Purification of recombinant and human apolipoprotein A-1 using surfactant micelles in aqueous two-phase systems: recycling of thermoseparating polymer and surfactant with temperature-induced phase separation.

An effective system has been developed for purification of apolipoprotein A-1 from Escherichia coli fermentation solution and human plasma using aqueous two-phase extraction and thermal-phase separation. The system included non-ionic surfactants (Triton or Tween) and as top phase-forming polymer a random copolymer of ethylene oxide (50%) and propylene oxide (50%), Breox PAG 50A 1000, was used. The bottom phase-forming polymer was either hydroxypropyl starch, Reppal PES 100 and PES 200, or hydroxyethyl starch, Solfarex A 85. The top-phase-forming polymer and the surfactants are thermoseparating in water solution, i.e., when heated a water phase and a polymer/surfactant phase are formed. Recombinant apolipoprotein A-1, the Milano variant, was extracted from E. coli fermentation solution in a primary Breox-starch phase system followed by thermal separation of the Breox phase where the target protein was recovered in the water phase. Both in the Breox-starch system and in the water-Breox system Triton X-100 was partitioned to the Breox phase. The addition of non-ionic surfactants to the Breox-starch system had strong effect on the purification and yield of the amphiphilic apolipoprotein A-1. In a system containing 17% Breox PAG 50A 1000, 12% Reppal PES 100 and addition of 1% Triton X-100 the purification factor was 7.2, and the yield 85% after thermal separation of the Breox phase. Recycling of copolymer and surfactant was possible after thermal separation of copolymer phase. Approximately 85% of the copolymer and surfactant could be recycled in each extraction cycle. DNA could be strongly partitioned to the starch phase in the primary-phase system. This resulted in a 1000-fold reduction of E. coli DNA in the apolipoprotein A-1 solution obtained after thermoseparation. In extraction from human plasma containing low concentrations of apolipoprotein A-1, it was possible to reach a purification factor of 420 with 98% yield. By reducing the volume ratio to 0.1 Apo A-1 could be concentrated in a small volume of top phase (concentration factor 10) with a yield of 85% and a purification factor of 110.     

Measurement of human apolipoprotein B-48 and B-100 kinetics in triglyceride-rich lipoproteins using [5,5,5-2H3]leucine.

A primed-constant infusion of deuterated leucine was used in humans to determine the maximal level of enrichment at plateau of apolipoprotein (apo)B-48 and apoB-100 which are synthesized in the intestine and liver, respectively, and to compare the kinetics of these two proteins under identical conditions. Eight normal subjects (four post-menopausal females and four males) over the age of 40 were studied in the constantly fed state over a 20-h period by providing small hourly feedings of identical composition. [5,5,5-2H3]Leucine (10 mumol/kg body weight followed by 10 mumol/kg body weight per hour) was infused over 15 h intravenously. The enrichment of deuterated leucine in apoB-48 and apoB-100 triglyceride-rich lipoproteins isolated by ultracentrifugation (d less than 1.006 g/ml) was determined during the entire infusion period. The plateau level of enrichment in triglyceride-rich lipoprotein apoB-48 was 3.96 +/- 1.41 tracer/tracee ratio (%) which was 39.7% of the plasma leucine enrichment level. The plateau level of enrichment in triglyceride-rich lipoprotein apoB-100 was 7.23 +/- 1.17 tracer/tracee ratio (%) which was 72.5% of the plasma leucine enrichment level. Mean fractional secretion rates of triglyceride-rich lipoprotein apoB-48 and apoB-100 were 4.39 +/- 2.00 and 5.39 +/- 1.98 pools per day, respectively, with estimated residence times of 5.47 and 4.45 hours, respectively. The data indicate that in the fed state there is about a twofold difference in the plateau enrichment of an intestinally derived protein, as compared to one of hepatic origin, most likely attributable to differences in the enrichment of the intracellular leucine in the two organs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calculation of stable isotope enrichment tracer kinetic procedures

