Accuracy of a method based on atomic absorption spectrometry to determine inorganic arsenic in food: Outcome of the collaborative trial IMEP-41.

A collaborative trial was conducted to determine the performance characteristics of an analytical method for the quantification of inorganic arsenic (iAs) in food. The method is based on (i) solubilisation of the protein matrix with concentrated hydrochloric acid to denature proteins and allow the release of all arsenic species into solution, and (ii) subsequent extraction of the inorganic arsenic present in the acid medium using chloroform followed by back-extraction to acidic medium. The final detection and quantification is done by flow injection hydride generation atomic absorption spectrometry (FI-HG-AAS). The seven test items used in this exercise were reference materials covering a broad range of matrices: mussels, cabbage, seaweed (hijiki), fish protein, rice, wheat, mushrooms, with concentrations ranging from 0.074 to 7.55mgkg(-1). The relative standard deviation for repeatability (RSDr) ranged from 4.1 to 10.3%, while the relative standard deviation for reproducibility (RSDR) ranged from 6.1 to 22.8%.

Is it possible to agree on a value for inorganic arsenic in food? The outcome of IMEP-112.

Two of the core tasks of the European Union Reference Laboratory for Heavy Metals in Feed and Food (EU-RL-HM) are to provide advice to the Directorate General for Health and Consumers (DG SANCO) on scientific matters and to organise proficiency tests among appointed National Reference Laboratories. This article presents the results of the 12th proficiency test organised by the EU-RL-HM (IMEP-112) that focused on the determination of total and inorganic arsenic in wheat, vegetable food and algae. The test items used in this exercise were: wheat sampled in a field with a high concentration of arsenic in the soil, spinach (SRM 1570a from NIST) and an algae candidate reference material. Participation in this exercise was open to laboratories from all around the world to be able to judge the state of the art of the determination of total and, more in particular, inorganic arsenic in several food commodities. Seventy-four laboratories from 31 countries registered to the exercise; 30 of them were European National Reference Laboratories. The assigned values for IMEP-112 were provided by a group of seven laboratories expert in the field of arsenic speciation analysis in food. Laboratory results were rated with z and ζ scores (zeta scores) in accordance with ISO 13528. Around 85 % of the participants performed satisfactorily for inorganic arsenic in vegetable food and 60 % did for inorganic arsenic in wheat, but only 20 % of the laboratories taking part in the exercise were able to report satisfactory results in the algae test material.

In vitro cytotoxicity of fungi spoiling maize silage.

Penicillium roqueforti, Penicillium paneum, Monascus ruber, Alternaria tenuissima, Fusarium graminearum, Fusarium avenaceum, Byssochlamys nivea and Aspergillus fumigatus have previously been identified as major fungal contaminants of Danish maize silage. In the present study their metabolite production and in vitro cytotoxicity have been determined for fungal agar and silage extracts. All 8 fungal species significantly affected Caco-2 cell viability in the resazurin assay, with large variations for each species and growth medium. The 50% inhibition concentrations (IC(50)) of the major P. roqueforti metabolites roquefortine C (48 µg/mL), andrastin A (>50 µg/mL), mycophenolic acid (>100 µg/mL) and 1-hydroxyeremophil-7(11),9(10)-dien-8-one (>280 µg/mL) were high. Fractionating of agar extracts identified PR-toxin as an important cytotoxic P. roqueforti metabolite, also detectable in maize silage. The strongly cytotoxic B. nivea and P. paneum agar extracts contained patulin above the IC(50) of 0.6 µg/mL, however inoculated onto maize silage B. nivea and P. paneum did not produce patulin (>371 µg/kg). Still B. nivea infected maize silage containing mycophenolic acid (∼50 mg/kg), byssochlamic acid and other metabolites, was cytotoxic. In contrast hot-spots of P. roqueforti, P. paneum, M. ruber and A. fumigatus were not more cytotoxic than uninoculated silage.

Multi-mycotoxin analysis of maize silage by LC-MS/MS.

