefore have central roles in the generation and regulation of fatigue, and the findings contribute with evidence to the concept of fatigue as a biological phenomenon signaled through specific molecular pathways., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Short-term survival after kidney transplantation is excellent, but long-term survival remains low and is equivalent to non-end-stage renal disease patients with many invasive malignancies. The aim of the study was to explore vitamin D status in the early phase after transplantation as a prognostic marker for long-term graft and patient survival., Methods: All first-time kidney transplant recipients between October 2007 and October 2012 in Norway were included. Vitamin D was measured 10 weeks post-transplant. Information on graft failure and death was obtained from the Norwegian Renal Registry., Results: Seven hundred and sixty-two first-time kidney transplant recipients were included, with a median age of 57 years and a median follow-up of 82 months. In the follow-up period, there were 172 graft failures (23%) and 118 deaths (15%). Eighty-six percent of the transplant recipients with sufficient vitamin D levels were alive with a well-functioning graft after 5 years using Kaplan-Meier survival estimates, compared with 79% and 76% of the patients with vitamin D deficiency and insufficiency, respectively (P = 0.006)., Conclusion: In a nation-wide cohort of 762 first-time kidney transplant recipients, long-term graft and patient survival were better in recipients with vitamin D sufficiency 10 weeks post-transplant compared with those with vitamin D deficiency and insufficiency., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Clinical mass spectrometry proteomics (cMSP) assays are being increasingly used in clinical laboratories for analyzing peptides and proteins. It has therefore become urgent to characterize and validate the methods available for liquid chromatography-tandem mass spectrometry (LC/MS-MS) targeted quantification of peptide and protein biomarkers in biological fluids in the context of in vitro diagnostics. LC-MS/MS for the detection of peptides and proteins is currently the main approach used in the field of cMSP. As a result of their selectivity, low reagent costs and the fact that these methods can be used for absolute quantification and multiplexing, they will likely eventually replace immunoassays. Although LC-MS/MS is known to be the main reference method involved in reference measurement procedures (RMPs), it needs to meet the requirements of in vitro diagnostic (IVD) regulations and standards. This review shows that cMSP is fully compatible with the regulatory IVD requirements and provides an overview of the characterization and validation of the use of LC-MS/MS targeted quantification of clinical protein biomarkers in biological fluids.

Background: LC-MS/MS methods for multiplexed analysis of steroid hormones in saliva are useful research tools in endocrinology, but require sensitive detection., Objective: To explore the use of hydrazide and hydrazine derivatization to improve sensitivity of detection for ketosteroids. On development and validation of a sample preparation method based on robot pipetting in the 96-well format we intended to then establish normal reference ranges for women and men, with saliva collected both in the early morning and late at night., Method: Four hydrazides and one hydrazine were evaluated for their effectiveness as derivatization reagents based on the comparative signal response and efficacy of LC separation of five ketosteroid hydrazones via a methanol gradient mixed with either 0.2% formic acid or 0.1% ammonium hydroxide. Processing of saliva via liquid-liquid extraction (LLE) was optimized by examining variations in both extraction solvent polarity and protein precipitation reagents intended to inhibit emulsion., Results: LLE using 10 % butanol in methyl tert -butyl ether (MTBE), with tannic acid as emulsion inhibitor, followed by 2-hydrazinopyridine (2-HP) derivatization enabled the multiplexed measurement of cortisol, cortisone, testosterone, dehydroepiandrosterone (DHEA), progesterone, and 17-alpha-hydroxyprogesterone (17-OHP) in the majority of saliva samples from women and men for this study., Conclusion: Tannic acid is a novel and effective protein precipitation reagent in bioanalytical sample preparation. The validated method was used in the establishment of reference ranges, the success of which indicated that the method is suitable for Cushing's screening and has potential as a novel analytical research tool., (© 2017 The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL). Published by Elsevier B.V.)

