In vitro comparison of antimicrobial activity of iodine and silver dressings against biofilms.

OBJECTIVE: To compare the antimicrobial effectiveness of silver- and iodine-containing wound dressings against preformed mature biofilms of pathogenic wound bacteria grown in vitro. METHOD: Biofilms of Pseudomonas aeruginosa and Staphylococcus aureus were grown within an in vitro flat bed perfusion biofilm model. Mature biofilms were removed and exposed to wound dressings containing either silver or iodine (Aquacel Ag and Iodozyme) within a static diffusion method, for up to 24 hours. This method was designed to reflect certain key features that determine antimicrobial activity within the wound. The numbers of viable bacteria surviving in the biofilms were determined at set time intervals over the test period. RESULTS: Both test dressings exerted an antimicrobial effect against the target species biofilms, although the iodine dressing was more efficacious under the experimental conditions employed. CONCLUSION: There are large and potentially significant differences (as measured in vitro) in the effectiveness of wound dressings containing broad-spectrum antimicrobial agents such as silver and iodine against specific types of bacterial biofilms.

Investigations by HPLC-electrospray mass spectrometry and NMR spectroscopy into the isomerisation of salinomycin.

HPLC-MS studies have indicated that certain polyether ionophore veterinary drugs are prone to degradation when stored as water-methanol solutions at ambient temperature. Salinomycin and narasin were particularly susceptible, disappearing completely within weeks to produce more polar species, which were identified as isomers of the original compounds. Lasalocid appeared to be stable under such conditions. Structural elucidation of the principal ultimate salinomycin isomerisation product was achieved by 2D NMR spectroscopy. This indicated that the isomerisation process consists of the opening of the spiro rings in the salinomycin structure with the concomitant formation of a furan moiety. The MS data indicated that the isomers retain the ability to complex alkali metal ions and may therefore retain their pharmacological activity. These discoveries may have implications both for the development of legislation covering acceptable levels of polyether ionophore residues in foodstuffs and also for analytical protocols designed to detect them.

Temperature conditioning of nasal air: effects of vasoactive agents and involvement of nitric oxide.

Nitric oxide (NO) is released into nasal air, but its function is unknown. We hypothesized that nasal vascular tone and/or flow influences temperature conditioning of nasal air and that NO participates in this process. We measured nasal air temperature (via a thermocouple) and exhaled nasal NO release (by chemiluminescence) in five humans and examined the effects of an aerosolized vasoconstrictor (oxymetazoline), a vasodilator (papaverine), N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase, or saline (control). Compared with saline (which caused no changes in nasal air temperature or exhaled NO release), oxymetazoline (0.05%) reduced nasal air temperature and NO release (130.8 +/- 15.1 to 81.3 +/- 12.8 nl. min(-1). m(-2); P < 0.01). Papaverine (0.01 M) increased nasal air temperature and NO release (131.8 +/- 13.1 to 157.2 +/- 17.4 nl. min(-1). m(-2); P < 0.03). N(G)-nitro-L-arginine methyl ester reduced nasal air temperature and NO release (123.7 +/- 14.2 to 44.2 +/- 23.7 nl. min(-1). m(-2); P < 0.01). The results suggest that vascular tone and/or flow modulates temperature conditioning and that NO may participate in that function.

Analysis of the Maillard reaction products of beta-lactoglobulin and lactose in skimmed milk powder by capillary electrophoresis and electrospray mass spectrometry.

When analysed by capillary electrophoresis, certain skimmed milk powders are seen to exhibit additional peaks migrating after the whey protein beta-lactoglobulin. Using a model reaction between beta-lactoglobulin and lactose, and studying the reaction products using electrospray mass spectrometry, it is demonstrated that these protein peaks are almost certainly due to a Maillard reaction between lactose and the epsilon-amino group of lysine. This results in the formation of a series of lactulose-protein conjugates exhibiting throughout molecular mass increments of 324, which is sufficient to allow their separation by capillary electrophoresis.

The characterisation of polyether ionophore veterinary drugs by HPLC-electrospray MS.

