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Analysis of drugs and metabolites in biological matrices such as blood or plasma by LC-MS is routinely challenged by the presence of large quantities of competing molecules for ionization in soft ionization sources, such as proteins and phospholipids. While the former can easily be removed by protein precipitation, pre-analytical extraction of the latter is necessary because they show very high retention in reversed-phase LC resulting in long analysis times or in ion suppression effects when not eluted before the next runs. A novel HILIC-based SPE approach, making use of silica cartridges and of acetone as organic solvent, is introduced as a potent alternative to current commercial methods for phospholipid removal. The methodology was developed and tested for a broad polarity range of pharmaceutical solutes (log P from 0 to 6.6) and broad applicability can therefore be envisaged. [ABSTRACT FROM AUTHOR]

1. A new assay procedure for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat liver microsomal preparations has been developed. Highly labeled [5-14C]3-hydroxy-3 methylglutaryl-CoA is incubated with rat liver microsomal suspensions in the presence of a NADPH regenerating system. After addition of carrier mevalonate the reaction is stopped by heating and mevalonate transformed into the lactone. After extraction the latter is converted into the more volatile trimethylsilyl ether, which is separated by gas liquid chromatography from accompanying radioactive materials and from an internal standard employed for quantitative mass determination. The eluate from the column after mass detection is continuously combusted to 14CO2, the radioactivity of which is detected in the flow-through cell of a scintillation spectrometer, i. e. mass and radioactivity of mevalolactonetrimethylsilyl ether are recorded almost synchronously. A formula is given for the calculation of specific activities from the large number of variables involved in the procedure. 2. A method is described for the determination of the specific radioactivities of the 3-hydroxy 3-methylglutaryl-CoA preparations used. It mainly consists of (a) a gaschromatographic procedure for determination of the ratio of the radioactivities of 3-methylglutaconic and 3-hydroxy 3-methylglutaric acids in a solution containing their enzymatically synthesized CoA-esters, and (b) an optical assay for hydroxymethylglutaryl-CoA, which involves the cleavage of that compound by hydroxymethyl-glutaryl-CoA acetoacetate lyase to acetoacetate and acetyl-CoA. The latter is determined in a citrate synthase test system. 3. Up to a certain enzyme concentration mevalonate formation from 3-hydroxy-3-methylglutaryl-CoA is proportional to the amount of 3-hydroxy-3-methylglutaryl-CoA reductase used in the assay. The Km for 3-hydroxy-3-methylglutaryl-CoA is determined to be 4.7 × 10-5 M. The assay system must include a thiolgroup carrier to protect enzyme sulfhydryl groups. 4. It is shown that the microsomal fraction also contains 3-hydroxy-3-methylglutaryl-CoA acetoacetate lyase which consumes the same substrate as the reductase and remains active in absence of Mg2+. In this way the lyase may interfere with the test for 3-hydroxy-3-methylglutaryl-CoA reductase, if the concentration of microsomal protein is too high. The microsomal protein concentration is low enough, if one obtains a linear relationship between mevalonate formation and the concentration of microsomal protein. [ABSTRACT FROM AUTHOR]

Fatty acid synthetase of yeast was strongly inhibited in vitro by long-chain acyl coenzyme A thioesters. Increasing the serum albumin concentration in the reaction mixture lowered, but did not prevent, this inhibition. Enzyme kinetic experiments showed that all long-chain acyl CoA compounds tested were competitive inhibitors of the substrate malonyl CoA. The inhibition with. respect to the substrate acetyl CoA and NADPH was non-competitive. These results support the hypothesis put forth earlier that the end products of the fatty acid synthetase are bound preferentially in the region of the thiol group which is part of the acyl-carrier-protein portion of the complex. The data indicate that the long-chain acyl CoA thioesters may be of importance in the regulation of de novo fatty acid biosynthesis via end product inhibition of the synthetase. [ABSTRACT FROM AUTHOR]

Fatty acid synthetase, a multienzyme complex from yeast, was analysed by small angle x-ray scattering. The molecular weight determined by this method (2.2 × 106) was in close agreement with the value determined earlier by ultracentrifugation. The experimental data indicate that the complex is contained in a ellipsoid volume with axial ratios 1:1:1.2. The relationship of radius of gyration to volume and the height of the second maximum the scattering curve suggest that the complex proteins enclose a hollow space. A model is proposed where the theoretical scattering curve closely agrees with the curve measured for the fatty acid synthetase. The model consists of a spherical shell, composed of spheres, having an average wall thickness of 20 Å and divided in half by a planar wall also 20 Å thick. The outer radius is 125 Å. On the basis of this model the catalysis of fatty acid synthesis by the synthetase is discussed. [ABSTRACT FROM AUTHOR]

