Stability assessment of FDA-approved ramucirumab monoclonal antibody; validated SE-HPLC method for degradation pattern evaluation.

Ramucirumab (RAMU) is a recently US Food and Drug Administration-approved monoclonal antibody that is included in various anticancer protocols. It has a structural complexity and high degradation risk that have a significant effect on its safety and effectiveness. The major aim of this work was to assess the degradation pattern of RAMU based on physicochemical characterization. Mechanical agitation, repeated freeze-thaw cycles, pH and temperature were the selected stress conditions to which RAMU samples were subjected. The SE-HPLC method was applied and validated to monitor the RAMU monomer along with its aggregates and/or fragments. The purity of the separated peaks together with system suitability parameters were determined through the calculation of percentage purity and percentage drop in RAMU concentration. The results were interpreted by correlating them with those of dynamic light scattering and reducing and non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Samples incubated at pH 2.0-10.0 and 37°C for up to 4 weeks were analysed, recording detection of reversed phase (RP) aggregates and low molecular weight peptide fragments. Similarly, samples under short-term storage conditions of 4 weeks at different temperatures (-20, 2-8, 25, 37 and 50°C) showed low molecular weight peptide fragments but to a lesser extent. These results highlight the alarming effect on RAMU multidose vial efficacy and safety.

Quality Assessment and Comparison of Plasma-Derived Extracellular Vesicles Separated by Three Commercial Kits for Prostate Cancer Diagnosis.

INTRODUCTION: Current standard biomarkers in clinic are not specific enough for prostate cancer (PCa) diagnosis. Extracellular vesicles (EVs) are nano-scale vesicles released by most mammalian cells. EVs are promising biomarker source for PCa liquid biopsy due to its minimal invasive approach, rich information and improved accuracy compared to the clinical standard prostate-specific antigen (PSA). However, current EV separation methods cannot separate pure EVs and the quality characteristics from these methods remain largely unknown. In this study, we evaluated the quality characteristics of human plasma-derived EVs by comparing three clinical suitable separation kits. METHODS: We combined EV separation by commercial kits with magnetic beads capture and flow cytometry analysis, and compared three kits including ExoQuick Ultra based on precipitation and qEV35 and qEV70 based on size exclusion chromatography (SEC). RESULTS: Our results indicated that two SEC kits provided higher EV purity and lower protein contamination compared to ExoQuick Ultra precipitation and that qEV35 demonstrated a higher EV yield but lower EV purity compared to qEV70. Particle number correlated very well particularly with CD9/81/63 positive EVs for all three kits, which confirms that particle number can be used as the estimate for EV amount. At last, we found that several EV metrics including total EVs and PSA-specific EVs could not differentiate PCa patients from health controls. CONCLUSION: We provided a systematic workflow for the comparison of three separation kits as well as a general analysis process in clinical laboratories for EV-based cancer diagnosis. Better EV-associated cancer biomarkers need to be explored in the future study with a larger cohort.

Conformation assessment of therapeutic monoclonal antibodies by SEC-MS: Unravelling analytical biases for application to quality control.

Immunogenicity related to the degradation of therapeutic monoclonal antibodies (mAbs) remains a major concern for their therapeutic efficacy and safety. Therefore, an analytical method allowing characterization and detection of mAbs degradation is mandatory. In this study, a simultaneous coupling of size exclusion chromatography (SEC) to native mass spectrometry (MS) and fluorescence detection (FLD) is proposed to detect degraded therapeutic mAbs and biases of structural changes (e.g. dimerization, denaturation) that may occur during native MS. A comprehensive study on infliximab behaviors have been performed under different mobile phase conditions (e.g. composition, pH, organic solvent, etc.) and MS parameters (e.g. gas temperatures, CID energies, etc.). Experimental conditions avoiding artificial denaturation and/ or dimerization have been defined. We have also demonstrated that under the developed conditions infliximab affinity towards its biological target TNFα is preserved. In addition, using this method dimers, denatured monomers and fragments could be detected in trastuzmab samples stressed by a long-term storage. These results were confirmed by using SEC coupled to ion mobility mass spectrometry as an orthogonal method for the detection of denatured monomer.

Assessment of separation methods for extracellular vesicles from human and mouse brain tissues and human cerebrospinal fluids.