Background: Mass spectrometry (MS) methods are being widely used these days in medical laboratories for quantifying many small molecular analytes as well as for microbiological purposes., Methods: Little use has been made so far, however, of MS for analyzing peptides and proteins in clinical laboratory (an approach known as clinical MS proteomics (cMSP)). The explanation for this situation may be that cMSP assays are more complex to implement than conventional assays, require large investments in terms of equipment and training, and have not yet been sufficiently validated for clinical applications. In addition, the protein analysis assays currently used in medical laboratories mostly meet both laboratory and clinical requirements in terms of analytical performances, ease of use, and turn-around-time., Results: With the spread of MS methods in laboratories, increasing interest seems to be focusing on the development of MS for quantifying new analytes. MALDI-TOF MS methods have already been replacing classical methods of bacterial classification in clinical laboratories, for example, and this can be said to be an important step in this direction., Conclusions: In this paper, the literature available on the topic of clinical MS proteomics is reviewed and the pre-analytical, analytical, and post-analytical challenges which will have to be met in connection with this approach are discussed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Diagnosis of diabetes and monitoring of long-term blood sugar are preferably done by measurement of glycated hemoglobin (HbA1c). Diabetic patients with end stage renal disease (ESRD) may have short-lived red blood cells due to hemodialysis (HD), and thus higher turnover of hemoglobin. The level of glycated hemoglobin (HbA1c) may be lower than expected for these patients, even at increased blood glucose, possibly making glycated albumin (GA) measurement a better alternative., Methods: The percentage of GA was measured by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Fast and efficient trypsin digestion of proteins in diluted serum or plasma resulted in a high number of proteotypic peptides from albumin, including KQTALVELVK which was detected both glycated and non-glycated by multiple reaction monitoring (MRM). The percentage of GA was estimated by neat peak area response of glycated peptide divided by the sum of glycated and non-glycated peptide., Results: Acceptable method reproducibility (6% CV), repeatability (2-6% CV), limit of quantification (0.75% GA), linearity (R(2) = 0.999) and recovery (79 ± 9%) was achieved without using calibration or isotope-labeled internal standard. GA was strongly correlated with HbA1c (r = 0.84) for patients without ESRD. The average ratio of GA/HbA1c was significantly higher (p = 0.0021) for ESRD patients (1.84 ± 0.38, n = 62) compared to other patients (1.67 ± 0.28, n = 225)., Conclusion: GA measurement by detecting glycation in KQTALVELVK with LC-MS/MS seems to be a useful supplement to HbA1c for detecting increased blood glucose in diabetic patients with ESRD.

The cerebrospinal fluid (CSF) proteome is of great interest for investigation of diseases and conditions involving the CNS. However, the presence of high-abundance proteins (HAPs) can interfere with the detection of low-abundance proteins, potentially hindering the discovery of new biomarkers. Therefore, an assessment of the CSF subproteome composition requires depletion strategies. Existing methods are time consuming, often involving multistep protocols. Here, we present a rapid, accurate, and reproducible method for preparing the CSF proteome, which allows the identification of a high number of proteins. This method involves acetonitrile (ACN) precipitation for depleting HAPs, followed by immediate trypsination. As an example, we demonstrate that this method allows discrimination between multiple sclerosis patients and healthy subjects., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Regression of cervical intraepithelial neoplasia (CIN) 2-3 to CIN 1 or less is associated with immune response as demonstrated by immunohistochemistry in formaldehyde-fixed paraffin-embedded (FFPE) biopsies. Proteomic analysis of water-soluble proteins in supernatants of biopsy samples with LC-MS (LTQ-Orbitrap) was used to identify proteins predictive of CIN2-3 lesions regression. CIN2-3 in the biopsies and persistence (CIN2-3) or regression (≤CIN1) in follow-up cone biopsies was validated histologically by two experienced pathologists. In a learning set of 20 CIN2-3 (10 regressions and 10 persistence cases), supernatants were depleted of seven high abundance proteins prior to unidimensional LC-MS/MS protein analysis. Mean protein concentration was 0.81 mg/mL (range: 0.55-1.14). Multivariate statistical methods were used to identify proteins that were able to discriminate between regressive and persistent CIN2-3. The findings were validated in an independent test set of 20 CIN2-3 (10 regressions and 10 persistence cases). Multistep identification criteria identified 165 proteins. In the learning set, zinc finger protein 441 and phospholipase D6 independently discriminated between regressive and persistent CIN2-3 lesions and correctly classified all 20 patients. Nine regression and all persistence cases were correctly classified in the validation set. Zinc finger protein 441 and phospholipase D6 in supernatant samples detected by LTQ-Orbitrap can predict regression of CIN2-3.

