High-performance liquid chromatography assay for N-acetylcysteine in biological samples following derivatization with N-(1-pyrenyl)maleimide

N-Acetylcysteine is a thiol antioxidant with expanding clinical importance. A sensitive, rapid method for determining reduced N-acetylcysteine (NAC) concentration in biological samples has been developed which uses a modified reversed-phase high-performance liquid chromatography (HPLC) technique in conjunction with the derivatizing agent N-(1-pyrenyl)maleimide (NPM). The NAC-NPM adduct was analyzed by HPLC with fluorescence detection. The calibration curve for NAC was linear over the range 8–2500 nM and the coefficient of variation obtained for the within-run precision and the between-run precision for 0.5 mM NAC was 1.5% and 2.7%, respectively. Relative recovery of NAC from biological materials ranged between 86% and 96% and the limit of quantitation from biological samples was 32 nM. These results suggest practical advantages relative to other widely-accepted methods of NAC measurement.     

Analysis of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine, the mercapturic acid derived from N,N-dimethylformamide

Human biotransformation of the industrial solvent N,N-dimethylformamide gives raise to N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) which has the longest half-life (about 23 h) among urinary metabolites of N,N-dimethylformamide. It could be used for monitoring industrial exposure over several workdays, by measuring it in urine samples collected at the end of the working week. This is consistent with the suggestions of the American Conference of Governmental Industrial Hygienists, which established a limit of 40 mg/l for the year 2000. An easy, cheap and user-friendly method has been developed for determination of urinary AMCC. Unlike currently available methods, it requires neither a time-consuming preparation phase nor gas chromatographic analysis with a nitrogen-phosphorus or mass detector. The method uses high-performance liquid chromatography (HPLC), with an UV detector at 436 nm. A 10-μl volume of urine is added to a carbonate–hydrogen carbonate buffer and mixed with a dabsyl chloride solution in acetonitrile. The reaction between AMCC and the reagent is performed at 70°C for 10 min. The ‘dabsylated’ product is stable for at least 12 h. After brief centrifugation, the solution is ready for HPLC analysis using a C₁₈ column (250×4.6 mm, 5 μm). The method is sensitive (detection limit 1.8 mg/l) and specific. It identified urinary AMCC in urine of 40 subjects not exposed to N,N-dimethylformamide with a median concentration of 3.9 mg/l. In urine samples from 20 workers exposed to N,N-dimethylformamide (5–40.8 mg/m³), AMCC concentrations ranged from 16 to 170 mg/l. Industrial toxicology laboratories with limited instrumentation will be able to use it in the biological monitoring of workers exposed to N,N-dimethylformamide.     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

A cellulosic exopolysaccharide produced from sugarcane molasses by a Zoogloea sp.

An extracellular polysaccharide producing bacterium Zoogloea sp. was isolated from an agro-industrial environment in the north-eastern region of Brazil. The extracellular polysaccharide produced from sugarcane molasses was hydrolysed with trifluoroacetic acid (mild and strong conditions) giving 88% of soluble material. The main monosaccharides present in the soluble fraction were glucose (87.6%), xylose (8.6%), mannose (0.8%), ribose (1.7%), galactose (0.1%), arabinose (0.4%) and glucuronic acid (0.8%). Methylation analysis of the polysaccharide showed mainly 2,3,6-tri-O-methylhexitol (74.7%) and 2,3,-di-O-methylhexitol (17.7%). Enzyme hydrolysis of the polysaccharide with a cellulase confirmed the presence of (1→4)-β- -glucopyranosyl residues.     

Peanut (Arachis hypogaea) lectin recognizes α-linked galactose, but not N-acetyl lactosamine in N-linked oligosaccharide terminals

Peanut (Arachis hypogaea) agglutinin (PNA) is extensively used as tumour marker as it strongly recognises the cancer specific T antigen (Galβ1→3GalNAc-), but not its sialylated version. However, an additional specificity towards Galβ1→4GlcNAc (LacNAc), which is not tumour specific, had been attributed to PNA. For correct interpretation of lectin histochemical results we examined PNA sugar specificity using naturally occurring or semi-synthetic glycoproteins, matrix-immobilised galactosides and lectin-binding tissue glycoproteins, rather than mono- or disaccharides as ligands. Dot-blots, transfer blots or polystyrene plate coatings of the soluble glycoconjugates were probed with horse-radish peroxidase (HRP) conjugates of PNA and other lectins of known specificity. Modifications of PNA-binding glycoproteins, including selective removal of O-linked oligosaccharides and treatment with glycosidases revealed that Galβ1→4GlcNAc (LacNAc) was ineffective while terminal α-linked galactose (TAG) as well as exposed T antigen (Galβ1→3 GalNAc-) was excellent as sugar moiety in glycoproteins for their recognition by PNA. When immobilised, melibiose was superior to lactose in PNA binding. Results were confirmed using TAG-specific human serum anti-α-galactoside antibody.     

