One-pot derivatization spectrofluorimetric method for detection of aspartame in sweetener commercial tablets and soft drinks: Greenness assessments of the methodology.

Aspartame is an artificial sweetener used in drinks and many foods. International Agency for Research on Cancer classified aspartame as possibly carcinogenic to humans (IARC Group 2B). In this study, a sensitive and selective spectrofluorimetric method was developed to detect aspartame. The method is based on switching on the fluorescence activity of aspartame upon its condensation with O-phthalaldehyde (Roth's reaction) in the presence of 2-mercaptoethanol. The reaction product was detected fluorometrically at λem of 438 nm after λex of 340 nm. All reaction conditions required to yield the optimal fluorescence intensity were observed and investigated. Furthermore, the approach was validated according to ICH guidelines. Upon plotting the concentrations of aspartame against their associated fluorescence intensity values, the relationship between the two variables was linear within the range of 0.5-3.0 µg/mL. Furthermore, the method was employed to analyze the quantity of aspartame in commercial packages and soft drinks with an acceptable level of recovery. In addition, the Green Solvents Selecting Tool, Complementary Green Analytical Procedure Index, and the Analytical Greenness Metric tool were used to evaluate the sustainability and the greenness of the developed methodology.

Identification of "modified OPDA (mo-OPDA)" as a Michael adduct of cis-OPDA.

cis-(+)-12-Oxo-phytodienoic acid (cis-OPDA) is a significant plant oxylipin, known as a biosynthetic precursor of the plant hormone jasmonoyl-l-isoleucine (JA-Ile), and a bioactive substance in plant environmental stresses. A recent study showed that a plant dioxygenase, Jasmonate Induced Dioxygenase 1 (JID1), converts cis-OPDA into an unidentified metabolite termed "modified-OPDA (mo-OPDA)" in Arabidopsis thaliana. Here, using ultra-performance liquid chromatography coupled with triple quad mass spectrometry (UPLC-MS/MS) experiment, the chemical identity of "mo-OPDA" was demonstrated and identified as a conjugate between cis-OPDA and 2-mercaptoethanol (cis-OPDA-2ME), an artifact produced by Michael addition during the JID1 digestion of cis-OPDA. However, previous reports demonstrated a decreased accumulation of cis-OPDA in the JID1-OE line, suggesting the existence of an unknown JID1-mediated mechanism regulating the level of cis-OPDA in A. thaliana.

The Michael addition of thiols to 13-oxo-octadecadienoate (13-oxo-ODE) with implications for LC-MS analysis of glutathione conjugation.

Unsaturated fatty acid ketones with αß,γδ conjugation are susceptible to Michael addition of thiols, with unresolved issues on the site of adduction and precise structures of the conjugates. Herein we reacted 13-keto-octadecadienoic acid (13-oxo-ODE or 13-KODE) with glutathione (GSH), N-acetyl-cysteine, and ß-mercaptoethanol and identified the adducts. HPLC-UV analyses indicated none of the products exhibit a conjugated enone UV chromophore, a result that conflicts with the literature and is relevant to the mass spectral interpretation of 1,4 versus 1,6 thiol adduction. Aided by the development of an HPLC solvent system that separates the GSH diastereomers and thus avoids overlap of signals in proton NMR experiments, we established the two major conjugates are formed by 1,6 addition of GSH at the 9-carbon of 13-oxo-ODE with the remaining double bond α to the thiol in the 10,11 position. N-acetyl cysteine reacts similarly, while ß-mercaptoethanol gives equal amounts of 1,4 and 1,6 addition products. Equine glutathione transferase catalyzed 1,6 addition of GSH to the two major diastereomers in 44:56 proportions. LC-MS in positive ion mode gives a product ion interpreted before as evidence of 1,4-thiol adduction, whereas here we find this ion using the authentic 1,6 adduct. LC-MS with negative ion APCI gave a fragment selective for 1,4 adduction. These results clarify the structures of thiol conjugates of a prototypical unsaturated keto-fatty acid and have relevance to the application of LC-MS for the structural analysis of keto-fatty acid glutathione conjugation.

