HPLC Mass Spectrometry Research Articles

Radical Trapping Study of the Relaxation of bis-Fe(IV) MauG

The di-heme enzyme, MauG, utilizes a high-valent, charge-resonance stabilized bis-Fe(IV) state to perform protein radical-based catalytic chemistry. Though the bis-Fe(IV) species is able to oxidize remote tryptophan residues on its substrate protein, it does not rapidly oxidize its own residues in the absence of substrate. The slow return of bis-Fe(IV) MauG to its resting di-ferric state occurs via up to two intermediates, one of which has been previously proposed by Ma et al. (Biochem J 2016; 473:1769) to be a methionine-based radical in a recent study. In this work, we pursue intermediates involved in the return of high-valent MauG to its resting state in the absence of the substrate by EPR spectroscopy and radical trapping. The bis-Fe(IV) MauG is shown by EPR, HPLC, UV-Vis, and high-resolution mass spectrometry to oxidize the trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to a radical species directly. Nitrosobenzene was also employed as a trapping agent and was shown to form an adduct with high-valent MauG species. The effects of DMPO and nitrosobenzene on the kinetics of the return to di-ferric MauG were both investigated. This work eliminates the possibility that a MauG-based methionine radical species accumulates during the self-reduction of bis-Fe(IV) MauG.

Lipoilcarnosine: synthesis, study of physico-chemical and antioxidant properties, biological activity

Synthesis of lipoilcarnosine (LipC) - a conjugated molecule based on two natural antioxidants, carnosine and a-lipoic acid, is described. Its physico-chemical, antioxidant properties and biological activity are characterized. According to reversed-phase HPLC with a UV detector, purity of the final product was 89.3%. The individuality of the obtained sodium salt of LipC was confirmed by tandem HPLC-mass spectrometry. High resistance of LipC to hydrolysis with serum carnosinase was demonstrated. The antioxidant activity of LipC measured by reaction with the formation of thiobarbituric acid reacting substances and kinetic parameters of iron-induced chemiluminescence was higher than that of carnosine and lipoic acid. LipC did not affect viability of SH-SY5Y human neuroblastoma culture cells, differentiated towards the dopaminergic type, at concentrations not exceeding 5 mM. At the concentration range of 0.1-0.25 mM LipC protected neuronal cells against 1-methyl-4-phenylpyridinium (MPP + )-induced toxicity.

Pharmacokinetic Analysis of Four Bioactive Iridoid and Secoiridoid Glycoside Components of Radix Gentianae Macrophyllae and Their Synergistic Excretion by HPLC-DAD Combined with Second-Order Calibration

An HPLC-DAD method combined with second-order calibration based on the alternating trilinear decomposition (ATLD) algorithm with the aid of region selection was developed to simultaneously and quantitatively characterize the synergistic relationships and cumulative excretion of the four bioactive ingredients of Radix Gentianae Macrophyllae in vivo. Although the analytes spectra substantially overlapped with that of the biological matrix, the overlapping profiles between analytes and co-eluting interferences can be successfully separated and accurately quantified by the ATLD method on the basis of the strength of region selection. The proposed approach not only determined the content change but also revealed the synergistic relationships and the cumulative excretion in vivo of the four ingredients in urine and feces samples collected at different excretion time intervals. In addition, several statistical parameters were employed to evaluate the accuracy and precision of the method. Quantitative results were confirmed by HPLC-mass spectrometry. Satisfactory results indicated that the proposed approach can be utilized to investigate the pharmacokinetics of Radix Gentianae Macrophyllae excretion in vivo.Graphical

Development and validation of a stability-indicating HPLC-UV method for the determination of triamcinolone acetonide and its degradation products in an ointment formulation