Mucoadhesive property and biocompatibility of methylated N-aryl chitosan derivatives

The methylated N-aryl chitosan derivatives, methylated N-(4-N,N-dimethylaminocinnamyl) chitosan chloride (MDMCMCh) and methylated N-(4-pyridylmethyl) chitosan chloride (MPyMeCh), were synthesized by two steps, the reductive amination and the methylation. The physicochemical properties of chitosan derivatives were determined by ATR-FTIR, NMR, X-ray diffraction (XRD) and thermogravimetric (TG) techniques. The XRD analysis showed that the crystallinity and thermal stability of methylated chitosan derivatives were lower than those of chitosan. The effects of degree of quaternization (DQ), polymer structure and positive charge location on mucoadhesive property and cytotoxicity were investigated by using a mucin particle method and MTT assay compared to N,N,N-trimethylammonium chitosan chloride (TMChC). It was found that the mucoadhesive property and cytotoxicity increased with increasing DQ. At the DQ of 65%, the mucoadhesive property of the MDMCMCh was twofold lower than that of the TMChC. However, this phenomenon did not affect the mucoadhesive property when the DQ was higher than 65%. Surprisingly, the MPyMeCh showed the lowest toxicity even with the high DQ. These could be due to the resonance effect of the positive charge in the pyridine ring and the molecular weight after methylation. Finally, our result revealed that the mucoadhesive property was dependent on the DQ and polymer structure whereas the cytotoxicity was dependent on the combination of the polymer structure, positive charge location and molecular weight after methylation.     

Alterations of asparagine-linked sugar chains of N-acetyl β- -hexosaminidase during human renal oncogenesis: a preliminary study using serial lectin affinity chromatography

Enzymatic properties and asparagine (Asn)-linked sugar-chain structures of N-acetyl β- -hexosaminidase A (Hex A) were compared in human tissues between normal renal cortex and renal cell carcinoma (RCC). No significant differences between the two Hex A preparations were observed with respect to enzymatic properties such as molecular mass, Michaelis–Menten value or optimal pH. With RCC preparations, relatively more Hex A passed through the concanavalin A (Con A) column, bound weakly to Con A, or bound strongly to Con A and also to the wheat germ agglutinin (WGA) column, than with preparations from normal renal cortex. In contrast, relatively less Hex A bound strongly to the Con A column, but passed through the WGA column with RCC preparations than with those from normal renal cortex. Asn-linked sugar-chain structures might apparently be altered during human renal oncogenesis.     

Simultaneous determination of two human urinary metabolites of N,N-dimethylformamide using gas chromatography–thermionic sensitive detection with mass spectrometric confirmation

Two human urinary metabolites of the industrial solvent N,N-dimethylformamide (DMF), N-hydroxymethyl-N-methylformamide (HMMF) and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), were assayed using a new analytical method (gas chromatography and thermionic sensitive detection). Clean-up of urine samples includes a liquid–liquid extraction step followed by a solid-phase extraction step to separate HMMF and AMCC from other urine components. During clean-up, AMCC is converted into ethyl-N-methylcarbamate (EMC), and during gas chromatography, HMMF is degraded in the injector to N-methylformamide (NMF). All the validation data necessary for a quantitative procedure are given. The method was applied to urine samples from workers exposed to DMF and from the general population. The results were confirmed by mass spectrometric determination. For this purpose a further liquid–liquid extraction step was introduced in the clean-up procedure. Background levels of AMCC in the general population were identified.     