This paper describes a method for determination of 27 mycotoxins and other secondary metabolites in maize silage. The method focuses on analytes which are known to be produced by common maize and maize-silage contaminants. A simple pH-buffered sample extraction was developed on the basis of a very fast and simple method for analysis of multiple pesticide residues in food known as QuEChERS. The buffering effectively ensured a stable pH in samples of both well-ensiled maize (pH < 4) and of hot spots with fungal infection (pH > 7). No further clean-up was performed before analysis using liquid chromatography-tandem mass spectrometry. The method was successfully validated for determination of eight analytes qualitatively and 19 quantitatively. Matrix-matched calibration standards were used giving recoveries ranging from 37% to 201% with the majority between 60% and 115%. Repeatability (5-27% RSD(r)) and intra-laboratory reproducibility (7-35% RSD(IR)) was determined. The limit of detection (LOD) for the quantitatively validated analytes ranged from 1 to 739 microg kg(-1). Validation results for citrinin, fumonisin B(1) and fumonisin B(2) were unsatisfying. The method was applied to 20 selected silage samples and alternariol monomethyl ether, andrastin A, alternariol, citreoisocoumarin, deoxynivalenol, enniatin B, fumigaclavine A, gliotoxin, marcfortine A and B, mycophenolic acid, nivalenol, roquefortine A and C and zearalenone were detected.

Production of brominated tiacumicin derivatives.

Several novel tiacumicin derivatives containing bromine have been produced by the addition of inorganic bromine to the fermentation both of Dactylosporangium aurantiacum subsp, hamdenensis. Structures were elucidated employing mass spectrometry and NMR spectroscopy. Antibacterial activity of the bromotiacumicins is comparable to that of the parent compounds.

Antifungal drug targets: Candida secreted aspartyl protease and fungal wall beta-glucan synthesis.

The incidence of severe, life-threatening fungal infections has increased dramatically over the last decade. Unfortunately, in practice the arsenal of antifungal drugs is limited to flucytosine, a few approved azoles, and polyenes, mainly amphotericin B. This situation is rather precarious in view of the extended spectrum of fungi causing severe disease in immunocompromised patients, development of resistance to some of the currently used agents, and the minimal fungicidal activity of the azoles. Although lagging behind the need for new antifungal agents, the study of fungal biochemistry, physiology, and genetics has undergone a resurgence to new heights of activity, thus providing a framework on which to build drug discovery programs in several new areas, two of which will be discussed in detail: the biology of Candida albicans secreted aspartyl protease with respect to inhibitor discovery, evaluation, and possible clinical utility; and the fungal cell wall beta-glucans with respect to the mechanism and regulation of synthesis and target sites for drug inhibition.

Fusacandins A and B; novel antifungal antibiotics of the papulacandin class from Fusarium sambucinum. I. Identity of the producing organism, fermentation and biological activity.

The fuscandins, antifungal agents of the papulacandin class, are produced by a strain of Fusarium sambucinum. Fermentation yielded 60 mg/liter of fusacandin A and minor amounts of fusacandin B. As expected, the fusacandins inhibit (1,3)-beta-glucan synthesis. Fusacandin A is slightly less active than papulacandin B against Candida albicans and, like papulacandin, loses activity in the presence of serum.

Macquarimicins, microbial metabolites from Micromonospora. I. Discovery, taxonomy, fermentation and biological properties.

A novel series of microbial metabolites were discovered in fermentation broths of two soil isolates. Both cultures were identified as strains of Micromonospora chalcea. Production of the metabolites, named macquarimicins, was monitored by an HPLC assay. A seven-day fermentation yielded 27 mg/liter of macquarimicin A. With MICs of 50 to 100 micrograms/ml, macquarimicin A has only very low activity against strains of Bacteroides and other anaerobes. Macquarimicin B has inhibitory activity against the leukemia cell line P-388.

Aselacins, novel compounds that inhibit binding of endothelin to its receptor. II. Isolation and elucidation of structures.

Three novel compounds, named the aselacins, which inhibit the binding of endothelin to its receptor have been isolated from two related Acremonium species of fungi grown in stationary culture. These compounds are cyclic pentapeptolides with a ring formed by cyclo[Gly-D-Ser-D-Trp-beta-Ala-L-Thr] and an additional exocyclic D-Gln to which is attached a functionalized long chain fatty acid. The aselacins differ in the functionalization of this acid. The structures of the aselacins were determined by amino acid analysis, mass spectrometry and evaluation of 1-D and 2-D homonuclear and heteronuclear 1H, 13C and 15N NMR spectra in protic and aprotic solvents. The stereochemistry of the amino acids present was elucidated by chiral HPLC of hydrolyzed compound.

