scholarly journals Extraction and LC-MS/MS Analysis of Ciguatoxins: A Semi-Targeted Approach Designed for Fish of Unknown Origin

Toxins ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 630
Author(s):  
Astrid Spielmeyer ◽  
Christopher R. Loeffler ◽  
Dorina Bodi
Keyword(s):  
Matrix Effects ◽  
Solid Phase ◽  
Unknown Origin ◽  
Fish Tissue ◽  
Enzymatic Digestion ◽  
In Vitro Cytotoxicity ◽  
Analysis Methods ◽  
Sodium Adducts ◽  
Freeze Dried

Ciguatoxins (CTXs) are polyether marine biotoxins that can cause ciguatera poisoning (CP) after the consumption of fish or invertebrates containing sub ppb levels; concentrations that present a challenge for current extraction and analysis methods. Here, a newly developed and (partly) validated single-day extraction protocol is presented. First, the fish sample is broken-down by enzymatic digestion, followed by extraction and extract clean-up by defatting and two solid-phase extractions. Final extracts were investigated using two different CTX-analysis methods; an in vitro cytotoxicity assay (N2a-assay) and by LC-MS/MS. Validation was performed for both fillet and freeze-dried samples of snapper, parrotfish, and grouper spiked with CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, and CTX3C. Based on recovery rates (35–88%) and matrix effects (66–116%) determined by LC-MS/MS, the enzyme protocol is applicable to various matrices. The protocol was applied to naturally contaminated fish tissue (Lutjanus bohar) obtained during a CP incident in Germany. Several potential CTX congeners were identified by a two-tier LC-MS/MS approach (screening of sodium adducts, high-resolution or low-resolution confirmation via ammonium adducts). Inclusion of >30 known CTX congeners into the LC-MS/MS methods and single-day sample preparation make the method suitable for analysis of ciguatera suspect samples at sub ppb levels also with undisclosed CTX profiles.

scholarly journals Iron Bioavailability from Multiple Biofortified Foods Using an in Vitro Digestion, Caco-2 Assay for Optimizing a Cyclic Menu for a Randomized Efficacy Trial (P10-029-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Bryan Gannon ◽  
Raymond Glahn ◽  
Saurabh Mehta
Keyword(s):  
Sweet Potato ◽  
Pearl Millet ◽  
Iron Uptake ◽  
Matrix Effects ◽  
Iron Concentration ◽  
In Vitro Digestion ◽  
Iron Bioavailability ◽  
Effect Estimate ◽  
Freeze Dried

Abstract Objectives A multiple biofortified food crop trial targeting iron, zinc, and vitamin A deficiencies among young children and their breastfeeding mothers is planned in India. We sought to determine iron bioavailability from biofortified and conventional crop mixes representative of planned meal components. Methods A 24-meal menu was developed based on pearl millet, sweet potato, and lentils targeted for a feeding trial. Crops were procured from India, cooked, and freeze-dried before two rounds of an established in vitro digestion/Caco-2 iron bioavailability assay. Samples used a fixed weight adjusted for sweet potato water content. Representative crop proportions were determined using k-means clustering, combined such that samples included either all biofortified or all control crop varieties, and analyzed in triplicate. Outcomes were Caco-2 iron uptake and uptake normalized to iron per sample for fractional bioavailability. Data were analyzed with generalized linear models in SAS accounting for crop proportions and variety. Results Across both experiments, biofortified pearl millet alone demonstrated higher iron uptake than conventional varieties (5.01 ± 1.66 vs. 2.17 ± 0.96 ng ferritin/mg protein, P ≤ 0.036). Addition of sweet potato to pearl millet did not change iron uptake for biofortified varieties (P ≥0.13), but increased control iron uptake for all amounts of sweet potato (P ≤ 0.006), which did not differ from biofortified varieties (P ≥ 0.08). Lentil proportion increased iron uptake (β = 4.6 ± 2.2, P = 0.009), with no effect of variety or a lentil by variety interaction (P ≥ 0.56). The overall effect estimate of biofortified vs. control was (β = 1.79 ± 0.91, P = 0.08). Iron uptake normalized to iron per sample was higher for control crops (P ≤ 0.02), and enhanced by sweet potato, while inhibited by pearl millet (both P < 0.001). Conclusions A Caco-2 assay predicts that biofortified pearl millet alone has greater iron bioavailability than control pearl millet. The addition of sweet potato and lentils increased overall and relative iron bioavailability, while reducing differences between biofortified and control varieties. Matrix effects, processing, and promoters/inhibitors of iron absorption should be considered in addition to total iron concentration when optimizing iron bioavailability. Funding Sources This work was supported by HarvestPlus and the USDA. Supporting Tables, Images and/or Graphs

scholarly journals New method for quantitative analysis of GD2 ganglioside in plasma of neuroblastoma patients.