We undertake the determination of a wide range of veterinary drug residues in a range of animal products. Various screening analyses are employed, followed by HPLC-API (atmospheric pressure ionisation)-MS for the unequivocal confirmation of significant positives. EU legislation for the use of GC-MS as a confirmatory technique requires the successful monitoring of at least four diagnostic ions and although no such requirement exists for HPLC-MS confirmation, a similar requirement would seem appropriate. Until recently, reports describing the electrospray MS confirmation of residues of the polyether ionophores have been based on monitoring one or two ions. We have found that the addition of ammonium acetate to the HPLC mobile phase, in conjunction with 'cone voltage' or 'skimmer' assisted fragmentation, is a convenient way of producing additional diagnostic ions from polyether ionophore compounds, without compromising the overall sensitivity. Results for lasalocid, the most widely used compound, are presented. Electrospray MS data and acquisition parameters for lasalocid, monensin, narasin and salinomycin are described. The advantage of this analytical approach is that it may be used to generate confirmatory data using a single quadrupole MS system, without the need for advanced MS instrumentation, e.g., MS-MS.

Metabolism of di-, tri- and tetrabromobiphenyls by hepatic microsomes isolated from control animals and animals treated with Aroclor 1254, a commercial mixture of polychlorinated biphenyls (PCBs).

1. The metabolism of di-, tri- and tetrabromobiphenyls (PBBs) by hepatic microsomes isolated from control animals and animals treated with Arochlor 1254 was studied. 2. Hepatic microsomes isolated from control rats expressed higher rates of oxidations than avians. 3. Treatment of rats and pigeons with Arochlor 1254 induced cytochrome P450 dependent monooxygenases leading to an increased regioselective metabolism of PBB isomer and congeneres. 4. There was an inverse relationship between the degree of halosubstitution and microsomal oxidation. Meta-para carbon atoms free of halosubstitution were the preferred side for oxidation.

Correlations between the molecular structures of polyhalogenated biphenyls and their metabolism by hepatic microsomal monooxygenases.

1. Collation of the data presented in the preceding papers (references 4,5) showed a significant correlation (r = 0.83; P < 0.001) between the molecular mass (and hence the extent of halosubstitution) of halogenated biphenyls and their rate of hydroxylation by hepatic microsomal monooxygenases. 2. There was no relationship between the extent of polyortho halosubstitution of biphenyl and the rate of metabolism. 3. A marginal correlation (r = 0.33; P < 0.001) was found when the number of adjacent unsubstituted meta-para positions were linked to the rate of metabolism of PCBs. This structural feature facilitates microsomal oxidation. 4. The results support the proposal that PCBs with meta-para hydrogen atoms are less enriched in tissues of animals and humans as this structural feature favours their metabolism by P450 isoenzymes.

Microsomal oxidation of bromo-, chloro- and fluorobiphenyls.

1. The metabolism of 2-, 3-, 4-bromo-, 2-, 4-chloro-, and 2-fluorobiphenyl by hepatic microsomes isolated from control and Aroclor 1254-treated rats and pigeons was studied. 2. Meta and para as well as dihydroxylated metabolites were detected, but para hydroxylation was the preferred route of metabolism with all of the substrates used. 3. The overall rates of hydroxylation were greater with hepatic microsomes from rats than from pigeons. 4. Treatment with Aroclor 1254, a potent inducer of hepatic monooxygenases, resulted in increased rates of metabolism and in the enhanced formation of diol metabolites. Metabolism of halobiphenyls by induced P450 isoenzymes altered the regioselective hydroxylation pathways. 5. Ortho- and meta halosubstituted biphenyls were less rapidly metabolised when compared with para substituted isomers.

Metabolism of di-, tri-, tetra-, penta- and hexachlorobiphenyls by hepatic microsomes isolated from control animals and animals treated with Aroclor 1254, a commercial mixture of polychlorinated biphenyls (PCBs).