Purification and properties of a wheat leaf N-acetyl-β-d-hexosaminidase

An N-acetyl-β-d-hexosaminidase has been purified from primary wheat leaves (Triticum aestivum L.) by freeze-thawing, (NH₄)₂SO₄ precipitation, methanol precipitation, gel filtration, cation exchange chromatography and affinity chromatography on concanavalin A-Sepharose. The activity of the purified preparations could be stabilised by addition of Triton X-100 and the enzyme was stored at -20°C without significant loss of activity. The enzyme hydrolysed pNP-β-d-GlcNAc (optimum pH 5.2, K_m 0.29 mM, V_max 2.56 μkat mg⁻¹) and pNP-β-d-GalNAc (optimum pH 4.4, K_m 0.27 mM, V_max 2.50 μkat mg⁻¹). Five major isozymes were identified, with isoelectric points in the range 5.13–5.36. All five isozymes possessed both N-acety-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activity. Inhibition studies and mixed substrate analysis suggested that both substrates are catalysed by the same active site. Both activities were inhibited by GlcNAc, 2-acetamido-2-deoxygluconolactone, GalNAc and the ions of mercury, silver and copper. The K_is for inhibition of N-acetyl-β-d-glucosaminidase activity were: GlcNAc (15.3 mM) and GalNAc (3.4mM). For inhibition of N-acety-β-d-galactosaminidase activity the corresponding values were: GlcNAc (18.2 mM) and GalNac (2.5 mM). The enzyme was considerably less active at releasing pNP from pNP-β-d-(GlcNAc)₂ and pNP-β-d-(GlcNAc)₃ than from pNP-β-d-GlcNAc. The ability of the N-acetyl-β-d-hexosaminidase to relase GlcNAc from chitin oligomers (GlcNAc)₂ (optimum pH 5.0) and (GlcNAc)₃₋₆ (optimum pH 4.4) was also low. Analysis of the reaction products revealed that the initial products from the hydrolysis of (GlcNAc)_n were predominantly (GlcNAc)_n−1 and GlcNAc.     

Synthesis and analgesic profile of conformationally constrained N-acylhydrazone analogues: Discovery of novel N-arylideneamino quinazolin-4(3H)-one compounds derived from natural safrole

In this work we reported the synthesis and evaluation of the analgesic, anti-inflammatory, and platelet anti-aggregating properties of new 3-(arylideneamino)-2-methyl-6,7-methylenedioxy-quinazolin-4(3H)-one derivatives (3a–j), designed as conformationally constrained analogues of analgesic 1,3-benzodioxolyl-N-acylhydrazones (1) previously developed at LASSBio. Target compounds were synthesized in very good yields exploiting abundant Brazilian natural product safrole (2) as starting material. The pharmacological assays lead us to identify compounds LASSBio-1240 (3b) and LASSBio-1272 (3d) as new analgesic prototypes, presenting an antinociceptive profile more potent and effective than dipyrone and indomethacin used, respectively, as standards in AcOH-induced abdominal constrictions assay and in the formalin test. These results confirmed the success in the exploitation of conformation restriction strategy for identification of novel cyclic N-acylhydrazone analogues with optimized analgesic profile.     

Application of capillary electrophoresis to the separation of structurally diverse N-(substituted)-glycine–peptoid combinatorial mixtures

The capillary electrophoresis (CE)-based separation of five N-(substituted)-glycine (NSG)–peptoid mixtures with a wide range of physical and chemical properties was studied. A CE separation, initially developed using a single representative peptoid mixture, with a backround electrolyte (BGE) modified by the addition of both methyl-β-cyclodextrin and heptane sulfonic acid was found to provide good separations of most of the combinatorial mixtures investigated. For those mixtures not separated well by this procedure, the use of SDS micelles in conjunction with methyl-β-cyclodextrin resulted in dramatic improvements in the separation. While no single set of separation conditions proved sufficient for all of the NSG–peptoid combinatorial mixtures, the two methods were able to provide separation sufficient for characterization of a set of mixtures with a wide range of physical and chemical properties. The efficiency of the CE-based separation of the combinatorial mixtures studied was compared to a reversed-phase liquid chromatographic method using gradient elution.     

Synthesis of N-monomethylagmatine, a decarboxylation product of N-monomethyl- -arginine

N^G-Monomethylagmatine, a decarboxylation product of N^G-monomethyl- -arginine, has been synthesized by reacting putrescine with N,S-dimethylthiopseudouronium iodide. The structural identity of the product was confirmed by proton NMR and mass spectroscopy, and its properties were determined on thin-layer and electrophoretic chromatography.     

Regioselective synthesis of mannobiose and mannotriose by reverse hydrolysis using a novel 1,6-α- -mannosidase from Aspergillus phoenicis

A novel 1,6-α- -mannosidase was produced by Aspergillus phoenicis grown on a commercial manno-oligosaccharide preparation in liquid culture. The enzyme hydrolysed only α- -Manp-(1→6)- -Manp and did not act on α- -Manp-(1→2)- -Manp, or α- -Manp-(1→3)- -Manp. The 1,6-α- -mannosidase was used for synthesis of manno-oligosaccharides by reverse hydrolysis reaction. The highest yields, expressed as percentages (w/w) of total sugar, were 21% mannobiose and 5% mannotriose, and they were obtained with 45% (w/w) initial mannose concentration at pH 4.5 after 12 days incubation at 55 °C. The disaccharide and trisaccharide products were separated and their structures determined by methylation analysis. Only 1–6 linkages were found in both of them.     