A stability indicating high performance liquid chromatography method has been developed for the determination of triamcinolone acetonide (TCA) and its main degradation products in ointment formulations. The method, based on extensive stress testing using metal salts, azobisisobutyronitrile, acid, base and peroxide, showed that TCA undergoes oxidative degradation. All degradation products were identified using HPLC mass spectrometry. Separation and quantification was achieved using an Altima C18 RP18 HP column (250×4.6mm2, with 5μm particles) using a mobile phase consisting of acetonitrile and water buffered at pH 7 using 10mM phosphate buffer. A gradient mode was operated at a flow rate of 1.5ml/min and detection was at 241nm. The method showed linearity for TCA and Impurity C in 0.02–125% of the workload, both square roots of the correlation coefficients were larger than 0.9999. Repeatability and intermediate precision were performed by six consecutive injections of both 1.25% and 125% of the work load for both TCA and Impurity C divided equally over two days. RSD were 0.6% and 0.7% for TCA and 0.5% and 0.1% for Impurity C respectively. Accuracy was determined as well, the average recoveries were 99.5% (±0.1%, n=3) for TCA and 96.9% (±1.3%, n=3) for impurity C respectively from spiked ointment samples. The robustness was also evaluated by variations of column (old vs new), mobile phase pH and filter retention. The applicability of the method was evaluated by analysis of a commercial ointment formulation. Interestingly, the extensive stress tests were able to predict all degradation products of TCA in a long term stability ointment sample.

Molecular characterization and bioactivity evaluation of two novel bombinin peptides from the skin secretion of Oriental fire-bellied toad, Bombina orientalis

Following the exploration of biochemicals in amphibian defensive skin secretion, great attention has been focused on the novel bioactive peptides with unique molecular structures and complicated features and functions. In this study, the skin secretion of Oriental fire-bellied toad, Bombina orientalis, was acquired to search peptides with therapeutic potential. Using "shotgun" cloning technique, a full-length peptide precursor co-encoding two novel bombinin peptides was cloned from the skin secretion-derived cDNA library of B. orientalis. The deduced peptides were identified as one bombinin-like peptide (BLP) (GIGSAILSAGKSIIKGLAKGLAEHF-NH2) and one bombinin H-type peptide (BH) (IIGPVLGLVGKALGGLL-NH2). The primary structures of both peptides were confirmed through reverse-phase HPLC fractionation and mass spectrometry. Secondary structural prediction revealed Bombinin-BO1 and Bombinin H-BO1 adopted α-helical structural features. In addition, the two peptides exhibited broad-spectrum antimicrobial effect against Gram-positive and Gram-negative bacteria and yeast. Meanwhile, the anticancer activity assay indicated both peptides exerted significant anticancer effects against human hepatoma cell lines tested (Hep G2/SK-HEP-1/Huh7). The peptides reported here for the first time may represent novel lead compounds for the design/development of new therapeutics for human infection and neoplastic disease.

Two International Round-Robin Studies Showed Good Comparability of 5-Methyltetrahydrofolate but Poor Comparability of Folic Acid Measured in Serum by Different High-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods

Background: Serum folate methods produce different results. The comparability of HPLC-mass spectrometry (MS)/MS methods is not well documented.Objective: We conducted an international "round-robin" investigation to assess the comparability, precision, and accuracy of serum folate HPLC-MS/MS methods.Methods: The CDC laboratory, 7 laboratories with independently developed methods (group 1), and 6 laboratories with an adapted CDC method (group 2) analyzed folate forms in 6 serum pools and 6 calibrators from the CDC (duplicate analysis over 2 d) and in two 3-level reference materials (duplicate analysis).Results: All laboratories measured 5-methyltetrahydrofolate (5-methylTHF) and folic acid; some measured additional folate forms. The geometric mean (range) concentrations (nanomoles per liter) for 5-methylTHF in the 6 serum pools were 18.3 nmol/L (CDC), 13.8-28.9 nmol/L (group 1), and 16.8-18.6 nmol/L (group 2); for folic acid the concentrations were 3.42 nmol/L (CDC), 1.09-4.74 nmol/L (group 1), and 1.74-2.90 nmol/L (group 2). The median imprecision (CV) for 5-methylTHF was 4.1% (CDC), 4.6-11% (group 1), and 1.7-6.0% (group 2); for folic acid it was 6.9% (CDC), 4.9-20% (group 1), and 3.9-23% (group 2). The mean ± SD (range) recovery of 5-methylTHF spiked into serum was 98% ± 27% (59-138%) for group 1 and 98% ± 10% (82-111%) for group 2; for folic acid it was 93% ± 29% (67-198%) for group 1 and 81% ± 16% (64-102%) for group 2. The mean relative bias for 5-methylTHF compared with the reference material certificate value was 12% (CDC), -24% to 30% (group 1), and -0.6% to 16% (group 2); for folic acid it was 73% (CDC), -47% to 578% (group 1), and -3.3% to 67% (group 2).Conclusions: For 5-methylTHF, group 2 laboratories demonstrated better agreement and precision, less variable spiking recovery, and less bias by using a reference material. Laboratory performance for folic acid was highly variable and needs improvement. Certified reference materials for serum folate forms and total folate are needed to improve method accuracy.

