Human Serum

The stability of interleukin 6 (IL-6), its soluble receptor (sIL-6R), IL-10 and CC16 or uteroglobin (an endogenous cytokine inhibitor) in human serum was examined using an accelerated stability testing protocol according to the Arrhenius equation. Further, the effect of time delay between blood sampling and sample processing, clotting temperature and repeated freeze-thaw cycles on serum levels of these proteins were determined. Paired serum samples were stored at 4 C, 20 C, 30 C and 40 C for 1 to 21 days. We found that IL-6 and CC16 concentrations did not change at 4 C, 20 C and 30 C. Interleukin-6 concentrations significantly declined after 11 days at 40 C. The concentrations of sIL-6R and IL-10 did not change at 4 C but significantly decreased at 20 C (after 21 and 14 days respectively), 30 C and 40 C (after 1 day at both temperatures for both cytokines). Arrhenius-plots indicated that sIL-6R and IL-10 are stable for at least several years at 20 C and 70 C, respectively. Since their relative stability, no Arrhenius-plot could be calculated for IL-6 and CC16. The concentrations of the proteins examined were not significantly altered by repeated freeze-thaw cycles, nor by extended clotting times at 4 C or 20 C. We conclude that serum samples for the determination of IL-6, sIL-6R and CC16 can be stored at 20 C for several years, but for IL-10 determinations, storage at 70 C is recommended.

Cisplatin is for a long time in clinical use as efficient antitumor drug. The success in cisplatin-based chemotherapy, however, strongly depends on how careful the drug's dosages are monitored in order to reduce severe side-effects and overcome cellular resistance. The use of micellar electrokinetic chromatography with direct UV detection is described for the determination of intact cisplatin in human serum. The main product of drug's hydrolytic metabolism, cis-diammineaquachloroplatinum(II), was quantified using capillary zone electrophoresis in combination with indirect UV detection and on-line transient isotachophoresis preconcentration. The detection limits of platinum species studied were about 2-3 mol l −1 that allows the proposed methods to be applied for quantification of the administered levels of cisplatin as well as drug's active metabolite. Changes in the speciation of cisplatin occurring after intravenous administration can also be monitored using these simple and convenient CE techniques.

Cholinergic loss is the single most replicated neurotransmitter deficiency in Alzheimer's disease (AD) and has led to the use of acetylcholinesterase inhibitors (AChE-Is) and unselective cholinesterase inhibitors (ChE-Is) as the mainstay of treatment. AChE-Is and ChE-Is, however, induce dose-limiting adverse effects. Recent studies indicate that selective butyrylcholinesterase inhibitors (BuChE-Is) elevate acetylcholine (ACh) in brain, augment long-term potentiation, and improve cognitive performance in rodents without the classic adverse actions of AChE-Is and ChE-Is. BuChE-Is thereby represent a new strategy to ameliorate AD, particularly since AChE activity is depleted in AD brain, in line with ACh levels, whereas BuChE activity is elevated. Our studies have focused on the design and development of cymserine analogues to induce selective time-dependent brain BuChE inhibition, and on the application of innovative and quantitative enzyme kinetic analyses to aid selection of drug candidates. The quantitative interaction of the novel inhibitor, dihydrobenzodioxepine cymserine (DHBDC), with human BuChE was characterized. DHBDC demonstrated potent concentration-dependent binding with BuChE. The IC 50 and specific new kinetic constants, such as K T50 , P PC , K T1/2 and R I , were determined at dual substrate concentrations of 0.10 and 0.60 mM butyrylthiocholine and reaction times, and are likely attainable in humans. Other classical kinetic parameters such as K ia , K ma , V ma and V mi were also determined. In synopsis, DHBDC proved to be a highly potent competitive inhibitor of human BuChE in comparison to its structural analogue, cymserine, and represents an interesting drug candidate for AD.