Biological activities and structure-activity relationship of substitution compounds of N-[2-(3-indolyl)ethyl]succinamic acid and N-[2-(1-naphthyl)ethyl]succinamic acid, derived from a new category of root-promoting substances, N-(phenethyl)succinamic acid analogs

In a previous study, it was demonstrated that N-(phenethyl)succinamic acid (PESA) derivatives form a new category of root-promoting substances which do not exhibit auxin-like activities, such as stem elongation and leaf epinasty (Soejima et al., 2000 [Plant Cell Physiol. 41s: 197]). In this study, N-[2-(3-indolyl)ethyl]succinamic acid (IESA) and N-[2-(1-naphthyl)ethyl]succinamic acid (NESA) were synthesized, and their biological activities were evaluated. In an adzuki root-promoting assay, IESA and NESA exhibited root-promoting activity equivalent to PESA. In adzuki stem elongation assays, elongation activity was not observed in the stem segments soaked in either an IESA or NESA aqueous solution, whereas the stem segments immersed in Indole-3-acetic acid (IAA) or 1-naphthylacetic acid (NAA) aqueous solution were clearly elongated. In an epinastic bending study, IAA and NAA exhibited leaf epinasty, whereas IESA and NESA did not, suggesting that the IESA and NESA derivatives belong to the same category of root-promoting substances as PESA derivatives and are different from auxin-like substances. In addition, eleven kinds of IESA derivatives and nineteen kinds of NESA derivatives were synthesized, and their root-promoting activities were measured. The activities of methyl ester derivatives were approximately three times higher than that of the acid compounds, with exceptions for some compounds. The partition coefficient (P) between 1-octanol and water for each IESA, NESA, and PESA derivative was measured in order to evaluate the hydrophobicity of their molecules and to determine their structure–activity relationship. The results indicate that the root-promoting activity of the acid compounds was significantly correlated with their hydrophobicity, whereas that of ester derivatives was not correlated.     

A facile synthesis, structure, and antimicrobial evaluation of novel 4-arylhydrazono-5-trifluoromethyl-2,4-dihydropyrazol-3-ones, their N- and N,O-bis-β-d-glucosides

Synthesis of some novel 4-arylhydrazono-5-trifluoromethyl-2,4-dihydropyrazol-3-ones, their N- and N,O-bis- β-d-glucosides is described. Antimicrobial evaluation of eight selected compounds against Aspergillus fumigatus RCMB 002008 (1), Penicillium italicum RCMB 001018 (1), Syncephalastrum racemosum RCMB 016001, Candida albicans RCMB 005003, Staphylococcus aureus RCMB 106-001 (1), Pseudomonas aeruginosa RCMB 102-002, Bacillus subtilis RCMB 101-001, and Escherichia coli RCMB 103-001 has been achieved. The screening results indicated that all the tested compounds exhibited different inhibitory effects against five to seven different organisms of the eight test organisms.     

Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [H7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-²H₇]α-ketoisocaproic acid ([²H₇]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5-²H₃]α-Ketoisocaproic acid ([²H₃]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [²H₃]KIC and [²H₇]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [²H₇]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [²H₇]KIC spiked to rat plasma in the range of 0.1 to 10 μM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [²H₇]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [²H₇]KIC after an intravenous administration of [²H₇]KIC in rat.     

Synthesis, (1-->3)-beta-D-glucanase-binding ability and phytoalexin-elicitor activity of (R)-2,3-epoxypropyl (1-->3)-beta-D-pentaglucoside

The (1-->3)-beta-D-pentaglucoside was synthesized as its (R)-2,3-epoxypropyl glycoside via 2+3 strategy. The disaccharide donor 8 was obtained by 3-selective coupling of 2 with 4, followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor 12 was prepared by coupling of 10 with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside. The results of these bioassays demonstrated that the (1-->3)-beta-D-glucanase was obviously inactivated by 15 with k(app)=3.79 x 10(-4) min(-1). At the same time, we found that the 15 was more active as compared to the laminaripentaose in eliciting phytoalexin accumulation in tobacco cotyledon tissue, and it could be kept longer time than laminaripentaose, which indicated it is much more stable than laminaripentaose.     

