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Reduction in MRSA environmental contamination with a portable HEPA-filtration unit.

There is renewed interest in the hospital environment as a potentially important factor for cross-infection with methicillin-resistant Staphylococcus aureus (MRSA) and other nosocomial pathogens. The aim of this study was to evaluate the effectiveness of a portable high-efficiency particulate air (HEPA)-filtration unit (IQAir Cleanroom H13, Incen AG, Goldach, Switzerland) at reducing MRSA environmental surface contamination within a clinical setting. The MRSA contamination rate on horizontal surfaces was assessed with agar settle plates in ward side-rooms of three patients who were heavy MRSA dispersers. Contamination rates were measured at different air filtration rates (60-235 m(3)/h) and compared with no air filtration using Poisson regression. Without air filtration, between 80% and 100% of settle plates were positive for MRSA, with the mean number of MRSA colony-forming units (cfu)/10-h exposure/plate ranging from 4.1 to 27.7. Air filtration at a rate of 140 m(3)/h (one patient) and 235 m(3)/h (two patients), resulted in a highly significant decrease in contamination rates compared with no air filtration (adjusted rate ratios 0.037, 0.099 and 0.248, respectively; P < 0.001 for each). A strong association was demonstrated between the rate of air filtration and the mean number of MRSA cfu/10-h exposure/plate (P for trend < 0.001). In conclusion, this portable HEPA-filtration unit can significantly reduce MRSA environmental contamination within patient isolation rooms, and this may prove to be a useful addition to existing MRSA infection control measures.

Adult↗

Reduced fungal contamination of the indoor environment with the Plasmair system (Airinspace).

Aspergillus spp. and other moulds cause life-threatening opportunistic infections in immunocompromised patients. Indoor contamination and construction work that liberate fungal spores are a major source of nosocomial aspergillosis. Dijon hospital is a tertiary care institution in northeast France undergoing construction work beside high-risk clinical units. To determine the impact of this activity, a surveillance programme was implemented one year before building work began in order to establish baseline levels of contamination. Air and surface fungal contamination in adult and paediatric haematology units were prospectively examined following use, or not, of a new air-treatment system with mobile Plasmair units (Airinspace). There were significant reductions in overall fungal contamination for the Plasmair treated rooms for air and surface samples in both clinical units. Plasmair treatment also significantly reduced A. fumigatus in the air. These data suggest that Plasmair units may provide an efficient method of reducing indoor fungal contamination in hospitals.

Air Microbiology↗

Seasonal and annual loads of hydrophobic organic contaminants from the Susquehanna River basin to the Chesapeake Bay.

Water from the Susquehanna River was collected and analyzed for polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyl (PCB) congeners to estimate seasonal and annual riverine loads to the Chesapeake Bay. Temporal variations in the chemical loads resulted from the large changes in the water flow rates and in the particle-associated contaminant concentrations. Concentrations of PCBs and PAHs in river particles (ng/g) were twice as great as those in the northern Chesapeake Bay, indicating that the Susquehanna River is an important source of these contaminants to the bay. The river carries a majority of its hydrophobic organic contaminants (HOCs) in the particulate phase. During periods of high flow, large amounts of suspended particles in the river result in elevated HOC levels and increased loadings of these contaminants to the bay. From 1997 to 1998, 60% of the total annual HOC loading occurred in the early spring coincident with high river flows. The total PCB and PAH annual loadings from the Susquehanna River to the Chesapeake Bay were 76 and 3160 kg/year, respectively and 75% of the loaded organic contaminants were in the particulate phase. Principal component analysis of PAH and PCB congener patterns in the particles reveals that the river suspended particles were dominated by autochthonous production in the summer and by resuspended sediment and watershed erosion during the winter and early spring.

Chromatography, Gas↗

Effects of hydrocarbon contamination on a free living marine nematode community: results from microcosm experiments.