2009 ◽  
Vol 56 (3) ◽  
Author(s):  
Dominik Czaplicki ◽  
Irena Horwacik ◽  
Aleksandra Kowalczyk ◽  
Aleksandra Wieczorek ◽  
Katarzyna Bolek-Marzec ◽  
...  
Keyword(s):  
Cancer Progression ◽  
High Performance ◽  
Neuroblastoma Cells ◽  
Unknown Origin ◽  
Enzymatic Digestion ◽  
Human Blood Plasma ◽  
Gd3 Ganglioside ◽  
Gd2 Ganglioside

Neuroblastoma, the most common extracranial solid tumour of childhood, is a malignancy of unknown origin and non-specific symptoms. One of the markers of the disease is GD2 ganglioside (disialoganglioside), which is abundantly expressed on the surface of neuroblastoma cells. Gangliosides are known to be shed by tumour cells and this phenomenon can be significant in cancer progression as they inhibit a number of immune responses both in vitro and in vivo. In search for novel markers useful in monitoring and prognosis of neuroblastoma, we developed and validated a new quantitative method of GD2 ganglioside analysis in human blood plasma. We evaluated the level of gangliosides in blood serum of 34 neuroblastoma patients using high-performance liquid chromatography. The technique was used to detect fluorescently labelled oligosaccharides derived from serum glycosphingolipids by enzymatic digestion with ceramide glycanase. The developed method allowed determination of GD2 concentrations at the picomole level and required only 40 microl of plasma, which should be particularly useful when the quantity of clinical material is limiting. Moreover, this method can be applied to study concentration of other gangliosides, as shown for GD3 ganglioside. Analysis of plasma samples from the 34 neuroblastoma patients did not reveal any correlations between the concentration of GD2 ganglioside and clinical parameters, including the results of therapy; it showed, however, that the concentration of GD2 ganglioside in the plasma of neuroblastoma patients decreased substantially in the course of treatment.

scholarly journals Freeze-Drying Ethylcellulose Microparticles Loaded with Etoposide for In Vitro Fast Dissolution and In Vitro Cytotoxicity against Cancer Cell Types, MCF-7 and Caco-2

2021 ◽  
Vol 11 (19) ◽  
pp. 9066
Author(s):  
Ahmed A. H. Abdellatif ◽  
Mashari A. Aldhafeeri ◽  
Waleed H. Alharbi ◽  
Fahad H. Alharbi ◽  
Waleed Almutiri ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Freeze Drying ◽  
Chemical Interaction ◽  
In Vitro Cytotoxicity ◽  
Dissolution Experiment ◽  
Freeze Dried ◽  
Fast Dissolution ◽  
Mcf 7

The aim of this study was to improve the solubility of etoposide–ethylcellulose (ET–ETO) microparticles using the freeze-drying technique. Ethylcellulose (EC) microparticles loaded with etoposide (ETO) were prepared with different drug–polymer molar ratios of 1:1, 1:3, 1:6, and 1:20 by the solvent evaporation method. The size of the prepared microparticles was 0.088 µm. The results showed that the amount of ETO encapsulated into the microparticles was 387.3, 365.0, 350.0, and 250 µg/50 mg microparticles for microparticles with drug–polymer ratios of 1:1, 1:3, 1:6, and 1:20, respectively. The FT-IR spectra showed no chemical interaction between ETO and the polymer in the solid state. The results obtained from the dissolution experiment showed that the freeze-dried microparticles were stable in 0.1 N HCl (gastric pH) for 2 h. At pH 7.4, the ETO release was 60 to 70% within the first 15 min and approximately 100% within 30 min. Results from the application of different dissolution models showed that the equations that best fit the dissolution data for the ET–ETO microparticles at pH 7.4 were the Higuchi and Peppas model equations. The in vitro cytotoxicity assay of free ETO and freeze-dried microspheres prepared in this study with a drug–polymer ratio of 1:1 was performed in two mammalian cancer cell lines, MCF-7 (for bone cancer of the mammary organ) and Caco-2 (for mammalian epithelial colorectal adenocarcinoma). The results showed that the half-maximal inhibitory concentrations (IC50 values) for ETO and freeze-dried ET–ETO microparticles were 18.6 µM and 27.1 µM, respectively. In conclusion, freeze-dried ET–ETO is a promising formulation for developing a fast-dissolving form of ETO with a significant antiproliferative activity against the tested cell lines used in this study. It is a promising formulation for local duodenal area targeting.

