The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.
22 Related JoVE Articles!
The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy
Institutions: University of Southampton.
Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.
Chemistry, Issue 85, gas chromatography, fatty acid, pregnancy, cholesteryl ester, solid phase extraction, polyunsaturated fatty acids
One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure
Institutions: Saint Louis University School of Medicine.
Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.
Biochemistry, Issue 40, metabolomics, gas chromatography/mass spectrometry, GC/MS, inborn errors, vitamin deficiency, BNA analyses, carbohydrate, amino acid, organic acid, urease
GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil
Institutions: University of Wisconsin, Madison, University of Wisconsin, Madison, University of Florida .
Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers1, 2
. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.
The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems3, 4
. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage5
Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications6, 7
. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives8
. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using δ13
C or ion fragments of these biomarkers in biogeochemical studies9, 10
Molecular Biology, Issue 63, Glucosamine, muramic acid, microbial residue, aldononitrile acetate derivatization, isotope incorporation, ion structure, electron ionization, GC, MS
Synthesis of Antiviral Tetrahydrocarbazole Derivatives by Photochemical and Acid-catalyzed C-H Functionalization via Intermediate Peroxides (CHIPS)
Institutions: Max-Planck-Institut fuer Kohlenforschung.
The direct functionalization of C-H bonds is an important and long standing goal in organic chemistry. Such transformations can be very powerful in order to streamline synthesis by saving steps, time and material compared to conventional methods that require the introduction and removal of activating or directing groups. Therefore, the functionalization of C-H bonds is also attractive for green chemistry. Under oxidative conditions, two C-H bonds or one C-H and one heteroatom-H bond can be transformed to C-C and C-heteroatom bonds, respectively. Often these oxidative coupling reactions require synthetic oxidants, expensive catalysts or high temperatures. Here, we describe a two-step procedure to functionalize indole derivatives, more specifically tetrahydrocarbazoles, by C-H amination using only elemental oxygen as oxidant. The reaction uses the principle of C-H functionalization via Intermediate PeroxideS (CHIPS). In the first step, a hydroperoxide is generated oxidatively using visible light, a photosensitizer and elemental oxygen. In the second step, the N-nucleophile, an aniline, is introduced by Brønsted-acid catalyzed activation of the hydroperoxide leaving group. The products of the first and second step often precipitate and can be conveniently filtered off. The synthesis of a biologically active compound is shown.
Chemistry, Issue 88, Catalysis, Photocatalysis, C-H functionalization, Oxygen, Peroxides, Indoles, Pharmaceuticals
Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure
Institutions: Grosshadern Hospital, Munich, Grosshadern Hospital, Munich, Hepacult LLC, Regensburg, Grosshadern Hospital, Munich.
The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77±10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.
Medicine, Issue 79, Cellular Biology, Biomedical Engineering, Anatomy, Physiology, Surgery, Life Sciences (General), Human hepatocyte isolation, human hepatocyte, collagenase, perfusion, collagenase perfusion, hepatocyte, liver, human, cell, isolation, clinical applications, clinical techniques
Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts
Institutions: Aix-Marseille Université, Aix-Marseille Université.
Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.
Neuroscience, Issue 91, metabolomics, brain tissue, rodents, neurochemistry, tissue extracts, NMR spectroscopy, quantitative metabolite analysis, cerebral metabolism, metabolic profile
A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat
Institutions: Université de Sherbrooke, Université de Sherbrooke, Université de Sherbrooke, Université de Sherbrooke.
We present a method for comparing the uptake of the brain's two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which 11
C-AcAc and 18
F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11
C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11
C-AcAc and 18
F-FDG brain uptake (cerebral metabolic rate; μmol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases.
Neuroscience, Issue 82, positron emission tomography (PET), 18F-fluorodeoxyglucose, 11C-acetoacetate, magnetic resonance imaging (MRI), kinetic modeling, cerebral metabolic rate, rat
A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
Institutions: Delft University of Technology, Delft University of Technology.