A sensitive optical method based on quantum dot (QD) technology is demonstrated for the detection of an important cancer marker, total prostate-specific antigen (TPSA) on a disposable carbon substrate surface. Immuno-recognition was carried out on a carbon substrate using a sandwich assay approach, where the primary antibody (Ab)-protein A complex covalently bound to the substrate surface, was allowed to capture TPSA. After the recognition event, the substrate was exposed to the biotinylated secondary Abs. After incubation with the QD streptavidin conjugates, QDs were captured on the substrate surface by the strong biotin-streptavidin affinity. Fluorescence imaging of the substrate surface illuminated the QDs, and provided a very sensitive tool for the detection of TPSA in undiluted human serum samples with a detection limit of 0.25ng/mL. The potential of this method for application as a simple and efficient diagnostic strategy for immunoassays is discussed.

The insecticide DDT is still used in specific areas of South Africa for indoor residual spray (IRS) to control malaria vectors. Local residents could be exposed to residues of DDT through various pathways including indoor air, dust, soil, food and water. The aims of this study were to determine the levels of DDT contamination, as a result of IRS, in representative homesteads, and to evaluate the possible routes of human exposure. Two villages, exposed (DV) and reference (TV) were selected. Sampling was done two months after the IRS process was completed. Twelve homesteads were selected in DV and nine in TV. Human serum, indoor air, floor dust, outside soil, potable water, leafy vegetables, and chicken samples (muscle, fat and liver) were collected and analyzed for both the o,p′-and p,p′-isomers of DDT, DDD and DDE. DDT was detected in all the media analyzed indicating a combination of potential dietary and non-dietary pathways of uptake. DV had the most samples with detectable levels of DDT and its metabolites, and with the exception of chicken muscle samples, DV also had higher mean levels for all the components analyzed compared to TV. Seventy-nine percent of participants from DV had serum levels of DDT (mean ∑DDT 7.3 μg g −1 lipid). These residues constituted mainly of p,p′-DDD and p,p′-DDE. ∑DDT levels were detected in all indoor air (mean ∑DDT 3900.0 ng m − 3) and floor dust (mean ∑DDT 1200.0 μg m − 2) samples. Levels were also detected in outside soil (mean ∑DDT 25.0 μg kg − 1) and potable water (mean ∑DDT 2.0 μg L − 1). Vegetable sample composition (mean ∑DDT 43.0 μg kg − 1) constituted mainly p,p′-DDT and p,p′-DDD. Chicken samples were highly contaminated with DDT (muscle mean ∑DDT 700.0 μg kg − 1 , fat mean ∑DDT 240,000.0 μg kg − 1 , liver mean ∑DDT 1600.0 μg kg − 1). The results of the current study raise concerns regarding the potential health effects in residents living in the immediate environment following DDT IRS.

The human serum resistance associated (SRA) gene isolated from a Ugandan strain of Trypanosoma brucei rhodesiense has been shown to be capable by itself of conferring the trait of human infectivity on T.b. brucei by transfection. This gene has also been identified in several other isolates of T.b. rhodesiense, but not in the other human pathogenic trypanosome in Africa, T.b. gambiense, casting doubt on its ubiquity and function. Here, we show that this gene occurs in T.b. rhodesiense from sleeping sickness foci throughout East Africa (Ethiopia, Uganda, Kenya, Tanzania, Zambia, Botswana), but is not found in T.b. brucei isolates or any other trypanosomes of subgenus Trypanozoon. SRA genes from 10 T.b. rhodesiense isolates from five disease foci were compared and were 97.9-99.7% homologous, with three minor sequence variants. PCR amplification of this gene forms the basis of a new test to identify T.b. rhodesiense. This is the first molecular marker identified for T.b. rhodesiense, despite intensive efforts over the past 20 years. It will be invaluable for identification of animal reservoir hosts and detection of T.b. rhodesiense in its tsetse fly vector. Strain typing using minisatellite markers showed considerable genetic heterogeneity between T.b. rhodesiense isolates, despite the presence of the conserved SRA gene. These results are consistent with the hypothesis that new T.b. rhodesiense strains arise by genetic exchange among T. brucei ssp. spreading the SRA gene and thereby the trait for human serum resistance and human infectivity.