Comparing the effect of using 2-Mercaptoethanol in the cell culture medium of different cell passages of human mesenchymal stem cells.

Preparing a suitable cell culture medium that supports the biological needs of the growing cells is crucial to enhancing the success rate of any in vitro and in vivo experiments and minimizing undesirable interferences.  Mesenchymal stem cells ( MSCs) which are powerful regenerative stem cells require being grown in proper culture media to preserve their stemness and therapeutic properties. MSCs are usually grown in Dulbecco's Modified Eagle low glucose Medium (DMEM low glucose) which contains 5.6 mmol/L of glucose and is supplemented with Fetal Bovine Serum (FBS), antibiotics, and 2-Mercaptoethanol. The addition of 2-Mercaptoethanol to the cell culture medium was proposed long ago and has continued to be used until now. Despite the positive effects of adding 2-Mercaptoethanol in the cell culture medium, its use is still controversial and needs continuous updates to limit its interference with experimental treatments. Herein, we found that 2-Mercaptoethanol is beneficial to enhancing the proliferation and survival of MSCs at higher passage numbers while its effect is negligible for earlier passages. This concise study provides updates regarding the suitable time to add 2-Mercaptoethanol which can minimize its intermeddling with the experimental design and treatments.

Synthesis of bimetallic core-shelled nanoparticles modified by 2-mercaptoethanol as SERS substrates for detecting ferbam and thiabendazole in apple puree.

Modification of surface-enhanced Raman spectroscopy (SERS) substrates with thiol ligands is an emerging approach in enhancing the stability and sensitivity of metal substrates due to their good affinity with metals such as Au, Ag, and Cu. Thus, in the current study, 2-mercaptoethanol was used to modify the surface of silver-coated gold nanoparticles to develop a novel SERS substrate for the rapid assessment of fungicide residues in fruit samples. Results showed that the substrate could achieve the detection of ferbam and thiabendazole residues in apple puree with limits of detection of approximately 0.0042 and 0.0064 ppm, high coefficients of determination of 0.9946 and 0.9968, good recoveries ranging from 80 to 105 and 81 to 107% and relative standard deviations of 3.5-7.5 and 3.8-7.9 %, respectively. Therefore, the substrate developed could potentially be utilised to assess other toxic agrochemicals in future.

2-Mercaptoethanol (2-ME)-based IATs or Polybrene method mitigates the interference of daratumumab on blood compatibility tests.

OBJECTIVES: Treating red blood cells (RBCs) with dithiothreitol (DTT) is a wildly-recommended to overcome the interference of the daratumumab (DARA) with blood compatibility testing. Nevertheless, DTT can be hard to obtain in the clinical laboratory, while its use in routine practice may be time-consuming. In the following study, we explored the feasibility of using a commercial 2-mercaptoethanol (2-ME) working solution or the time-saving Polybrene method to mitigate DARA interference. METHODS: Antibody screening and cross-matching were performed using 2-ME or DTT-based indirect antiglobulin tests (IATs) and Polybrene method (with human IgG anti-E same IATs titer as DARA as positive control) on 37 samples. Most clinically important blood group antigens on RBCs were detected after treatment with 2-ME or DTT. RESULTS: Treating RBCs with 2-ME eliminates the DARA interference with the antibody screening or cross-matching; yet, K antigen is denatured during treatment. DARA does not interfere with antibody screening and cross-matching via Polybrene method, while 2+ agglutinations of anti-E antibody with the same titer (IATs method) as DARA could be observed in the positive controls via this method. CONCLUSION: 2-ME-based IATs or Polybrene method could replace DTT-based IATs to mitigate DARA interference.

Crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP.