This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli
and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9
addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli
to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2
) of the alkane hydroxylase system from Gordonia
were transformed into E. coli
. For the conversion of long-chain alkanes (C15-C36), theladA
gene from Geobacillus thermodenitrificans
was implemented. For the required further steps of the degradation process, ADH
and ALDH (
originating from G. thermodenitrificans
) were introduced10,11
. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli
K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii
OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g.
under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n
-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli
to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.
Bioengineering, Issue 68, Microbiology, Biochemistry, Chemistry, Chemical Engineering, Oil remediation, alkane metabolism, alkane hydroxylase system, resting cell assay, prefoldin, Escherichia coli, synthetic biology, homologous interaction mapping, mathematical model, BioBrick, iGEM
Human Skeletal Muscle Biopsy Procedures Using the Modified Bergström Technique
Institutions: Appalacian State University, Appalachian State University, Carolinas Medical Center NorthEast.
The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergström needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergström needle consists of an outer cannula with a small opening (‘window’) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90° and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.
Medicine, Issue 91, percutaneous muscle biopsy, needle biopsy, suction-modified, metabolism, enzyme activity, mRNA, gene function, fiber type, histology, metabolomics, skeletal muscle function, humans
Ovariectomy and 17β-estradiol Replacement in Rats and Mice: A Visual Demonstration
Institutions: Linköping University.
Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17β-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17β-estradiol level/administration method profoundly influences the experimental results1-3
. We have in a series of studies characterized the different modes of 17β-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17β-estradiol4-6
. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration7
, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17β-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.
Medicine, Issue 64, Physiology, Oophorectomy, Rat, Mouse, 17β-estradiol, Administration, Silastic capsules, Nutella
Oral Administration of Rotenone using a Gavage and Image Analysis of Alpha-synuclein Inclusions in the Enteric Nervous System
Institutions: Technische Universität Dresden.
In Parkinson's disease (PD) patients, the associated pathology follows a characteristic pattern involving inter alia the enteric nervous system (ENS) 1,2
, the olfactory bulb (OB), the dorsal motor nucleus of the vagus (DMV)3
, the intermediolateral nucleus of the spinal cord 4
and the substantia nigra, providing the basis for the neuropathological staging of the disease4,5
. The ENS and the OB are the most exposed nervous structures and the first ones to be affected. Interestingly, PD has been related to pesticide exposure6-8
. Here we show in detail two methods used in our previous study 9
. In order to analyze the effects of rotenone acting locally on the ENS, we administered rotenone using a gavage to one-year old C57/BL6 mice. Rotenone is a widely used pesticide that strongly inhibits mitochondrial Complex I 10
. It is highly lipophylic and poorly absorbed in the gastrointestinal tract 11
. Our results showed that the administration of 5 mg/kg of rotenone did not inhibit mitochondrial Complex I activity in the muscle or the brain. Thus, suggesting that using our administration method rotenone did not cross the hepatoportal system and was acting solely on the ENS. Here we show a method to administer pesticides using a gavage and the image analysis protocol used to analyze the effects of the pesticide in alpha-synuclein accumulation in the ENS. The first part shows a method that allows intragastric administration of pesticides (rotenone) at a desired precise concentration. The second method shows a semi-automatic image analysis protocol to analyze alpha-synuclein accumulation in the ENS using an image analysis software.
Neuroscience, Issue 44, neurogical disorders, Parkinson's disease, animal model, mouse, rotenone, gavage, image analysis
Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer
Institutions: University of Victoria, University of Victoria.
Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.
Basic Protocol, Issue 81, eye, molecular imaging, chemistry technique, analytical, mass spectrometry, matrix assisted laser desorption/ionization (MALDI), tandem mass spectrometry, lipid, tissue imaging, bovine lens, dithranol, matrix, FTICR (Fourier Transform Ion Cyclotron Resonance)
Manual Restraint and Common Compound Administration Routes in Mice and Rats
Institutions: Charles River , Charles River.
Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.