Extracellular vesicle (EV) is a unified terminology of membrane-enclosed vesicular species ubiquitously secreted by almost every cell type and present in all body fluids. They carry a cargo of lipids, metabolites, nucleic acids and proteins for their clearance from cells as well as for cell-to-cell communications. The exact composition of EVs and their specific functions are not well understood due to the underdevelopment of the separation protocols, especially those from the central nervous system including animal and human brain tissues as well as cerebrospinal fluids, and the low yield of proteins in the separated EVs. To understand their exact molecular composition and their functional roles, development of the reliable protocols for EV separation is necessary. Here we report the methods for EV separation from human and mouse unfixed frozen brain tissues by a sucrose step gradient ultracentrifugation method, and from human cerebrospinal fluids by an affinity capture method. The separated EVs were assessed for morphological, biophysical and proteomic properties of separated EVs by nanoparticle tracking analysis, transmission electron microscopy, and labeled and label-free mass spectrometry for protein profiling with step-by-step protocols for each assessment.

A Streamlined Bioanalytical Approach to Select a Compatible Primary Container System Early in Drug Development: A Toolbox for the Biopharmaceutical Industry.

To ensure drug efficacy and patient safety, the importance of interaction between primary container and a biological drug product must not be ignored. The United States Food and Drug Administration guidance on development and manufacturing of combination products (e.g., the biologic and the primary container) encourages careful selection and stability testing of the drug and its performance in the marketed primary container. With various options available for primary container type (vials, prefilled syringes, and cartridges), material (e.g., glass or plastic), and lubricants/coatings, we designed a platform consisting of several bioanalytical methods that can simplify selection of a compatible primary container. We tested the stability of a commercially available monoclonal antibody (mAb) in 3 syringe types under 3 conditions: cold storage, high temperature, and agitation induced stress, respectively. Under each condition, dynamic fluid imaging was sensitive enough to differentiate mAb stability as measured by aggregate formation in different syringe systems, followed by size exclusion-high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis but only at high temperature. With this platform, we identified a primary container that provided higher mAb stability under cold storage as well as stress conditions. We recommend that such an approach should be applied early in drug development stage to identify a superior primary container system to maintain drug stability and quality.

Biochemical and biophysical characterization of a prokaryotic Mg2+ ion channel: Implications for cost-effective purification of membrane proteins.

Although magnesium is the second most abundant cation present in the cell, the transport mechanism of Mg2+ across membranes is poorly understood. Importantly, the prokaryotic MgtE Mg2+ channel is related to mammalian SLC41A1 transporters and, therefore, biochemical and biophysical characterization of MgtE and its orthologs assumes significance. To date, the purification and structure determination of MgtE from Thermus thermophilus has been carried out using the widely used nonionic detergent, n-dodecyl-ß-d-maltopyranoside (DDM). However, DDM is an expensive detergent and alternative methods to produce high-quality proteins in stable and functional form will be practically advantageous to carry out structural studies in a cost-effective manner. In this work, we have utilized 'dual-detergent strategy' to successfully purify MgtE channel in a stable and functional form by employing relatively inexpensive detergents (Triton X-100 and Anzergent 3-14) for membrane solubilization and subsequently changed to DDM during purification. Our results show that Triton X-100 and Anzergent 3-14 extract MgtE well and the quality of purified protein is comparable to DDM-extracted MgtE. Interestingly, addition of high concentration of salt and glycerol during solubilization does not significantly affect the quantity and quality of MgtE. Importantly, limited proteolysis assay, circular dichroism spectroscopy and ensemble tryptophan fluorescence strongly support the use of Triton X-100, in particular, as an inexpensive, alternative detergent for the purification of MgtE without compromising the structural integrity of the channel and Mg2+-induced gating-related conformational dynamics. Overall, these results are relevant for the cost-effective purification of stable and functional membrane proteins in general, and magnesium channels, in particular.

Critical assessment of different methods for quantitative measurement of metallodrug-protein associations.

Quantitative screening for potential drug-protein binding is an essential step in developing novel metal-based anticancer drugs. ICP-MS approaches are at the core of this task; however, many applications lack in the capability of large-scale high-throughput screenings and proper validation. In this work, we critically discuss the analytical figures of merit and the potential method-based quantitative differences applying four different ICP-MS strategies to ex vivo drug-serum incubations. Two candidate drugs, more specifically, two Pt(IV) complexes with known differences of binding affinity towards serum proteins were selected. The study integrated centrifugal ultrafiltration followed by flow injection analysis, turbulent flow chromatography (TFC), and size exclusion chromatography (SEC), all combined with inductively coupled plasma-mass spectrometry (ICP-MS). As a novelty, for the first time, UHPLC SEC-ICP-MS was implemented to enable rapid protein separation to be performed within a few minutes at > 90% column recovery for protein adducts and small molecules. Graphical abstract Quantitative screening for potential drug-protein binding is an essential step in developingnovel metal-based anticancer drugs.