Anthropogenic inputs of crude and refined petroleum hydrocarbons into the sea require knowledge of the effects of these contaminants on the receiving assemblages of organisms. A microcosm experiment was carried out to study the influence of diesel on a free living nematode community of a Tunisian lagoon. Sediments were contaminated by diesel that ranged in concentration from 0.5 to 20 mg diesel kg(-1) dry weight (dw), and effects were examined after 90 days. Gradual changes in community structure were revealed depending on the quantity of diesel administrated. In the medium (1 mg diesel kg(-1) and 5 mg diesel kg(-1) (dw)) and high (10 mg diesel kg(-1), 15 mg diesel kg(-1) and 20 mg kg(-1) (dw)) treated microcosms, most univariate measures, including diversity and species richness, decreased significantly with increasing level of diesel contamination whereas nematode assemblage from the low treated microcosm (0.5 mg diesel kg(-1) (dw)) remained unaffected. Results from multivariate analyses of the species abundance data demonstrated that responses of nematode species to the diesel treatments were varied: Chaetonema sp. was eliminated at all doses tested and seemed to be intolerant species to diesel contamination; Pomponema sp. and Oncholaimus campylocercoïdes were significantly affected at all diesel contamination levels but they were not eliminated, these species were categorized as "diesel-sensitive"; Hypodontolaimus colesi, Daptonema trabeculosum and Daptonema fallax which significantly increased respectively at 0.5, 1 and 5 mg diesel kg(-1) (dw) concentrations and appeared to be "opportunistic" species at these doses whereas Marylynnia stekhoveni which increased at all high doses (10, 15 and 20 mg diesel kg(-1) (dw)) seemed to be a "diesel-resistant" species.

Animals↗

Increased phagocytic activity in contaminated seastars (Asterias rubens) collected in the Southern Bight of the North Sea.

In invertebrates, the phagocytosis exerted by immune cells constitutes the main line of internal defence against offending microorganisms. This study assessed during two consecutive years the phagocytic activity in seastars (Asterias rubens) collected in stations along the Belgian and Dutch coasts. The contamination of these seastars by metals and PCBs were measured in parallel. Increased phagocytic activities were found in seastars collected in the plume of the Scheldt river. This correlated with the contamination of seastars by metals, mainly Pb, but not with the contamination by the analysed PCB congeners. Furthermore, the relationship between phagocytosis and metal contamination was reproducible from one year to another. The possible mechanisms explaining this effect are discussed in light of a direct or indirect link between phagocytic activity and metal contamination of seastars.

Analysis of Variance↗

Studies of a contaminated brackish marsh in the Hackensack Meadowlands of northeastern New Jersey: an assessment of natural recovery.

Eight-Day Swamp is known to be contaminated with heavy metals, especially mercury. Sediment cores were collected to approximately 32 cm at 17 sites on four transects and analyzed at 1 or 2 cm intervals for seven metals and organic matter. Very high metal levels were found throughout the site. Long and Morgan's "effects range-median" (ER-M) was exceeded in more than 50% of slices for all elements except As. Hg had the highest concentration relative to ER-M; median Hg concentration was 72 times its ER-M. On the marsh plain, all metals showed enrichment at 14-20 cm depth of 10-40X over surficial sediments. 137Cs analysis showed sedimentation rates ranging from 0.33 to 0.50 cm yr(-1) over the last approximately 40 yr. These rates indicate that metal contamination peaks occurred in sediments deposited in the early 1960s. Thus, newer, less contaminated sediments are burying older, more contaminated layers and peak levels of contaminants are becoming less available to benthos.

Cesium Radioisotopes↗

Contamination by metallic elements released from joint prostheses.

When a metallic implant is in contact with human tissues, the organism reacts and a corrosion process starts. Consequently, we might observe liberation of metallic debris and wear. Our purpose is to measure the contamination and the migration of these metallic elements in the surrounding tissues of the implant. Two types of samples have been studied. First type is sample taken on post-mortem tissues around prostheses to study contamination gradients. Second type is sample taken on pathologic joints on periprosthetic capsular tissues in surgical conditions. These allow estimating contamination degree. The experiments were made on a Van de Graaff accelerator located at CERI (Centre d'Etude et de Recherche par Irradiation, Orléans, France). We measure elemental concentrations resulting from the contamination of the surface of each sample. Results are analysed in function of the pathology and the type of implants. According to the pathology and the location of the sampling, these measurements show a very heterogeneous contamination by metallic elements under particles and/or ionic species which can migrate through soft tissues by various mechanisms.