A rapid, simple and sensitive assay was developed for determination of captopril in human serum. We employed silica gel cartridge for efficient extraction of captopril adduct from human serum. Captopril was trapped with p-bromophenacyl bromide (pBPB) to give captopril-pBPB adduct. A 4-ml benzene extract of 1 ml acidified serum was passed through 1 ml silica gel cartridge. Potential interfering compounds were removed with 4-ml benzene wash. The captopril-pBPB adduct was eluted with 0.5 ml acetonitrile. Of this acetonitrile solution (100 ~tl) was injected on an ODS reverse phase HPLC column (chromatography conditions; mobile phase; acetonitrile water acetic acid (225:270:5, v/v/v), flow rate; 1 ml rain ~, detection; UV at 263 nm). It is found that this method is accurate and does not require time consuming evaporation-concentration steps. Recovery exceeds 94% and analytical responses are linear over captopril concentration range from 50 up to 1000 ng ml-i. The coefficients of variation from 108 ng ml t to 605 ng ml ~ ~ varied between 3.7-7.7% and the relative error did not exceed 3.7%. Therefore, this method can be used for routine clinical monitoring and in pharmacokinetic studies of captopril.

The concentrations of retinol, a-tocopherol, and trans-b-carotene in lyophilized serum stored at 2 258C and 2 808C have been monitored for 10 years. There was no evidence of degradation of any of these compounds over the 10-year period. Retinol, a-tocopherol, and trans-b-carotene were less stable at 2 258C in liquid-frozen serum than they were in lyophilized serum. At 2 808C, trans-b-carotene levels were stable for up to 3 years of storage in liquid-frozen serum. Both retinol and a-tocopherol appeared stable in liquid-frozen serum for at least 5 years at 2 808C. The effect of repeated freeze / thaw cycles on retinol, a-tocopherol, trans-lycopene, and trans-bcarotene in liquid-frozen and reconstituted lyophilized serum both stored at 2 208C was also studied. Retinol, a-tocopherol, trans-lycopene, and trans-b-carotene in reconstituted lyophilized serum stored at 2 208C were stable for at least 3 days with minimal ( , 5) freeze / thaw cycles.

Entacapone has a relatively low oral bioavailability which may, in part, be due to its low aqueous solubility at low pH and/or its hydrophilic character at neutral pH. Various novel N -alkyl and N,Ndialkyl carbamate esters of entacapone were synthesized as possible prodrugs of entacapone in order to increase its aqueous solubility at an acidic pH and to increase its lipophilicity at neutral pH. Oral bioavailability of entacapone and selected carbamate esters were investigated in rats. Both N -alkyl and N,N -dialkyl carbamate esters were relatively stable against chemical hydrolysis at pH 7.4 (t 1/2 ϭ 14.9Ð 20.7 h), but hydrolyzed rapidly (t 1/2 ϭ 0.8Ð2.7 h) in human serum. However, in contrast to N -alkyl carbamates, N,N -dialkyl carbamates did not release entacapone in in vitro enzymatic hydrolysis (human serum) studies. N -Alkyl carbamates, 2a-c, showed increased aqueous solubility at pH 7.4, of which 2a and 2c also show increased aqueous solubility at pH 5.0, compared to entacapone. In addition to increased aqueous solubility, 2c showed increased lipophilicity at pH 7.4. However, two N -alkyl carbamates of entacapone did not increase the oral bioavailability of the parent drug in rats. Thus, it can be concluded that the relatively low lipophilicity of entacapone is not the cause of its low bioavailability.