The purified multienzyme complex, fatty acid synthetase of yeast, produces palmityl-CoA and stearyl-CoA in equal amounts under standard conditions, with adequate NADPH, and a 1:2 ratio of acetyl-CoA and malonyl-CoA. Based on the known enzymatic properties of fatty acid synthetase a model which rationalizes the chain termination at the level of C16 and C18-acid is proposed. The model is based on two assumptions that are supported by experimental evidence: (a) the probability of any covalently enzyme-bound saturated acyl residue forming a product by transfer to CoA is determined by the relative velocities of the condensing and transferring reactions. (b) The growing alkan chain interacts with the enzyme protein only after a chain length of 13 carbon atoms has been attained; this interaction changes the relative velocities in favour of product formation by an energy increment of —0.9 kcal for each additional methylene group beyond the 13th. To calculate the probability of product release at a particular chain length, an equation was derived from the model describing quantitatively the observed product distribution. The formula suggests conditions under which either short acyl-CoA derivatives or exclusively stearyl-CoA can be produced. Synthesis under these conditions was examined experimentally and results indicated that the formula can be applied to a wide range of experimental conditions. Because the condensation reaction in fatty acid synthesis is the rate limiting step under normal conditions, saturated acyl residues have the longest life-time as intermediates and consequently the highest probability of leaving the enzyme complex as an endproduct. The reduction reactions can be made rate limiting when the NADPH level drops. Under such circumstances β-ketoacyl-CoA's and α,β-unsaturated acyl-CoA's appear as endproducts. With NADH as electron donor the first reduction is slowed to such an extent that predominantly β-ketoacyl-CoA's derivatives are synthesized. The possibility that the enzyme complex catalyses synthesis of short chain fatty acids and and β-ketoacids in vivo is discussed. [ABSTRACT FROM AUTHOR]

[14C]Pantetheine fatty acid synthetase from baker's yeast was denatured in guanidine hydrochloride. Then the protein components of the multienzyme complex were subjected to a Sephadex filtration in guanidine hydrochloride and a preparative polyacrylamide gel electrophoresis in a phenol containing medium. It could be demonstrated that the [14C]pantetheine label was covalently bound to one of the smallest polypeptide chains of the multienzyme complex. In analogy to the soluble acyl carrier protein which was first, isolated from Escherichia coli and contained 4'-phosphopantetheine as a prosthetic group, the pantetheine-protein component of the yeast multienzyme complex was denoted as yeast acyl carrier protein. Its molecular weight seemed to be higher and its amino acid composition different, from the E. coli acyl carrier protein. [ABSTRACT FROM AUTHOR]

If no NADPH is available for reduction, fatty acid biosynthesis is blocked at the stage of the acetoacetyl-acyl carrier protein intermediate. In this case highly purified fatty acid synthetase from baker's yeast catalyzes, in a derailment reaction, the formation of triacetic acid lactone from acetyl- and malonyl-CoA. The identity, of the product was shown by the enzymatic incorporation of radioactivity from [1-14acetyl-CoA and from recrystallization to constant. specific radioactivity. In paper chromatography, thin layer chromatography, and ionophoresis both the chemically synthesized triacetic acid lactone and the enzymatically formed compound migrated with the same RF-values and electrophoretic mobility. By an oxidation with chromic acid according to the procedure of Kuhn-Roth it could be demonstrated that the radioactivity of [1-14C]acetyl-CoA is incorporated only into the C-6 position of triacetic acid lactone. Free triaceti acid and tetraacetic acid lactone do not seem to be formed by fatty acid synthetase. The ratio of the malonyl-CoA utilization for the synthesis of palmitoyl- and stearoyl-CoA to that for the synthesis of triacetic acid lactone was found to be about 90:1. The significance, of these findings is discussed. A chemical mechanism for the formation of triacetic acid lactone by fatty acid. synthetase is proposed. [ABSTRACT FROM AUTHOR]

