A Mass Spectrometric Study for Comparative Analysis and Evaluation of Metabolite Recovery from Plasma by Various Solvent Systems

A solvent system that extracts a maximum number of metabolites belonging to diverse chemical classes from complex biofluids, such as plasma, may offer useful inputs to understand the metabolic and physiological state of an individual. The present study compared seven solvent systems for extraction of metabolites from plasma. The extracts were analyzed by mass spectrometry (MS) and MS/MS (MS2) using a quadrupole time-of-flight liquid chromatography/MS system in positive and negative modes of ionization. Metabolites with molecular mass below 400 were identified using Human Metabolome Database MS2 and MS search interfaces. The acetone/isopropanol (2:1) system yielded promising results in positive ionization mode, as the maximum number of MS and MS2 features was detected in the extract. It was found to be superior in extraction of various classes of metabolites, especially organic acids, nucleosides and nucleoside derivatives, and heterocyclic molecules. Glycerophosphocholines in the mass range of 400–700 were found to be efficiently extracted by the methanol/chloroform/water (8:1:1) system. In negative mode as well, the maximum number of MS2 features was detected in methanol/chloroform/water and acetone/isopropanol extracts. The fingerprints of molecular features obtained in the negative and positive modes differed from each other to a significant extent.     

Lipase-catalyzed transamidation of non-activated amides in organic solvent

A novel biocatalytic reaction of transamidation of non-activated amides with amines is reported. Among 45 different lipolytic and proteolytic enzymes tested, only the lipase from Candida antarcticawas able to catalyze this reaction. The reaction proceeded with up to ca. 80% conversion in anhydrous methyl tert-butyl ether and worked with both N-substituted and unsubstituted amides. The biocatalytic transamidation is an equilibrium process and, therefore, higher conversions to the desired amide were achieved by using increased concentrations of the amine nucleophile.     

Replacing xylene with n-heptane for paraffin embedding

Abstract

In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.     

From Life Cycle Assessment to Life Cycle Management

Life cycle assessment (LCA) is a widely accepted methodology to support decision‐making processes in which one compares alternatives, and that helps prevent shifting of environmental burdens along the value chain or among impact categories. According to regulation in the European Union (EU), the movement of waste needs to be reduced and, if unavoidable, the environmental gain from a specific waste treatment option requiring transport must be larger than the losses arising from transport. The EU explicitly recommends the use of LCA or life cycle thinking for the formulation of new waste management plans. In the last two revisions of the Industrial Waste Management Programme of Catalonia (PROGRIC), the use of a life cycle thinking approach to waste policy was mandated. In this article we explain the process developed to arrive at practical life cycle management (LCM) from what started as an LCA project. LCM principles we have labeled the “3/3” principle or the “good enough is best” principle were found to be essential to obtain simplified models that are easy to understand for legislators and industries, useful in waste management regulation, and, ultimately, feasible. In this article, we present the four models of options for the management of waste solvent to be addressed under Catalan industrial waste management regulation. All involved actors concluded that the models are sufficiently robust, are easy to apply, and accomplish the aim of limiting the transport of waste outside Catalonia, according to the principles of proximity and sufficiency.     

Extraction and fractionation of complex lipid mixtures with dense carbon dioxide on a micro scale

The effects of pressure, temperature and some organic solvents on the recovery of various lipid classes from plant and animal tissues can be assessed by fractional extraction with dense carbon dioxide and consecutive analysis by thin-layer chromatography.     

Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media

Subtilisin 72 was immobilized on cryogel of poly(vinyl alcohol), the macroporous carrier prepared by the freeze-thaw-treatment of concentrated aqueous solution of the polymer. The obtained biocatalyst was active and stable in aqueous, aqueous-organic, as well as in low water media. The stability of immobilized biocatalyst was substantially higher than that of native enzyme in all mixtures especially in aqueous buffer containing 5–8 M Urea and in acetonitrile/60–90%DMF mixtures. The ability of native and immobilized subtilisin to catalyze peptide bond formation between Z-Ala-Ala-Leu-OMe and Phe-pNA was studied in non-aqueous media. Considerable enzyme stabilization in acetonitrile/90%DMF mixture, induced by the immobilization, resulted in higher product yield (57%) than in case of native subtilisin suspension (32%). Detailed study of synthesis reaction revealed that notable increase in product yield could be reached using increase in both substrate concentrations up to 200 mM.     