Characterization of 30 therapeutic antibodies and related products by size exclusion chromatography: Feasibility assessment for future mass spectrometry hyphenation.

Despite the popularity of targeted and immune therapies, the number of studies dealing with the quantitation of aggregates for Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved mAb and related products are still very scarce in literature. In this work, 30 therapeutic proteins including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), Fc-fusion proteins and a bi-specific antibody (bsAb) were investigated using size exclusion chromatography (SEC). Their levels of high molecular weight species (HMWS) were experimentally estimated between 0.1% and 13.1%. Except for blinatumomab, etanercept and pembrolizumab, the HMWS amount for the other antibodies was well below the limit of 5% usually set a specification for therapeutic mAbs in the biopharmaceutical industry. The main chromatographic peak shape of 24 therapeutic antibodies and the NIST mAb [1] was found suitable (0.8

A rapid expression and purification condition screening protocol for membrane protein structural biology.

Membrane proteins control a large number of vital biological processes and are often medically important-not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high-resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents. This is normally a time-consuming and highly iterative process since only one or a few conditions can be tested at a time. In this article, we describe a rapid screening protocol in a 96-well format that largely mimics standard membrane protein purification procedures, but eliminates the ultracentrifugation and membrane preparation steps. Moreover, we show that the results are robustly translatable to large-scale production of detergent-solubilized protein for structural studies. We have applied this protocol to 60 proteins from an E. coli membrane protein library, in order to find the optimal expression, solubilization and purification conditions for each protein. With guidance from the obtained screening data, we have also performed successful large-scale purifications of several of the proteins. The protocol provides a rapid, low cost solution to one of the major bottlenecks in structural biology, making membrane protein structures attainable even for the small laboratory.

Quantification and Assessment of the Chemical Form of Residual Gadolinium in the Brain After Repeated Administration of Gadolinium-Based Contrast Agents: Comparative Study in Rats.

OBJECTIVE: Multiple clinical and preclinical studies have reported a signal intensity increase and the presence of gadolinium (Gd) in the brain after repeated administration of Gd-based contrast agents (GBCAs). This bioanalytical study in rat brain tissue was initiated to investigate whether the residual Gd is present as intact GBCA or in other chemical forms by using tissue fractionation and chromatography. MATERIALS AND METHODS: Rats were divided randomly in 6 groups of 10 animals each. They received 10 daily injections of 2.5 mmol/kg bodyweight of 1 of 5 different GBCAs: linear GBCAs such as gadodiamide (Omniscan; GE Healthcare), gadopentetate dimeglumine (Gd-DTPA, Magnevist; Bayer), or gadobenate dimeglumine (Multihance; Bracco) and macrocyclic GBCAs such as gadobutrol (Gadovist; Bayer) and gadoterate meglumine (Gd-DOTA, Dotarem; Guerbet) or saline. On days 3 and 24 after the last injection (p.i.), 5 randomly chosen animals of each group were killed by exsanguination, and their brains were excised and divided into cerebrum, pons, and cerebellum. The brain sections were homogenized by sonication in ice-cold buffer at pH 7.4. Soluble and insoluble fractions were separated by centrifugation, and the soluble fractions were further separated by gel permeation chromatography (GPC). The Gd concentration in all tissue fractions and in the GPC eluate was measured by inductively coupled plasma-mass spectrometry. In a recovery control experiment, all GBCAs were spiked to blank brain tissue and more than 94% recovery of Gd in the tissue fractions was demonstrated. RESULTS: Only traces of the administered Gd were found in the rat brain tissue on day 3 and day 24 p.i. In the animals treated with macrocyclic GBCAs, Gd was found only in the soluble brain fraction and was present solely as low molecular weight molecules, most likely the intact GBCA. In the animals treated with linear GBCAs Gd was found to a large extent in the insoluble tissue fraction. The Gd concentration in the soluble fraction was comparable to the macrocyclic agents. According to GPC, a smaller portion of the Gd in the soluble fraction of the linear GBCAs groups was bound to macromolecules larger than 250 to 300 kDa. The nature of the Gd-containing macromolecules and the insoluble species were not determined, but they appeared to be saturable with Gd. The excretion of the soluble Gd species in the linear and macrocyclic GBCA groups was still ongoing between days 3 and 24 p.i. This was also observed for the macromolecular Gd species in the linear GBCA groups, but at a slower rate. CONCLUSIONS: The residual Gd found in the rat brain after repeated administration of all 3 linear GBCAs was present in at least 3 distinctive forms-soluble small molecules, including the intact GBCA, soluble macromolecules, and to a large extent in insoluble form. The latter 2 are most likely responsible for the prolonged signal intensity enhancement in brain structures observed in magnetic resonance imaging. No relevant differences between the 3 linear GBCAs were observed. The Gd concentrations in the brain after administration of macrocyclic GBCAs are lower, and the Gd is only present in soluble small molecules, which were slowly excreted. This underlines the crucial importance of the kinetic inertness of macrocyclic agents in the prevention of potential retention of Gd in the brain compared with the 3 linear, kinetically less restricted GBCAs.