1. The metabolism of a wide range of di-, tri-, tetra-, penta- and hexachlorobiphenyls by hepatic microsomes isolated from control animals and animals treated with Aroclor 1254 was studied. 2. Hepatic microsomes isolated from control rats expressed higher rates of oxidations than avians. 3. Treatment of rats and pigeons with Aroclor 1254 induced cytochrome P450 dependent monooxygenases leading to an increased regioselective metabolism of PCB isomer and congeneres. 4. There was an inverse relationship between the degree of halosubstitution and microsomal oxidation. Meta-para carbon atoms free of halosubstitution were the preferred side for oxidation. 5. A good correlation was found between the in vitro metabolism of PCBs and their relative abundance in tissue extracts, thus suggesting oxidative metabolism to be the major route of metabolic disposal.

Distribution and elimination in vivo of polychlorinated biphenyl (PCB) isomers and congeners in the pigeon.

1. Pigeons were injected with a single dose of commercial PCB mixtures (Aroclor 1248 plus Aroclor 1260), killed 120 h later and the abundance of individual PCBs was determined in adipose tissue, gonads, liver, brain, kidney, heart, muscle and blood. 2. Elimination factors for individual PCBs were calculated. Values of greater than 1 were obtained for PCBs with meta-para-unsubstituted carbon atoms in at least one ring, indicating that elimination exceeded accumulation in all or most tissues. By contrast, ortho-meta unsubstituted PCBs had elimination factors less than 1, thus indicating their impaired removal. 3. Tissues with high microsomal monooxygenase activity had the highest elimination factors for individual PCBs (i.e. liver greater than kidney greater than muscle greater than heart). 4. Distribution of individual PCBs was independent of sex and of ortho-chlorine substitution and showed that 90% of total PCBs in cadavers was present in adipose tissue, 2% in kidneys, 1% each in brain, muscle and heart and less than 0.1% in blood. 5. The distribution of the highly toxic non-ortho and mono-ortho substituted PCBs did not differ amongst all tissues analysed. 6. The present studies indicate that elimination of PCBs in vivo is favoured by the molecular feature of unsubstituted meta-para carbon atoms in the biphenyl moiety.

Metabolism of [14C]4-chlorobiphenyl by hepatic microsomes isolated from razorbills, pigeons and rats.

1. Analysis of individual PCB-isomers and congeners in extracts of adipose tissue from N = 12 razorbills suggested that 4-chlorobiphenyl was subjected to metabolism. 2. In vitro metabolism studies using [14C]-4-chlorobiphenyl as substrate showed that razorbills metabolise this substrate to [14C]4-chloro-4'-hydroxybiphenyl at an average rate of 20 pmol/mg microsomal protein/min. For comparison, the metabolism of [14C]-4-chlorobiphenyl by pigeons and rats was also studied, and average rates in the formation of [14C]-4-chloro-4'-hydroxybiphenyl of 12 pmol/mg microsomal protein min and 342 pmol/mg microsomal protein min were estimated. 3. A comparison of the hepatic drug metabolising enzyme system of razorbills and pigeons showed similar concentrations of cytochrome P-450, cytochrome b5 and comparable catalytic activities of cytochrome P-450-dependent monooxygenase, when assessed for HHDN epoxidase, PROD, EROD and the Phase II enzymes glutathiones-S-transferase, but were significantly lower, when compared with rats. The results obtained suggest fundamental differences in the catalytic activities of cytochrome P-450-dependent monooxygenase between avian and mammalian species. 4. The present study, however, provides evidence that fish-eating seabirds have the ability to metabolically dispose of certain PCB isomers and congeners, which are amongst the most ubiquitously distributed pollutants in the ecosystem.

In vitro metabolism of [14C]4-chlorobiphenyl and [14C]2,2',5,5'-tetrachlorobiphenyl by hepatic microsomes from rats and pigeons. Evidence against an obligatory arene oxide in aromatic hydroxylation reactions.