Anti-inflammatory effect of the taffy mu yeot, made from the Korean radish Raphanus sativus L. in a lipopolysaccharide-induced murine model of pulmonary inflammation

Background: Korean radish (Raphanus sativus L.) is used in kimchi, a staple of Korean cuisine. A taffy, mu yeot, that includes mainly radish together with other herbs (Bulbus Allii Sativi, Fructus Zizyphi, Semen Zizyphi, Rhizoma Zingberis, and Radix Glycyrrihizae) is used traditionally in Korea to improve pulmonary symptoms. However, to our knowledge, these effects of the taffy have not yet been studied systematically. Objectives: To elucidate the anti-inflammatory effects of radish taffy on macrophages and a murine model of pulmonary inflammation, and to suggest the most likely candidate for the effects. Methods: A macrophage cell line, RAW264.7, was used to examine effects of the radish taffy in vitro. Pulmonary inflammation was assessed 24 h after oral instillation of lipopolysaccharide in mice treated with radish taffy for 10 days. We determined the chemical components of radish taffy quantitatively using tandem HPLC mass spectroscopy. Results: Radish taffy had no apparent effect on the RAW264.7 cells. Treatment of the mice with radish taffy significantly reduced the recruitment of neutrophils to the lung, and the level of myeloperoxidase in bronchoalveolar lavage fluid (BALF). ELISAs showed that the treatment significantly decreased the level of IL 6 in BALF induced by LPS, but not the levels of IFN?, TNF?, IL 10, or IL 12. Nor did the taffy change the levels of those cytokines or NF-?B activation in lung homogenates. HPLC-MS suggested glycyrrhizin as the most likely candidate for the anti-inflammatory effects. Conclusions: Radish taffy suppresses neutrophil recruitment to lungs of mice, possibly by reducing IL 6 levels, which may lead to reduced pulmonary inflammation. Keywords: IL 6, lipopolysaccharide, neutrophil, pulmonary inflammation, radish taffy

A simple high performance liquid chromatography–mass spectrometry method for Therapeutic Drug Monitoring of isavuconazole and four other antifungal drugs in human plasma samples

Triazoles chanced the prevention and treatment of invasive fungal infections, but their pharmacokinetic properties are still unclear. In particular, isavuconazole (ISC) is a new broad-spectrum antifungal triazole approved in 2015 as first-line treatment for intravenous and oral use against invasive aspergillosis and for mucormycosis. Nowadays, the optimal management of the treatments with triazoles requires the use of Therapeutic Drug Monitoring (TDM), in order to prevent sub-therapeutic or toxic concentrations. In turn, the routine use of TDM requires reliable quantification methods The aim of this work was the development and full validation of a HPLC-mass spectrometry assay for the simultaneous quantification of fluconazole, itraconazole, isavuconazole, posaconazole and voriconazole in human samples. Both standards and quality controls were prepared in human plasma. After the addition of internal standard (6,7-dimethyl-2,3-di(2-pyridyl)quinaxoline for voriconazole, posaconazole and itraconazole; stable isotope labeled compounds for fluconazole and isavuconazole), protein precipitation with acetonitrile and dilution with water were performed. Chromatographic separation was performed on Atlantis® T3 5μm 4.6×150mm column, with a gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra-day and inter-day imprecision fitted FDA and EMA guidelines, while matrix effects and recoveries resulted stable between samples for each analyte. Stability results were in accordance with previously published data. Finally, we tested this method by monitoring plasma concentrations in real patients and using external quality controls with good results. This method resulted very simple, fast, cheap and very useful for TDM application, to improve clinical management of antifungal therapy in critically ill patients.