Arthroplasty↗

DNA damage measured by the single-cell gel electrophoresis (Comet) assay in mammals fed with mussels contaminated by the 'Erika' oil-spill.

This research aimed to estimate potential genotoxicity for consumers resulting from the ingestion of seafood contaminated with polycyclic aromatic hydrocarbons (PAHs) released into the marine environment after the 'Erika' shipwreck along the coasts of south Brittany, in France. Mussels (Mytilus sp.) collected from sites on the Atlantic coast that were affected by the oil slick in various degrees, were used to feed rats daily for 2 and 4 weeks. DNA damage was measured by use of the Comet assay in the liver, bone marrow and blood of rats receiving food contaminated with 312 microg of 16PAHs/kg dry weight (d.w.) equivalent to 33.8 microg TEQs (toxic equivalent quantities to benzo(a)pyrene (BaP))/kg d.w. mussels, 569 microg/kg d.w. (83.6 microg TEQs/kg) and 870 microg/kg d.w. (180.7 microg TEQs/kg). A dose-effect-time relationship was observed between the amount of DNA damage in the liver and bone marrow of the rats and the PAH contamination level of the mussels. Genotoxicity increased during the period between 15 and 30 days in rats that received food at the highest two PAH levels. On the other hand, no significant change in liver and bone marrow of rats fed with mussels containing 33.8 microg TEQs/kg d.w. was recorded at 30 days compared with 15 days, indicating efficient DNA repair capacity at low levels of exposure. No signs of genotoxicity were found in peripheral blood. Globally, the observed effects were rather moderate. These results show that oil-contaminated food caused DNA damage in predators, and underline the bioavailability to consumers of pollutants in mussels contaminated with fuel oil. The usefulness of the Comet assay as a sensitive tool in biomonitoring studies analyzing responses of PAH transfer through food webs was also confirmed.

Animals↗

Cytogenetic effects of combined radioactive (137Cs) and chemical (Cd, Pb, and 2,4-D herbicide) contamination on spring barley intercalar meristem cells.

The frequency of cytogenetic effects in spring barley intercalar meristem cells was studied in the presence of a range of different stressors. There was a non-linear dependence on the concentrations of 137Cs, Cd, Pb, and dichlorophenoxyacetic acid (2,4-D) herbicide contamination in the exposure ranges used. The frequency of cytogenetic effects increased at the lower concentrations of the pollutants more rapidly than at the higher concentrations. Contamination of the soil by lead at a concentration that meets the current standards for permissible content in soil, and by 2,4-D herbicide at the application levels recommended for agricultural use resulted in a significant increase in aberrant cell frequency. In these cases, the extent of the observed cytogenetic effects was comparable with the effect induced by a 137Cs soil contamination of 49.2 kBq/kg, a level that exceeds by 10-fold the maximum level permitted in radionuclide-contaminated areas where people are resident. In most cases, the experimentally observed combined effects of the pollutants studied differed from those expected from an additive hypothesis. When combined with 137Cs contamination, antagonistic effects became increasingly stronger when the second stressor was changed from cadmium to lead, and then to the herbicide, as measured both by tests of the 'frequency of aberrant cells' and the 'aberrations per cell'. Data from this study and previous reported literature suggest that synergistic increases in cytogenetic effects can be induced by the simultaneous influence of several stressors even at low intensities. This indicates that there is a capability for mutual intensification of the effects of environmental factors that actually occur in situations of low-level exposure.

2,4-Dichlorophenoxyacetic Acid↗

Mutagens in contaminated soil: a review.