The choice of method of expressing isotopic enrichment in tracer kinetic experiments utilizing stable isotopes was found to affect the calculation of tracee pool size and half-life. The most commonly used definition, the difference between enriched and natural abundance, i.e. atom percent excess, was found to result in significant error in model systems when the dose of tracer was 10% of the pool size. Errors in determining first-order rate constants of efflux and in pool sizes decreased with decreasing ratio of tracer to tracee. Error in determining pool size increased with longer 'sampling' periods, while error in determining the rate constant increased with shorter sampling periods. Of three less frequently used expressions of isotopic enrichment two were found to yield the exact answers in model systems. The correct expressions of isotopic enrichment were linear functions of the quantity of tracer in the system. A practical example demonstrated the effect of choice of expression of enrichment on estimates of whole body copper pool size and turnover in dairy cattle.     

Cortisol production rate measurement by stable isotope dilution using gas chromatography-negative ion chemical ionization mass spectrometry.

Presented here is a stable isotope dilution technique for determining cortisol production rate (CPR). The method involves extraction and derivatization of cortisol isoforms from serum (0.5 ml), separation of derivatives by gas chromatography, and detection by using negative ion chemical ionization mass spectrometry. This method provides 50-100-fold greater sensitivity than positive ion mass spectrometry and allows for estimations of cortisol production rate with the use of small amounts of pooled serum, even in the presence of high concentrations of lipophilic contaminants. The area under the curve for the total selected ion chromatogram of fluoroacyl derivatives of cortisol (d0, m/z 782) and deuterated cortisol (d3, m/z 785) were used to determine the isotopic dilution ratio in three types of samples: 1) standards: d0/d3 ratios ranging from 1 to 8%; 2) controls: d3-cortisol added to serum with known cortisol concentration; 3) subjects: 24-h pooled serum samples (q 30 min over 24 h) from healthy children (male 10-13 years; female 7-11 years) receiving continuous infusions of d3-cortisol at 2-4% of their estimated CPR. Recovery after the solid phase extraction and derivatization process was >90%, as determined by thin-layer chromatography. Expected versus measured ratios for d3/d0 in standards and serum controls were highly correlated (r2(standard) = 0.99; r2(control) = 0.99) over a wide range of d3-cortisol enrichment (1.0-10.0%). Mean 24-h CPRs were 4.8 +/- 0.6 mg/m2/24 h (mean +/- SEM, n = 7) in male children and 4.4 +/- 0.5 mg/m2/24 h in female children (n = 4). These CPR values are lower than those derived by radio tracer methods, but are in agreement with previous isotopic dilution studies. This technique is an important tool for assessing CPRs in a wide range of disease states affecting cortisol production.     

A quantitative analysis of Arabidopsis plasma membrane using trypsin-catalyzed (18)O labeling

Typical mass spectrometry-based protein lists from purified fractions are confounded by the absence of tools for evaluating contaminants. In this report, we compare the results of a standard survey experiment using an ion trap mass spectrometer with those obtained using dual isotope labeling and a Q-TOF mass spectrometer to quantify the degree of enrichment of proteins in purified subcellular fractions of Arabidopsis plasma membrane. Incorporation of a stable isotope, either H(2)(18)O or H(2)(16)O, during trypsinization allowed relative quantification of the degree of enrichment of proteins within membranes after phase partitioning with polyethylene glycol/dextran mixtures. The ratios allowed the quantification of 174 membrane-associated proteins with 70 showing plasma membrane enrichment equal to or greater than ATP-dependent proton pumps, canonical plasma membrane proteins. Enriched proteins included several hallmark plasma membrane proteins, such as H(+)-ATPases, aquaporins, receptor-like kinases, and various transporters, as well as a number of proteins with unknown functions. Most importantly, a comparison of the datasets from a sequencing "survey" analysis using the ion trap mass spectrometer with that from the quantitative dual isotope labeling ratio method indicates that as many as one-fourth of the putative survey identifications are biological contaminants rather than bona fide plasma membrane proteins.     

Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification

In this study we show that proteins can be partitioned and separated in a novel aqueous two-phase system composed of only one polymer in water solution. This system represents an attractive alternative to traditional two-phase systems which uses either two polymers (e.g., PEG/dextran) or one polymer in high-salt concentration (e.g., PEG/salt). The polymer in the new system is a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups (C(14)H(29)) at both ends (HM-EOPO). This polymer thermoseparates in water, with a cloud point at 14 degrees C. The HM-EOPO polymer forms an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO in water when separated at 17-30 degrees C. The copolymer is self-associating and forms micellar-like structures with a CMC at 12 microM (0.01%). The partitioning behavior of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied, as well as the effect of various ions, pH, and temperature on protein partitioning. The amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO-rich phase within a broad-temperature range. The partitioning of hydrophobic proteins can be directed with addition of salt. Below the isoelectric point (pI) BSA was partitioned to the HM-EOPO-rich phase and above the pI to the water phase when NaClO(4)was added to the system. Lysozyme was directed to the HM-EOPO phase with NaClO(4), and to the water phase with Na-phosphate. The possibility to direct protein partitioning between water and copolymer phases shows that this system can be used for protein separations. This was tested on purification of apolipoprotein A-1 from human plasma and Escherichia coli extract. Apolipoprotein A-1 could be recovered in the HM-EOPO-rich phase and the majority of contaminating proteins in the water phase. By adding a new water/buffer phase at higher pH and with 100 mM NaClO(4), and raising the temperature for separation, the apolipoprotein A-1 could be back-extracted from the HM-EOPO phase into the new water phase. This novel system has a strong potential for use in biotechnical extractions as it uses only one polymer and can be operated at moderate temperatures and salt concentrations and furthermore, the copolymer can be recovered.     

Laboratory contamination over time during low‐biomass sample analysis

Bacteria are not only ubiquitous on earth but can also be incredibly diverse within clean laboratories and reagents. The presence of both living and dead bacteria in laboratory environments and reagents is especially problematic when examining samples with low endogenous content (e.g., skin swabs, tissue biopsies, ice, water, degraded forensic samples or ancient material), where contaminants can outnumber endogenous microorganisms within samples. The contribution of contaminants within high‐throughput studies remains poorly understood because of the relatively low number of contaminant surveys. Here, we examined 144 negative control samples (extraction blank and no‐template amplification controls) collected in both typical molecular laboratories and an ultraclean ancient DNA laboratory over 5 years to characterize long‐term contaminant diversity. We additionally compared the contaminant content within a home‐made silica‐based extraction method, commonly used to analyse low endogenous content samples, with a widely used commercial DNA extraction kit. The contaminant taxonomic profile of the ultraclean ancient DNA laboratory was unique compared to modern molecular biology laboratories, and changed over time according to researcher, month and season. The commercial kit also contained higher microbial diversity and several human‐associated taxa in comparison to the home‐made silica extraction protocol. We recommend a minimum of two strategies to reduce the impacts of laboratory contaminants within low‐biomass metagenomic studies: (a) extraction blank controls should be included and sequenced with every batch of extractions and (b) the contributions of laboratory contamination should be assessed and reported in each high‐throughput metagenomic study.     

Using the dual isotope method to assess cecal amino acid absorption of goat whey protein in rats,a pilot study

Measurement of ileal amino acids (AA) bioavailability is recommended to evaluate protein quality. A dual isotope tracer method, based on plasma isotopic enrichment ratios, has been proposed to determine true digestibility in humans. In a pilot study, we aimed to evaluate whether this method could be implemented in rats to determine AA bioavailability based on isotopic enrichment ratios measured in cecal digesta or plasma samples. Goat milk proteins were intrinsically labeled with ¹⁵N and ²H. Wistar rats were fed a meal containing the doubly labeled goat whey proteins and a tracer dose of ¹³C-spirulina. Blood samples were collected 0, 1 h and 3 h after meal ingestion from the tail vein. The rats were euthanized 4 h (n?=?6) or 6 h (n?=?6) after meal to collect plasma and intestinal contents. True orocecal protein digestibility and AA bioavailability were assessed by means of ¹⁵N and ²H enrichment in cecum content and compared with absorption indexes determined at the plasma or cecum level using isotopic ratios. Plasma kinetics of isotopic enrichment could not be completed due to the limited quantity of plasma obtained with sequential blood collection. However, the absorption indexes determined from cecal ¹⁵N or ²H/¹³C ratios gave coherent values with true orocecal AA bioavailability. This dual isotope approach with measurements of isotopic ratios in digestive content could be an interesting strategy to determine true AA bioavailability in ileal digesta of rats.