Polar lipid composition of bioactive dairy co-products buttermilk and butterserum: Emphasis on sphingolipid and ceramide isoforms

Bioactive lipids of the milk fat globule membrane become concentrated in two co-products of the butter industry, buttermilk and butterserum. Their lipid composition is detailed here with special emphasis on sphingolipid composition of nutritional interest, determined using GC, HPLC and tandem mass spectrometry. Butterserum was 2.5 times more concentrated in total fat than buttermilk, with 7.7±1.5vs 19.5±2.9wt% and even more concentrated in polar lipids, with 1.4±0.2vs 8.5±1.1wt%. Both ingredients constitute concentrated sources of sphingomyelin (3.4–21mg/g dry matter) and contained low amounts of bioactive ceramides in a ratio to sphingomyelin of 1:5mol% in buttermilk and 1:10mol% in butterserum. Compared to other natural lecithins, these two co-products are rich in long and saturated fatty acids (C22:0-C24:0), contain cholesterol and could have interesting applications in neonatal nutrition, but also as brain-protective, hepatoprotective and cholesterol lowering ingredients.

Diagnosis of respiratory diseases using the proteomic analysis of exhaled breath condensate

Investigation of exhaled breath condensate (EBC) is a noninvasive diagnostic method in respiratory diseases. The objective of this study was to compare EBC protein spectrum in healthy volunteers and in patients with chronic obstructive pulmonary disease (COPD), pneumonia and lung cancer (NSCLC), as well as to assess a role of proteomic analysis of EBC for diagnosis and differential diagnosis of these diseases. Methods. We examined 18 patients with COPD, 13 patients with community-acquired pneumonia, 26 patients with lung cancer and 24 healthy non-smoking volunteers. EBC was collected using ECoScreen system (VIASYS Healthcare, Germany) and a standardized method. EBC-samples were lyophilized, hydrolyzed and analyzed by HPLC and tandem mass spectrometry. To identify proteins, we used Mascot (Matrix Science, UK) and IPI-human (version 3.82) databases provided by the European Bioinformatics Institute. Results. Proteomic analysis of EBC identified more than 300 different proteins; most of them were types I and II cytoskeletal keratins. Cytokeratin 5, 6, and 14 concentrations in EBC of NSCLC patients were significantly higher than that in healthy volunteers. Dermcidin, immunoglobulin alpha, kininogen, cytoplasmic actin, serum albumin, and Zn-alpha2-glycoprotein were identified in EBC of healthy volunteers and patients with COPD and pneumonia. High concentration of peroxiredoxin in EBC of COPD patients could be due to severe oxidative stress. High levels of acute-phase and hypoxia proteins (annexins A1 and A2, HSP90B, cystatins M and B, collagen and histones fragments) were detected in EBC of pneumonia patients. Also, β- и α-subunit of hemoglobin, nuclear ubiquitin casein (NUCKS), POTEE, high mobility group protein (HMG-I/HMG-Y) and lactoferrin were identified in EBC of NSCLC patients. Conclusion. We found that EBC in healthy nonsmokers and in patients with COPD, pneumonia and NSCLC had characteristic protein spectrum. Most of the identified proteins could be used for diagnosis of these diseases.