The progressive loss of motor control due to reduction of dopamine-producing neurons in the substantia nigra pars compacta and decreased striatal dopamine levels are the classically described features of Parkinson disease (PD). Neuronal damage also progresses to other regions of the brain, and additional non-motor dysfunctions are common. Accumulation of environmental toxins, such as pesticides and metals, are suggested risk factors for the development of typical late onset PD, although genetic factors seem to be substantial in early onset cases. Mutations of DJ-1 are known to cause a form of recessive early onset Parkinson disease, highlighting an important functional role for DJ-1 in early disease prevention. This study identifies human DJ-1 as a metal-binding protein able to evidently bind copper as well as toxic mercury ions in vitro. The study further characterizes the cytoprotective function of DJ-1 and PD-mutated variants of DJ-1 with respect to induced metal cytotoxicity. The results show that expression of DJ-1 enhances the cells' protective mechanisms against induced metal toxicity and that this protection is lost for DJ-1 PD mutations A104T and D149A. The study also shows that oxidation site-mutated DJ-1 C106A retains its ability to protect cells. We also show that concomitant addition of dopamine exposure sensitizes cells to metal-induced cytotoxicity. We also confirm that redox-active dopamine adducts enhance metal-catalyzed oxidation of intracellular proteins in vivo by use of live cell imaging of redox-sensitive S3roGFP. The study indicates that even a small genetic alteration can sensitize cells to metal-induced cell death, a finding that may revive the interest in exogenous factors in the etiology of PD.

Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in 'functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP).

Extracts, subfractions, isolated anthocyanins and isolated procyanidins B2, B5 and C1 from the berries and bark of Aronia melanocarpa were investigated for their antioxidant and enzyme inhibitory activities. Four different bioassays were used, namely scavenging of the diphenylpicrylhydrazyl (DPPH) radical, inhibition of 15-lipoxygenase (15-LO), inhibition of xanthine oxidase (XO) and inhibition of α-glucosidase. Among the anthocyanins, cyanidin 3-arabinoside possessed the strongest and cyanidin 3-xyloside the weakest radical scavenging and enzyme inhibitory activity. These effects seem to be influenced by the sugar units linked to the anthocyanidin. Subfractions enriched in procyanidins were found to be potent α-glucosidase inhibitors; they possessed high radical scavenging properties, strong inhibitory activity towards 15-LO and moderate inhibitory activity towards XO. Trimeric procyanidin C1 showed higher activity in the biological assays compared to the dimeric procyanidins B2 and B5. This study suggests that different polyphenolic compounds of A. melanocarpa can have beneficial effects in reducing blood glucose levels due to inhibition of α-glucosidase and may have a potential to alleviate oxidative stress.

Background: Oxidative stress has been associated with many diseases and can among others be assessed as increased levels of advanced oxidation protein products (AOPP). Current AOPP methods suffer from poor reproducibility and accuracy due to precipitation of lipids in plasma samples. We therefore aimed to develop a robust method in which plasma lipids are solubilized., Methods: Plasma was diluted with citric acid, and AOPP measured as absorbance at 340 nm. The method was optimized and validated, and then used to analyze AOPP levels in plasma from healthy control subjects (HC), and in three patients groups; chronic kidney disease (CKD), primary Sjögren's Syndrome (pSS) and systemic lupus erythematosus (SLE)., Results: AOPP was detected with improved precision compared to established methods where lipids precipitate. Within- and between days variations were less than 1.4% and 2.2%, respectively. A control chart was established and the long-term reproducibility followed over six months., Conclusions: This improved method detects plasma AOPP with significantly better reproducibility and accuracy compared to previously reported methods. Solubilization of plasma lipids before spectrophotometric measure of AOPP levels is novel. It prevents both loss of lipoproteins due to precipitation and overestimation as a result of light scattering., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Several studies have shown an association between vitamin D deficiency and cardiovascular risk. Vitamin D status is assessed by determination of 25-hydroxyvitamin D [25(OH)D] in serum., Methods: We assessed the prognostic utility of 25(OH)D in 982 chest-pain patients with suspected acute coronary syndrome (ACS) from Salta, Northern Argentina. 2-year follow-up data including all-cause mortality, cardiac death and sudden cardiac death were analyzed in quartiles of 25(OH)D, applying univariate and multivariate analysis., Results: There were statistically significant changes in seasonal 25(OH)D levels. At follow-up, 119 patients had died. The mean 25(OH)D levels were significantly lower among patients dying than in long-term survivors, both in the total population and in patients with a troponin T (TnT) release (n = 388). When comparing 25(OH)D in the highest quartile to the lowest quartile in a multivariable Cox regression model for all-cause mortality, the hazard ratio (HR) for cardiac death and sudden cardiac death in the total population was 0.37 (95% CI, 0.19-0.73), p = 0.004, 0.23 (95% CI, 0.08-0.67), p = 0.007, and 0.32 (95% CI, 0.11-0.94), p = 0.038, respectively. In patients with TnT release, the respective HR was 0.24 (95% CI, 0.10-0.54), p = 0.001, 0.18 (95% CI, 0.05-0.60), p = 0.006 and 0.25 (95% CI, 0.07-0.89), p = 0.033. 25(OH)D had no prognostic value in patients with no TnT release., Conclusion: Vitamin D was shown to be a useful biomarker for prediction of mortality when obtained at admission in chest pain patients with suspected ACS., Trial Registration: ClinicalTrials.gov NCT01377402.