The intentional and accidental discharges of toxic pollutants into the lithosphere results in soil contamination. In some cases (e.g., wood preserving wastes, coal-tar, airborne combustion by-products), the contaminated soil constitutes a genotoxic hazard. This work is a comprehensive review of published information on soil mutagenicity. In total, 1312 assessments of genotoxic activity from 118 works were examined. The majority of the assessments (37.6%) employed the Salmonella mutagenicity test with strains TA98 and/or TA100. An additional 37.6% of the assessments employed a variety of plant species (e.g., Tradescantia clone 4430, Vicia faba, Zea mays, Allium cepa) to assess mutagenic activity. The compiled data on Salmonella mutagenicity indicates significant differences (p<0.0001) in mean potency (revertents per gram dry weight) between industrial, urban, and rural/agricultural sites. Additional analyses showed significant empirical relationships between S9-activated TA98 mutagenicity and soil polycyclic aromatic hydrocarbon (PAH) concentration (r2=0.19 to 0.25, p<0.0001), and between direct-acting TA98 mutagenicity and soil dinitropyrene (DNP) concentration (r2=0.87, p<0.0001). The plant assay data revealed excellent response ranges and significant differences between heavily contaminated, industrial, rural/agricultural, and reference sites, for the anaphase aberration in Allium cepa (direct soil contact) and the waxy locus mutation assay in Zea mays (direct soil contact). The Tradescantia assays appeared to be less responsive, particularly for exposures to aqueous soil leachates. Additional data analyses showed empirical relationships between anaphase aberrations in Allium, or mutations in Arabidopsis, and the 137Cs contamination of soils. Induction of micronuclei in Tradescantia is significantly related to the soil concentration of several metals (e.g., Sb, Cu, Cr, As, Pb, Cd, Ni, Zn). Review of published remediation exercises showed effective removal of genotoxic petrochemical wastes within one year. Remediation of more refractory genotoxic material (e.g., explosives, creosote) frequently showed increases in mutagenic hazard that remained for extended periods. Despite substantial contamination and mutagenic hazards, the risk of adverse effect (e.g., mutation, cancer) in humans or terrestrial biota is difficult to quantify.

Industrial Waste↗

Amygdala activity in obsessive-compulsive disorder with contamination fear: a study with oxygen-15 water positron emission tomography.

Previous imaging studies of obsessive-compulsive symptom states have implicated frontal-striatal and limbic regions in the pathophysiology of obsessive-compulsive disorder (OCD). Functional imaging studies, however, have yielded inconsistent results, presumably due to methodological differences (patient inclusion criteria, stimulus paradigm, imaging technique, and absence of control groups). In the present study, randomized presentation of contamination-related and neutral visual stimuli was used to investigate the neurophysiological correlates of contamination fear in a group of medication-free OCD patients with washing behaviors and healthy controls. A total of 21 subjects (11 OCD patients and 10 healthy controls) were scanned using H(2)(15)O positron emission tomography (PET). Subjects were presented with pictures of clean and dirty surroundings and were requested to make indoor/outdoor decisions to control for attention differences. State anxiety and obsessionality were rated after each scan using visual analogue scales. Main effects of stimulus type (contamination vs. neutral) were found in bilateral occipital cortex in both groups. A significant group interaction effect was observed in the left amygdala reflecting enhanced activity in response to contamination stimuli in OCD patients. Sensitization effects were observed in the right amygdala in the OCD group; these paralleled an increase in levels of distress and obsessionality as well as a decrease in dorsolateral prefrontal activity. The findings of the present study are consistent with the hypothesis of decreased frontal-striatal control of limbic structures, specifically the amygdala, resulting in an inadequate fear response in OCD patients with contamination fear.

Adult↗

Spectroscopic study on the zinc-contaminated soils for the determination of zinc speciation.

The purpose of this paper reported the zinc species in the highly contaminated soils from metallurgical area around Erh-Jen River in the southern of Taiwan. FTIR and XPS were used to investigate the zinc species in the contaminated soils. Through a FTIR analysis, appreciable of zinc bound by organic matter was found and their bonding structure consisted of antisymmetric and symmetric stretchings, Zn-HSsym and Zn-HSasym. With XPS analysis, the Angstrom scale depth scan was surveyed for investigation the zinc speciation. No signal of zinc was detected in the depth of 900A for the larger particle contaminated soils whereas the detectable signal of zinc was determined in the XPS experiment. With further regression and fitting results, the major zinc species presented in the surface of larger and fine particle contaminated soils consisted of reductive and oxidative zinc species. In the deeper depth of contaminated soils, an inverse spinel zinc ferrite was found in the case of larger particle soil. No inverse spinel zinc ferrite was detected in the case of fine particle soil and could be attributed to the relatively small particle size.

