Denaturation Of Serum Proteins Research Articles

Comparative study on microbiological, physicochemical and nutritional properties of whole cow milk by thermal and non-thermal processing technologies

The majority of the bioactive components in milk exhibit sensitivity to heat treatments. This research is the first systematically compare the effects of thermal (low-temperature long time pasteurization, LTLT; high-temperature short time pasteurization, HTST; ultra-high temperature, UHT; microwave-assisted heating; retort sterilization) and non-thermal (ultraviolet-C, UV-C; high hydrostatic pressure processing, HPP; filtered sterilization; high-intensity ultrasound, HIUS) treatments on the physicochemical properties and immune-active serum proteins of whole milk aliquots under the equivalent microbicidal efficacy. LTLT and HTST had little impact on whole milk. HPP at 400 MPa for 5 min was an optimal alternative to pasteurized milk in terms of equivalent microbicidal efficacy, higher nutrient retention (∼6% and 16.2% loss for IgG and lactoferrin) and fewer oxidation products (0.69 nmol/mg protein carbonyls). The serum protein denaturation of 600 MPa HPP-treated milk (79.4% and 31.36% loss for IgG and lactoferrin) was significantly higher than in pasteurized milk, but still lower than in UHT milk (97.16% and 70.85% loss for IgG and lactoferrin). Since photo-oxidation induced by high-dose irradiation was detrimental to the organoleptic properties, relying on UV-C as stand-alone replacement to pasteurization is not advisable. Combined with existing pasteurization methods, UV-C can achieve superior microbial inactivation at mild sterilization temperatures. HIUS, a promising novel tool, should be further optimized regarding the relevant standards to enable it to be used in industrial-scale processing and reproducibility. Compared with traditional thermal treatments, non-thermal strategies offer a significant reduction in the microbial load of raw milk while better preserving bioactive proteins, However, optimizing processing parameters remains essential to strike a balance between microbial inactivation and retention of active substances.

Lab-in-syringe automated protein precipitation and salting-out homogenous liquid-liquid extraction coupled online to UHPLC-MS/MS for the determination of beta-blockers in serum

A sample preparation method involving tandem implementation of protein precipitation and salting-out homogenous liquid-liquid extraction was developed for the determination of beta-blockers in serum. The entire procedure was automated using a computer-controlled syringe pump following the Lab-In-Syringe approach. It is based on the denaturation of serum proteins with acetonitrile followed by salt-induced phase separation upon which the proteins accumulate as a compact layer at the interphase of the solutions. The extract is then separated and diluted in-syringe before being submitted to online coupled UHPLC-MS/MS. A 1 mL glass syringe containing a small stir bar for solution mixing at up to 3000 rpm, was used to deal with sample volumes as small as 100 μL. A sample throughput of 7 h−1 was achieved by performing the chromatographic run and sample preparation procedure in parallel. Linear working ranges were obtained for all analytes between 5 and 100 ng mL−1, with LOD values ranging from 0.4 to 1.5 ng mL−1. Accuracy values in the range of 88.2–106% and high precision of <11% RSD suggest applicability for routine analysis that can be further improved using deuterated standards.

Bioprospective studies of pigmented ink from Sepioteuthis lessoniana and its molecular identification using CO1 gene

5BackgroundThe present-day world is teeming with numerous diseases due to the changing environment. The enormous growth of population has overburdened the prevailing resources of drugs; hence, drug manufacturers are in the lookout to develop effective and safe drugs in the pharmaceutical field. Marine environment is well known for its secondary metabolites, having a high potential in the research world of medicines. Several successful researches have explored the bioactivities of the marine organisms. In this regard, this study highlights the bioprospective activities of squid ink and identification of the organism using CO1 gene marker.ResultsIn the present study, anti-inflammatory activity evaluated by human red blood cell (HRBC) membrane stabilization assay revealed protection of human blood cells in hypotonic solution confirming ant-inflammatory property of squid ink extract. Bovine serum protein denaturation method for investigating in vitro anti-arthritic activity proved that the ink extract has appreciable inhibitory effect on denatured proteins. The in vitro antioxidative property of the squid ink disclosed remarkable free radical scavenging activity. The squid ink exhibited potent antibacterial activity against three microbial pathogens such as Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. From the molecular study using CO1 gene sequencing, it was found that the given species of squid showed 100% similarity with a species in NCBI GenBank and it was identified to be Sepioteuthis lessoniana.ConclusionsIt is evident from the study that squid ink extract is a good source of anti-inflammatory, antioxidative, anti-arthritic and antimicrobial agents which would replace the existing cost of effective investigations intending to purify these active compounds and its identification of new molecular skeleton can give idea to the development of either the base or a new drug itself in the future.