CRM1 is a nuclear export receptor that has been intensively targeted over the last decade for the development of antitumor and antiviral drugs. Structural analysis of several inhibitor compounds bound to CRM1 revealed that their mechanism of action relies on the covalent modification of a critical cysteine residue (Cys528 in the human receptor) located in the nuclear export signal-binding cleft. This study presents the crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP at 2.58 Šresolution. The results demonstrate that buffer components can interfere with the characterization of cysteine-dependent inhibitor compounds.

Effect of the drug cyclophosphamide on the activity of porcine kidney betaine aldehyde dehydrogenase.

The enzyme betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the synthesis of glycine betaine (GB), an osmolyte and osmoprotectant. Also, it participates in several metabolic pathways in humans. All BADHs known have cysteine in the active site involved in the aldehyde binding, whereas the porcine kidney enzyme (pkBADH) also has a neighborhood cysteine, both sensitive to oxidation. The antineoplastic and immuno-suppressant pre-drug cyclophosphamide (CTX), and its bioactivation products, have two highly oxidating chlorine atoms. This work aimed to analyze the effect of CTX in the activity of porcine kidney betaine aldehyde dehydrogenase. PkBADH was incubated with varying CTX concentration (0 to 2.0 mM) at 25 °C and lost 50 % of its activity with 2.0 mM CTX. The presence of the coenzyme NAD+ (0.5 mM) decreased 95% the activity in 2.0 mM CTX. The substrate betaine aldehyde (0.05 and 0.4 mM, and the products NADH (0.1-0.5 mM) and GB (1 and 10 mM) did not have an effect on the enzyme inactivation by CTX. The reducing agents, dithiothreitol and ß-mercaptoethanol, reverted the pkBADH inactivation, but reduced glutathione (GSH) was unable to restore the enzyme activity. Molecular docking showed that CTX could enter at the enzyme active site, where its chlorine atoms may interact with the catalytic and the neighboring cysteines. The results obtained show that CTX inactivates the pkBADH due to oxidation of the catalytic cysteine or because it oxidizes catalytic and neighborhood cysteine, forming a disulfide bridge with a concomitant decrease in the activity of the enzyme.

Citric acid promotes disulfide bond formation of whey protein isolate in non-acidic aqueous system.

Impacts of citric acid (CA) treatment under non-acidic conditions (pH 7.0, 8.0 and 9.0) on whey protein isolate (WPI) were examined in this study. Size exclusion chromatography and SDS-PAGE indicated that molecular size and weight of WPI-CA became larger at pH 7.0, 8.0 and 9.0 with CA ranged from 0 to 15 mg/mL, but the protein aggregates disappeared after ß-mercaptoethanol was added. The free SH groups of WPI-CA gradually decreased. This could be deduced that CA could promote disulfide bond formation of WPI at the non-acidic pH values. Furthermore, fourier transform infra-red (FTIR) spectroscopy and fluorescence spectroscopy data confirmed the conformational changes of secondary and tertiary structures of CA-modified WPI, respectively. Therefore, these results suggested that disulfide bond formation of WPI occurred at citric acid treatment under non-acidic conditions, being contributed to production of its larger molecular size substances and alteration of its structural characteristics.

Synthesis and biological activity of 2-cyanoacrylamide derivatives tethered to imidazopyridine as TAK1 inhibitors.

The importance of transforming growth factor beta-activated kinase 1 (TAK1) to cell survival has been demonstrated in many studies. TAK1 regulates signalling cascades, the NF-κB pathway and the mitogen-activated protein kinase (MAPK) pathway. TAK1 inhibitors can induce the apoptosis of cancerous cells, and irreversible inhibitors such as (5Z)-7-oxozeaenol are highly potent. However, they can react non-specifically with cysteine residues in proteins, which may have serious adverse effects. Reversible covalent inhibitors have been suggested as alternatives. We synthesised imidazopyridine derivatives as novel TAK1 inhibitors, which have 2-cyanoacrylamide moiety that can form reversible covalent bonding. A derivative with 2-cyano-3-(6-methylpyridin-2-yl)acrylamide (13h) exhibited potent TAK1 inhibitory activity with an IC50 of 27 nM. It showed a reversible reaction with ß-mercaptoethanol, which supports its potential as a reversible covalent inhibitor.

