NMR investigation of enzymatic coupling of sulfonamide antimicrobials with humic substances

Phenoloxidases mediate the oxidative transformation of soil phenolic constituents, contributing to the formation of humic substances and the chemical incorporation of some xenobiotic organic compounds into natural organic matter. We previously demonstrated phenoloxidase-mediated covalent coupling of sulfonamide antimicrobials with model humic constituents. Here, we investigate fungal peroxidase-mediated covalent coupling of 13C-sulfamethazine and 15N-sulfapyridine to humic substances. 1H-13C heteronuclear single quantum correlation (HSQC) nuclear magnetic resonance spectroscopy provided an initial indication of peroxidase-mediated covalent binding of 13C-sulfamethazine to humic acid. To confirm the role of the sulfonamide anilinic nitrogen in coupling to humic acid and to determine the nature of the covalent linkage, we incubated 15N-sulfapyridine with humic acid and peroxidase and examined reaction products in 1H-15N heteronuclear multiple bond (HMBC) experiments. The HMBC spectra revealed the presence of Michael adducts (i.e., anilinohydroquinones, anilinoquinones) and possibly other covalent linkages. No evidence for Schiff base formation was observed. Analogous experiments with the model humic constituent catechol provided corroborating evidence for these assignments. Michael adducts are expected to exhibit greater environmental stability than imine linkages that can form between sulfonamides and 2,6-dimethoxyphenols. Because the free anilinic nitrogen is required for the bioactivity of sulfonamide antimicrobials, nucleophilic addition occurring through this moiety could result in the biochemical inactivation of these compounds.     

Laccase-mediated michael addition of 15N-sulfapyridine to a model humic constituent

Chemical incorporation of sulfonamide antimicrobials into natural organic matter may represent an important process influencing the fate of these synthetic, primarily agents in soil and sediment environments. We previously demonstrated that a fungal peroxidase mediates covalent coupling of sulfonamide antimicrobials to model humic constituents; reactions with the 2,6-dimethoxyphenol syringic acid produced Schiff bases (Bialk et al. Environ. Sci. TechnoL 2005, 39, 4436-4473). Here, we show that fungal laccase-mediated reaction of sulfapyridine with the orthodihydroxyphenol protocatechuic acid yields a Michael adduct. We synthesized 15N-enriched sulfapyridine to facilitate determination of the covalent linkage(s) formed between sulfapyridine and protocatechuic acid by NMR spectroscopy. 1H-(15)N heteronuclear multiple bond correlation experiments and tandem mass spectrometry demonstrated that the sulfapyridine anilinic nitrogen engaged in a Michael addition reaction to oxidized protocatechuic acid to form an anilinoquinone. Michael adducts are more stable than the previously reported imine linkages between sulfonamides and 2,6-dimethoxyphenols. Michael addition to quinone-like structures in soil organic matter is expected to diminish the mobility and biological activity of sulfonamide antimicrobials.     

Birnessite-induced binding of phenolic monomers to soil humic substances and nature of the bound residues

The nature of the abiotic birnessite (δ-MnO(2))-catalyzed transformation products of phenolic compounds in the presence of soil organic matter is crucial for understanding the fate and stability of ubiquitous phenolic carbon in the environment. (14)C-radioactive and (13)C-stable-isotope tracers were used to study the mineralization and transformation by δ-MnO(2) of two typical humus and lignin phenolic monomers-catechol and p-coumaric acid-in the presence and absence of agricultural and forest soil humic acids (HAs) at pH 5-8. Mineralization decreased with increasing solution pH, and catechol was markedly more mineralized than p-coumaric acid. In the presence of HAs, the mineralization was strongly reduced, and considerable amounts of phenolic residues were bound to the HAs, independent of the solution pH. The HA-bound residues were homogeneously distributed within the humic molecules, and most still contained the unchanged aromatic ring as revealed by (13)C NMR analysis, indicating that the residues were probably bound via ester or ether bonds. The study provides important information on δ-MnO(2) stimulation of phenolic carbon binding to humic substances and the molecular distribution and chemical structure of the bound residues, which is essential for understanding the environmental fates of both naturally occurring and anthropogenic phenolic compounds.     

