Simple GC-FID method development and validation for determination of α-tocopherol (vitamin E) in human plasma

This paper describes the development and validation of a novel GC-FID method for the determination of α-tocopherol concentration in human plasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. To determine the concentration of α-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. α-tocopherol was extracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min^− 1. Calibration curves were linear over the concentration range 1–30 μg ml^− 1 (for standard solutions and solutions without endogenous α-tocopherol in plasma) and 5–34 μg ml^− 1 (for solutions with endogenous α-tocopherol in plasma). Absolute recovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of α-tocopherol from plasma averaged 97.44%. The limit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 μg.ml^− 1 and 0.30 μg.ml^− 1, respectively. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) was better than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of α-tocopherol. The endogenous α-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with this method.     

Synthesis and in vitro α-chymotrypsin inhibitory activity of 6-chlorobenzimidazole derivatives

A library of benzimidazole derivatives 1–20 were synthesized, and studied for their α-chymotrypsin (α-CT) inhibitory activity in vitro. Kinetics and molecular docking studies were performed to identify the type of inhibition. Compound 1 was found to be a good inhibitor of α-chymotrypsin enzyme (IC₅₀ = 14.8 ± 0.1 μM, K_i = 16.4 μM), when compared with standard chymostatin (IC₅₀ = 5.7 ± 0.13 μM). Compounds 2–8, 15, 17, and 18 showed significant inhibitory activities. All the inhibitors were found to be competitive inhibitors, except compound 17, which was a mixed type inhibitor. The substituents (R) in para and ortho positions of phenyl ring B, apparently played a key role in the inhibitory potential of the series. Compounds 1–20 were also studied for their cytotoxicity profile by using 3T3 mouse fibroblast cells and compounds 3, 5, 6, 8, 12–14, 16, 17, 19, and 20 were found to be cytotoxic. Molecular docking was performed on the most active members of the series in comparison to the standard compound, chymostatin, to identify the most likely binding modes. The compounds reported here can serve as templates for further studies for new inhibitors of α-chymotrypsin and other chymotrypsin-like serine proteases enzymes.

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Analysis of naltrexone and its metabolite 6-ß-naltrexol in serum with high-performance liquid chromatography

ABSTRACT: BACKGROUND: Naltrexone has been proven to be an effective treatment option for the treatment of alcohol dependency. In this article we introduce a reliable and simple method developed for the simultaneous determination of naltrexone and 6-beta-naltrexol in human serum by using high-performance liquid chromatography (HPLC). FINDINGS: Liquid-liquid extraction with butyl acetate from basic solutions (pH 9) was chosen for extraction with nalorphine as an internal standard (IS). Analytes were back-extracted from organic solvent into perchloric acid. The acid extract was chromatographed by HPLC with a reverse-phase ODS-column and electrochemical detector. The mobile phase was a NaH2PO4-solution with acetonitrile as an organic modifier and octanesulphonic acid and tetraethylammonium hydrogen sulphate as ion-pair reagents. The recovery of the extraction method was 48 % for naltrexone and 75 % for 6-beta-naltrexol. The limit of quantification was 5.0 ng/ml for naltrexone and 1.0 ng/ml for 6-beta-naltrexol. The analysed concentrations of naltrexone differed from the theoretic concentrations by 0.7 to 2.3 % and those of 6-beta-naltrexol by 2.6 %. The relative standard deviation of within-day assay was from 0.9 to 5.7 % for naltrexone and from 0.8 to 4.2 % for 6-beta-naltrexol; for the between-day assay it was 5.7 % and 4.2 %, respectively. CONCLUSIONS: Our results indicate that the developed method is suitable for determination of naltrexone and 6-beta-naltrexol in human serum.     

The role of the delocalized α,α′-carbanion in vitamin B6 and Al(III) catalyzed transamination of α-amino and α-keto acids

The rates of the transamination reactions of α-amino acids and α-keto acids were followed by measurement of the 200 MHz proton NMR spectra of solution species as a function of time. Reaction systems measured in D₂O at 10 °C consisted of 1:1:1 molar ratios of pyridoxal:α-amino acid:Al(III) or pyridoxamine:α-keto acid:Al(III). Amino and keto acids employed are alanine, α-aminoisobutyric acid, valine, phenylglycine, pyruvic acid, and α-ketobutyric acid. A negatively charged deprotonated Schiff base coordinated to Al(III) was detected in all systems that undergo transamination (i.e., except α-aminoisobutyric acid). The intermediate resembles the aldimine Al(III) chelate with NMR resonances shifted upfield in accordance with its greater negative charge. Its equilibrium concentration is reached in the time required to reach transamination equilibrium and is maintained in solution at a ca. 10–20% of the aldimine Schiff base concentration.     