Free-Radical-Mediated Formation of Trans-Cardiolipin Isomers, Analytical Approaches for Lipidomics and Consequences of the Structural Organization of Membranes.

Free-radical-mediated processes, such as peroxidation, isomerization and hydrogenation affecting fatty acid integrity and biological functions, have a trans-disciplinary relevance. Cardiolipins (CL, (1,3-diphosphatidyl-sn-glycerol)) and tetra-linoleoyl-CL are complex phospholipids, exclusively present in the Inner Mitochondrial Membrane (IMM) lipids, where they maintain membrane integrity and regulate enzyme functionalities. Peroxidation pathways and fatty acid remodeling are known causes of mitochondrial disfunctions and pathologies, including cancer. Free-radical-mediated isomerization with the change of the cis CL into geometrical trans isomers is an unknown process with possible consequences on the supramolecular membrane lipid organization. Here, the formation of mono-trans CL (MT-CL) and other trans CL isomers (T-CL) is reported using CL from bovine heart mitochondria and thiyl radicals generated by UV-photolysis from 2-mercaptoethanol. Analytical approaches for CL isomer separation and identification via 1H/13C NMR are provided, together with the chemical study of CL derivatization to fatty acid methyl esters (FAME), useful for lipidomics and metabolomics research. Kinetics information of the radical chain isomerization process was obtained using γ-irradiation conditions. The CL isomerization affected the structural organization of membranes, as tested by the reduction in unilamellar liposome diameter, and accompanied the well-known process of oxidative consumption induced by Fenton reagents. These results highlight a potential new molecular modification pathway of mitochondrial lipids with wide applications to membrane functions and biological consequences.

An improved and highly selective fluorescence assay for measuring phosphatidylserine decarboxylase activity.

Phosphatidylserine decarboxylases (PSDs) catalyze the conversion of phosphatidylserine (PS) to phosphatidylethanolamine (PE), a critical step in membrane biogenesis and a potential target for development of antimicrobial and anti-cancer drugs. PSD activity has typically been quantified using radioactive substrates and products. Recently, we described a fluorescence-based assay that measures the PSD reaction using distyrylbenzene-bis-aldehyde (DSB-3), whose reaction with PE produces a fluorescence signal. However, DSB-3 is not widely available and also reacts with PSD's substrate, PS, producing an adduct with lower fluorescence yield than that of PE. Here, we report a new fluorescence-based assay that is specific for PSD and in which the presence of PS causes only negligible background. This new assay uses 1,2-diacetyl benzene/ß-mercaptoethanol, which forms a fluorescent iso-indole-mercaptide conjugate with PE. PE detection with this method is very sensitive and comparable with detection by radiochemical methods. Model reactions examining adduct formation with ethanolamine produced stable products of exact masses (m/z) of 342.119 and 264.105. The assay is robust, with a signal/background ratio of 24, and can readily detect formation of 100 pmol of PE produced from Escherichia coli membranes, Candida albicans mitochondria, or HeLa cell mitochondria. PSD activity can easily be quantified by sequential reagent additions in 96- or 384-well plates, making it readily adaptable to high-throughput screening for PSD inhibitors. This new assay now enables straightforward large-scale screening for PSD inhibitors against pathogenic fungi, antibiotic-resistant bacteria, and neoplastic mammalian cells.

Multiplexed in-gel microfluidic immunoassays: characterizing protein target loss during reprobing of benzophenone-modified hydrogels.