Blood serum denaturation profile examined by differential scanning calorimetry reflects the effort put into ultramarathon by amateur long-distance runners

The impact of participation in the ultramarathon on the health and mental and physical condition is very complex. Undoubtedly, exercise brings many benefits but also involves health risks. Especially such an extreme effort as the one associated with finishing the ultramarathon run, can be dangerous to the health of the runner. With the variety of possible biomarkers of excessive fatigue that threaten health and life, a question arises which of them are the best and which should be considered in amateur long-distance runners showing particularly high individual variability. In this study differential scanning calorimetry (DSC) has been applied to show the overall effect of the 12-h run on blood sera of participants. Serum samples were obtained from the blood of ten male amateur long-distance runners, collected before and immediately after the run. Distinct changes in the shape of DSC curves have been observed for serum after finishing the run relative to pre-race serum. Statistically significant differences between stages "before" and "after" ultramarathon running have been found for parameters of the endothermic transition associated with denaturation of serum proteins. An increase in the temperature (from 70.9±0.9 to 75.8±2.9°C) and excess heat capacity (from 0.859±0.201 to 1.102±0.226 Jg-1 °C-1) at peak maximum, the enthalpy of serum denaturation (from 18.55±6.52 to 22.08±5.61 Jg-1) and the first moment of the thermal transition with respect to the temperature (from the value of 67.0±2.1 to 72.6±2.1°C) has been observed. These results show a clear impact of running an ultramarathon on the participant's blood serum.

Design, Synthesis, In Silico and In Vitro Studies for New Nitric Oxide-Releasing Indomethacin Derivatives with 1,3,4-oxadiazole-2-thiol Scaffold.

Starting from indomethacin (IND), one of the most prescribed non-steroidal anti-inflammatory drugs (NSAIDs), new nitric oxide-releasing indomethacin derivatives with 1,3,4-oxadiazole-2-thiol scaffold (NO-IND-OXDs, 8a–p) have been developed as a safer and more efficient multitarget therapeutic strategy. The successful synthesis of designed compounds (intermediaries and finals) was proved by complete spectroscopic analyses. In order to study the in silico interaction of NO-IND-OXDs with cyclooxygenase isoenzymes, a molecular docking study, using AutoDock 4.2.6 software, was performed. Moreover, their biological characterization, based on in vitro assays, in terms of thermal denaturation of serum proteins, antioxidant effects and the NO releasing capacity, was also performed. Based on docking results, 8k, 8l and 8m proved to be the best interaction for the COX-2 (cyclooxygense-2) target site, with an improved docking score compared with celecoxib. Referring to the thermal denaturation of serum proteins and antioxidant effects, all the tested compounds were more active than IND and aspirin, used as references. In addition, the compounds 8c, 8h, 8i, 8m, 8n and 8o showed increased capacity to release NO, which means they are safer in terms of gastrointestinal side effects.