Discovery and structure–activity relationships of 6-(benzoylamino)benzoxaboroles as orally active anti-inflammatory agents

Structure–activity relationships of 6-(benzoylamino)benzoxaborole analogs were investigated for the inhibition of TNF-α, IL-1β, and IL-6 from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compound 1q showed potent activity against all three cytokines with IC₅₀ values between 0.19 and 0.50 μM, inhibited LPS-induced TNF-α and IL-6 elevation in mice and improved collagen-induced arthritis in mice. Compound 1q (AN4161) is considered to be a promising lead for novel anti-inflammatory agent with an excellent pharmacokinetic profile.     

Structure–activity relationships of 6-(aminomethylphenoxy)-benzoxaborole derivatives as anti-inflammatory agent

A series of novel 6-(aminomethylphenoxy)benzoxaborole analogs was synthesized for the investigation of the structure–activity relationship of the inhibition of TNF-alpha, IL-1beta, and IL-6, from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compounds 9d and 9e showed potent activity against all three cytokines with IC₅₀ values between 33 and 83 nM. Chloro substituted analog 9e (AN3485) is considered to be a promising lead for novel anti-inflammatory agent with a favorable pharmacokinetic profile.     

In vitro Formation of Triple-Stranded DNA Structures in the Human α1-Antitrypsin Gene

We analyzed the possibility of triplex formation in the human 1-antitrypsin gene. Conditions and nucleotide sequence dependence of triplex formation and thermostability of the product were determined.     

Synthesis,in vitro β-glucuronidase inhibitory activity and in silico studies of novel (E)-4-Aryl-2-(2-(pyren-1-ylmethylene)hydrazinyl)thiazoles

Current research is based on the synthesis of novel (E)-4-aryl-2-(2-(pyren-1-ylmethylene)hydrazinyl)thiazole derivatives (3–15) by adopting two steps route. First step was the condensation between the pyrene-1-carbaldehyde (1) with the thiosemicarbazide to afford pyrene-1-thiosemicarbazone intermediate (2). While in second step, cyclization between the intermediate (2) and phenacyl bromide derivatives or 2-bromo ethyl acetate was carried out. Synthetic derivatives were structurally characterized by spectroscopic techniques such as EI-MS, ¹H NMR and ¹³C NMR. Stereochemistry of the iminic double bond was confirmed by NOESY analysis. All pure compounds 2–15 were subjected for in vitro β-glucuronidase inhibitory activity. All molecules were exhibited excellent inhibition in the range of IC₅₀ = 3.10 ± 0.10–40.10 ± 0.90 μM and found to be even more potent than the standard d-saccharic acid 1,4-lactone (IC₅₀ = 48.38 ± 1.05 μM). Molecular docking studies were carried out to verify the structure-activity relationship. A good correlation was perceived between the docking study and biological evaluation of active compounds.     

Actions of terazosin and its enantiomers at subtypes of α- and α2-Adrenoceptors in vitro

Abstract

Terazosin and its enantiomers, antagonists of α1-adrenoceptors, were studied in radioligand binding and functional assays to determine relative potencies at subtypes of α1- and α2-adrenoceptors in vitro. The racemic compound and its enantiomers showed high and apparently equal affinity for subtypes of α1-adrenoceptors with K values in the low nanomolar range, and showed potent antagonism of α1-adrenoceptors in isolated tissues, with the enantiomers approximately equipotent to the racemate at each α1-adrenoceptor subtype. At α2b sites, R(+) terazosin bound less potently than either the S(-) enantiomer or racemate. R(+) terazosin was also less potent than the S(-) enantiomer or the racemate at rat atrial α2B receptors. These agents were not significantly different in their potencies at α2a or α2A sites. Since the high affinity for α2B sites of quinazoline-type α-adrenoceptor antagonists has been used to differentiate α2-adrenoceptor subtypes, the low affinity of R(+) terazosin for these sites was unexpected. Because terazosin or its enantiomers are approximately equipotent at α1 -adrenoceptor subtypes, the lower potency of R(+) terazosin at α2B receptors indicates a somewhat greater selectivity for α1- compared to α2B adrenoceptor subtypes. The possible pharmacological significance of this observation is discussed.     