Background: Cervical intraepithelial neoplasia (CIN) grades 2 and 3 are usually grouped and treated in the same way as "high grade", in spite of their different risk to cancer progression and spontaneous regression rates. CIN2-3 is usually diagnosed in formaldehyde-fixed paraffin embedded (FFPE) punch biopsies. This procedure virtually eliminates the availability of water-soluble proteins which could have diagnostic and prognostic value., Aim: To investigate whether a water-soluble protein-saving biopsy processing method followed by a proteomic analysis of supernatant samples using LC-MS/MS (LTQ Orbitrap) can be used to distinguish between CIN2 and CIN3., Methods: Fresh cervical punch biopsies from 20 women were incubated in RPMI1640 medium for 24 hours at 4°C for protein extraction and subsequently subjected to standard FFPE processing. P16 and Ki67-supported histologic consensus review CIN grade (CIN2, n = 10, CIN3, n = 10) was assessed by independent gynecological pathologists. The biopsy supernatants were depleted of 7 high abundance proteins prior to uni-dimensional LC-MS/MS analysis for protein identifications., Results: The age of the patients ranged from 25-40 years (median 29.7), and mean protein concentration was 0.81 mg/ml (range 0.55 - 1.14). After application of multistep identification criteria, 114 proteins were identified, including proteins like vimentin, actin, transthyretin, apolipoprotein A-1, Heat Shock protein beta 1, vitamin D binding protein and different cytokeratins. The identified proteins are annotated to metabolic processes (36%), signal transduction (27%), cell cycle processes (15%) and trafficking/transport (9%). Using binary logistic regression, Cytokeratin 2 was found to have the strongest independent discriminatory power resulting in 90% overall correct classification., Conclusions: 114 proteins were identified in supernatants from fresh cervical biopsies and many differed between CIN2 and 3. Cytokeratin 2 is the strongest discriminator with 90% overall correct classifications.

Six European plum cultivars ( Prunus domestica L.) grown in Norway have been studied with respect to phenolic composition. Neochlorogenic acid was found to be the most important phenolic acid in all cultivars. Together with other phenolic acids, this compound varied significantly in amount among the cultivars. Cyanidin 3-rutinoside was found to account for >60% of the total anthocyanin content. Minor amounts of flavonols (rutin and quercetin 3-glucoside) were detected in all cultivars. Total antioxidant capacity varied from 814 to 290 micromol of Trolox 100 g(-1) of fresh weight. Measurement of total phenolic content in terms of Prussian blue complex formation revealed a method failure of magnitude order compared to results obtained by HPLC. Comparison of the response factors of a range of phenolic compounds obtained upon analysis by the Prussian blue and Folin-Ciocalteu assays revealed that the latter method returned higher yields in terms of gallic acid (GAE).

The flavonoid pathway is known to be up-regulated by different environmental stress factors. Down-regulation of the pathway is much less studied and is emphasized in the present work. Flavonoid accumulation was induced by exposing plants for 1 week to nitrogen depletion at 10 degrees C, giving high levels of anthocyanins and 3-glucoside-7-rhamnosides, 3,7-di-rhamnosides and 3-rutinoside-7-rhamnosides of kaempferol and quercetin. Flavonol accumulation as influenced by temperatures and nitrogen supply was not related to the glycosylation patterns but to the classification as quercetin and kaempferol. When nitrogen was re-supplied, transcripts for main regulators of the pathway, PAP1/GL3 and PAP2/MYB12, fell to less than 1 and 0.1% of initial values, respectively, during 24 h in the 15-30 degrees C temperature range. Anthocyanins showed a half-life of approximately 1 d, while the degradation of flavonols was much slower. Interestingly, the initial fluxes of anthocyanin and flavonol degradations were found to be temperature-independent. A kinetic model for the flavonoid pathway was constructed. In order to get the observed concentration-temperature profiles as well as the temperature compensation in the flavonoid degradation flux, the model predicts that the flavonoid pathway shows an increased temperature sensitivity at the end of the pathway, where the up-regulation by PAP/GL3 has been found to be largest.