Rates of oxidative coupling of humic phenolic monomers catalyzed by a biomimetic iron-porphyrin

A synthetic water-soluble meso-tetra(2,6-dichloro-3-sulfonatophenyl)porphyrinate of iron(lll) chloride (FeP) was used as biomimetic catalyst in the oxidative coupling of three monomeric phenols (catechol, caffeic, and p-coumaric acids), which are common constituents of natural humic substances. The extent of oxidation induced by the FeP catalyst in solutions of phenolic monomers was followed in the presence of an oxygen donor such as hydrogen peroxide or dissolved oxygen under daylight radiation. Both UV- and fluorescence-detected liquid chromatograms indicated that primary oxidation products had a larger electronic conjugation and molecular mass than the original phenols, thereby suggesting that the biomimetic oxidative catalysis produced covalently linked phenylene and oxyphenylene oligomers. However, the polyphenolic products were further oxidized in the progress of the catalytic reaction to possible undetectable aliphatic acids or even to complete mineralization. Rate constants describing the initial reaction period were larger for the catalyzed oxidation with hydrogen peroxide than those for the noncatalyzed control solutions under autoxidation or hydrogen peroxide treatment. However, the rate constants measured for the phenol solutions treated with just the FeP catalyst showed that the presence of dissolved oxygen and the action of the daylight radiation were sufficient to significantly increase the reaction rate in respect to control solutions. These results confirmed previous findings, showing that humic materials may undergo oxidative coupling catalyzed by metal-porphyrins in the presence of either an oxygen donor or, simply, dissolved molecular oxygen under daylight. The increase of molecular mass of natural humic and polyphenolic substances by this biomimetic technology may have useful applications in environmental chemistry.     

Studies on enzymic browning of potatoes (Solanum tuberosum). I. Phenoloxidases and phenolic compounds from different varieties (author's transl)]

31 samples of potato varieties with slow, medium and fast rates of browning were studies. Characteristic enzyme patterns were obtained from polyacrylamide gel electrophoresic of the phenoloxidases of varieties with different discolouration rates. The differences lie mainly in the intensities of the enzyme bands. The qualitative determinaiton of the phenols showed no significant differences. Tyrosine, chlorogenic acid and caffeic acid produce coloured oxidation products; the characteristic colour gradations of in vivo browning were only observed in the presence of tyrosine. It is concluded that the same reactions take place during the discolouration of all the varieties.     

European proficiency testing of national reference laboratories for the confirmation of sulfonamide residues in muscle and milk

Two interlaboratory studies were organized in 2002-2003 in order to check the proficiency of laboratories in confirming the presence of sulfonamide residues in muscle and milk. These studies involved 25 EU National Reference Laboratories (NRLs) from 21 different European countries in charge of statutory monitoring of antimicrobial residues in food of animal origin at a national level. The study was conducted according to international and national guidelines by the Community Reference Laboratory (CRL) in charge of antimicrobial substances. Four different test matrices of sheep muscle and four different test matrices of bovine milk containing different sulfonamide substances were prepared and sent to the participants. Each participant was asked to use his own routine confirmatory method and to analyse each sample in triplicate within a period of about six weeks during which the stability of the materials was checked by the organizer. The sulfonamide content of each material was determined by calculating the robust means of all the results and the deviation of the results from the assigned values was assessed by calculating Z-scores. Overall, results were satisfactory, particularly considering that it was the first proficiency test dealing with sulfonamides organised by the Community Reference Laboratory.     

Comparison of Cd(II), Cu(II), and Pb(II) biouptake by green algae in the presence of humic acid

The present study examines the role of humic acid, as a representative of dissolved organic matter, in Cd(II), Cu(II), and Pb(II) speciation and biouptake by green microalgae. Cellular and intracellular metal fractions were compared in the presence of citric and humic acids. The results demonstrated that Cd and Cu uptake in the presence of 10 mg L(-1) humic acid was consistent with that predicted from measured free metal concentrations, while Pb biouptake was higher. By comparing Cd, Cu, and Pb cellular concentrations in the absence and presence of humic acid, it was found that the influence of the increased negative algal surface charge, resulting from humic acid adsorption, on cellular metal was negligible. Moreover, the experimental results for all three metals were in good agreement with the ternary complex hypothesis. Given that metal has much higher affinity with algal sites than humic acid adsorbed to algae, the contribution of the ternary complex to metal bioavailability was negligible in the case of Cd (II) and Cu (II). In contrast, the ternary complex contributed to over 90% of total cellular metal for Pb(II), due to the comparable affinity of Pb to algal sites in comparison with humic acid adsorbed to algae. Therefore, the extension of the biotic ligand model by including the formation of the ternary complex between the metal, humic acid, and algal surface would help to avoid underestimation of Pb biouptake in the presence of humic substances by green algae Chlorella kesslerii.     