     

An integral equation approach to measuring turnover in nonsteady compartmental and distributed systems

Physiological systems are often modelled by a set of compartments. Alternatively they can be described by the diffusion-convection-reaction equations governing distributed systems. The problem considered here is that of identifying a continuously changing input of some metabolite )tracee), endogenous to the system and hence inaccessible, when a nonlinear or time-varying component is also introduced into the loss parameter, as for example through feedback mechanisms. A tracer is used to determine the steady-state impulse response under time-invariant, linear conditions. A known input of tracer is also administered when the system is driven out of steady state. The integral equations developed utilize the predetermined impulse response, the measured concentrations of both tracer and tracee (output) in some region of the system to estimate the changing loss parameter and the unknown input in a continuous fashion.     

Measurement of exchange of trace metals between the body and its surroundings with the use of trace methodology

Application of general tracer theory to the problem of estimating fluxes of tracee between the gastrointestinal tract and the body proper, from observations of the movement of tracer, shows that a number of assumptions must be fulfilled. Two specific sets of assumptions are discussed and, in both cases, measurement of tracer fluxes yields information on the integrated absorption of the tracee.     

Human apolipoprotein A-I kinetics within triglyceride-rich lipoproteins and high density lipoproteins.

Stable isotope methodology was used to determine the kinetic behavior of apolipoprotein (apo) A-I within the triglyceride-rich lipoprotein (TRL) fraction and to compare TRL apoA-I kinetics with that of apoA-I in high density lipoprotein (HDL) and TRL apoB-48. Eight subjects (5 males and 3 females) over the age of 40 were placed on a baseline average American diet and after 6 weeks received a primed-constant infusion of [5,5,5-(2)H(3)]-l-leucine for 15 h while consuming small hourly meals of identical composition. HDL and TRL apoA-I and TRL apoB-48 tracer/tracee enrichment curves were obtained by gas chromatography;-mass spectrometry. Data were fitted to a compartmental model to determine the fractional secretion rates of apoA-I and apoB-48 within each lipoprotein fraction. Mean plasma apoA-I levels in TRL and HDL fractions were 0. 204 +/- 0.057 and 134 +/- 15 mg/dl, respectively. The mean fractional catabolic rate (FCR) of TRL apoA-I was 0.250 +/- 0.069 and HDL apoA-I was 0.239 +/- 0.054 pools/day, with mean estimated residence times (RT) of 4.27 and 4.37 days, respectively. The mean TRL apoB-48 FCR was 5.2 +/- 2.0 pools/day and the estimated mean RT was 5.1 +/- 1.8 h. Our results indicate that apoA-I is catabolized at a slower rate than apoB-48 within TRL, and that apoA-I within TRL and HDL fractions are catabolized at similar rates.     

Studying apolipoprotein turnover with stable isotope tracers: correct analysis is by modeling enrichments

Lipoprotein kinetic parameters are determined from mass spectrometry data after administering mass isotopes of amino acids, which label proteins endogenously. The standard procedure is to model the isotopic content of the labeled precursor amino acid and of proteins of interest as tracer-to-tracee ratio (TTR). It is shown here that even though the administered tracer alters amino acid mass and turnover, apolipoprotein synthesis is unaltered and hence the apolipoprotein system is in a steady state, with the total (labeled plus unlabeled) masses and fluxes remaining constant. The correct model formulation for apolipoprotein kinetics is shown to be in terms of tracer enrichment, not of TTR. The needed mathematical equations are derived. A theoretical error analysis is carried out to calculate the magnitude of error in published results using TTR modeling. It is shown that TTR modeling leads to a consistent underestimation of the fractional synthetic rate. In constant-infusion studies, the bias error percent is shown to equal approximately the plateau enrichment, generally <10%. It is shown that, in bolus studies, the underestimation error can be larger. Thus, for mass isotope studies with endogenous tracers, apolipoproteins are in a steady state and the data should be fitted by modeling enrichments.     