1. The catalytic activities of cytochromes P-450IA1 and P-450IIB1 in control and Aroclor 1254 treated rats and pigeons (1 mmol/kg) were assessed using [14C]4-chloro- and [14C]2,2',5,5'-tetrachlorobiphenyl as substrates. Treatment of rats resulted in increases of the total amount of chloroform-extractable metabolites of [14C]4-chlorobiphenyl from 37.2 (control) to 199.4 and 221.6 nmol/hr per mg microsomal protein at 48 and 120 hr post treatment. The portion of [14C]4-chloro-3',4'-dihydroxybiphenyl (M4) and of a second unidentified dihydroxylated metabolite (M3) increased during these incubations from 13.7% for controls to 53.5% at 48 hr and 69.12% at 120 hr post treatment. 2. [14C]4-chloro-3'-hydroxybiphenyl (M1) and [14C]4-chloro-4'-hydroxybiphenyl (M2) were the major metabolites formed by pigeon hepatic microsomes; however, the amounts formed were 38.7- and 29.3-fold less, respectively, than in untreated rats. Treatment of pigeons with Aroclor 1254 increased the metabolite formation from 1.0 (control) to 13.6 and 22.4 nmol/hr per mg microsomal protein at 48 hr and 120 hr post treatment respectively; however, only small amounts of metabolites M3 (0.5 nmol/hr per mg protein) and M4 (2.0 nmol/hr per mg protein) were detected. 3. Treatment of rats with Aroclor 1254 resulted in an approximately two-fold increase in the rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl, and the ratio of 3- to 4-hydroxylation increased from 0.45 (control) to 0.6 and 0.8 at 48 hr and 120 hr post treatment respectively. The rate of metabolism of [14C]2,2',5,5'-tetrachlorobiphenyl by control and Aroclor 1254 treated pigeons was up to 23-fold lower than in rats and there was no evidence for the formation of the diol metabolite M3. However, as with rats, the ratio of meta- to para-carbon atom hydroxylation increased from 0.58 (controls) to 0.72 at 120 hr post treatment. 4. From the evidence presented, it is suggested that cytochromes P-450IA1 and P-450IIB1 may not metabolize PCB-congeneric substrates via an obligatory arene oxide intermediate.

Effects of molecular substitution patterns on the cytochrome P-450-dependent metabolism of 2,2',3,5,5',6- and 2,2',3,4,4',6-hexachlorobiphenyl by rat liver microsomal monooxygenases.

The metabolism by rat hepatic microsomal cytochrome P-450-dependent monooxygenases of several model substrates that are specific for individual isoforms of cytochrome P-450 and the metabolism by these monooxygenases of two structurally related isomers of hexachlorobiphenyl was studied. The most striking result was that 2,2',3,5,5',6-hexachlorobiphenyl was metabolised in vitro at the rate of 4.5 pmol/mg microsomal protein per min, whereas the other isomer 2,2',3,4,4',6-hexachlorobiphenyl was not metabolised at detectable rates. This finding provides strong evidence for a regioselective oxidative attack by cytochrome P-450-dependent monooxygenase with preferential insertion of oxygen at meta-para unsubstituted carbon atoms. Investigations into the mechanism of this oxidative attack suggest that the ortho hydrogen atom at carbon atom C-6' of 2,2',3,4,4',6-hexachlorobiphenyl was associated with a lower charge (0.075 e) compared with the meta or para hydrogen atoms at carbon atom C-3' and C-4' of 2,2',3,5,5',6-hexachlorobiphenyl (0.086 e). In addition, measurement of the main C-C bond length using MOPAC calculations and X-ray crystalographic data suggests significant differences in the bond-length distance, with the main bond lengths of 1.390, 1.385 and 1.374 A, respectively, for bridgehead to ortho (C1-C2), for ortho to meta (C2-C3), and for meta to para bonds. These results provide evidence that the preferential meta-para oxidative attack is linked to a shorter carbon-carbon bond length and a more positive charge distribution of the corresponding hydrogen atoms.

Transport and cellular uptake of polychlorinated biphenyls (PCBs)--I. Association of individual PCB isomers and congeners with plasma lipoproteins and proteins in the pigeon.