Physicochemical evaluation, in vitro anti-inflammatory, in vitro anti-arthritic activities and GC-MS analysis of the oil from the leaves of Gaultheria fragrantissima Wall of Meghalaya

The aim of our present study is to assess the in vitro anti-inflammatory activity against denaturation of proteins whereby the test oil sample at different concentrations was incubated with egg albumin and the absorbance was determined at 660 nm. The anti-arthritic activity was investigated by two methods; firstly, in-vitro method by bovine serum protein denaturation and the absorbance was measured at 255 nm and secondly in vitro method by egg albumin denaturation where the absorbance was measured at 660 nm. In all the studies diclofenac sodium was used as thestandard drug. The physicochemical parameters like colour, solubility, refractive index, boiling point, specific gravity, carbon residue, iodine value, acid value, ester value were evaluated. The different components of the volatile oil were determined by GC-MS analysis. Ten organic compounds were identified out of which Methyl salicylate C8H8O6 was found to be the most dominant organic compound of Gaultheria fragrantissima oil (97.7%). The present results exhibited a concentration dependent inhibition of protein (albumin) denaturation by the test oil. The study reveals that diclofenac sodium was less effective when compared with the test oil. From the present findings it can be concluded that the essential oil of Gaultheria fragrantissima from Meghalaya possessed significant anti-inflammatory and anti- arthritic effects against the denaturation of protein in vitro. This effect could be due to the high content of methyl salicylate (97.7%) which is an inflammation-fighting compound. The high altitude and conducive climatic conditions of Meghalaya make wintergreen from this region far more superior in comparison to the ones grown in other parts of the world. Keywords: Gaultheria fragrantissima, anti-inflammatory, anti-arthritic, GC-MS, methyl salicylate.

A sensitive antibody-free 2D-LC-MS/MS assay for the quantitation of myostatin in the serum of different species.

Aim: Myostatin (MSTN) is an attractive therapeutic target for the treatment of muscle degeneration-related diseases and is being evaluated as a target engagement biomarker. Methods: A sensitive 2D-LC-MS/MS assay was developed to quantify MSTN in different animal species. Sample preparation involved SDS denaturation of serum proteins followed by tryptic digestion and peptide enrichment by SPE. Results: The assay was validated with LLOQ of 2.5ng/ml in rat and monkey serum. The precision was within 13.7%, and the bias was within±12.6% for all quality control samples in authentic matrices. Conclusion: This new assay was successfully applied to measure MSTN in mouse, rat, monkey and human serum. The total MSTN in rat and monkey serum was elevated following administration of an MSTN inhibitor.

Policosanol composition, antioxidant and anti-arthritic activities of milk thistle (Silybium marianum L.) oil at different seed maturity stages

BackgroundSeveral anti-arthritic drugs and synthetic antioxidants have wide pharmaceutical uses and are often associated with various side effects on the human health. Dietary seed oils and their minor components like policosanol may offer an effective alternative treatment for arthritic and oxidative-stress related diseases. The biological effects of seed oils were affected by different parameters such as the stage of seed maturity. Hence, this study seeks to determine the policosanol content, antioxidant and anti-arthritic activities of milk thistle (Silybium marianum L.) oil extracted at various stages of seed maturation.MethodsMilk thistle oil samples were extracted from seeds collected at three maturation stages (immature, intermediate, and mature). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) radical scavenging assays were used to determine the antioxidant activity of the extracted oils. The anti-arthritic activity of oil samples was evaluated with bovine serum protein denaturation and egg albumin denaturation methods. Gas chromatography coupled to mass spectrometry (GC-MS) was employed to determine the policosanol profile.ResultsPolicosanol profile, antioxidant and anti-arthritic activities of milk thistle oil were influenced by the seed maturity stages. The oil extracted from the immature seeds had the highest total policosanol content (987.68 mg/kg of oil) and displayed the maximum antiradical activity (96.42% and 90.35% for DPPH test and ABTS assay, respectively). Nine aliphatic alcohols were identified in the milk thistle oil. The dominant poliosanol in the mature seed oil was octacosanol (75.44%), while triacontanol was the major compound (40.25%) in the immature seed oil. Additionally, the maximum inhibition of bovine serum protein denaturation (92.53%) and egg albumin denaturation (86.36%) were observed in immature seed oil as compared to mature seed oil. A high correlation was found between the total policosanol content, anti-arthritic activity and antioxidant capacity of oil.ConclusionsThe milk thistle oil exhibited a potential anti-arthritic and antioxidant activities and that it might contribute to the protection of humans from a variety of diseases like rheumatoid arthritis. Also, it could serve as natural antioxidant and anti-arthritic agents for application in the food industries and pharmaceutic. Policosanol level in the seed oils might contribute to their anti-arthritic and antioxidant activities.