Synthesis and regulation of acute phase plasma proteins in primary cultures of mouse hepatocytes

Adult mouse hepatocytes respond in vivo to experimentally induced acute inflammation by an increased synthesis and secretion of alpha 1-acid glycoprotein, haptoglobin, hemopexin, and serum amyloid A. Concurrently, the production of albumin and apolipoprotein A-1 is reduced. To define possible mediators of this response and to study their action in tissue culture, we established primary cultures of hepatocytes. Various hormones and factors that have been proposed to regulate the hepatic acute phase reaction were tested for their ability to modulate the expression of plasma proteins in these cells. Acute phase plasma and conditioned medium from activated monocytes influenced the production of most acute phase plasma proteins, and the regulation appears to occur at the level of functional mRNA. Purified hormones produced a significant anabolic response in only a few cases: dexamethasone was found to be effective in maintaining differentiated expression of the cells; and glucagon produced a specific inhibition of haptoglobin synthesis. When cells were treated with a combination of conditioned monocyte medium and dexamethasone, secretion of proteins was markedly reduced. The carbohydrate moieties of all plasma glycoproteins were incompletely modified, apparently as a result of decreased intracellular transport of newly synthesized plasma proteins. Although primary hepatocytes were not phenotypically stable in tissue culture, the cells nevertheless retained a broad response spectrum to exogenous signals. We propose this as a useful system to study the production of plasma proteins and thereby pinpoint the nature and activity of effectors mediating the hepatic acute phase reaction.     

尿液蛋白富集方法的比较

人尿液中蛋白含量低,在进行质谱分析时易被高丰度蛋白掩盖。因此,发展高效和高选择性的富集方法,是实现尿蛋白标记物深度覆盖的必要前提。探究不同实验方法对尿液蛋白富集和尿蛋白质组的影响尤为重要。本研究采用超滤法、硝酸纤维素膜富集法和饱和硫酸铵沉淀法,等体积各处理5例健康志愿者和膀胱癌患者10 mL尿液样本,富集尿液蛋白,SDS-PAGE分离尿蛋白,比较不同方法纯化的效率;通过质谱分析,比较不同纯化方法的肽段鉴定效果,确定针对尿液蛋白质组蛋白的最佳富集方法。相对于超滤和硝酸纤维素膜富集法,饱和硫酸铵沉淀法成功地应用于健康人尿蛋白的富集和质谱检测,在保证回收蛋白质量的前提下,可减少高丰度白蛋白的干扰,富集更多低丰度蛋白,提高了质谱鉴定的灵敏度。综上所述,饱和硫酸铵提取尿蛋白的效果较好,该方法具有大规模处理尿液、提高蛋白质组学筛选临床诊断标记物研究的应用潜力。     

Mass kinetics of apolipoprotein A-I in interstitial fluid after administration of intravenous apolipoprotein A-I/lecithin discs in humans

Apolipoprotein kinetics are customarily determined by modeling time curves of specific radioactivity or isotopic enrichment in plasma after intravenous infusion of radiolabeled lipoproteins or stable isotope-enriched amino acids. However, this provides no information on the fractional rate of transfer of the apolipoprotein from plasma to interstitial fluid (k(p-if)) or its mean residence time in interstitial fluid (MRT(if)). To determine these parameters for a pharmacologic dose of exogenous apolipoprotein A-I (apoA-I) given intravenously as apoA-I/lecithin discs, we measured apoA-I in plasma and prenodal leg lymph in five healthy men before, during, and after a 4 h infusion at 10 mg/kg/h. ApoA-I concentrations in plasma and lymph were modeled by linear compartmental models (SAAM II version 1.1), using lymph albumin to adjust for the effects of variations in lymph flow rate. k(p-if) averaged 0.75%/h (range, 0.33-1.32), and MRT(if) averaged 29.1 h (14.1-40.0). Neither parameter was correlated with the distribution volume (57-105 ml/kg) or the fractional elimination rate (1.44-2.91%/h) of apoA-I, determined by modeling plasma apoA-I concentration alone. Although used here to study the mass kinetics of apoA-I, if combined with infusion of a tracer, analysis of lymph could also expand the modeling of endogenous apolipoprotein kinetics.     