Thiolase was purified 600–700 fold from a crude extract of pig heart by use of differential adsorption on Al(OH)3 gel, ammonium sulfate fractionation and ion exchange chromatography on DEAE­Sephadex and phosphorylated cellulose. The enzyme preparation is homogenous by the following criteria: sedimentation analysis and electrophoresis on polyacrylamide gel and on cellulose acetate membranes. The isoelectric poit of the protein was found to be approximately pH 7.2–7.3. The enzyme crystallized from ammonium sulfate solutions as thin plates of irregular shape. Crystalline thiolase stored for 18 months at –10° did not show any loss of activity. The thiolytic activity and the acyl transferase activity of the enzyme could be demonstrated separately. The thiolytic activity was demontrated by the specific incorporation of 14C from [1 ­14C]­acetyl­CoA into the carboxyl position of unlabelled acetoacetyl­CoA on incubation wtih the enzyme. The acyl transferase activity eas studied using either [1­14C]­CoA and patethein as substrates or 14C­labelled CoA and acyl­CoA derivatives of varius chain length. A remarkable specificity was found for the transfer of the acetyl group. Thiolase activity and acyl transferase activity of the enzyme were diminished with the same kinetics during heat treatment or incubation with N ­ethylmaleeimide, ρ­cholomercuribenzoate or iodoacetamide. this indicates that both activities belong to the same protein. Inhibition of thiolase and acyltranferase activities by iodoacetamide (5 x 10­5 could be prevented by preicubation of the enzyme with acetoacetyl­CoA or acetyl­CoA. The acyl transfer is the rate determining step when the thiolase reaction operates in the direction of chain cleavage. Incubation of thiolase with acetyl­Coa results in binding of acetyl groups to the protein. The uptake of acetyl groups is dependent upon the specific activity of the enzyme and corresponds to 3 acetyl residues per molecule of protein (molecular weight 170,000) for the highest enzymatic activity obtained. both incorporation of acetyl groups into the enzyme and thiolase activity are inhibited to the same extent by iodoacetamide. the incorportion of radioactivity into the enztme during incubation with iodo­[1­14C]­acetamide can be divided into two different reactions: a fast incorporation with accompanying loss of enzymatic and a much slower process which continues after complete inactivation. From the initial rate of incorporation, it was calculaetd that incorporation of 3.1 carbamoylmethyl residues per molecule of protein would completely inhibit the enzyme withthe highest obtained activity. this ration cmpares well with the results obtained by acetyl binding studies and indicates a minimum of 3 active sites per molecule of enzyme. Enzyme labelled with iodo­[1­14C]acetamide was hydrolyzed with 6 N HCl. Approximately 95% of the radioactivity in the hydrolysate was found to coincide with S­carboxymethyl­L­cystine on electrophoresis and paper chromatography. Repeated crystallization with authentic carboxymethyl­cystine did not result in a decrease of specific radioactivity. [ABSTRACT FROM AUTHOR]

Temperature-responsive liquid chromatography (TRLC) offers a promising alternative to reversed-phase liquid chromatography (RPLC) for environmentally friendly analytical techniques by utilizing pure water as a mobile phase, eliminating the need for harmful organic solvents. TRLC columns, packed with temperature-responsive polymers coupled to silica particles, exhibit a unique retention mechanism influenced by temperature-induced polymer hydration. An investigation of the physicochemical parameters driving separation at high and low temperatures is crucial for better column manufacturing and selectivity control. Assessment of predictability using a dataset of 139 molecules analyzed at different temperatures elucidated the molecular descriptors (MDs) relevant to retention mechanisms. Linear regression, support vector regression (SVR), and tree-based ensemble models were evaluated, with no standout performer. The precision, accuracy, and robustness of models were validated through metrics, such as r and mean absolute error (MAE), and statistical analysis. At 45 ∘ C , logP predominantly influenced retention, akin to reversed-phase columns, while at 5 ∘ C , complex interactions with lipophilic and negative MDs, along with specific functional groups, dictated retention. These findings provide deeper insights into TRLC mechanisms, facilitating method development and maximizing column potential., (© 2024. The Author(s).)