Tissue and serum α2-3- and α2-6-linkage specific sialylation changes in oral carcinogenesis

Increased sialylation of cell surface glycoconjugates is among the key molecular changes associated with malignant transformation and cancer progression. We investigated significance of linkage-specific sialylation changes in oral carcinogenesis. Tissue and serum levels of total sialic acid (TSA), linkage-specific sialyltransferases (ST) and sialoproteins were analyzed from patients with oral precancerous conditions (OPC) and oral cancer as well as the post-treatment follow-up blood samples of oral cancer patients. TSA levels were measured using a spectrophotometric method. The linkage-specific lectins, Sambusus nigra (SNA) and Maackia amurensis (MAM) detects α2-6- and α2-3-linked sialic acid, respectively, were used to analyze ST activity and sialoproteins. Malignant tissues showed significantly higher levels of TSA, reactivity of SNA and MAM, and α2,3-ST activity compared to the adjacent normal tissues. α2,6-ST was also higher in malignant tissues. Similarly, the marker levels were higher in precancerous tissues than their adjacent normal tissues. Serum levels of TSA, TSA/ total proteins, α2-6-sialoproteins and α2,6-ST were markedly increased in untreated oral cancer patients compared to the controls and OPC as well as responder (CR) patients. Serum levels of the markers were higher or comparable between untreated oral cancer patients and non-responders (NR). Serum levels of α2-3-sialylation were elevated in non-responders compared with the responders. Further, the observed sialylation changes in tissue and serum were found to be associated with various clinicopathological features and disease progression. Thus, the data suggest potential utility of sialylation markers in early detection, prognostication and treatment monitoring of oral cancer.     

Modulation of α-synuclein aggregation by dopamine in the presence of MPTP and its metabolite

The neurotransmitter dopamine has been shown to inhibit fibrillation of α-synuclein by promoting the formation of nonamyloidogenic oligomers. Fibrillation of α-synuclein is accelerated in the presence of pesticides and the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The aim of this study was to determine whether dopamine continues to have an adverse effect on the fibrillation of α-synuclein in the presence of MPTP and its metabolite 1-methyl-4-phenylpyridinum ion (MPP(+) ). We also attempted to answer the ambiguous question of whether conversion of MPTP to MPP(+) is required for the fibrillation of α-synuclein. For this, α-synuclein was incubated in the presence of MPTP and MPP(+) along with dopamine. The fibrillation of α-synuclein was monitored by Thioflavin T fluorescence and immunoblotting. The morphology of the aggregates formed was observed using scanning electron microscopy. The concentrations of the neurotoxin and its metabolite were estimated by reverse phase HPLC. We found definitive evidence that the conversion of MPTP to MPP(+) is not required for aggregation of α-synuclein. MPP(+) was found to accelerate the rate of α-synuclein aggregation even in the absence of components of mitochondrial complex I. In contrast to the effect of dopamine on the aggregation of α-synuclein alone, in the presence of MPTP or MPP(+) , the aggregates formed are Thioflavin T-positive and amyloidogenic. Thus, the effect of dopamine on the nature of aggregates formed in case of α-synuclein alone and in the presence of MPTP/MPP(+) is different.     

Exploring the thermal stability and activity of α-chymotrypsin in the presence of spermine

Polyamines such as spermine can have interaction with protein. The aim of the present study was to investigate how spermine could influence the structure, thermal stability, and the activity of α-chymotrypsin. Kinetics, thermodynamics, molecular dynamics (MD), and docking simulations studies were conducted to investigate the effect of spermine on the activity and structure of α-Chymotrypsin (α-Chy) in 50 mM Tris–HCl buffer, with the pH 8, using different spectroscopic techniques as well as molecular docking and MD simulations. The stability and activity of α-Chy were increased in the presence of spermine. The results of the kinetic study showed that the activity of spermine was increased. Enzyme activation was accompanied by changes on the α-Chy conformation. Fluorescence intensity changes showed dynamic quenching during spermine binding. The fluorescence quenching of the α-Chy suggested the more polar location of Trp residues. Near-UV and Far-UV circular dichroism studies also demonstrated the transfer of Trp, Phe, and Tyr residues to a more flexible environment. The increase in the absorption of α-Chy in the presence of spermine was as a result of the formation of spermine–α-Chy complex. Molecular docking results revealed the presence of one binding site with a negative value for the Gibbs free energy of the binding of spermine to α-Chy. Docking study also revealed that van der Waals interactions and hydrogen bonds played a major role in stabilizing the complex.     