Industrial Waste↗

Biological assessment of contaminated land using earthworm biomarkers in support of chemical analysis.

Biological indicators can be used to assess polluted sites but their success depends on the availability of suitable assays. The aim of this study was to investigate the performance of two earthworm biomarkers, lysosomal membrane stability measured using the neutral red retention assay (NRR-T) and the total immune activity (TIA) assay, that have previously been established as responsive to chemical exposure. Responses of the two assays were measured following in situ exposure to complexly contaminated field soils at three industrial sites as well as urban and rural controls. The industrial sites were contaminated with a range of metal (cadmium, copper, lead, zinc, nickel and cobalt) and organic (including polycyclic aromatic hydrocarbons) contaminants, but at concentrations below the 'New Dutch List' Intervention concentrations. Exposed earthworms accumulated both metals and organic compounds at the contaminated sites, indicating that there was significant exposure. No effect on earthworm survival was found at any of the sites. Biomarker measurements, however, indicated significant effects, with lower NRR-T and TIA found in the contaminated soils when compared to the two controls. The results demonstrate that a comparison of soil pollutant concentrations with guideline values would not have unequivocally identified chemical exposure and toxic effect for soil organisms living in these soils. However, the earthworm biomarkers successfully identified significant exposure and biological effects caused by the mixture of chemicals present.

Animals↗

Use of iodide to enhance the phytoextraction of mercury-contaminated soil.

Low bioavailability of mercury (Hg) in soil is a restricting factor in phytoextraction of Hg-contaminated soil. Iodide is known to mobilize Hg in soil and, therefore, the aim of this study was to investigate the possibility to use iodide to increase phytoextraction of Hg. The sensitivity of willow to iodide was investigated in both hydroponics with 0-10 mM KI and in soil with 0.2 and 1 mM KI addition. The capacity to accumulate HgI(2) was compared with that of Hg(NO(3))(2) in hydroponics. Soil extractions with 0-2 mM KI were used to study mobilization of Hg in aged Hg-spiked soil. Additions of KI (0.2-1 mM) were used in pot tests with aged Hg-spiked soil as well as in field trials in an industrial Hg-contaminated soil to study whether iodide addition increased the accumulation of Hg from contaminated soil and the translocation of Hg to the shoots. The total Hg contents in plants, soils and extracts were analyzed by CVAAS. The results showed that too high KI concentration was toxic to plants. Moreover, KI was toxic to plants at lower concentrations in hydroponics than in soil additions. KI (85 microM) in hydroponics gave 50% growth decrease in terms of dry weight of shoot biomass, whereas 0.2 mM KI in soil addition could be tolerated by the plant without growth disturbance. Willow accumulated Hg from HgI(2) solution slower than that from Hg(NO(3))(2) solution. KI mobilized Hg in contaminated soil and thereby increased the bioavailability of Hg in soil. Up to 1 mM KI addition increased the Hg concentrations to about 5, 3 and 8 times, respectively, in the leaves, branches and roots. However, the Hg translocation to the shoots did not show an increase with KI addition. Only less than 1% of the total Hg accumulated in the plant was distributed in the shoots. We may conclude that iodide addition enhances the phytoextraction of Hg, however, the translocation of Hg to the shoots is still too low and therefore it will not be realistic to use this method for phytoextraction of Hg-contaminated soil in practice.

Biodegradation, Environmental↗

Accumulation of Pb, Cu, and Zn in native plants growing on a contaminated Florida site.