Isolation and Characterization of Bioactive Moiety from Leaf Extract of Ipomoea mauritiana and Evaluation of Anti-Inflammatory Activity by Carrageenan Induced Rat Paw Edema Test

Ipomoea mauritiana Jacq. is a member of family Convolvulaceae and commonly called as Ksheervidari is widely used in various traditional medicines for the treatment of many diseases. In the present work the column fractions of leaf extracts of I. mauritiana obtained by Soxhlet exrtaction was subjected to TLC, column chromatography, HPLC and liquid chromatography and mass spectroscopy (LC-MS). The compounds like caffeic acid and β-amyric acetate were tentatively identified to be present in one of the purified fraction. The column fraction was also tested for anti-inflammatory activity by carrageenan induced rat paw edema assay. The column fraction tested showed a very good anti inflammatory activity.

Production of Mycosporine-Like Amino Acids from Gracilaria vermiculophylla (Rhodophyta) Cultured Through One Year in an Integrated Multi-trophic Aquaculture (IMTA) System.

This study evaluates the production of biomass and mycosporine-like amino acids (MAAs) throughout the year in Gracilaria vermiculophylla (Rhodophyta) collected in Ria de Aveiro (Portugal). The algae were grown in outdoor tanks in seawater with the addition of fishpond effluents under two different water flows (100 and 200Lh-1) in an integrated multi-trophic aquaculture (IMTA) system (tanks 1200L; 1.5m2) and different algal densities (3, 5, and 7kgm-2). MAA content in IMTA seaweeds was significantly affected by the interaction of time and stocking density, but not by the water flow. The highest MAA content was observed in April (about 3.13mgg-1 DW) followed by May (1.79mgg-1 DW). Seaweed biomass productivity was higher in May (372.06gDWm-2week-1) than in April (353.40gDWm-2week-1). Four MAAs were identified by HPLC and electrospray ionization mass spectrometry (ESI-MS) in G. vermiculophylla: Porphyra-334, Shinorine, Palythine and Asterina-330. The highest levels of Porphyra-334 and Shinorine were reached from November to January and the Palythine + Asterina-330 from April to August. Taking into account the average biomass and MAA production of G. vermiculophylla growing in this IMTA system (8.56g of MAA in 18m2 culture along 8months; 35.5% produced in April), a total amount of 71.33gMAAyear-1 could be produced in this system by scaling up to 100m2. MAAs could be further used as photoprotector and antioxidant compounds in cosmetic products.

Targeted Modification of a Novel Amphibian Antimicrobial Peptide from Phyllomedusa tarsius to Enhance Its Activity against MRSA and Microbial Biofilm.

Antimicrobial peptides (AMPs) in the skin secretions of amphibians are fundamental components of a unique defense system that has evolved to protect these hosts from microbial invasion. Medusins constitute a recently-discovered AMP family from phyllomedusine leaf frog skin and exhibit highly-conserved structural characteristics. Here, we report a novel medusin, medusin-PT, from the skin secretion of the Tarsier Leaf Frog, Phyllomedusa tarsius. The mature peptide was initially identified from its cloned biosynthetic precursor-encoding cDNA as obtained by the rapid amplification of cDNA ends (RACE) method. Reverse-phase HPLC and tandem mass spectrometry confirmed both the presence of medusin-PT in the skin secretion and its primary structure. In a range of bioassays, medusin-PT exhibited antimicrobial activity against only the Gram-positive bacterium Staphylococcus aureus at 64 μg/ml. However, after directed changes to enhance the cationicity and amphipathicity of the peptide structure, three analog showed more potent antimicrobial activity against several additional bacteria including the antibiotic-resistant bacterium MRSA. In addition, these analog exhibited activity against microbial biofilm (minimum biofilm inhibitory and eradication concentrations of 32 μg/ml and over 64 μg/ml, respectively). These data provide evidence that medusins might be promising candidates as novel antibiotic leads and that the targeted modification of a natural AMP can both improve its efficacy so as to provide new insights into antibiotic design and development.