From whole tissues to single-cell lysate, heterogeneous immunoassays are widely utilized for analysis of protein targets in complex biospecimens. Recently, benzophenone-functionalized hydrogel scaffolds have been used to immobilize target protein for immunoassay detection with fluorescent antibody probes. In benzophenone-functionalized hydrogels, multiplex target detection occurs via serial rounds of chemical stripping (incubation with sodium-dodecyl-sulfate (SDS) and ß-mercaptoethanol at 50-60 °C for ≥1 h), followed by reprobing (interrogation with additional antibody probes). Although benzophenone facilitates covalent immobilization of proteins to the hydrogel, we observe 50% immunoassay signal loss of immobilized protein targets during stripping rounds. Here, we identify and characterize signal loss mechanisms during stripping and reprobing. We posit that loss of immobilized target is responsible for ≥50% of immunoassay signal loss, and that target loss is attributable to disruption of protein immobilization by denaturing detergents (SDS) and incubation at elevated temperatures. Furthermore, our study suggests that protein losses under non-denaturing conditions are more sensitive to protein structure (i.e., hydrodynamic radius), than to molecular mass (size). We formulate design guidance for multiplexed in-gel immunoassays, including that low-abundance proteins be immunoprobed first, even when targets are covalently immobilized to the gel. We also recommend careful scrutiny of the order of proteins targets detected via multiple immunoprobing cycles, based on the protein immobilization buffer composition.

Spectral signatures of five hydroxymethyl chlorophyll a derivatives chemically derived from chlorophyll b or chlorophyll f.

Chlorophylls (Chls) are pigments involved in light capture and light reactions in photosynthesis. Chl a, Chl b, Chl d, and Chl f are characterized by unique absorbance maxima in the blue (Soret) and red (Qy) regions with Chl b, Chl d, and Chl f each possessing a single formyl group at a unique position. Relative to Chl a the Qy absorbance maximum of Chl b is blue-shifted while Chl d and Chl f are red-shifted with the shifts attributable to the relative positions of the formyl substitutions. Reduction of a formyl group of Chl b to form 7-hydroxymethyl Chl a, or oxidation of the vinyl group of Chl a into a formyl group to form Chl d was achieved using sodium borohydride (NaBH4) or ß-mercaptoethanol (BME/O2), respectively. During the consecutive reactions of Chl b and Chl f using a three-step procedure (1. NaBH4, 2. BME/O2, and 3. NaBH4) two new 7-hydroxymethyl Chl a species were prepared possessing the 3-formyl or 3-hydroxymethyl groups and three new 2-hydroxymethyl Chl a species possessing the 3-vinyl, 3-formyl, or 3-hydroxymethyl groups, respectively. Identification of the spectral properties of 2-hydroxymethyl Chl a may be biologically significant for deducing the latter stages of Chl f biosynthesis if the mechanism parallels Chl b biosynthesis. The spectral features and chromatographic properties of these modified Chls are important for identifying potential intermediates in the biosynthesis of Chls such as Chl f and Chl d and for identification of any new Chls in nature.

Quantification of 2-aminoisobutyric acid impurity in enzalutamide bulk drug substance using hydrophilic interaction chromatography with fluorescence detection.

A rapid procedure for the determination of 2-aminoisobutyric acid in enzalutamide bulk drug substance based on hydrophilic interaction chromatography with fluorescence detection was developed. Fluorescence detection after postcolumn derivatization with o-phthaldialdehyde/2-mercaptoethanol was carried out at excitation and emission wavelength of 345 nm and 450 nm, respectively. The postcolumn reaction conditions such as reaction temperature, mobile phase and derivatization reagent flow rate and the reagents concentrations were studied and optimized due to steric hindrance of amino group of 2-aminoisobutyric acid. The derivatization reaction was applied for the hydrophilic interaction chromatography method which was based on COSMOSIL HILIC column with a mobile phase consisting of a mixture of 25 mmol/L acetic acid adjusted to pH 5.5 (using 1 mol/L potassium hydroxide) and acetonitrile using an isocratic elution (28:72, ν/ν). The benefit of the reported approach consists in a simple sample pretreatment and a quick and sensitive hydrophilic interaction chromatography method. The developed method was validated in terms of linearity, limit of detection, limit of quantification, accuracy, precision and selectivity according to the International Conference on Harmonisation guidelines. The developed method was demonstrated to be applied for the analysis of 2-AIBA in routine quality control evaluation of commercial samples of enzalutamide bulk drug substance.