Sorption and manganese-induced oxidative coupling of hydroxylated aromatic compounds by natural geosorbents

The sorption/desorption equilibria and solvent extractabilities of phenol, o-cresol, and p-chlorophenol with respect to natural sorbents having different types of soil organic matter were investigated. Parallel tests in systems amended with birnessite (delta-MnO2), a solid-phase oxidative coupling catalyst, were also conducted. Sorption/desorption isotherms and solvent extraction data reveal that the relative isotherm linearities, desorption hysteresis, and extractabilities of these compounds are related to the geochemical nature of the sorbent organic matter and to the existence of system conditions that promote oxidative coupling reactions. When suitable coupling catalysts are present, soils containing primarily diagenetically "young" and highly amorphous organic matter (e.g., humic materials) are more likely to retain those solutes than are those containing primarily diagenetically "old" and more condensed organic matter (e.g., kerogens). The sorption/desorption properties of the solutes were significantly altered in the presence of birnessite as a result of both cross-coupling reactions with reactive soil organic matter components and self-coupling reactions with each other to form polymeric species. Under appropriate conditions, mineral-catalyzed oxidative coupling may exert a dominant influence on the sorption and transport of hydroxylated aromatic compounds in soil and sediment systems.     

15N NMR investigation of the covalent binding of reduced TNT amines to soil humic acid,model compounds,and lignocellulose

The five major reductive degradation products of TNT-4ADNT (4-amino-2,6-dinitrotoluene), 2ADNT (2-amino-4,6-dinitrotoluene), 2,4DANT (2,4-diamino-6-nitrotoluene), 2,6DANT (2,6-diamino-4-nitrotoluene), and TAT (2,4,6-triaminotoluene)-labeled with 15N in the amine positions, were reacted with the IHSS soil humic acid and analyzed by 15N NMR spectrometry. In the absence of catalysts, all five amines underwent nucleophilic addition reactions with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and nonheterocyclic condensation products. Imine formation via 1,2-addition of the amines to quinone groups in the soil humic acid was significant with the diamines and TAT but not the monoamines. Horseradish peroxidase (HRP) catalyzed an increase in the incorporation of all five amines into the humic acid. In the case of the diamines and TAT, HRP also shifted the binding away from heterocyclic condensation product toward imine formation. A comparison of quantitative liquid phase with solid-state CP/MAS 15N NMR indicated that the CP experiment underestimated imine and heterocyclic nitrogens in humic acid, even with contact times optimal for observation of these nitrogens. Covalent binding of the mono- and diamines to 4-methylcatechol, the HRP catalyzed condensation of 4ADNT and 2,4DANT to coniferyl alcohol, and the binding of 2,4DANT to lignocellulose with and without birnessite were also examined.     

Effects of feeding pasteurized waste milk to dairy calves on phenotypes and genotypes of antimicrobial resistance in fecal Escherichia coli isolates before and after weaning

The aim of this study was to evaluate the effects of feeding pasteurized waste milk (pWM) to calves on antimicrobial resistance of fecal Escherichia coli at both phenotypic and genotypic levels. Fifty-two Holstein female calves (3 ± 1.3 d of age) were fed 1 of the 2 different types of milk: milk replacer (MR) without antimicrobials or pWM with β-lactam residues until weaning at 49 d of age. Fecal swabs of all calves were obtained on d 0, 35, and 56 of the study and 3 E. coli isolates per sample were studied. Phenotypic resistance was tested by the disk diffusion method against a panel of 12 antimicrobials. A total of 13 resistance genes consisting of β-lactam, sulfonamide, tetracycline, and aminoglycoside families were examined by PCR. Feeding pWM to calves increased the presence of phenotypic resistance to ampicillin, cephalotin, ceftiofur, and florfenicol in fecal E. coli compared with MR-fed calves. However, the presence of resistance to sulfonamides, tetracyclines, and aminoglycosides was common in dairy calves independent of their milk-feeding source, suggesting other factors apart from the feeding source are involved in the emergence of antimicrobial resistance.     