Structural changes induced by high-pressure processing in micellar casein and milk protein concentrates.

Reconstituted micellar casein concentrates and milk protein concentrates of 2.5 and 10% (wt/vol) protein concentration were subjected to high-pressure processing at pressures from 150 to 450 MPa, for 15 min, at ambient temperature. The structural changes induced in milk proteins by high-pressure processing were investigated using a range of physical, physicochemical, and chemical methods, including dynamic light scattering, rheology, mid-infrared spectroscopy, scanning electron microscopy, proteomics, and soluble mineral analyses. The experimental data clearly indicate pressure-induced changes of casein micelles, as well as denaturation of serum proteins. Calcium-binding αS1- and αS2-casein levels increased in the soluble phase after all pressure treatments. Pressurization up to 350 MPa also increased levels of soluble calcium and phosphorus, in all samples and concentrations, whereas treatment at 450 MPa reduced the levels of soluble Ca and P. Experimental data suggest dissociation of calcium phosphate and subsequent casein micelle destabilization as a result of pressure treatment. Treatment of 10% micellar casein concentrate and 10% milk protein concentrate samples at 450 MPa resulted in weak, physical gels, which featured aggregates of uniformly distributed, casein substructures of 15 to 20 nm in diameter. Serum proteins were significantly denatured by pressures above 250 MPa. These results provide information on pressure-induced changes in high-concentration protein systems, and may inform the development on new milk protein-based foods with novel textures and potentially high nutritional quality, of particular interest being the soft gel structures formed at high pressure levels.

The influence of ultra-pasteurization by indirect heating versus direct steam injection on skim and 2% fat milks

Fluid milk is traditionally pasteurized by high temperature, short time (HTST) pasteurization, which requires heating to at least 72°C for 15 s. Ultra-pasteurization (UP) extends milk shelf life and is defined as heating to at least 138°C for 2 s. The UP process can be done by indirect heating (IND) or by direct steam injection (DSI). The influence of these 2 UP methods on milk flavor has not been widely investigated. The objective of this study was to compare the effect of HTST, IND-UP, and DSI-UP on sensory perception of fluid milk. Raw skim and standardized 2% milks were pasteurized at 140°C for 2.3 s by IND or DSI or by HTST (78°C, 15 s) and homogenized at 20.7 MPa. The processed milks were stored in light-shielded opaque high-density polyethylene containers at 4°C and examined by descriptive analysis and microbial analysis on d 3, 7, and 14. Furosine and serum protein denaturation analyses were performed on d 0 and 14 as an indicator of heat treatment. Last, consumer acceptance testing was conducted at d 10, with adults (n = 250) and children (ages 8 to13 y, n = 100) who were self-reported consumers of skim or 2% milk; consumers only received samples for either skim or 2% milk. The entire experiment was repeated in triplicate. Milks treated by HTST had lower cooked flavor than either UP milk. Milks heated by DSI-UP were characterized by sulfur or eggy and cooked flavors, whereas IND-UP milks had higher sweet aromatic and sweet taste compared with DSI-UP milk. Aromatic flavor intensities of all milks decreased across 14 d of storage. Furosine concentrations and serum protein denaturation were highest for the IND treatments, followed by DSI and HTST. Furosine content in both skim and 2% milk increased with time, but the increase was faster in IND-UP skim milk. Adult and child consumers preferred HTST milk over either UP milk, regardless of fat content. Ultra-pasteurization by IND or DSI did not affect consumer acceptance at 10 d postprocessing, but traditional HTST milks were preferred by consumers of all ages.