Diffuse reflectance UV (DR-UV) spectroscopy was used for the first time to quantity the amount of creatinine adsorbed onto mordenite type zeolite from two different media: Phosphate Buffered Saline solution (D-PBS) and D-PBS solution containing albumin at a concentration corresponding to human serum conditions (50 g/L). The UV spectra of creatinine adsorbed onto mordenite zeolites was quantitatively treated using the Kubelka-Munk formalism. Quantification by diffuse reflectance UV spectroscopy after microporous solids extraction was linearly correlated with creatinine concentration whatever the medium. This study evidences that DR-UV spectroscopy may be an efficient method to directly quantify the amount of chosen molecules present in a liquid medium by pre-concentrating them on a specific solid adsorbent like in the solid phase extraction (SPE) procedure. The results on hand also prove that this is even possible in rather complex conditions. The method is simple, fast, and with a low detection threshold.

The enzymatic hydrolysis of phenyl acetate, catalysed by arylesterase/paraoxonase (EC 3.1.8.1) was studied at 37 • C in Tris buffer, pH 8, by spectrophotometry and flow microcalorimetry, using an enzyme purified from human serum. After correction for buffer protonation and product ionization, the hydrolysis reaction was found to be slightly endothermic, with H = 8.2 kJ mol −1 . Microcalorimetric data were analysed with the integrated Michaelis equation to give the kinetic parameters of the enzyme: Michaelis constant K m = 2.4 mM, catalytic constant k cat = 2.4 × 10 3 s −1 , bimolecular rate constant k s = 1.0 × 10 6 M −1 s −1 . These results were in agreement with the spectrophotometric method. This study confirms the usefulness of microcalorimetry in the field of enzyme kinetics.

Oxidative modification of low-density lipoprotein (LDL) represents an important factor in atherogenesis. In the present study, we have investigated the antioxidant capability of diphenyl diselenide (PhSe) 2 , a simple organoseleno compound, against copper (Cu 2+ ) and peroxyl radical-induced human LDL oxidation in vitro. In initial studies using human serum, (PhSe) 2 caused a dose-dependent inhibition of Cu 2+induced lipid peroxidation, which was correlated to thiol consumption. (PhSe) 2 increased lipid peroxidation lag phase and decreased lipid peroxidation rate in isolated human LDL, evaluated by measuring both conjugated diene (CD) and thiobarbituric acid reactive substances (TBARS) levels. Consistent with these observations, (PhSe) 2 showed a marked inhibitory effect on 2,2-azobis(2-amidinopropane dihydrochloride) (AAPH)-induced oxidation of LDL or parinaric acid (PnA) incorporated into LDL. (PhSe) 2 also displayed a dose-dependent protective effect against Cu 2+ -induced lipid peroxidation in rat aortic slices. Interestingly, besides the antioxidant effects of (PhSe) 2 toward the lipid moieties of LDL, which was related to its thiol-peroxidase activity, protein moieties from human isolated LDL were also protected against Cu 2+ -induced oxidation. The results presented herein are the first to show that (i) (PhSe) 2 inhibits lipid peroxidation in human isolated LDL in vitro, (ii) this phenomenon is related to its thiol-peroxidase activity, and (iii) this chalcogen also prevents the oxidation of protein moieties of human LDL. Taken together, such data render (PhSe) 2 a promising molecule for pharmacological studies with respect to the atherogenic process.

Complement-dependent cellular cytotoxicity: Lymphoblastoid lines that activate complement component 3 (C3) and express C3 receptors have increased sensitivity to lymphocyte-mediated lysis in the presence of fresh human serum (spontaneous cytotoxicity)

In 1999, a campaign of the Flemish Ministry of Health, Belgium was set up to assess pollutant concentrations and related health effect biomarkers in humans living in two regions of Flanders. The study was called the 'Flemish Environment and Health Study' (FLEHS). Concentrations of selected organochiorine pesticides, polychlorinated biphenyls (PCB) and polychlorinated dibenzo-p-dioxins (PCDD) and flirans (PCDF) were measured by gas chromatography-mass spectrometry in 47 pooled human serum samples originating from 200 individual women between 50 and 65 years living in two Flemish regions. The CALUX ® (Chemical-Activated Luciferase gene eXpression) bioassay was assessed on the same pools. The correlation between CALUX-TEQ and total TEQ (sum of PCDD/ PCDF, non-and mono-ortho PCBs) varied from 0.43 to 0.73 for the rural and urban region, respectively. The mean value for the total TBQ (75 pg WHO-TEQ/g fat) was two times higher than the mean TEQ value determined with the CALUX bioassay (36 pg TEQ/g fat). This shows that the assessment of dioxin-like exposure by these two measurements was different. However, regional differences in concentrations were observed for neither total TEQs, nor CALUX-TEQs. It was concluded that the CALUX ® can be an alternative screening tool for biomonitoring purposes, especially when the objective is to compare different groups of people (e.g. living in different regions).