A High-Throughput Bioluminescent Assay to Monitor the Deamidation of Asparagine and Isomerization of Aspartate Residues in Therapeutic Proteins and Antibodies

Since the introduction of Herceptin and Rituximab in 1986, therapeutic antibodies have gained tremendous momentum in the treatment of broad range of several diseases such as cancer and inflammation. Selection of the clinical candidate mAb usually starts with large-scale in vitro screening and profiling of multiple mAbs to identify candidates that show high in vitro or in vivo activity, and thus it is necessarily to identify and eliminate potentially unstable mAbs during the lead selection process. Antibodies undergo a variety of degradation reactions that may result in compromised bioactivity and safety profile. The nonenzymatic post-translational modification of both deamidation of asparagine and isomerization of aspartate residues is one of the major chemical reactions occurring in proteins during production and storage resulting in formation of protein variants that may affect the quality, safety, and functionality of the therapeutic proteins. Current methods (HPLC and liquid chromatography and mass spectrometry) for monitoring isoaspartate (isoAsp) formation are time consuming, require specialized equipment and trained personnel, and are not amenable to high-throughput scaling. We have developed a robust, homogenous, high-throughput formatted, and sensitive assay to accurately monitor the formation of isoAsp under several conditions, such as new formulations, storage periods, and temperature.

Green and Efficient Extraction Method to Determine Polyphenols in Cocoa and Cocoa Products

Cocoa and cocoa products have broad and well-proven health benefits, most of which are associated with the high antioxidant capacity of cocoa flavonoids. Most extraction methods for flavonoids in cocoa products use several steps including a defatting step and large amount of organic solvents. Moreover, they are labor-intensive and time-consuming. In this work, a pressurized hot water extraction (PHWE) method has been compared to conventional sonication extraction (CSE) method. The contribution of individual compounds to the total antioxidant capacity of the extracts was evaluated by developing an analytical technique consisting of high-performance liquid chromatography (HPLC) with photodiode array coupled to electrochemical and to charged aerosol detectors and HPLC-mass spectrometry for the confirmation of the identity of compounds present. Additionally, procyanidins were analyzed by fluorescence detection. PHWE turned out to be more efficient in extracting phenolics and methylxanthines, as compared to the conventional method, in addition to being more “green” in terms of using less organic solvents.

Proteomic Analysis of the Bovine and Human Ciliary Zonule.

PurposeThe zonule of Zinn (ciliary zonule) is a system of fibers that centers the crystalline lens on the optical axis of the eye. Mutations in zonule components underlie syndromic conditions associated with a broad range of ocular pathologies, including microspherophakia and ectopia lentis. Here, we used HPLC–mass spectrometry to determine the molecular composition of the zonule.MethodsTryptic digests of human and bovine zonular samples were analyzed by HPLC–mass spectrometry. The distribution of selected components was confirmed by immunofluorescence confocal microscopy. In bovine samples, the composition of the equatorial zonule was compared to that of the hyaloid zonule and vitreous humor.ResultsThe 52 proteins common to the zonules of both species accounted for >95% of the zonular protein. Glycoproteins constituted the main structural components, with two proteins, FBN1 and LTBP2, constituting 70%–80% of the protein. Other abundant components were MFAP2, EMILIN-1, and ADAMTSL-6. Lysyl oxidase-like 1, a crosslinking enzyme implicated in collagen and elastin biogenesis, was detected at significant levels. The equatorial and hyaloid zonular samples were compositionally similar to each other, although the hyaloid sample was relatively enriched in the proteoglycan opticin and the fibrillar collagens COL2A1, COL11A1, COL5A2, and COL5A3.ConclusionsThe zonular proteome was surprisingly complex. In addition to structural components, it contained signaling proteins, protease inhibitors, and crosslinking enzymes. The equatorial and hyaloid zonules were similar in composition, but the latter may form part of a composite structure, the hyaloid membrane, that stabilizes the vitreous face.

Identification of phenolic compounds by liquid chromatography-mass spectrometry in seventeen species of wild mushrooms in Central Mexico and determination of their antioxidant activity and bioactive compounds.