Polychlorinated biphenyls (PCBs) are abundant and persistent pollutants in the ecosystem. Commercial mixtures (e.g. Aroclor 1254) can contain up to 80 different isomers and congeners, many of which accumulate in biological systems by the ingestion of PCB-contaminated lipid components of food chains. PCBs are lipophilic and lipid-rich lipoproteins provide an excellent system to transport PCBs to tissues. We report here the distribution of PCBs between plasma fractions in the pigeon. Twenty-four hours after injection, [14C]4-monochlorobiphenyl and [14C]2,2',5,5'-tetrachlorobiphenyl were associated with the protein-rich HDL fraction and the lipoprotein-poor fraction (predominantly albumin), rather than with the lipid-rich VLDL and LDL fractions. Five days after injection with the commercial PCB mixture Aroclor 1254, there was a distinctive distribution between the plasma fractions of the 41 congeners detected. Avian species have a poorly developed lymphatic system and dietary lipids are secreted into the portal vein. To emphasize this route of entry, the lipoprotein particles formed are termed portomicrons rather than chylomicrons. The most striking result was that the lipid-rich portomicron and the VLDL fraction was associated almost exclusively with only one congener (2,2',4,4'5,5'-hexachlorobiphenyl), whereas the other isomers and congeners were distributed amongst the LDL, HDL and the lipoprotein-poor (predominantly albumin) fractions. Thirteen of the congeners detected accounted for 74, 53 and 54%, respectively, of the total amount of PCBs in the LDL, HDL and lipoprotein-poor protein fractions. Five congeners that are highly toxic were enriched in the latter fraction. The distribution of PCBs is more complex than can be explained solely by their solubility in the lipid components of plasma fractions, and may suggest a complex association with apolipoproteins and plasma proteins that are important in transporting PCB to tissues. The identification of individual PCBs in lipoprotein fraction provides evidence for their role in the transport of lipophilic xenobiotics in blood and it is suggested that PCBs associated with lipoproteins are taken up by cells as lipoprotein-PCB complexes.

Evidence for the induction of cytochrome P-452 in rat liver by Aroclor 1254, a commercial mixture of polychlorinated biphenyls.

We have examined the ability of a commercial mixture of polychlorinated biphenyls (Aroclor 1254) to induce hepatic cytochrome P-452-linked enzyme activities in rat and pigeon liver five days after its intraperitoneal injection. The results provide evidence that, at the doses used, Aroclor 1254 induces cytochrome P-452-linked enzyme activities in rats, but not in pigeons. This inductive effect was previously regarded as being specific for hypolipidemic drugs and phthalate ester plasticisers.

Saw-toothed grain beetleOryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) : Collection, identification, and bioassay of attractive volatiles from beetles and oats.

Over 200 beetle- and food-produced volatiles were collected from cultures of the saw-toothed grain beetleOryzaephilus surinamensis (L.) on oats. It proved possible to develop the electroantennogram recording technique for these beetles, despite their small size, allowing volatiles causing antennal responses to be identified by coupled GC-EAG and subsequent GCMS techniques. Three beetle-produced macrolide lactones were identified as (Z,Z)-3,6-dodecadien-11-olide, (Z,Z)-3,6-dodecadienolide, and (Z,Z)-5,8-tetradecadien-13-olide in an average ratio of 4.4∶1∶2. These have been reported as components of the aggregation pheromone from a different population of this species, although the ratio of the components produced was different. Three food volatiles with EAG activity were also identified: 1-octen-3-ol, 3-octanone, and nonanal. A mixture of the six identified volatiles produced similar levels of attraction, in a behavioral assay, to the entire mixture of collected volatiles.

Attractancy toOryzaephilus surinamensis (L.) of volatile materials isolated from vacuum distillate of heat-treated carobs.

Vacuum distillation of heat-treated carobs gave an aqueous, colorless, sweet-smelling distillate which was tested over a wide range of concentrations and found to be highly attractive to adultOryzaephilus surinamensis (L.). The materials responsible for the aroma were isolated from the distillate by saturating with sodium chloride and extracting into diethyl ether as separate acidic, neutral, and basic fractions. The extraction efficiency was checked by recombining portions of the three fractions and replacing the diethyl ether with water to give a "reconstituted distillate;" this was almost as attractive as the original distillate. Bioassay of aqueous solutions of the three separate fractions showed that the acidic was attractive, while the neutral and basic had little effect. The five major components of the acidic fraction were found to be acetic, isobutyric,n-butyric, 2-methylbutyric, and hexanoic (caproic) acids. Bioassay of these in aqueous solution, both separately and combined, showed that hexanoic acid was the most attractive and may be responsible for both the longer-lasting attractive effect of the carob distillate and for the effectiveness of carobs themselves used in bait bags to detect stored product insects.