Occurrence and sorption behavior of sulfonamides, macrolides, and trimethoprim in activated sludge treatment

The occurrence of sulfonamide and macrolide antimicrobials, as well as trimethoprim, was investigated in conventional activated sludge treatment. Average daily loads in untreated wastewater correlated well with those estimated from annual consumption data and pharmacokinetic behavior. Considerable variations were found during a day, and seasonal differences seem to occur for the macrolides, probably caused by a higher consumption of these substances in winter. The most predominant macrolide and sulfonamide antimicrobials were clarithromycin and sulfamethoxazole, respectively. In the case of sulfamethoxazole, the main human metabolite, N4-acetylsulfamethoxazole, was included as an analyte, accounting for up to 86% of the total load in untreated wastewater. The results obtained illustrate the importance of considering retransformable substances, for example human metabolites, when investigating the behavior and fate of pharmaceuticals. Average concentrations of sulfapyridine, sulfamethoxazole, trimethoprim, azithromycin, and clarithromycin in activated sludge ranged between 28 and 68 microg/kg of dry weight. Overall the sorption to activated sludge was shown to be low for the investigated antimicrobials, with estimated sorption constants for activated sludge below 500 L/kg. Elimination in activated sludge treatment was found to be incomplete for all investigated compounds. In final effluents, the median concentrations for sulfamethoxazole and clarithromycin were 290 and 240 ng/L, respectively.     

Thin-layer Chromatographic Method for Quantification of Sulfonamides in Chicken Meat

The sulfonamides are a widely used group of antimicrobials in veterinary practice especially in developing countries due to their broad spectrum of activity, availability, and low cost. The objective of this study was to establish a thin-layer chromatographic (TLC) method to detect residues of commonly used sulfonamides in Sri Lanka in chicken. The TLC method separated sulfadiazine (SDZ), sulfadoxine (SD), sulfamethazine (SMZ), sulfathiazole (STZ), and sulfaquinoxaline (SQ) on silica gel plates using chloroform: n_butanol (90:10). Sulfonamides densitometric measurements were conducted by derivatising with fluorescamine and scanning the separated bands at 366 nm. The quantity of the sulfonamide in samples was calculated using a standard calibration curve obtained by plotting values of peak area against sulfonamide concentrations at 200, 150, 100, 50, 30, and 25 ng/g. The mean percentage recoveries were 80–120% while the intra-day and inter-day precision was 3–22% and 6–42% respectively. The SD had the highest limit of detection (LOD) and limit of quantification (LOQ) values of 60 and 100 ng/g respectively. The LOD of all the other analytes were 40 ng/g, and STZ had the lowest LOQ values of 50 ng/g. A reliable, inexpensive, simple, liquid-liquid extraction method for the rapid analysis of five sulfonamides at maximum residue limit (MRL) in chicken using TLC was developed.     

Reactions of a sulfonamide antimicrobial with model humic constituents: assessing pathways and stability of covalent bonding

The mechanism of covalent bond formation of the model sulfonamide sulfathiazole (STZ) and the stronger nucleophile para-ethoxyaniline was studied in reactions with model humic acid constituents (quinones and other carbonyl compounds) in the absence and presence of laccase. As revealed by high resolution mass spectrometry, the initial bonding of STZ occurred by 1,2- and 1,4-nucleophilic additions of the aromatic amino group to quinones resulting in imine and anilinoquinone formation, respectively. Experiments using the radical scavenger tert-butyl-alcohol provided the same products and similar formation rates as those without scavenger indicating that probably not radical coupling reactions were responsible for the initial covalent bond formation. No addition with nonquinone carbonyl compounds occurred within 76 days except for a slow 1,4-addition to the β-unsaturated carbonyl 1-penten-3-one. The stability of covalent bonds against desorption and pressurized liquid extraction (PLE) was assessed. The recovery rates showed no systematic differences in STZ extractability between the two product types. This suggests that the strength of bonding is not controlled by the initial type of bond, but by the extent of subsequent incorporation of the reaction product into the formed polymer. This incorporation was monitored for (15)N aniline by (1)H-(15)N HMBC NMR spectroscopy. The initial 1,2- and 1,4-addition bonds were replaced by stronger heterocyclic forms with increasing incubation time. These processes could also hold true for soils, and a slow nonextractable residue formation with time could be related to a slow increase of the amount of covalently bound sulfonamide and the strength of bonding.     