Experimental and Theoretical Study on the Synergistic Inhibition Effect of Pyridine Derivatives and Sulfur-Containing Compounds on the Corrosion of Carbon Steel in CO2-Saturated 3.5 wt.% NaCl Solution.

The corrosion inhibition performance of pyridine derivatives (4-methylpyridine and its quaternary ammonium salts) and sulfur-containing compounds (thiourea and mercaptoethanol) with different molar ratios on carbon steel in CO2-saturated 3.5 wt.% NaCl solution was investigated by weight loss, potentiodynamic polarization, electrochemical impedance spectroscopy, and scanning electron microscopy. The synergistic corrosion inhibition mechanism of mixed inhibitors was elucidated by the theoretical calculation and simulation. The molecules of pyridine derivative compounds with a larger volume has priority to adsorb on the metal surface, while the molecules of sulfur-containing compounds with a smaller volume fill in vacancies. A dense adsorption film would be formed when 4-PQ and sulfur-containing compounds are added at a proper mole ratio.

User and environment friendly direct agglutination test for the sero-diagnosis of visceral leishmaniasis: exclusion of formaldehyde and ß-mercaptoethanol in test execution.

PURPOSE: Based on world-wide evaluation, the direct agglutination test (DAT) is now generally acknowledged as one of the leading diagnostics for visceral leishmaniasis (VL). To enhance more routine and mass application, but simultaneously ensure safety to both user and environment, further improvements need to be introduced. METHODOLOGY: In the current format, a two-sixfold titre decrease was observed due to using formaldehyde as an antigen preservative in DAT. Successful formaldehyde preservative exclusion was achieved by increasing its concentration to 3 % (wt/vol) for conserving promastigote status after ß-mercaptoethanol (ß-ME) treatment and repeating exposure of the parasite to the fixative after Coomassie Brilliant Blue staining. RESULTS: Microbial contamination was not observed in any of the antigen aliquots preserved in 0.05 % (wt/vol) sodium dichloroisocyanurate (chlorine) instead of formaldehyde for 6 months or longer. By excluding formaldehyde, restoring the normal antibody level, prior to treatment of sera with ß-ME only minimally influenced the test outcome. A comparable successful reduction in non-specific agglutination, as with ß-ME, was achieved by incorporating urea (0.3 % wt/vol) in the improved DAT procedure (P=0.646; T=23.0). As with the current procedure, the improved equivalent (formaldehyde and ß-ME free) showed good reliability for VL detection (VL - Fr=52.39, W=0.70, P<0.001; and non-VL - Fr=65.97, W=0.83, P<0.001). A much lower cut-off (titre 1 : 400 versus 1 : 3200) for VL diagnosis can be adopted if urea is integrated in the improved procedure. CONCLUSIONS: By introducing the modifications mentioned, we think we have succeeded to a reasonable degree in increasing the DAT potential for VL control.

[Difficult immunofixation electrophoresis interpretation of serum proteins]. / Interprétation délicate de l'immunofixation des protéines sériques.

Immunofixation is currently very used in medical laboratories. The interpretation of the results is usually easy, but some cases raise interpretative problems. We here report two cases difficult to interpret. In the first case, we report a case of nonspecific precipitation of the protein on each track, in the second case we report a case of double monoclonal band on immunofixation electrophoresis. Reducing agent such as ß2-mercaptoethanol used in these two cases allowed to solve the problem and to make a diagnosis. A comparison between clinical radiological and laboratory test data is necessary before making a diagnosis of monoclonal immunoglobulin.