scholarly journals Less Polar Compounds and Targeted Antioxidant Potential (In Vitro and In Vivo) of Codium adhaerens C. Agardh 1822

2021 ◽  
Vol 14 (9) ◽  
pp. 944
Author(s):  
Sanja Radman ◽  
Ana-Marija Cikoš ◽  
Ivana Flanjak ◽  
Sanja Babić ◽  
Lara Čižmek ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Solid Phase ◽  
Antioxidant Properties ◽  
Polar Compounds ◽  
Antioxidant Potential ◽  
In Vitro Assays ◽  
Pheophytin A ◽  
Freeze Dried

Codium adhaerens from the Adriatic Sea (Croatia) was comprehensively investigated regarding less polar compounds for the first time. Although there are several phytochemical studies on C. adhaerens from other regions, this is the first report on volatile organic compounds (VOCs) from fresh (FrCa) and air-dried (DrCa) samples. The novelty is also related to its targeted antioxidant potential in vitro and in vivo. The main aims were to: (a) identify and compare VOCs of FrCa and DrCa obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD); (b) determine fatty acid (FA) composition of freeze-dried sample (FdCa); (c) determine the composition of less polar fractions of FdCa by high-performance liquid chromatography–high-resolution mass spectrometry with electrospray ionisation (UHPLC-ESI-HRMS); and (d) comprehensively evaluate the antioxidant activity of the fractions by four in vitro assays and in vivo zebrafish model (including embryotoxicity). Significant changes of VOCs were found after air drying. ω6 FAs were present in higher content than ω3 FAs indicating C. adhaerens as a good source of dietary polyunsaturated FAs. The results obtained in vivo correlate well with in vitro methods and both fractions exerted similar antioxidative responses which is in agreement with the high abundance of present biomolecules with known antioxidant properties (e.g., fucoxanthin, pheophytin a, and pheophorbide a). These results suggest that C. adhaerens might be a potent source of natural antioxidants that could be further used in the research of oxidative stress-related diseases.

scholarly journals Iron Bioavailability from Multiple Biofortified Foods Using an In Vitro Digestion, Caco-2 Assay for Optimizing a Cyclical Menu for a Randomized Efficacy Trial

2021 ◽  
Vol 5 (9) ◽  
Author(s):  
Bryan M Gannon ◽  
Raymond P Glahn ◽  
Saurabh Mehta
Keyword(s):  
Sweet Potato ◽  
Iron Uptake ◽  
Matrix Effects ◽  
Human Study ◽  
In Vitro Digestion ◽  
Iron Bioavailability ◽  
Total Iron ◽  
Cell Protein ◽  
Freeze Dried

ABSTRACT Background Inadequate nutritional status contributes to substantial losses in human health and productivity globally. A multiple biofortified food crop trial targeting iron, zinc, and vitamin A deficiencies among young children and their breastfeeding mothers is being conducted in India. Objective We sought to determine the relative iron bioavailability from biofortified and conventional crops and crop combinations representative of a cyclical menu using crops targeted for inclusion in the feeding trial. Methods Crops were procured from India, cooked, freeze-dried, and analyzed with an established in vitro digestion/Caco-2 iron bioavailability assay using a fixed sample weight. Crop proportions representative of meals planned for the human study were determined and combined such that samples included either all biofortified or all control crops. Crops were analyzed as single crops (n = 4) or crop combinations (n = 7) by variety (biofortified or control) in triplicate. The primary outcome was iron uptake measured by Caco-2 ferritin production normalized to total Caco-2 protein (nanograms of ferritin/milligrams of cell protein) analyzed for effects of crop variety and crop proportion using generalized linear models. Results Biofortified pearl millet alone demonstrated higher iron uptake than conventional varieties (5.01 ± 1.66 vs. 2.17 ± 0.96; P = 0.036). Addition of sweet potato or sweet potato + pulse improved iron uptake for all proportions tested in control varieties and select proportions for biofortified varieties (P ≤ 0.05). Two multiple crop combinations demonstrated modestly higher iron uptake from biofortified crops. Conclusions Optimizing total iron delivery should consider matrix effects, processing, and promoters/inhibitors of iron absorption in addition to total iron concentration. Future directions include evaluating recipes as prepared for consumption and comparison against human iron bioavailability studies.

scholarly journals Biomimetic Composite Scaffold Based on Naturally Derived Biomaterials