Role of bacterial biomass in the sorption of Ni by biomass-birnessite assemblages

Birnessites precipitated by bacteria are typically poorly crystalline Mn(IV) oxides enmeshed within biofilms to form complex biomass-birnessite assemblages. The strong sorption affinity of bacteriogenic birnessites for environmentally important trace metals is relatively well understood mechanistically, but the role of bacterial cells and extracellular polymeric substances appears to vary among trace metals. To assess the role of biomass definitively, comparison between metal sorption by biomass at high metal loadings in the presence and absence of birnessite is required. We investigated the biomass effect on Ni sorption through laboratory experiments utilizing the birnessite produced by the model bacterium, Pseudomonas putida. Surface excess measurements at pH 6-8 showed that birnessite significantly enhanced Ni sorption at high loadings (up to nearly 4-fold) relative to biomass alone. This apparent large difference in affinity for Ni between the organic and mineral components was confirmed by extended X-ray absorption fine structure spectroscopy, which revealed preferential Ni binding to birnessite cation vacancy sites. At pH ≥ 7, Ni sorption involved both adsorption and precipitation reactions. Our results thus support the view that the biofilm does not block reactive mineral surface sites; instead, the organic material contributes to metal sorption once high-affinity sites on the mineral are saturated.     

Effect of humic substance photoalteration on lead bioavailability to freshwater microalgae

The present study provides results on the influence of humic substance (HS) photoalteration on lead availability to the freshwater microalga Chlorella kesslerii . The evolution of the free lead-ion concentrations measured by the ion exchange technique [Pb](IET) and intracellular lead contents was explored in the presence of Suwannee River humic (SRHA) and fulvic (SRFA) acids, as well as Aldrich humic acid (AHA) exposed at increasing radiance doses under a solar simulator. Modifications of HS characteristics highly relevant to Pb complexation and accumulation of HS to algal surfaces, including Fourier transform infrared spectroscopy, were followed. It was demonstrated that simulated sunlight exposure of HS increased [Pb](IET) in the medium for SRFA and SRHA, but had no effect for AHA. No clear relationship was observed between the changes in free lead-ion concentrations and intracellular content in alga for all studied HS, suggesting that HS photodegradation products also exhibit Pb complexation properties, and that direct interactions between HS and alga are affected. Indeed, photoalteration of humic substances reduced the adsorption of HS to the algal surface; the effect was more pronounced for SRFA and AHA and less significant for SRHA. The bioavailability results were consistent with the characterization of the phototransformation of humic substances: Pb speciation changes followed the modification of the relative abundance of the carboxylic groups and their molecular environment, while the reduced HS adsorption to the alga correlated with losses of the double bond abundance and aromaticity.     

Role of humic acid and ouinone model compounds in bromate reduction by zerovalent iron

Experiments were conducted to examine the role of humic acid and quinone model compounds in bromate reduction by Fe(0). The reactivity of Fe(0) toward bromate declined by a factor of 1.3-2.0 in the presence of humic acid. Evidence was obtained that the quick complexation of humic acid with iron species and its adsorption passivated the iron surface and decreased the rate of bromate reduction by Fe(0). On the other hand, in the long run, the reduced functional groups present in humic acid were observed to regenerate Fe(II) and reduce bromate abiotically. Compared with the case of humic acid only, the simultaneous presence of Fe(II) and humic acid significantly increased the bromate removal rate. Fe(III)/Fe(II) acted as a catalyst in the oxidation of humic acid by bromate. Anthraquinone-2,6-disulfonate (AQDS) and lawsone did not cause any significant effect on the bromate reduction rate by Fe(0). However, the redox reactivity of lawsone in the presence of Fe(III) was evident, while AQDS did not show any under the tested conditions. The difference was attributable to the presence/ absence of reducing functional groups in the model compounds. The electron spin resonance further demonstrated that the redox functional groups in humic acid are most likely quinone-phenol moieties. Although the bromate reduction rate by regenerated Fe(II) is a few times slower than that by Fe(0), the reactive Fe(II) can be, alternatively, reductively formed to maintain iron surface activation and bromate reduction to prolong the lifetime of the zerovalent iron.     