5-N-acetylardeemin, a novel heterocyclic compound which reverses multiple drug resistance in tumor cells. I. Taxonomy and fermentation of the producing organism and biological activity.

The ardeemins are a new family of secondary metabolites produced by submerged fermentation of a fungus which was isolated from a soil sample collected in Brazil. Based on taxonomic studies, the producing culture was identified as Aspergillus fischeri var. brasiliensis strain AB 1826M-35. 5-N-Acetylardeemin potentiated the cytotoxicity of the anticancer agent vinblastine in multidrug resistant human tumor cells.

Calbistrins, novel antifungal agents produced by Penicillium restrictum. II. Isolation and elucidation of structure.

The novel antifungal agents, calbistrins A, B, C and D have been isolated from a strain of Penicillium restrictum (AB 1875C-28). The four congeners were separated by bioactivity directed fractionation using countercurrent chromatography and preparative-HPLC. NMR studies revealed that the calbistrins each contain a carboxylic acid conjugated tetraene attached through an aliphatic ester linkage to a hexahydronaphthalene system.

A colorimetric microassay for the detection of agents that interact with DNA.

A simple microtiter assay for the detection of compounds that bind DNA is described. Agents that displace methyl green from DNA are detected spectrophotometrically by a decrease in absorbance at 630 nm. The feasibility of using the assay for detecting DNA-active compounds in fermentation extracts was assessed, and the activities of reference compounds in the methyl green assay and an ethidium bromide displacement method were compared.

Pacidamycins, a novel series of antibiotics with anti-Pseudomonas aeruginosa activity. III. Microbiologic profile.

Pacidamycins are nucleosidyl-peptide antibiotics which have activity only against Pseudomonas aeruginosa. Their MICs for other organisms such as Enterobacteriaceae, Staphylococcus aureus, most Streptococci and other Pseudomonas species are greater than 100 micrograms/ml. These compounds had no activity against erythromycin-susceptible Streptococci. The MICs for Streptococcus pyogenes with constitutive- and inducible-type of macrolide-lincosamide-streptogramin resistance were 12.5 and 25 micrograms/ml, respectively. The MICs against P. aeruginosa ranged from 8 to 64 micrograms/ml. The activity of these compounds was 1 to 2-fold less in serum than broth. Time-kill curves were performed using 4 and 8 times the MIC of pacidamycin 1. It was bactericidal against P. aeruginosa (3 log10 decrease in 4 to 6 hours). At 24 hours, resistant mutants were found in the cultures. The MICs of piperacillin and gentamicin for these mutants were the same as for the parent strain. The frequency of resistance to these compounds was less than 3.5 x 10(-6). The resistant mutants were stable after 10 transfers in antibiotic-free medium. The pacidamycins were inactive against P. aeruginosa in mouse protection test. After a single subcutaneous injection of 25 mg/kg of pacidamycin 1, the Cmax was approximately 50 micrograms/ml and the serum half-life was 0.5 hour.

Coloradocin, an antibiotic from a new Actinoplanes. II. Identity with luminamicin and elucidation of structure.

Coloradocin was isolated from a fermentation broth by adsorption onto Amberlite XAD-2. The activity was eluted in MeOH and purified by gel filtration on Shephadex LH-20, followed by liquid-liquid chromatography on diol-bonded silica gel. The last two steps in the purification of this antibiotic included reverse-phase chromatography on C18-bonded silica gel and countercurrent chromatography on an Ito Coil Planet Centrifuge to give material of 90% purity. Analytically pure material was obtained by preparative HPLC. As a result of extensive homo and heteronuclear two-dimensional NMR studies, a structure was proposed for coloradocin. The structure consists of a decalin ring system fused to a 10-membered macrolactone which in turn is fused to a 14-membered macrolactone possessing an enol ether in conjugation with an unsaturated cyclic anhydride functionality. Coloradocin is related to a small class of antibiotics which include nodusmicin and nargenicin and was shown to be identical to luminamicin for which no structure has been reported.

Preparative low-pressure chromatography of antibiotics on a column of diol-bonded silica gel.