Non-antioxidant properties of α-tocopherol reduce the anticancer activity of several protein kinase inhibitors in vitro

The antioxidant properties of α-tocopherol have been proposed to play a beneficial chemopreventive role against cancer. However, emerging data also indicate that it may exert contrasting effects on the efficacy of chemotherapeutic treatments when given as dietary supplement, being in that case harmful for patients. This dual role of α-tocopherol and, in particular, its effects on the efficacy of anticancer drugs remains poorly documented. For this purpose, we studied here, using high throughput flow cytometry, the direct impact of α-tocopherol on apoptosis and cell cycle arrest induced by different cytotoxic agents on various models of cancer cell lines in vitro. Our results indicate that physiologically relevant concentrations of α-tocopherol strongly compromise the cytotoxic and cytostatic action of various protein kinase inhibitors (KI), while other classes of chemotherapeutic agents or apoptosis inducers are unaffected by this vitamin. Interestingly, these anti-chemotherapeutic effects of α-tocopherol appear to be unrelated to its antioxidant properties since a variety of other antioxidants were completely neutral toward KI-induced cell cycle arrest and cell death. In conclusion, our data suggest that dietary α-tocopherol could limit KI effects on tumour cells, and, by extent, that this could result in a reduction of the clinical efficacy of anti-cancer treatments based on KI molecules.     

Synthesis and in vitro antiproliferative evaluation of d-secooxime derivatives of 13β- and 13α-estrone

d-Secooximes were synthesized from the d-secoaldehydes in the 13β- and 13α-estrone series. The oximes were modified at three sites in the molecule: the oxime function was transformed into an oxime ether, oxime ester or nitrile group, the propenyl side-chain was saturated and the 3-benzyl ether was removed in order to obtain a phenolic hydroxy function. Triazoles were formed via Cu(I)-catalysed azide–alkyne cycloaddition (CuAAC) from 3-(prop-2-yniloxy)-d-secooximes and benzyl azides. All the products were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7, A2780 and A431). Some of them exhibited activities with submicromolar IC₅₀ values, better than that of the reference agent cisplatin. The structural modifications led to significant differences in the cytostatic properties. Flow cytometry indicated that one of the most potent agents resulted in a cell cycle blockade.     

Molecular cloning and characterization of the sheep α-TTP gene and its expression in response to different vitamin E status

The α-tocopherol transfer protein (α-TTP) is a ~ 32 kDa protein that exhibits a marked ligand specificity and selectively recognizes of α-tocopherol, which is the most active form of vitamin E. The α-TTP gene has been cloned and its physiological functions have been studied in numbers of species, however, the understanding of sheep α-TTP is still in his infancy. In this study, the full-length cDNA of sheep α-TTP gene was cloned from sheep liver by using of rapid amplification of complementary DNA ends (RACE). As a result, the sheep α-TTP gene was 1098 bp in nucleotide which contained 23 bp 5'-untranslated region (UTR), 226 bp 3'-UTR and 849 bp open reading frame (ORF) that encoded a basic protein of 282 amino acids. Further bioinformatic analysis indicated that the sheep α-TTP gene had a high homologous of both nucleotide and amino acid sequences compared with that of other species and had a Sec14p-like lipid-binding domain which called the CRAL-TRIO domain. Moreover, the expression of sheep α-TTP mRNA and protein in response to different vitamin E supplemented levels were observed according to quantitative real-time PCR (qRT-PCR) and Western blotting analysis. The results showed that dietary vitamin E levels did not affect α-TTP mRNA expression significantly while the low vitamin E supplemented level groups of sheep had significantly higher α-TTP protein compared to high-vitamin E groups.