ОТРИМАННЯ МІКРОПАРТИКУЛЯТУ З КОНЦЕНТРАТУ БІЛКІВ МОЛОЧНОЇ СИРОВАТКИ

The article shows the problem of reducing calorie food rations and materials scientific studies the feasibility of using a modified whey protein concentrate as a microparticulate as fat in the production of culinary products are presented. This problem is achieved under conditions of severe mechanical shear on production equipment - dispersant at a temperature higher than the temperature of denaturation of serum proteins. The possibility of obtaining microparticulate, and technological scheme production in the restaurant industry is presented. Functional properties of the resulting product of whey protein , its therapeutic and preventive effects on the human body and high organoleptic properties are defined. The rheological properties of the resulting microparticulate are shown, namely viscosity and strain rate dependence of the shear stress. The possibility of the use Bingham equation to calculate the process yield microparticulate is proved.

Optimized determination of polybrominated diphenyl ethers and polychlorinated biphenyls in sheep serum by solid-phase extraction–gas chromatography–mass spectrometry

We describe a solid-phase extraction (SPE) method, followed by gas chromatography–mass spectrometry (GC–MS), for the simultaneous determination of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in sheep serum samples. The denaturation of serum proteins by formic acid, water-1-propanol and water-2-propanol were compared and optimized. Seven different solid-phase sorbents were tested and it was found that Strata-X cartridge (200mg, 6mL) gave the best recoveries (92–106%, SD<6%, n=3) for all the target analytes. The different extraction solvents (iso-hexane and dichloromethane), either alone or in combination, were used to extract these persistent organic compounds from spiked serum samples by SPE. Removal of co-extracted biogenic materials was achieved using adsorption chromatography with acid modified silica and activated silica. Iso-hexane was found to be the most appropriate solvent for clean-up providing good recoveries and clear chromatographic separation; its use is preferable to that of DCM because it is less environmentally toxic. The limits of detection (LOD) of the proposed method were 47–105pgg−1 and 16–24pgg−1 for the different PBDEs and PCBs studied, respectively. The developed method was linear over the range from 0.05 to 30ngg−1, for all PBDEs except PBDE 183 (0.10–30ngg−1), and from 0.02 to 30ngg−1 for all tested PCB congeners. The established method was successfully applied to sheep serum samples from Scotland, UK, for the determination of the target PBDEs and PCBs.

FALSE-NEGATIVE HISTOPLASMA ANTIGEN IN ACUTE PULMONARY HISTOPLASMOSIS

We report an infant with localized pulmonary histoplasmosis in whom Histoplasma antibody assays, quantitative Histoplasma urine and serum antigen concentrations, and histopathologic findings of a mediastinal mass were nondiagnostic. A provisional diagnosis of histoplasmosis was established by using laboratory methods that increase the sensitivity of the antigen assay using ultrafiltration of urine and ethylenediaminetetraacetic acid/heat denaturation of serum proteins.

Simultaneous determination of bisphenol A, triclosan, and tetrabromobisphenol A in human serum using solid-phase extraction and gas chromatography-electron capture negative-ionization mass spectrometry

This study aimed at optimizing and validating a sensitive method for simultaneous determination of bisphenol A (BPA), triclosan (TCS), and tetrabromobisphenol A (TBBPA) in human serum using solid-phase extraction (SPE) and gas chromatography coupled to electron-capture negative-ionization mass spectrometry (GC-ECNI/MS). Sample preparation involved denaturation of serum proteins with formic acid followed by SPE on an Oasis HLB cartridge. Fractionation was performed on Florisil from which the phenolic compounds were eluted with methanol-dichloromethane (DCM) (5:1, v/v). The phenolic fraction was further derivatized with pentafluoropropionic acid anhydride (30 min at 70 degrees C). Further liquid-liquid partitioning using hexane-DCM (4:1, v/v) and K(2)CO(3) 3% aqueous solution was used to eliminate excess reagent and acidic by-products formed during derivatization. The cleaned extract was injected into a GC-ECNI/MS system operated in selected ion monitoring mode. For thorough method validation, each step of the procedure was rigorously optimized. The method limits of quantitation for BPA, TCS, and TBBPA were 0.28 ng mL(-1), 0.09 ng mL(-1) and 0.05 ng mL(-1), respectively. Furthermore, the method was applied to 21 Belgian human serum samples. The median concentrations obtained for BPA (0.71 ng mL(-1)) and TCS (0.52 ng mL(-1)) in Belgian human serum samples were similar to previously reported data for human fluids. Slightly higher levels of TBBPA (0.08 ng mL(-1)) were found in Belgium samples compared to Norwegian serum.