Background: The chemical composition and biological activities of Eucalyptus essential oils (EOs) have been documented in numerous studies against multiple infectious diseases. The antibacterial activity of individual Eucalyptus EOs against strains that cause ear infections was investigated in our previous study. The study's antibacterial activity was promising, which prompted us to explore this activity further with EO blends., Methods: We tested 15 combinations (9 binary combinations and 6 combinations of binary combinations) of Eucalyptus EOs extracted by hydrodistillation from eight Tunisian Eucalyptus species dried leaves against six bacterial strains responsible for ear infections: three bacterial isolates (Haemophilus influenzae, Haemophilus parainfluenzae, and Klebsiella pneumoniae) and three reference bacteria strains (Pseudomonas aeruginosa, ATTC 9027; Staphylococcus aureus, ATCC 6538; and Escherichia coli, ATCC 8739). The EOs were analyzed using GC/FID and GC/MS. The major compounds, as well as all values obtained from the bacterial growth inhibition assay, were utilized for statistical analysis., Results: The antibacterial activity of the EO blends exhibited significant variation within Eucalyptus species, bacterial strains, and the applied methods. Principal component analysis (PCA) and hierarchical cluster analysis (HCA), based on the diameters of the inhibition zone, facilitated the identification of two major groups and ten subgroups based on the level of antibacterial activity. The highest antibacterial activity was observed for the mixture of EOs extracted from E. panctata, E. accedens, and E. cladoclayx (paac) as well as E. panctata, E. wandoo, E. accedens, and E. cladoclayx (pwac) using the disc diffusion method. Additionally, significant activity was noted with EOs extracted from E. panctata, E. wandoo (pw) and E. panctata, E. accedens (pa) using the broth microdilution method., Conclusion: Our findings suggest that certain EO combinations (paac, pwac, pw, and pa) could be considered as potential alternative treatment for ear infections due to their demonstrated highly promising antibacterial activities., (© 2024. The Author(s).)

Implementing effective environmental management strategies requires a comprehensive understanding of the chemical composition of environmental pollutants, particularly in complex mixtures. Utilizing innovative analytical techniques, such as high-resolution mass spectrometry and predictive retention index models, can provide valuable insights into the molecular structures of environmental contaminants. Liquid Chromatography-High-Resolution Mass Spectrometry is a powerful tool for the identification of isomeric structures in complex samples. However, there are some limitations that can prevent accurate isomeric structure identification, particularly in cases where the isomers have similar mass and fragmentation patterns. Liquid chromatographic retention, determined by the size, shape, and polarity of the analyte and its interactions with the stationary phase, contains valuable 3D structural information that is vastly underutilized. Therefore, a predictive retention index model is developed which is transferrable to LC-HRMS systems and can assist in the structural elucidation of unknowns. The approach is currently restricted to carbon, hydrogen, and oxygen-based molecules <500 g mol -1 . The methodology facilitates the acceptance of accurate structural formulas and the exclusion of erroneous hypothetical structural representations by leveraging retention time estimations, thereby providing a permissible tolerance range for a given elemental composition and experimental retention time. This approach serves as a proof of concept for the development of a Quantitative Structure-Retention Relationship model using a generic gradient LC approach. The use of a widely used reversed-phase (U)HPLC column and a relatively large set of training (101) and test compounds (14) demonstrates the feasibility and potential applicability of this approach for predicting the retention behaviour of compounds in complex mixtures. By providing a standard operating procedure, this approach can be easily replicated and applied to various analytical challenges, further supporting its potential for broader implementation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: The chemical composition and biological activity of Eucalyptus essential oils have been studied extensively (EOs). A few of them were tested for antibacterial effectiveness against otitis strains. The chemical composition and antibacterial activity of the EOs of eight Tunisian Eucalyptus species were assessed in the present study., Methods: Hydrodistillation was used to extract EOs from the dried leaves of eight Eucalyptus species: Eucalyptus accedens, Eucalyptus punctata, Eucalyptus robusta, Eucalyptus bosistoana, Eucalyptus cladocalyx, Eucalyptus lesouefii, Eucalyptus melliodora and Eucalyptus wandoo. They are assessed by GC/MS and GC/FID and evaluated for antibacterial activity using agar diffusion and broth microdilution techniques against three bacterial isolates (Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiella pneumoniae) and three reference bacteria strains (Pseudomonas aeruginosa, ATTC 9027; Staphylococcus aureus, ATCC 6538; and Escherichia coli, ATCC 8739). Furthermore, the selected twenty-one major compounds and all values of the inhibition zone diameters were subjected to further statistical analysis using PCA and HCA., Results: The EO yields of the studied Eucalyptus species range from 1.4 ± 0.4% to 5.2 ± 0.3%. Among all the species studied, E. lesouefii had the greatest mean percentage of EOs. The identification of 128 components by GC (RI) and GC/MS allowed for 93.6% - 97.7% of the total oil to be identified. 1,8-cineole was the most abundant component found, followed by α-pinene, p-cymene, and globulol. The chemical components of the eight EOs, extracted from the leaves of Eucalyptus species, were clustered into seven groups using PCA and HCA analyses, with each group forming a chemotype. The PCA and HCA analyses of antibacterial activity, on the other hand, identified five groups., Conclusion: The oils of E. melliodora, E. bosistoana, and E. robusta show promise as antibiotic alternatives in the treatment of otitis media., (© 2021. The Author(s).)