Increased concentration of tracee affects estimates of muscle protein synthesis

Muscle protein synthesis was measured by infusion of L-[2H(5)]phenylalanine in two groups of anesthetized dogs, before and during infusion of insulin with euaminoacidemia, and with differing concentrations of unlabeled phenylalanine (tracee). With the infusion of insulin, muscle protein synthesis increased 39 +/- 12% based on phenylalanyl-tRNA. Calculation with plasma phenylalanine enrichment overestimated insulin stimulation by 40% (56 +/- 12 vs. 39 +/- 12%). Raising the concentration of plasma phenylalanine twofold during infusion of insulin further increased the apparent stimulation of muscle protein synthesis based on plasma relative to phenylalanyl-tRNA by 225% (65 +/- 19 vs. 20 +/- 14%, P < 0.001). In both experiments, the stimulation of synthesis rates calculated from phenylalanine enrichment within the muscle was closer to that from phenylalanyl-tRNA (48 +/- 19%, experiment 1; 30 +/- 14%, experiment 2). Results indicate that the enrichment of a labeled amino acid within plasma and tissue amino acid pools is affected by the concentration of tracee infused. Increasing the concentration of tracee overestimates the insulin-mediated stimulation of muscle protein synthesis when amino acid pools other than aminoacyl-tRNA are used as the precursor enrichment.     

Equations for Lipid Normalization of Carbon Stable Isotope Ratios in Aquatic Bird Eggs

Stable isotope ratios are biogeochemical tracers that can be used to determine the source of nutrients and contaminants in avian eggs. However, the interpretation of stable carbon ratios in lipid-rich eggs is complicated because ¹³C is depleted in lipids. Variation in ¹³C abundance can therefore be obscured by variation in percent lipids. Past attempts to establish an algebraic equation to correct carbon isotope ratios for lipid content in eggs have been unsuccessful, possibly because they relied partly on data from coastal or migratory species that may obtain egg lipids from different habitats than egg protein. We measured carbon, nitrogen and sulphur stable isotope ratios in 175 eggs from eight species of aquatic birds. Carbon, nitrogen and sulphur isotopes were enriched in lipid-extracted egg samples compared with non extracted egg samples. A logarithmic equation using the C∶N ratio and carbon isotope ratio from the non extracted egg tissue calculated 90% of the lipid-extracted carbon isotope ratios within ±0.5‰. Calculating separate equations for eggs laid by species in different habitats (pelagic, offshore and terrestrial-influenced) improved the fit. A logarithmic equation, rather than a linear equation as often used for muscle, was necessary to accurately correct for lipid content because the relatively high lipid content of eggs compared with muscle meant that a linear relationship did not accurately approximate the relationship between percent lipids and the C∶N ratio. Because lipid extraction alters sulphur and nitrogen isotope ratios (and cannot be corrected algebraically), we suggest that isotopic measurement on bulk tissue followed by algebraic lipid normalization of carbon stable isotope ratio is often a good solution for homogenated eggs, at least when it is not possible to complete separate chemical analyses for each isotope.     