Contamination of heavy metals represents one of the most pressing threats to water and soil resources as well as human health. Phytoremediation can be potentially used to remediate metal-contaminated sites. This study evaluated the potential of 36plants (17species) growing on a contaminated site in North Florida. Plants and the associated soil samples were collected and analyzed for total metal concentrations. While total soil Pb, Cu, and Zn concentrations varied from 90 to 4100, 20 to 990, and 195 to 2200mg kg(-1), those in the plants ranged from 2.0 to 1183, 6.0 to 460, and 17 to 598mg kg(-1), respectively. None of the plants were suitable for phytoextraction because no hyperaccumulator was identified. However, plants with a high bioconcentration factor (BCF, metal concentration ratio of plant roots to soil) and low translocation factor (TF, metal concentration ratio of plant shoots to roots) have the potential for phytostabilization. Among the plants, Phyla nodiflora was the most efficient in accumulating Cu and Zn in its shoots (TF=12 and 6.3) while Gentiana pennelliana was most suitable for phytostabilization of sites contaminated with Pb, Cu and Zn (BCF=11, 22 and 2.6). Plant uptake of the three metals was highly correlated, whereas translocation of Pb was negatively correlated with Cu and Zn though translocation of Cu and Zn were correlated. Our study showed that native plant species growing on contaminated sites may have the potential for phytoremediation.

Biodegradation, Environmental↗

Arsenic groundwater contamination and its health effects in the state of Uttar Pradesh (UP) in upper and middle Ganga plain, India: a severe danger.

This communication presents results of our 2-year survey on groundwater arsenic contamination in three districts Ballia, Varanasi and Gazipur of Uttar Pradesh (UP) in the upper and middle Ganga plain, India. Analyses of 4,780 tubewell water samples revealed that arsenic concentrations in 46.5% exceeded 10 microg/L, in 26.7%, 50 microg/L and in 10% 300 microg/L limits. Arsenic concentrations up to 3,192 microg//L were observed. The age of tubewells (n=1,881) ranged from less than a year to 32 years, with an average of 6.5 years. Our study shows that older tubewells had a greater chance of contamination. Depth of tubewells (n=3,810) varied from 6 to 60.5 m with a mean of 25.75 m. A detailed study in three administrative units within Ballia district, i.e. block, Gram Panchayet, and village was carried out to assess the magnitude of the contamination. Before our survey the affected villagers were not aware that they were suffering from arsenical toxicity through contaminated drinking water. A preliminary clinical examination in 11 affected villages (10 from Ballia and 1 from Gazipur district) revealed typical arsenical skin lesions ranging from melanosis, keratosis to Bowens (suspected). Out of 989 villagers (691 adults, and 298 children) screened, 137 (19.8%) of the adults and 17 (5.7%) of the children were diagnosed to have typical arsenical skin lesions. Arsenical neuropathy and adverse obstetric outcome were also observed, indicating severity of exposure. The range of arsenic concentrations in hair, nail and urine was 137-10,900, 764-19,700 microg/kg, and 23-4,030 microg/L, respectively. The urine, hair and nail concentrations of arsenic correlated significantly (r=0.76, 0.61, and 0.55, respectively) with drinking water arsenic concentrations. The similarity to previous studies on arsenic contamination in West Bengal, Bihar and Bangladesh indicates that people from a significant part of the surveyed areas in UP are suffering and this will spread unless drives to raise awareness of arsenic toxicity are undertaken and an arsenic safe water supply is immediately introduced.

Adult↗

Molecular epidemiology of microbial contamination in the operating room environment: Is there a risk for infection?