Polymers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1161
Author(s):  
Ionela Andreea Neacsu ◽  
Adriana Petruta Serban ◽  
Adrian Ionut Nicoara ◽  
Roxana Trusca ◽  
Vladimir Lucian Ene ◽  
...  
Keyword(s):  
Cellular Response ◽  
Composite Scaffold ◽  
Healing Process ◽  
Organic Matrix ◽  
In Vitro Cytotoxicity ◽  
Eggshell Membrane ◽  
Bovine Bone ◽  
Freeze Dried ◽  
Biomimetic Hydroxyapatite

This paper proposes the development of a biomimetic composite based on naturally derived biomaterials. This freeze-dried scaffold contains a microwave-synthesized form of biomimetic hydroxyapatite (HAp), using the interwoven hierarchical structure of eggshell membrane (ESM) as bio-template. The bone regeneration capacity of the scaffold is enhanced with the help of added tricalcium phosphate from bovine Bone ash (BA). With the addition of Gelatin (Gel) and Chitosan (CS) as organic matrix, the obtained composite is characterized by the ability to stimulate the cellular response and might accelerate the bone healing process. Structural characterization of the synthesized HAp (ESM) confirms the presence of both hydroxyapatite and monetite phases, in accordance with the spectroscopy results on the ESM before and after the microwave thermal treatment (the presence of phosphate group). Morphology studies on all individual components and final scaffold, highlight their morphology and porous structure, characteristics that influence the biocompatibility of the scaffold. Porosity, swelling rate and the in vitro cytotoxicity assays performed on amniotic fluid stem cells (AFSC), demonstrate the effective biocompatibility of the obtained materials. The experimental results presented in this paper highlight an original biocomposite scaffold obtained from naturally derived materials, in a nontoxic manner.

scholarly journals Solid-Phase Synthesis and In Vitro Cytotoxicity of New Peptide Functionalized Cyclencarboxymethylen and L-DOPA Hybrids

2017 ◽  
Vol 7 (12) ◽  
Author(s):  
L Arabuli ◽  
R Jezek ◽  
T Macek ◽  
P Lovecka ◽  
E Nikoleishvili ◽  
...  
Keyword(s):  
Solid Phase Synthesis ◽  
Solid Phase ◽  
In Vitro Cytotoxicity ◽  
Phase Synthesis

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

Patients with fever of unknown origin (FUO): diagnosis by nuclear medicine imaging

2001 ◽  
Vol 40 (03) ◽  
pp. 59-70 ◽  
Author(s):  
W. Becker ◽  
J. Meiler
Keyword(s):  
Nuclear Medicine ◽  
Fever Of Unknown Origin ◽  
Tumor Imaging ◽  
White Blood Cells ◽  
Unknown Origin ◽  
Final Diagnosis ◽  
Recurrent Fever ◽  
Nuclear Medicine Imaging

SummaryFever of unknown origin (FUO) in immunocompetent and non neutropenic patients is defined as recurrent fever of 38,3° C or greater, lasting 2-3 weeks or longer, and undiagnosed after 1 week of appropriate evaluation. The underlying diseases of FUO are numerous and infection accounts for only 20-40% of them. The majority of FUO-patients have autoimmunity and collagen vascular disease and neoplasm, which are responsible for about 50-60% of all cases. In this respect FOU in its classical definition is clearly separated from postoperative and neutropenic fever where inflammation and infection are more common. Although methods that use in-vitro or in-vivo labeled white blood cells (WBCs) have a high diagnostic accuracy in the detection and exclusion of granulocytic pathology, they are only of limited value in FUO-patients in establishing the final diagnosis due to the low prevalence of purulent processes in this collective. WBCs are more suited in evaluation of the focus in occult sepsis. Ga-67 citrate is the only commercially available gamma emitter which images acute, chronic, granulomatous and autoimmune inflammation and also various malignant diseases. Therefore Ga-67 citrate is currently considered to be the tracer of choice in the diagnostic work-up of FUO. The number of Ga-67-scans contributing to the final diagnosis was found to be higher outside Germany than it has been reported for labeled WBCs. F-l 8-2’-deoxy-2-fluoro-D-glucose (FDG) has been used extensively for tumor imaging with PET. Inflammatory processes accumulate the tracer by similar mechanisms. First results of FDG imaging demonstrated, that FDG may be superior to other nuclear medicine imaging modalities which may be explained by the preferable tracer kinetics of the small F-l 8-FDG molecule and by a better spatial resolution of coincidence imaging in comparison to a conventional gamma camera.

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