增强型固相萃取净化-高效液相色谱-串联质谱法测定鲈鱼中18种磺胺类药物的残留量

建立了增强型脂质去除固相萃取净化高效液相色谱串联质谱法同时检测鲈鱼中18种磺胺类药物残留的分析方法。鱼肉样品经1%甲酸乙腈提取,增强型脂质去除固相萃取柱进行净化,用CNW Athena C₁₈-WP色谱柱（2.1 mm×100 mm,3 μm）进行分离,以甲醇和0.1%甲酸水溶液为流动相梯度洗脱,ESI正离子模式扫描,采用多反应监测模式测定,内标法定量。结果表明:18种磺胺类药物在5～200 ng/mL范围内具有良好的线性关系（r≥0.9962）,方法的检出限和定量限分别为0.11～0.47和0.37～1.57 μg/kg。在3个不同浓度添加水平下,样品回收率在73.30%～116.9%之间,相对标准偏差为0.29%～7.74%（n=6）。该方法操作简单,准确快速,灵敏度高,可用于鲈鱼中多种磺胺类药物残留的检测。     

Chlorination byproduct formation in the presence of humic acid, model nitrogenous organic compounds, ammonia, and bromide

The formation of trihalomethanes (THMs), haloacetic acids (HAAs), and cyanogen halides (CNXs) after chlorination of synthetic solutions containing humic acid, nitrogenous organic (N-organic) compounds, ammonia, and bromide ions was studied. Humic acid (from Aldrich) was used to provide the source of the precursors. Glycine was chosen as the primary model N-organic compound and other four model N-organic compounds (including glutamic acid, glycylglycine, diethylamine, and methylamine) were also evaluated for comparison. The formation of THMs and HAAs was found to decrease with increasing glycine and ammonia concentrations but to increase with increasing bromide ion concentration. CNX formation was found to be highly sensitive to free chlorine to glycine ratios, and its formation trends were significantly affected bythe presence/absence of ammonia. The incorporation of bromine changed the byproducts speciation toward brominated species and enhanced the yields of total THMs, HAAs, and CNXs. Different model N-organic compounds exerted different effects on the formation of THMs, HAAs, and CNXs. Their effects on the formation of THMs and HAAs were likely dependent on their reactivity to chlorine in competing with the humic acid chlorination reactions. The difference in the CNCI yields was attributable to the variations in the compound structures.     

Immobilizing humic acid in a sol-gel matrix: a new tool to study humic-contaminants sorption interactions

Humic substances originated from aquatic, soil, or sediment environments are mixtures of humic compounds with various characteristics. Sorption interactions with isolated, well defined humic fractions can be studied either in an aqueous phase ("dissolved humic substances"), or in a solid-phase, by coating mineral particles with the humic materials, or simply by working with humic acid particles (powder) at low pH to minimize dissolution. Each attitude, by definition, can be studied by different experimental techniques and has a different meaning for understanding natural environmental processes. In this study, a new tool for studying sorption interactions is presented. Sol-gel was used as an inert matrix to immobilize (entrap) various humic acids (HAs), and then used to study the interactions of several polycyclic aromatic hydrocarbons (PAHs) with the entrapped HA. Linear and nonlinear sorption coefficients were highly correlated with contaminant hydrophobicity. Sorption of pyrene to immobilized HA was in the order of soil HA > Aldrich HA approximately = peat HA. It was concluded that the entrapped HAs retained their original properties in the gel matrix and were accessible to the external contaminant through the pore network. Additionally, binding coefficients of pyreneto dissolved humic substances and to dissolved organic matter (DOM) were determined from the reduction in pyrene sorption to immobilized HA in the presence of dissolved humic material or DOM in solution. Binding coefficients of pyrene were in the order of the following: dissolved Aldrich HA > dissolved peat fulvic acid (FA) > DOM derived from mature compost > DOM derived from fresh compost.