Several schemes are presented which illustrate the general utility of column chromatography on diol-bonded silica gel for the purification of various antibiotics. The antibiotics include rosarimicin, rosarimicin dimethylacetal, everninomicin D, Juvenimicin A4, coloradocin and the benzanthrins. A set of partition coefficients, determined in different two-phase solvent systems, for a given antibiotic or antibiotic complex, can be used in selecting appropriate solvent systems for this chromatography as well as for semi-preparative counter-current chromatography on the Ito coil planet centrifuge.

Benzanthrins A and B, a new class of quinone antibiotics. I. Discovery, fermentation and antibacterial activity.

Nocardia lurida has been shown to produce two novel quinone antibiotics, benzanthrins A and B. The antibiotics were discovered in concentrated butanol extracts of fermentation broths and were separated by TLC and HPLC. Benzanthrins A and B were produced in a fermentation medium consisting of glucose, yeast, selected peptones and CaCO3. The antibiotics were present primarily at 66 hours in shake flask fermentations and from 66 to 162 hours in 14-liter fermentors. Benzanthrins A and B inhibited a number of Gram-positive pathogenic bacteria but were inactive against Gram-negative bacteria.

Benzanthrins A and B, a new class of quinone antibiotics. II. Isolation, elucidation of structure and potential antitumor activity.

The benzanthrins, which were produced by Nocardia lurida, were extracted from the fermentation broth with CH2Cl2. Subsequent purification on Sephadex LH-20 and diolbonded silica gel, followed by countercurrent chromatography, afforded analytically pure benzanthrins A and B. FAB-MS revealed that benzanthrins A and B were isomeric. It was demonstrated through degradative and spectroscopic studies that the benzanthrins were di-glycosides of a trihydroxy benz[a]anthraquinone chromophore where one of the sugars was linked through carbon and the other through oxygen. Benzanthrins A and B differed in the stereochemistry of the O-glycosidic sugar. Both compounds inhibited the growth of Gram-positive bacteria and 9KB, 9PS and 9ASK tumor cells in tissue culture.

Receptor recognition of maleyl-albumin induces chemotaxis in human monocytes.

We demonstrate here that the exceptionally active maleyl-albumin receptor of human monocytes functions in vitro as a chemoattractant receptor. Chemotaxis of human monocytes occurs at an effective median dose of 3-4 microM maleyl-albumin, a concentration representing 1% of the total albumin in the adult human. Computerized analyses by LIGAND of the saturable binding of maleyl-albumin to human monocytes reveal two classes of binding sites, described by dissociation constants of 37 nM and 5.3 microM with maximal binding of 1.6 and 23 pmol maleyl-albumin/mg cellular protein, respectively. Chemotaxis of human monocytes thus occurs at concentrations of maleyl-albumin promoting binding to the lower-affinity sites. We propose that conformational isomers of albumin that are chemotactic may form in vivo and that albumin, in addition to receptor-independent plasma transport functions, may also play an important role in the receptor-mediated recruitment and accumulation of phagocytic cells at sites of inflammation and injury.

Two distinct receptors account for recognition of maleyl-albumin in human monocytes during differentiation in vitro.

A comparison of the receptor-mediated interaction of malondialdehyde-low density lipoprotein and maleyl-albumin has been examined in human monocytes during differentiation in vitro. The recognition of both ligands by the scavenger receptor of these cells has been confirmed. We now report that human monocytes express a second cellular surface receptor for maleyl-albumin that is distinct from the scavenger receptor. The activity of the maleyl-albumin receptor, determined by both binding and lysosomal hydrolytic assays, substantially exceeds that of the scavenger receptor in freshly isolated monocytes. A dramatic and rapid decline in the activity of the maleyl-albumin receptor occurs within 72 to 96 h during differentiation in vitro. At day 7, while only 5-10% of the original activity of the maleyl-albumin receptor remains, it is similar to that of the maximally expressed scavenger receptor. Both the binding and hydrolysis of ligand mediated by the maleyl-albumin receptor are specifically inhibited by alpha-casein and alkaline-treated albumin; neither of these proteins is recognized by the scavenger receptor. The occurrence of the exceptionally active maleyl-albumin receptor on freshly isolated human monocytes suggests that it participates in processes necessary to the function of the cells that diminish in importance after differentiation of the monocytes into macrophages in vitro. Furthermore, while maleyl-albumin is a useful adjunct to studies of cellular events mediated by the scavenger receptor, the presence of a second receptor for maleyl-albumin must be taken into account as a potential contributing and complicating event.