Analytical comparison of a US generic enoxaparin with the originator product: The focus on comparative assessment of antithrombin-binding components.

Enoxaparin sodium, a low-molecular-weight heparin (LMWH) prepared from porcine intestinal heparin, is widely used for the prevention and treatment of venous thromboembolism. The antithrombotic activity of heparin is mediated mainly through its activation of antithrombin (AT) and subsequent inhibition of coagulation factors. Heparin is a complex heteropolymer and the sulfation pattern of its alternating uronic acid and glucosamine sugar units is a major factor influencing its biological activity. The manufacturing process itself is associated with the introduction of exogenous microheterogeneities that may further affect its biological efficacy. This is important since enoxaparin is prepared by depolymerizing the heparin with the aim of optimizing its biological activity and safety. Changes during its manufacture could thus affect its biological activity and safety. The current study was performed to assess potential differences between the originator enoxaparin and a new generic enoxaparin commercialized by Teva. Heparinase digestion, AT affinity chromatography, gel permeation chromatography, anion exchange chromatography, and nuclear magnetic resonance methodologies were used. The results indicated differences in oligosaccharides related to the cleavage selectivity around the heparin AT-binding sequences of the Teva Enoxaparin Sodium Injection, USP and the originator Sanofi enoxaparin. These differences influence the strength of the AT-binding affinity of the individual oligosaccharides, their ability to activate AT and, therefore, the inhibitory potency on the proteases of the coagulation cascade. This study, together with other published analytical reports, describes specific compositional differences between generics and originator LWMHs. However, it is yet to be established whether such variations might have any clinical relevance.

On-line coupling of surface plasmon resonance optical sensing to size-exclusion chromatography for affinity assessment of antibody samples.

Surface plasmon resonance (SPR) is an optical technique that measures biomolecular interactions. Stand-alone SPR cannot distinguish different binding components present in one sample. Moreover, sample matrix components may show non-specific binding to the sensor surface, leading to detection interferences. This study describes the development of coupled size-exclusion chromatography (SEC) SPR sensing for the separation of sample components prior to their on-line bio-interaction analysis. A heterogeneous polyclonal human serum albumin antibody (anti-HSA) sample, which was characterized by proteomics analysis, was used as test sample. The proposed SEC-SPR coupling was optimized by studying system parameters, such as injection volume, flow rate and sample concentration, using immobilized HSA on the sensor chip. Automated switch valves were used for on-line regeneration of the SPR sensor chip in between injections and for potential chromatographic heart cutting experiments, allowing SPR detection of individual components. The performance of the SEC-SPR system was evaluated by the analysis of papain-digested anti-HSA sampled at different incubation time points. The new on-line SEC-SPR methodology allows specific label-free analysis of real-time interactions of eluting antibody sample constituents towards their antigenic target.

Rapid formulation assessment of filgrastim therapeutics by a thermal stress test.

The biosimilar versions of recombinant methionyl human granulocyte colony-stimulating factor (rh-Met-G-CSF, filgrastim) are now widely available. Because changes to the formulation often lead to subtle differences, there is a critical need to define techniques to test and insure the quality of these products. The present study was designed to compare formulation and thermal stress stability of filgrastim products. The formulation ingredients including acetate, polysorbate 80, and sorbitol were determined using state-of-the-art validated analytical methods. The formulation pH and osmolality were also measured. Moreover, the stability profiles of 8 filgrastim products using thermal stress at 57 °C for 4 h were assessed by size-exclusion high-performance liquid chromatography (SE-HPLC) and in vitro biological assay. The products had different stability profiles. More stable products were within the specification for formulation and less stable products were beyond the specification limits. Altogether, the results suggest that a short-time stress study at 57 °C and analysis of filgrastim by SE-HPLC could unveil formulation problems and is potentially useful for comparability studies.