Solid-phase analytical derivatization (SPAD) with bis(trimethylsilyl)trifluoroacetamide (BSTFA) has successfully been used as a sample preparation method for determination of (APs) in fish bile treated with beta-glucuronidase and sulfatase. Derivatized APs were analysed by gas chromatography-mass spectrometry in the electron ionization mode (GC-EI-MS). Overall limits of detection (LODs) ranged from 5 to 18ng/g bile for 19 out of 21 investigated compounds. LODs were not determined for 4-methylphenol and 4-tert-octylphenol due to high background levels in control bile. Recoveries ranged from 83 to 109%. The analysed APs vary in degree of alkylation from methyl (C(1)) to nonyl (C(9)), and represent various pollution sources, including produced water (PW) discharge from the offshore oil industry. The applicability and sensitivity of the method has been demonstrated by analysis of bile taken from Atlantic cod (Gadus morhua L.) exposed to two dilutions of PW (1:500 and 1:1500) in a continuous flow system.

Within the BEEP project (Biological Effects of Environmental Pollution in Marine Ecosystems) the Work Package 1 was addressed to the development of new and more sensitive biomarkers of exposure in several sentinel organisms. Within this framework, common mesocosm exposures of organic pollutants relevant for marine ecosystems were conducted in the facilities of Akvamiljø a/s (Stavanger, Norway). In the first experiment, Atlantic cod (Gadus morhua), turbot (Scophthalmus maximus) and shore crab (Carcinus maenas) were exposed to nonylphenol, North Sea crude oil and a combination of crude oil and alkylated phenols. Mussels (Mytilus edulis) were exposed to North Sea crude oil and a combination of crude oil, alkylated phenols and PAHs. In the second experiment, Atlantic cod, turbot, mussel and spider crab (Hyas araneus) were exposed to the plasticizers bisphenol A and diallyl phatalate and the brominated flame retardant BDE-47. The main purpose of the present study was to provide the 30 participating Institutes with samples which had been exposed to defined contaminant concentrations in a controlled laboratory exposure for 3 weeks. This paper describes the mesocosm experimental design, the transplantation and treatment of the organisms, and the contaminant exposures.

A procedure used by the Norwegian Food Safety Authority for surveillance of contaminants from plastic food contact materials (polyolefin drinking bottles, water boilers, polyamide cooking utensils and plastic multi-layer materials) is described. It is based on gas chromatographic-mass spectrometric (GC/MS) analysis of food simulants exposed to plastic materials. Most migrants were substances not-intentionally added to the plastic (degradation products, impurities) or originated from non-plastic components, such as printing inks, adhesives, not-listed additives, solvents and coatings. Hence, the majority of the identified migrants were regulated by the general statements in the EU Framework Regulation, which neither specify limits nor requirements regarding risk assessment, rather than by specific migration controls. Risk assessment has been carried out for selected non-authorized substances. The analysis and the management of these substances and materials with respect to safety represents a challenge to the food authorities.

This review article presents an overview of halogen-specific detection in gas chromatography (GC) and liquid chromatography (LC). Attention is primarily focused on the use of plasma emission spectroscopy and plasma mass spectrometry as detectors, but other halogen-selective detection principles are also mentioned. Different instrumental configurations are discussed both with respect to technical set-up and performance, the principal reasons for halogen-selective detection are highlighted, and recent applications are reviewed from areas such as environmental chemistry, petroleum characterization, and drug analysis.

Solid phase analytical derivatization with trifluoroacetic anhydride has been introduced as sample preparation for the determination of primary aromatic amines in water by gas chromatography coupled with mass spectrometry. Water was used as a food simulant for testing migration from laminated flexible food packaging materials. The method was evaluated for 8 primary aromatic amines in 200 ml water samples, which resulted in detection limits in the 0.1-0.4 microg/l range, relative standard deviations in the 4-17% range and acceptable linearity (R2 = 0.997-1.000). Detectable levels of 2,4-diaminotoluene, 2,6-diaminotoluene and 4,4'-methylenedianiline were found in water food simulant from some of the investigated food packaging materials.

The on-line supercritical fluid extraction-supercritical fluid chromatography-gas chromatography method was applied to the determination of volatile compounds of raw and baked Baltic herring (Clupea harengus membras). The analytes were extracted with supercritical carbon dioxide at 45 degrees C and 10 MPa pressure. After extraction, the volatiles and coeluted lipids were separated on-line using supercritical fluid chromatography and the volatile fraction was introduced directly into a gas chromatograph. In all, 30 compounds were identified from fish samples with mass spectrometry. The most abundant compounds in the fresh Baltic herrings were heptadecane and 1-heptadecene. When the fish were stored for 3-6 days at 6 degrees C, the total peak area of the volatiles began to increase and the proportions of short chain acids (acetic, propanoic, 2-methylpropanoic, and 3-methylbutanoic) also increased. After 8-9 days of storage, 3-methylbutanoic acid comprised about 36 and 40% of all volatiles in raw and baked herring, respectively.