Different experimental protocols for decontamination affect the cleaning of medical devices. A preliminary electron microscopy analysis

The aim of the present study was to examine the efficiency of different decontamination-cleaning protocols on blood-soiled catheters used for interventional cardiology. Electrophysiology and cardiac ablation disposable devices were contaminated with bacteria-spiked human blood and underwent four different pre-sterilization protocols, including a chlorine-releasing agent, a polyphenolic emulsion, and an enzymatic detergent. Treated samples were examined by optical microscopy, scanning electron microscopy and transmission electron microscopy to identify and characterize biological and inorganic residuals. The use of chlorine as a first treatment caused denaturation of serum proteins and adherence of blood components to the surface of the device, thus hindering the cleaning efficiency of subsequent treatments with enzymatic detergents. An enzymatic/chlorine protocol was more efficient, but was considered to be a greater risk to healthcare staff. Polyphenolic-based treatments had the highest level of efficiency in bioburden removal, but interaction and adsorption of this class of chemicals onto biopolymers might lead to serious concerns about toxicity on subsequent reuse. Adequate pre-sterilization cleaning is fundamental for sterilization success and high-resolution electron microscopy can provide significant and detailed information about the efficiency of chemicals used for cleaning a blood-soiled device.

A Blank Slate? Layer-by-Layer Deposition of Hyaluronic Acid and Chitosan onto Various Surfaces

Although poly(alpha-hydroxy esters), especially the PLGA family of lactic acid/glycolic acid copolymers, have many properties which make them promising materials for tissue engineering, the inherent chemistry of surfaces made from these particular polymers is problematic. In vivo, they promote a strong foreign-body response as a result of nonspecific adsorption and denaturation of serum proteins, which generally results in the formation of a nonfunctional fibrous capsule. Surface modification post-production of the scaffolds is an often-utilized approach to solving this problem, conceptually allowing the formation of a scaffold with mechanical properties defined by the bulk material and molecular-level interactions defined by the modified surface properties. A promising concept is the so-called "blank slate": essentially a surface that is rendered resistant to nonspecific protein adsorption but can be readily activated to covalently bind bio-functional molecules such as extracellular matrix proteins, growth factors or polysaccharides. This study focuses on the use of the quartz crystal microbalance (QCM) to follow the layer-by-layer (LbL) electrostatic deposition of high molecular weight hyaluronic acid and chitosan onto PLGA surfaces rendered positively charged by aminolysis, to form a robust, protein-resistant coating. We further show that this surface may be further functionalized via the covalent attachment of collagen IV, which may then be used as a template for the self-assembly of basement membrane components from dilute Matrigel. The response of NIH-3T3 fibroblasts to these surfaces was also followed and shown to closely parallel the results observed in the QCM.

Involvement of oxygen free radicals in the serum‐mediated increase of benzoquinone genotoxicity