Ultrafast molecular dynamics of biofuel extraction for microalgae and bacteria milking: blocking membrane folding pathways to damaged lipid-bilayer conformations with nanomicelles

Cell milking is a 100% renewable green energy for CO₂ by extraction of biofuels inside the cytosol of photosynthetic micro-organisms as microalgae and bacteria. The cells are exposed to a hydrophobic solvent forming holes and cracks through their membranes from which the biofuels can leak out. In protein folding, the goal would be to find pathways to the unique functional protein conformer. However, in the lipid-bilayer interaction with the solvent for milking, the objective is to block the pathways for damaged membrane conformations of low free energy with undesired nanostructures, using the solvent properties, as shown with an ab initio structural bioinformatic model. Statistical thermodynamics is used to compute the free energy (including entropy) from the molecular dynamics trajectory of the biomolecular system with many conformational changes. This model can be extended to the general problem of biomolecules folding as for proteins and nucleic acids. Using an adaptation of the Einstein diffusion law, the conformational change dynamics of the lipid bilayer depends on the two diffusion coefficients of the solvent: D₁ before the irreversible folding transition time and the much smaller D₂ thereafter. In contrast to the n-hexane and n-heptane hydrocarbons of smaller size, the residual D₂?=?4.7?×?10^?7?cm²/s of the n-decane solvent, with the highest partition coefficient among the three extractors, is the only to present a D₂ value that is significantly below the critical threshold of 10^?6?cm²/s. Therefore, the membrane would resist to long hydrocarbons and the exposed cells would remain viable for milking.     

Candida Antarctica Lipase B-Catalysed Synthesis Of Dihydroxyacetone Fatty Acid Esters

The enzymatic syntheses of 1-lauroyl-dihydroxyacetone and 1, 3-dilauroyl-dihydroxyacetone were investigated. Lipase B from Candida Antarctica (SP435) was used to catalyse the acylation of dihydroxyacetone (DHA) with lauric acid in organic solvent media at controlled water activity. High conversions of dihydroxyacetone (< 90%) are achieved when the water activity is 0.11 or below in solvents of various hydrophobicities, such as diethyl ether, methyl-terr-butyl ether (MTBE) and diphenyl ether. The main product in the esterification of DHA with lauric acid is 1-lauroyl-DHA, while the amount of 1, 3-dilauroyl-DHA that is produced can be increased by changing the reaction conditions. Thus, hasing the water activity from 0.75 to 0.06 resulted in an increase in the total yield of 1, 3-dilauroyl-DHA from 3% to 20%. Solvents which have high logP values favoured the acylation of 1-lauroyl-DHA and thereby the formation of 1, 3-dilauroyl-DHA. Thus, when diphenyl ether was used in this reaction, the yield of 1, 3-dilauroyl-DHA was 45%. Complete acylation to 1, 3-dilauroyl-DHA was achieved when a fatty acid vinyl ester was used as acyl donor in a closed reactor.     

Enzymatic Peptide Synthesis in Organic Media. Synthesis of CCK-8 Dipeptide Fragments

The enzymatic synthesis of the seven consecutive dipeptide fragments of the cholecysto kinin C-terminal octapeptide (CCK-8) in organic media is reported. The influence of the reaction medium composition, the protease, and the structure of N-α and C-α protecting groups of both carboxyl and amino components was evaluated. α-Chymotrypsin, papain and thermolysin adsorbed on Celite were used as catalysts, under thermodynamic and kinetic control. The carboxamidomethyl, methyl and allyl ester derivatives of acetyl, benzyloxycarbonyl, tert-butyloxycarbonyl and fluoren-9-ylmethoxycarbonyl amino acids, were assayed as carboxy components. Amino acid amide and ester derivatives were employed as nucleophiles with a preference for the latter, since the dipeptide product obtained could be used directly, without any further chemical modification, as acyl-donor in subsequent coupling steps. All dipeptides selected were successfully synthesized, using the optimal combination of protecting groups, reaction media and enzyme different for each coupling reaction. The information gained with this study should be instrumental in designing an optimal strategy for the total enzymatic synthesis of cholecystokinin C-terminal octapeptide (CCK-8).