Basic Protocols, Issue 67, Anatomy, Medicine, Rodents, training, handling, restraint, injections, oral gavage
Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency
Institutions: Environmental Health Centre.
mutations arise mostly in the male germline and may contribute to adverse health outcomes in subsequent generations. Traditional methods for assessing the induction of germ cell mutations require the use of large numbers of animals, making them impractical. As such, germ cell mutagenicity is rarely assessed during chemical testing and risk assessment. Herein, we describe an in vivo
male germ cell mutation assay using a transgenic rodent model that is based on a recently approved Organisation for Economic Co-operation and Development (OECD) test guideline. This method uses an in vitro
positive selection assay to measure in vivo
mutations induced in a transgenic λgt10 vector bearing a reporter gene directly in the germ cells of exposed males. We further describe how the detection of mutations in the transgene recovered from germ cells can be used to characterize the stage-specific sensitivity of the various spermatogenic cell types to mutagen exposure by controlling three experimental parameters: the duration of exposure (administration time), the time between exposure and sample collection (sampling time), and the cell population collected for analysis. Because a large number of germ cells can be assayed from a single male, this method has superior sensitivity compared with traditional methods, requires fewer animals and therefore much less time and resources.
Genetics, Issue 90, sperm, spermatogonia, male germ cells, spermatogenesis, de novo mutation, OECD TG 488, transgenic rodent mutation assay, N-ethyl-N-nitrosourea, genetic toxicology
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
A Strategy for Sensitive, Large Scale Quantitative Metabolomics
Institutions: Cornell University, Cornell University.
Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously. Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.
Chemistry, Issue 87, high-resolution mass spectrometry, metabolomics, positive/negative switching, low mass calibration, Orbitrap
Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis
Institutions: National Jewish Health, University of Colorado Denver.
Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes. Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 µl. Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.
Bioengineering, Issue 89, plasma, chemistry techniques, analytical, solid phase extraction, mass spectrometry, metabolomics, fluids and secretions, profiling, small molecules, lipids, liquid chromatography, liquid-liquid extraction, cerebrospinal fluid, bronchoalveolar lavage fluid
Conducting Miller-Urey Experiments
Institutions: Georgia Institute of Technology, Tokyo Institute of Technology, Institute for Advanced Study, NASA Johnson Space Center, NASA Goddard Space Flight Center, University of California at San Diego.
In 1953, Stanley Miller reported the production of biomolecules from simple gaseous starting materials, using an apparatus constructed to simulate the primordial Earth's atmosphere-ocean system. Miller introduced 200 ml of water, 100 mmHg of H2
, 200 mmHg of CH4
, and 200 mmHg of NH3
into the apparatus, then subjected this mixture, under reflux, to an electric discharge for a week, while the water was simultaneously heated. The purpose of this manuscript is to provide the reader with a general experimental protocol that can be used to conduct a Miller-Urey type spark discharge experiment, using a simplified 3 L reaction flask. Since the experiment involves exposing inflammable gases to a high voltage electric discharge, it is worth highlighting important steps that reduce the risk of explosion. The general procedures described in this work can be extrapolated to design and conduct a wide variety of electric discharge experiments simulating primitive planetary environments.
Chemistry, Issue 83, Geosciences (General), Exobiology, Miller-Urey, Prebiotic chemistry, amino acids, spark discharge
Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans
Institutions: Deakin University.
Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual's oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual's ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.
Neuroscience, Issue 88, taste, overweight and obesity, dietary fat, fatty acid, diet, fatty food liking, detection threshold
Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites
Institutions: Royal Military College of Canada, Queen's University.
The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g.
carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.
Environmental Sciences, Issue 93, biochar, characterization, carbon sequestration, remediation, International Biochar Initiative (IBI), soil amendment
A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window
Institutions: University of California, Los Angeles.
The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.
Neuroscience, Issue 12, red blood cell, cortex, fluorescein, rhodamine, dextran, two-photon, 2-photon, capillary
Monitoring Plant Hormones During Stress Responses
Institutions: University of Texas.
Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.
Plant Biology, Issue 28, Jasmonic acid, salicylic acid, abscisic acid, plant hormones, GC/MS, vapor phase extraction