Human serum paraoxonase (PON1, EC 3.1.8.1.) is a high-density lipid (HDL)-associated, calcium-dependent enzyme; its physiological substrates are not known. In this study, a new purification strategy for human PON1 enzyme was developed using two-step procedures, namely ammonium sulfate precipitation and sepharose-4B-L-tyrosine-1-napthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. Overall purification rate of our method was found 227fold. The V max and K m of the purified enzyme were determined 227.27 EU and 4.16 mM, respectively. The in vitro effects of commonly used antibiotics, namely gentamycin sulfate and cefazolin sodium was also investigated on the purified human serum PON1 enzyme and human liver PON1 enzyme from human hepatoma cell (HepG2). Gentamycin sulfate and cefazolin sodium caused a dose-and time-dependent decrease on PON1 activity in HepG2 cells. Moreover, gentamycin sulfate and cefazolin sodium were effective inhibitors on purified human serum PON1 activity with IC 50 of 0.887 and 0.0084 values, respectively. The kinetics of interaction of gentamycin sulfate and cefazolin sodium with the purified human serum PON1 indicated a different inhibition pattern. Cefazolin sodium showed a competitive inhibition with K i of 0.012 ± 0.00065 mM. However, Gentamycin sulfate was inhibited in non-competitive manner with K i of 0.026 ± 0.015.

Memory impairment is a process associated with alterations in neuronal plasticity, synapses formation, and stabilization. As the neural cell adhesion molecule (NCAM) plays a key role in synaptic bond stabilization, we analyzed the usefulness of soluble NCAM isoforms in the diagnosis of patients with dementia of the Alzheimer type (DAT).NCAM was measured in the sera of 70 control subjects and

J Bras Patol Med Lab • v. 42 • n. 5 • p. 345-350 • outubro 2006 key words y y ey o ds y y key words y y y y y y y y y y y unitermos unitermos unitermos u te os unitermos unitermos abstract Thiol groups have been described as the main responsible for antioxidative effects of plasmatic proteins. Also, thiol serum levels have shown a positive correlation with total antioxidant capacity (TAC) in many studies. Measurement of TAC by substract oxidation-based methods have been widely used as a reference to measure antioxidant status; however, in many cases these methods are inexact or imprecise, usually when performed by manual procedures. In this paper we describe a simple automated procedure for the determination of total thiols in serum, which was based on Ellman's method. It was correlated with several markers of oxidative/antioxidative status, such as TAC and thiobarbituric acid reactive substance test (TBARs). Serum thiol levels were correlated positively with TAC (r = 0.298, r p < 0.001) and negatively p with TBARs levels (r = -0.330, r p < 0.001). The novel automated procedure for thiol groups measurement p can be a great tool in estimation of antioxidant status together with TAC assay. This procedure makes the determination of total thiol groups in large scale possible in clinical chemistry or research laboratories where this approach is necessary. Thiol groups Total antioxidant capacity TBARs Oxidative stress Automation resumo Os tióis são descritos como os principais responsáveis pelos efeitos antioxidantes das proteínas plasmáticas. Além disso, diversos estudos mostram uma correlação positiva entre os níveis séricos de tióis e a capacidade antioxidante total (CAT). A medida da CAT por métodos baseados na oxidação de substratos tem sido amplamente usada como referência na estimativa da capacidade antioxidante em amostras biológicas; porém, em muitos casos esses métodos são inexatos e imprecisos, principalmente quando realizados por procedimentos não-automatizados. Neste artigo descrevemos um procedimento automatizado simples para a determinação de tióis totais no soro, com base no conhecido método de Ellman. A dosagem dos tióis foi correlacionada com diversos marcadores da capacidade oxidante/antioxidante, como a CAT, o teste das substâncias reativas ao ácido tiobarbitúrico (TBARs) e os níveis de peróxidos totais. Os tióis correlacionaram-se positivamente com a CAT (r ( ( = 0,298; r p < 0,001) e negativamente com os níveis de TBARs (r ( ( = -0,330; r p < 0,001). O procedimento aqui descrito para a dosagem de tióis pode ser uma ferramenta importante na medida da capacidade antioxidante juntamente com a dosagem da CAT. A utilização desse novo procedimento possibilita a determinação dos tióis totais em larga escala por laboratórios clínicos e de pesquisa onde essa demanda é necessária. Tióis Capacidade antioxidante total TBARs Estresse oxidativo Automação