BACKGROUND: Modern operating rooms are considered to be aseptic environments. The use of surgical mask, frequent air exchanges, and architectural barriers are used to reduce airborne microbial populations. Breaks in surgical technique, host contamination, or hematogenous seeding are suggested as causal factors in these infections. This study implicates contamination of the operating room air as an additional etiology of infection. METHODS: To investigate the potential sources of perioperative contamination, an innovative in situ air-sampling analysis was conducted during an 18-month period involving 70 separate vascular surgical procedures. Air-sample cultures were obtained from multiple points within the operating room, ranging from 0.5 to 4 m from the surgical wound. Selected microbial clonality was determined by pulse-field gel electrophoresis. In a separate series of studies microbial nasopharyngeal shedding was evaluated under controlled environmental conditions in the presence and absence of a surgical mask. RESULTS: Coagulase-negative staphylococci were recovered from 86% of air samples, 51% from within 0.5 m of the surgical wound, whereas Staphylococcus aureus was recovered from 64% of air samples, 39% within 0.5 m from the wound. Anterior nares swabs were obtained from 11 members of the vascular team, clonality was observed between 8 strains of S epidermidis, and 2 strains of S aureus were recovered from selected team members and air-samples collected throughout the operating room environment. Miscellaneous Gram-negative isolates were recovered less frequently (<33%); however, 7 isolates expressed multiple patterns of antimicrobial resistance. The traditional surgical mask demonstrated limited effectiveness at curtailing microbial shedding, especially during symptomatic periods of rhinorrhea. CONCLUSIONS: Gram-positive staphylococcal isolates were frequently isolated from air samples obtained throughout the operating room, including areas adjacent to the operative field. Nasopharyngeal shedding from person participating in the operation was identified as the source of many of these airborne contaminants. Failure of the traditional surgical mask to prevent microbial shedding is likely associated with an increased risk of perioperative contamination of biomedical implants, especially in procedures lasting longer than 90 minutes.

Air Microbiology↗

Albumin contamination of a purified human alpha 1-antitrypsin preparation does not affect either structural conformation or the electrophoretic mobility of the inhibitor.

A partially purified preparation of human alpha 1-antitrypsin (alpha 1-AT) shown to be 60% active as an inhibitor of bovine trypsin, was chosen as starting material to investigate the nature and extent of contamination by human serum albumin (HSA) and to see whether or not such a contamination was responsible for both the reduced inhibitory activity and the slower migratory rate of the proteinase inhibitor in SDS-PAGE. Immunoblotting analysis revealed the presence of HSA in the unprocessed preparation of alpha 1-AT which, both in denaturing and non-denaturing PAGEs, had the same mobility as HSA, appearing as a single band of 65 kDa. By submitting the unprocessed alpha 1-AT preparation to affinity chromatography on an Affi-Gel Blue chromatography column, an apparently highly purified and homogeneous form of alpha 1-AT was obtained, as confirmed by measurement of inhibitory activity and densitometric scanning of SDS-PAGE in non-reducing conditions. However, immunoblotting analysis still revealed the presence of HSA in the most active fractions of the inhibitor eluted from the column, and regardless of purification degree, the molecular mass of the inhibitor was always 65 kDa. Treatment with beta-mercaptoethanol led to separation in SDS-PAGE of HSA as a distinct band of about 10 kDa higher than the alpha 1-AT band, which instead maintained the same mobility as in non-reducing conditions. The results indicate that HSA has not been completely removed from alpha 1-AT, and its presence does not affect the electrophoretic mobility of the inhibitor. The possibility that the structural conformation of the alpha 1-AT, rather than contamination with HSA, was responsible for its abnormal slower migratory rate was therefore tested. For this purpose alpha 1-AT preparations of different degrees of purification were submitted to heat treatment to induce a non-inhibitory conformation such as loop-sheet polymerization. Polymerization was followed both by the appearance in SDS- and non-denaturing PAGEs of high molecular weight bands, which were mostly present in less purified preparations of the inhibitor, and by a decrease in inhibitory activity. A higher degree of polymerization with complete loss of inhibitory activity was observed in the unprocessed alpha 1-AT preparation when dissolved in Na-phosphate buffer at acidic pH, and after dialysis. After heat treatment, the purified alpha 1-AT was shown to run faster in the gel and, in both reducing and non-reducing conditions, the calculated mass of the inhibitor was that expected of about 54 kDa. After reducing treatment, high molecular weight polymers in SDS-PAGEs were reduced, strongly suggesting that disulphide bridges are also involved in the polymerization of alpha 1-AT. Results indicate that the mobility of alpha 1-AT in SDS-PAGE is crucially dependent on its structural conformation which dictates the extent of SDS binding. HSA contaminating the alpha 1-AT preparation does not affect the inhibitor conformation although at a higher degree of contamination and in favourable conditions, it does reduce the inhibitor activity.

Animals↗