A combination of magnetic permeability detection with nanometer-scaled superparamagnetic tracer and its application for one-step detection of human urinary albumin in undiluted urine

A rapid (6.5 min) and simple one-step magnetic immunoassay (MIA) has been developed for analysis of human urinary albumin in near patient settings. Polyclonal rabbit anti-human albumin was used as a capture antibody and monoclonal mouse anti-human albumin as a detection antibody in a two-site immunometric assay requiring no additional washing procedures. The polyclonal anti-human albumin was conjugated to silica microparticles (solid phase) and the monoclonal antibody to dextran-coated nanoscaled superparamagnetic particles (tracer). Quantification of human albumin in undiluted urine was performed by adding 2 μL urine to a measuring vial containing solid-phase, superparamagnetic tracer and reaction buffer and then inverting the vial by hand for 20 s. The measuring vial was allowed to stand for 6 min prior to detection, in order for the solid-phase sediment to form at the bottom of the vial. Lastly, the measuring vial was placed into a magnetic permeability detector, which measured the enrichment of superparamagnetic tracer in the sediment due to complex formation with human albumin. Total analysis time was 6.5 min. A linear response was obtained for 0–400 mg/L albumin with a detection limit of 5 mg/L. The total coefficient of variation (CV) was 11% calculated from four consecutive runs on a urine sample containing 11.1 mg/L human albumin during 3 consecutive days. Human urinary albumin analysis was performed on 149 patient samples using the MIA technique and the obtained results showed good correlation with the hospital immunoturbidimetric reference method (y = 1.004x + 4.01, R² = 0.978, N = 149) and a commercially available point of care albumin analysis provided by HemoCue Inc. (y = 0.98x + 5.8, R² = 0.833, N = 90).     

Identification of Metals Contamination in Firing-Range Soil Using Geochemical Correlation Evaluation

Lead, antimony, copper, and zinc are expected contaminants in firing-range soil due to their presence in bullets, shells, etc. These elements are also naturally occurring, so it is important to distinguish between naturally high background concentrations and actual contamination during site investigations. A data visualization technique based on geochemical principles has been successfully applied during range investigations to identify contaminated samples and confirm the success of remediation efforts. For example, at some locations, lead has a natural affinity to adsorb on manganese oxides, yielding positive correlations between lead and manganese concentrations and consistent Pb/Mn ratios in uncontaminated samples. Contaminated samples are identified by anomalously high Pb/Mn ratios. Plots of copper or zinc versus lead provide supporting evidence for a contaminant source; Cu/Pb or Zn/Pb ratios in oxic soils are distinctly different in uncontaminated samples versus samples co-contaminated with these metals. Two case studies are presented from facilities in the United States.     

The influence of breeding colony and sex on mercury,selenium and lead levels and carbon and nitrogen stable isotope signatures in summer and winter feathers of <Emphasis Type="Italic">Calonectris</Emphasis> shearwaters

Contamination in marine foodwebs is nowadays of great environmental concern owing to the increasing levels of pollution in marine ecosystems from different anthropogenic sources. Seabirds can be used as indicators of regional contaminant patterns across large temporal and spatial scales. We analysed Hg, Se and Pb levels as well as stable isotope ratios of C (¹³C/¹²C, δ¹³C) and N (¹⁵N/¹⁴N, δ¹⁵N) in breeding- and winter-season feathers on males and females of two related shearwater species, providing information on spatiotemporal patterns of contaminants as well as the influence of the trophic ecology of these seabirds on contaminant levels. During the breeding season, Se and Pb concentrations were highest at the Cape Verde archipelago, showing no differences among the other colonies or between the sexes. However, Hg levels varied among colonies, being highest in the Mediterranean, probably resulting from the larger emissions and fallout of this pollutant in Europe. Feathers grown during breeding also showed sexual differences in Hg concentrations and δ¹³C. Differences in Hg concentration between sexes are mainly due to egg-laying decontamination in females. In contrast, differences in Hg among colonies are probably related to differences in trophic ecology, as indicated by δ¹³C and δ¹⁵N measurements. Contaminant concentrations in winter-grown feathers did not show any relationship with stable isotope values but were affected by contaminant loads associated with the breeding season. These findings suggest that the interpretation of contaminant levels of migratory species from feathers moulted out of the breeding season should be made with caution because those values could reflect exposures to contaminants acquired during the breeding season. We conclude that factors other than feeding ecology may play an important role in the interpretation of contaminant levels and their annual dynamics at several spatial scales. Consideration of the relevant temporal context provided by isotopic signatures and contaminant concentrations is important in deciphering contaminant information based on various tissues. Xavier Ruiz: deceased 27 April 2008.