In this paper, a novel graft copolymer, poly-(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) with part of the PEG chains carrying a terminal nitrilotriacetic acid group (NTA) was synthesized. Through electrostatic interactions, these polycationic graft co-polymers assemble spontaneously from aqueous solution onto negatively charged surfaces, forming polymeric monolayers that present NTA groups at controlled surface densities on a highly PEGylated background. The NTA-functionalized PLL-g-PEG surfaces proved to be highly resistant to nonspecific adsorption in contact with human serum while allowing the specific and reversible surface binding of GFPuv-6His and &#946;-lactamase-6His in native conformation. Micropatterns consisting of NTA-functionalized PLL-g-PEG in a background of PLL-g-PEG were produced using the &quot;molecular assembly patterning by lift-off&quot; technique. Exposure to Ni&lt;sup&gt;2+&lt;/sup&gt;and GFPuv-6His resulted in a protein pattern of excellent contrast a...

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Background: The Dutch project&quot; Calibration 2000&quot; aims at harmonization of laboratory results via calibration by development of commutable, matrix-based, secondary reference materials. An alternative approach to the NCCLS EP14 protocol for studying commutability ...

The rapid and accurate determination of specific metabolites present in biofluids is a very demanding task which is essential in both medicine and chemistry. Lcarnitine (3-hydroxy-4-N-trimethylammonium butyrate) is an important metabolite which participates in a series of biological paths and therefore its determination is of diagnostic importance. A single quantum coherence filtering 1 H NMR methodology was used for the accurate and rapid determination of L-carnitine in human serum samples. The methodology is based on spectral simplification, and specifically on the distinction of the N-methyl proton signal of L-carnitine that is greatly overlapped in the 1 H-NMR spectrum of serum. The quantitative results provided by the proposed method are in excellent agreement with those obtained by the enzymatic method, which is widely used. The proposed method is rapid (~20 min of experimental time), selective, sensitive, and has good analytical characteristics (accuracy, reproducibility). Selected protein precipitation methods were also investigated and sample pretreatment with EtOH is suggested.

Fluorescence spectroscopy and circular dichroism (CD) spectroscopy were used to investigate the interaction of coumarin-3-carboxylic acid with human serum albumin (HSA) and bovine serum albumin (BSA) under physiological conditions in a buffer solution of pH 7.4. Quenching constants were determined using the Lineweaver-Burk equation to provide a measure of the binding affinity of coumarin-3-carboxylic acid to HSA/BSA. Binding studies concerning