The genotoxicity of benzoquinone (BQ), a toxic benzene metabolite, is greatly enhanced by the presence of fetal calf serum (FCS) in the incubation medium. The FCS effect is abolished by heat denaturation of serum proteins and is slightly decreased by dialysis. In the present study, we have further investigated the serum effect on BQ genotoxicity by measuring DNA damage produced in peripheral blood mononuclear cells (PBMCs) using the Comet assay. We have also evaluated the effect of human serum and rat liver post-mitochondrial fraction (S9) on the DNA damage produced by BQ. Both human serum and a rat liver S9 enhanced the genotoxicity of BQ in a manner similar to FCS. Gel filtration experiments showed that all the enhancing activity of the serum eluted with the high molecular weight fractions, suggesting that low molecular weight serum constituents do not play an important role in modulating genotoxicity. The genotoxicity-enhancing activity of serum was inhibited by the iron chelator deferoxamine and by superoxide dismutase and catalase. Incubating PBMCs with BQ in the presence of FCS also resulted in the accumulation of intracellular peroxides as demonstrated by loading the cells with 2',7'-dichlorofluorescin diacetate and analyzing for peroxide formation by flow cytometry. These results indicate that oxygen free radicals are involved in the enhancement of BQ-induced DNA damage by serum. We hypothesize that enzyme activities that reduce BQ by transferring single electrons could be the source of the oxygen free radicals.

Simple solid-phase extraction method for determination of polychlorinated biphenyls and selected organochlorine pesticides in human serum

A simple off-line solid-phase extraction (SPE) method for isolation of polychlorinated biphenyls (PCBs) and selected organochlorine pesticides (OCPs) from human serum has been developed. The procedure includes denaturation of serum proteins by a mixture of water–1-propanol, application of the sample by aspiration twice repeatedly through the SPE column and elution with a mixture of n-hexane–dichlormethane. After final clean-up the compounds of interest were analysed by gas chromatography with micro-electron capture detection (GC–μECD). The recoveries achieved for PCB congeners using spiked porcine serum samples were 99–120% and for OCPs 88–115%. Relative standard deviations (RSD) ranged from 3 to 7%. The method was applied to real human serum samples and the recoveries of analytes in the serum were proportionally recalculated considering the recovery of the internal standard PCB-174. PCB-103 served as a syringe standard to correct volume of samples analysed. The aim of this study was to develop an effective off-line SPE procedure by optimization of existing SPE methods to supply laborious, solvent- and time-consuming liquid–liquid extraction (LLE) in routine analytical process.

The oxidation-reduction state of serum proteins in multiple sclerosis patients: effect of Interferon beta-1b

The concentration of reduction equivalents in serum was studied in a cohort of healthy individuals, in a group of multiple sclerosis (MS) patients undergoing treatment with interferon beta-1b and another group of MS patients who refused treatment with interferon beta-1b. Two classes of sulfhydryl groups were detectable in serum: (1) the uncovered sulfhydryls, accessible to the oxidation-reduction substrate 5,5-dithiobis-(-2-nitrobenzoic acid) (DTNB); and (2) the hidden sulfhydryls that required previous heat denaturation of serum proteins to become accessible to DTNB. The concentration of the reduced form of both the uncovered- and hidden-type of sulfhydryls was higher in the serum of MS patients than in healthy individuals. Interferon beta-1b lowered the plasma concentration of the uncovered reduced sulfhydryls after 3 months of treatment. This was in contrast to a minor effect of interferon beta-1b in the hidden-form of sulfhydryl groups. The results suggest that the concentration of reduced sulfhydryls is a biochemical marker of the in vivo oxidation/reduction reactions in MS.

Improved sample preparation method for selected persistent organochlorine pollutants in human serum using solid-phase disk extraction with gas chromatographic analysis

An improved solid-phase extraction (SPE) method was developed to isolate and concentrate trace levels of selected POPs (persistent organochlorine pollutants) in human serum prior to GC–MS in SIM mode or GC–ECD quantitation. The extraction involves denaturation of serum proteins with formic acid, SPE using C 18 Empore™ disk cartridges, followed by elimination of lipid interferences using a sulfuric acid wash of the eluate. Use of the SPE disk improved assay throughput and gave a cleaner analytical matrix compared with previously reported solid-phase and liquid–liquid extraction techniques. The extraction method provided consistent recoveries at three fortification levels using 13C 12 PCB 149 as internal standard. Recoveries ranged from 48 to 140% for organochlorine pesticides (6.25, 12.5 and 25 ng/ml) and 71 to 126% for polychlorinated biphenyls (0.625, 1.25 and 2.5 ng/ml).