Biobleaching of industrial important dyes with peroxidase partially purified from garlic.

An acidic peroxidase was extracted from garlic (Allium sativum) and was partially purified threefold by ammonium sulphate precipitation, dialysis, and gel filtration chromatography using sephadex G-200. The specific activity of the enzyme increased from 4.89 U/mg after ammonium sulphate precipitation to 25.26 U/mg after gel filtration chromatography. The optimum temperature and pH of the enzyme were 50°C and 5.0, respectively. The Km and V max for H2O2 and o-dianisidine were 0.026 mM and 0.8 U/min, and 25 mM and 0.75 U/min, respectively. Peroxidase from garlic was effective in decolourizing Vat Yellow 2, Vat Orange 11, and Vat Black 27 better than Vat Green 9 dye. For all the parameters monitored, the decolourization was more effective at a pH range, temperature, H2O2 concentration, and enzyme concentration of 4.5-5.0, 50°C, 0.6 mM, and 0.20 U/mL, respectively. The observed properties of the enzyme together with its low cost of extraction (from local sources) show the potential of this enzyme for practical application in industrial wastewater treatment especially with hydrogen peroxide. These Vat dyes also exhibited potentials of acting as peroxidase inhibitors at alkaline pH range.

Polydispersity in size-exclusion chromatography: a stochastic approach.

We investigate the impact of polydispersity of the sample molecules on the separation process and on the efficiency of size-exclusion chromatography. Polydispersity was integrated into the molecular (stochastic) model of chromatography; the characteristic function, the band profile and the most important moments of the elution profiles were calculated for several kind of pore structures. We investigated the parameters affected by polydispersity on the separation for a number of pore shapes. Our results demonstrate that even a small distribution in the molecular size (i.e. polydispersity) can contribute substantially to the total width of the chromatographic peak. The pure effect of polydispersity can only be investigated via mathematical modeling, because its contribution to an experimental chromatogram cannot be separated from other band-broadening effects.

Konjac glucomannan microspheres for low-cost desalting of protein solution.

In this study, low-cost konjac glucomannan (KGM) microspheres used for desalting were developed by an inverse dispersion method. High concentration of KGM was pretreated to reduce viscosity by acid hydrolysis method under the condition of high temperature and pressure. The selectivity of the obtained cross-linked KGM gels with different degree of crosslinking was studied by constructing calibration curves (K(av)) of standard molecular weight substances. The stability of the gels was investigated, which showed that the KGM microspheres are tolerant to a wide range of pH and stable in all commonly used aqueous buffers, and insensitive to autoclaving as well. Furthermore, protein-desalting performances of GM-1250, a cross-linked KGM microsphere, were evaluated with two proteins, bovine serum albumin and filamentous hemagglutinin, which turned out that GM-1250 is comparable to the widely used commercial product--Sephadex G25 Fine. From economic considerations, KGM gel is a prospective alternative for dextran gels in protein desalting process.

Assessment of immunoglobulin concentrates on thrombogenic activity by thrombin generation assay, prekallikrein activator assay, and size-exclusion chromatography.

BACKGROUND: Immunoglobulin G (IgG) concentrates have recently been found to be contaminated with procoagulant impurities causing thromboembolic events (TEEs) in vivo. In this study the question was raised whether a thrombin generation assay (TGA) will be able to characterize IgG samples from the Austrian market with regard to their thrombogenic potential. STUDY DESIGN AND METHODS: A total of 44 IgG concentrates have been assayed by TGA employing pooled normal plasma and Factor (F)XI-deficient plasma (FXIdp). Furthermore, the prekallikrein activator assay including determination of blank values, size-exclusion chromatography, and further test systems required for batch release testing of IgG concentrates according to the European Pharmacopeia (Pharm. Eur.) were carried out. RESULTS: All samples complied with acceptance criteria stated in the Plarm. Eur. and/or prescribed by the marketing approval. One intravenous immunoglobulin (IVIG) involved in TEEs exceeded a threshold level of 350 nmol peak thrombin, which was not exceeded after change of manufacture and by all the other IVIGs tested. Two hyperimmune globulins revealed elevated peak thrombin levels of up to 810 nmol in FXI and up to 285 nmol in FXIdp. CONCLUSION: The study indicates that the TGA is able to reliably predict procoagulant activities probably associated with the presence of FXIa and potential thrombogenicity. Comparison of thrombin generation with product-specific acceptance criteria as well as variables from other test systems as amidolytic activity and molecular size can help to monitor IgG quality and manufacturing changes with regard to thrombogenicity.