A very sensitive analytical method is proposed for the determination of lithium based on the reaction of Li + ion wiht 1,4-dihydroxyanthraquinone (Quinizarin). In dimethylsulfoxide medium (90%) and in the presence of sodium hydroxide and sodium carbonate, a bluish-violet color (2ma x = 601 nm) develops and is stable over a period of 30 min to 2.5 h. The NaOH and Quinizarin concentrations were optimized simultaneously using the response surface methodology from sequential experimental Doehlert designs. Beer's law is obeyed in the concentration range 14-250 ppb Li + in aqueous and serum matrices, and the errors (RSD) in the determination of 100 ppb Li ÷ are 4.0% and 3.9°/,, respectively. The proposed procedure was satisfactorily applied to the determination of lithium in drugs and human serum (no deproteinization is required). substituents -OH in one ring and -COOH, -PO(OH)2 or -AsO(OH)2 in the second ring have been used as complexing agents of Li ÷ (Thoron, Quinazolinazo, Nitroantranylazo, Phosphonazo R, Arsenazo III) in photometric determinations [3]. Among these, Thoron (o-(2-hydroxy-3, 6-disulfo-1 -naphthylazo) benzenearsonic acid) has been the most used. Proposed by Kuznetsov [4], it gives, together with Li ÷ ion, an orange color in a strong basic medium. The spectrophotometric method using this reagent, developed by Thomason [5], has been used for the determination of lithium in high-purity beryllium and beryllium oxide [6], sea water [7], mollusc shells [8] and blood serum .

A protocol that utilises data (trueness/recovery, precision and robustness) from validation tests to calculate measurement uncertainty was described and applied to a sector field inductively coupled plasma mass spectrometry (SF-ICP-MS)-based method for the determination of Be, Cd, Hg, Ir, Pb, Pd, Pt, Rh, Sb, U, Tl and W in human serum. The method was validated according to criteria issued by international bodies such as AOAC, Eurachem and ISO and the uncertainty in the analytical measurements was estimated following the Eurachem/Citac guide. The methodology was based on dilution of human serum with water and analysis by serum-matched standard calibration. The method quantification limits ranged 0.02 mg/L (Tl, Ir) to 0.26 mg/L (Hg). The coefficients of regression were greater than 0.9991 over a range of two orders of magnitude of concentration. The mean trueness was 101% and the mean recovery on three levels of fortification (1-, 1.5-, and 2-times the baseline serum level) ranged between 93.3% and 106%. The maximum relative standard deviation values for repeatability and within-laboratory reproducibility were 12.8% and 13.5%. The method was robust to slight variations of some critical factors relevant to the sample preparation and SF-ICP-MS instrumentation. The relative expanded uncertainty over three levels of concentration ranged from 11.6% (Hg) to 27.6% (Pt), and the uncertainty on the within-laboratory reproducibility, which included factors such as time, analyst and calibration, represented the main contribution to the overall uncertainty.

In this paper, we represent a label-free biosensor based on immobilization of serum antibodies for rapid detection of viral antigens. Human serum containing specific antibodies against Japanese encephalitis virus (JEV) was immobilized on a silanized surface of an interdigitated sensor via protein A/glutaraldehyde for electrical detection of JEV antigens. The effective immobilization of serum antibodies on the sensor surface was verified by Fourier transform infrared spectrometry and fluorescence microscopy. The signal of the biosensor obtained by the differential voltage converted from the change into non-Faradic impedance resulting from the specific binding of JEV antigens on the surface of the sensor. The detection analyzed indicates that the detection range of this biosensor is 1-10 g/ml JEV antigens, with a detection limit of 0.75 g/ml and that stable signals are measured in about 20 min. This study presents a useful biosensor with a high selectivity for rapid and simple detection of JEV antigens, and it also proposes the biosensor as a future diagnostic tool for rapid and direct detection of viral antigens in clinical samples for preliminary pathogenic screenings in the case of possible outbreaks.

A periodate-resorcinol microassay for the quantitation of total sialic acid (TSA) has been developed. A 96-well microtiter plate format was used as a single reagent mixing well. The principle of this newly-developed assay is the periodate-resorcinol procedure. Conditions for the quantitation of sialic acid in serum, such as the concentration of periodate, resorcinol, incubation time and human serum volume, were optimized. It was found that the optimal concentration of periodic acid and resorcinol reagent were 1.3 mM and 0.6%, respectively. An incubation time of 60 minutes for the reaction of periodic acid and resorcinol with samples was found to give the highest absorbance. These new procedures were used for the quantitation of sialic acid in human serum. It was found that only 5 µL samples were needed to give a varied coefficient of the intra- and inter-assay of 0.79% and 4.68%, respectively. Furthermore, this method demonstrated the recovery percentage of 94.25% by the addition of...