Wild mushrooms are important for the diet of some communities in Mexico. However, limited information exists on their chemical composition, contribution to the diet, and health effects. We characterized seventeen wild mushroom species growing in the state of Queretaro in Central Mexico. Most species analyzed were edible, but also included nonedible, medicinal, poisonous and toxic specimens. Whole mushrooms (caps and stipes) were characterized for water content, color, and total content of phenolic compounds, flavonoids and anthocyanins. In vitro antioxidant capacity was measured by FRAP and DPPH assays. Phenolic compounds were identified and quantified by HPLC-mass spectrometry. All species analyzed were found to possess antioxidant activity in vitro and a wide range of phenolic and organic compounds were identified. Our results add to the limited information available on the composition and potential nutritional and health value of wild mushrooms. Further analyses of their bioactivities are warranted.

Flavonoid Profiles of Wild Grapes Native to Japan: <i>Vitis coignetiae </i>Pulliat and<i> Vitis ficifolia </i>Bunge var. <i>ganebu</i> Hatusima

Flavonoids are a group of natural compounds in plants with versatile health benefits for humans. Grapes are a dietary source of flavonoids and the flavonoid components in grape berries can depend on the grape species and cultivar. In this experiment, proanthocyanidins, flavonols, and anthocyanins were analyzed in Vitis coignetiae and V. ficifolia var. ganebu, wild grapes native to Japan, and compared with those in V. labruscana cv. Muscat Bailey A, to evaluate the potential of the wild grapes as a grape resource. Proanthocyanidin contents in seeds were lower in the two wild grapes than in Muscat Bailey A. However, the skin of V. ficifolia var. ganebu was the richest source of proanthocyanidins. Flavonol levels in the skins of the two wild grapes were lower than that in the skin of Muscat Bailey A. Colorimetry determined that the total anthocyanin content in the skin of V. ficifolia var. ganebu was 6 times and 7 times higher, respectively, than those of V. coignetiae and Muscat Bailey A. Although monoglucoside anthocyanin levels analyzed by high-pressure liquid chromatography (HPLC) were in the order Muscat Bailey A > V. ficifolia var. ganebu > V. coignetiae, most of the diglucoside and acylated monoglucoside and diglucoside anthocyanin levels identified by HPLC-mass spectrometry were highest in V. ficifolia var. ganebu. These data suggest that V. ficifolia var. ganebu might be a novel source of flavonoids and superior to V. coignetiae as a source of flavonoids.

Exploring the efficacy of immersion analgesics in zebrafish using an integrative approach

Home Office guidelines recommend the use of analgesics for all protected species, including fish during scientific procedures likely to result in pain, suffering, distress and lasting harm. In larger fish species some drugs have shown efficacy in terms of perioperative analgesia when administered intramuscularly or at the site of tissue damage. However, this is impractical in very small species (<1g) and when large numbers need to be treated. Immersion is a commonly used route of administering anaesthesia in fish and may be a less invasive alternative to administering analgesia. Therefore, this study examined the efficacy of three water soluble analgesic drugs (buffered acetylsalicylic acid, butorphanol tartrate and lidocaine) provided pre- and post-surgery (tail fin clip) through addition to the tank water. The effectiveness of these drugs was measured as the reduction of changes in pain-related post-surgical behaviour and since pain is inherently stressful, HPI axis activity, quantified as whole body cortisol levels. To determine uptake of each drug, whole body concentrations were determined by HPLC-mass spectroscopy. Administration of both lidocaine and acetylsalicylic acid resulted in a significant reduction of post-nociceptive behavioural patterns, compared with animals exposed to saline solution. There was no effect of treatment on whole body cortisol. Finally, uptake was verified for lidocaine with whole-body concentrations showing clear dose dependency, whereas the other two compounds were not detectable. As the only drug with both a behavioural sparing effect and pharmacokinetic validation in this study, low-dose lidocaine immersions can be recommended for the first time to aid perioperative analgesia and provide a valuable refinement for reducing the impact of invasive procedures in zebrafish thereby improving welfare.

Proteomics reveals the effects of sustained weight loss on the human plasma proteome.

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill‐defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry‐based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual‐specific protein levels with wide‐ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte‐secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of −16% and +37%, respectively (P < 10−13), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten‐protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly evaluates and monitors intervention in metabolic diseases.