Design, synthesis, and evaluation of a novel series of alpha-substituted phenylpropanoic acid derivatives as human peroxisome proliferator-activated receptor (PPAR) alpha/delta dual agonists for the treatment of metabolic syndrome

A series of alpha-alkyl-substituted phenylpropanoic acids was prepared as dual agonists of peroxisome proliferator-activated receptors alpha and delta (PPARalpha/delta). Structure-activity relationship studies indicated that the shape of the linking group and the shape of the substituent at the distal benzene ring play key roles in determining the potency and the selectivity of PPAR subtype transactivation. Structure-activity relationships among the amide series (10) and the reversed amide series (13) are similar, but not identical, especially in the case of the compounds bearing a bulky hydrophobic substituent at the distal benzene ring, indicating that the hydrophobic tail part of the molecules in these two series binds at somewhat different positions in the large binding pocket of PPAR. alpha-Alkyl-substituted phenylpropanoic acids of (S)-configuration were identified as potent human PPARalpha/delta dual agonists. Representative compounds exhibited marked nuclear receptor selectivity for PPARalpha and PPARdelta. Subtype-selective PPAR activation was also examined by analysis of the mRNA expression of PPAR-regulated genes.     

Turnover of N-acetylglutamate in isolated rat hepatocytes

Isolated hepatocytes from starved rats were loaded with N-[¹⁴C]acetylglutamate by preincubating them with [¹⁴C]bicarbonate, oleate, NH₃, ornithine and lactate. Turnover of N-acetylglutamate in these cells was subsequently measured in an unlabelled medium under conditions of minimal flux (oleate alone present) and maximal flux (oleate, NH₃, ornithine and lactate present) through the urea cycle. 1. Direct measurement of the distribution of N-[¹⁴C]acetylglutamate across the mitochondrial membrane in the hepatocytes showed that, under the conditions studied, the rate of degradation of total intracellular N-[¹⁴C]acetylglutamate was about equal to the rate of efflux of N-acetylglutamate from the mitochondria. 2. In the presence of oleate alone, intramitochondrial N-acetylglutamate decreased because mitochondrial N-acetylglutamate efflux predominated over the synthesis of N-acetylglutamate in the mitochondria. 3. In the presence of oleate, NH₃, ornithine and lactate both the rate of synthesis of N-acetylglutamate and the rate of its transport out of the mitochondria were increased when compared with the condition with oleate alone. However, the intramitochondrial concentration of N-acetylglutamate increased because initially the rate of its synthesis exceeded that of its efflux from the mitochondria. Finally, a steady state was reached in which both rates were equal. 4. The data indicate that in hepatocytes from starved rats N-acetylglutamate transport out of the mitochondria takes place at a rate proportional to its intramitochondrial concentration. It is concluded that transport of N-acetylglutamate either occurs by diffusion or is mediated by a transport system with a high K_m for intramitochondrial N-acetylglutamate.     

Mutational spectrum induced in Saccharomyces cerevisiae by the carcinogen N-2-acetylaminofluorene

The spectrum of mutations induced by the carcinogen N-2-acetylaminofluorene (AAF) was analysed in Saccharomyces cerevisiae using a forward mutation assay, namely the inactivation of the URA3 gene. The URA3 gene, carried on a yeast/bacterial shuttle vector, was randomly modified in vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF. The binding spectrum of AAF to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF. Independent Ura^– mutants were selected in vivo after transformation of the modified plasmid into a ura3 yeast strain. Plasmid survival decreased as a function of AAF modification, leading to one lethal hit (37% relative survival) for an average of 50 AAF adducts per plasmid molecule. At this level of modification the mutation frequency was equal to 70 × 10^–4, i.e. 50-fold above the background mutation frequency. UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicating the lack of an SOS-like mutagenic response in yeast. Sequence analysis of the URA3 mutants revealed 48% frameshifts, 44% base substitutions and 8 % complex events. While most base substitutions (74%) were found to be targeted at G residues where AAF is known to form covalent C8 adducts, frameshift mutations were observed at GC base pairs in only 24% of cases. Indeed, more than 60% of frameshift events occurred at sequences such as 5-(A/T)_nG-3 where a short (n = 2 or 3) monotonous run of As or Ts is located on the 5' side of a guanine residue. We refer to these mutations as semi-targeted events and present a potential mechanism that explains their occurrence.