Assessment of recombinant human parathyroid hormone: correlation of LC methods with bioassays.

Reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography (SE-LC) methods were validated for the assessment of recombinant human parathyroid hormone (rhPTH 1-34). The gradient RP-LC method was carried out on a Zorbax 300 SB C(18) column (150 mm × 4.6 mm i.d.), maintained at 40 °C. The mobile phase A consisted of 0.1 M sodium sulphate buffer, pH 2.3, and the mobile phase B was acetonitrile. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.), maintained at 25 °C. The mobile phase consisted of 0.1 M phosphoric acid buffer, pH 2.5, run isocratically at a flow rate of 0.7 mL min(-1). Chromatographic separation was obtained with retention times of 12.2 min, and 13.2 min, and was linear over the concentration range of 1-250 µg mL(-1) (r(2) = 0.9997) and 2-300 µg mL(-1) (r(2) = 0.9993), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 214 nm. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.49% and 100.22%, with bias lower than 1.12% and 0.81% respectively. Moreover, the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed significant differences (p < 0.05). Chromatographic methods were applied for the content/potency assessment of rhPTH and related proteins in biopharmaceutical injectable dosage forms, and the results were correlated with those of in vitro and in vivo bioassays. It is concluded that the employment of the methods in conjunction allows a great improvement in monitoring stability, contributing to evaluate alternatives which improve the quality control and thereby assure the therapeutic efficacy of the biotechnology-derived medicine.

Biological response of free-living mouse Mus spretus from Doñana National Park under environmental stress based on assessment of metal-binding biomolecules by SEC-ICP-MS.

A metallomic approach based on the use of size-exclusion chromatography (Superdex-75) with inductively coupled plasma mass spectrometry (ICP-MS) detection is combined with anion or cation exchange chromatography to characterize the biological response of the free-living mouse Mus spretus. The approach has been applied to contaminated and non-contaminated areas from Doñana National Park (southwest Spain) and the surroundings. Several areas affected by differential contamination from mining, industrial, and agricultural activities have been considered. The high presence of Mn, Cu, and Zn in liver and As and Cd in kidney is remarkable, especially in contaminated areas. The size exclusion chromatograms traced by Mn in liver cytosolic extracts are more intense than in kidney; a Mn-peak matching with the standard of 32 kDa (superoxide dismutase) is present in these organs, and its intensity is correlated with the concentration of Mn in the extracts. High-intensity peaks traced by Cu, Zn, and Cd at 7 kDa (matching with metallothionein I standard) in liver extract are triggered by the presence of contaminants. Other peaks related with molecules of 32 and 67 kDa traced by Cu and Zn can also be observed, although their intensity is higher in sites with low contamination. In kidney extracts, the presence of a Cd-peak with Mr of 7 kDa (tentatively Cd-metallothionein) with high intensity under the action of contaminants was observed, but high biological responses are also proven in the protected area of the Park, which denotes a progressive increase of diffuse contamination.

A simple and sensitive method for lipoprotein and lipids profiles analysis of individual micro-liter scale serum samples.

A simple and sensitive method to determine lipoprotein and lipids profiles in micro-liter scale individual serum sample is not presently available. Traditional lipoprotein separation techniques either by ultra-centrifugation or by liquid chromatography methods have their disadvantages in both lipoprotein separation and lipids component quantification. In this study we used small volume needing size-exclusion fast protein liquid chromatography to separate different lipoprotein subclasses in 50µL serum. And lipids contents, such as cholesterol, cholesterol ester and triacylglycerol, were measured by using two different fluorescence-based lipid detection methods. With this method, very low density lipoprotein, low density lipoprotein and high density lipoprotein could be easily separated, and follow-up lipid detection was completed by simple kinds of reactions. Serum lipoprotein and lipids profiling from C57BL/6 mice (n=5) and human (n=5) were analyzed. The elution profiles of five individuals were highly reproducible, and there were lipoprotein and lipids distribution variations between C57BL/6 mice and human beings. In conclusion, this method which combined small volume needing size-exclusion fast protein liquid chromatography and fluorescence-based lipids measurement, provided a simple, efficient, integrity and reproducible procedure for determining serum lipoprotein and lipids profiles in micro-liter scale levels. It becomes possible that determination of lipoprotein profiles and gaining information of lipids in different lipoproteins can be accomplished simultaneously.