An amperometric lactate biosensor was developed based on a conducting polymer, poly-5,2 0 -5 0 ,2 00 -terthiophene-3 0 -carboxylic acid (pTTCA), and multiwall carbon nanotube (MWNT) composite on a gold electrode. Lactate dehydrogenase (LDH) and the oxidized form of nicotinamide adenine dinucleotide (NAD + ) were subsequently immobilized onto the pTTCA/MWNT composite film. The modified electrode was characterized by quartz crystal microbalance (QCM), scanning electron microscopy (SEM), and electrochemical experiments. The detection signal was amplified by the pTTCA/MWNT assembly onto which a sufficient amount of enzyme was immobilized and stabilized by the covalent bond formation between the amine groups of enzyme and the carboxylic acid groups of the pTTCA/MWNT film. Experimental parameters affecting the sensor responses, such as applied potential, pH, and temperature, were assessed and optimized. Analytical performances and dynamic ranges of the sensor were determined, and the results showed that the sensitivity, stability, and reproducibility of the sensor improved significantly using pTTCA/MWNT composite film. The calibration plot was linear (r 2 = 0.9995) over the range of 5 to 90 lM. The sensitivity was approximately 0.0106 lA/lM, with a detection limit of 1 lM, based on a signal/noise ratio of 3. The applicability of the sensor for the analysis of L-lactate concentration in commercial milk and human serum samples was demonstrated successfully.

For the past 25 years the National Institute of Standards and Technology (NIST) has developed certified reference materials (CRMs), known as standard reference materials (SRMs), for determination of organic contaminants in environmental matrices. Assignment of certified concentrations has usually been based on combining results from two or more independent analytical methods. The first-generation environmental-matrix SRMs were issued with certified concentrations for a limited number (5 to 10) of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Improvements in the analytical certification approach significantly expanded the number and classes of contaminants determined. Environmental-matrix SRMs currently available include air and diesel particulate matter, coal tar, marine and river sediment, mussel tissue, fish oil and tissue, and human serum, with concentrations typically assigned for 50 to 90 organic contaminants, for example PAHs, nitro-substituted PAHs, PCBs, chlorinated pesticides, and polybrominated diphenyl ethers (PBDEs).

It would be desirable to establish and standardize methods that can measure the total antioxidant capacity level directly from vegetable extracts containing phenolics. Antioxidant capacity assays may be broadly classified as electron transfer (ET)− and hydrogen atom transfer (HAT)−based assays. The majority of HAT assays are kineticsbased, and involve a competitive reaction scheme in which antioxidant and substrate compete for peroxyl radicals thermally generated through the decomposition of azo compounds. ET−based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes colour when reduced. ET assays include the ABTS/TEAC, CUPRAC, DPPH, Folin-Ciocalteu and FRAP methods, each using different chromogenic redox reagents with different standard potentials. This review intends to offer a critical evaluation of existing antioxidant assays applied to phenolics, and reports the development by our research group of a simple and low-cost antioxidant capacity assay for dietary polyphenols, vitamins C and E, and human serum antioxidants, utilizing the copper(II)neocuproine reagent as the chromogenic oxidizing agent, which we haved named the CUPRAC (cupric ion reducing antioxidant capacity) method. This method offers distinct advantages over other ET−based assays, namely the selection of working pH at Molecules 2007, 12 1497 physiological pH (as opposed to the Folin and FRAP methods, which work at alkaline and acidic pHs, respectively), applicability to both hydrophilic and lipophilic antioxidants (unlike Folin and DPPH), completion of the redox reactions for most common flavonoids (unlike FRAP), selective oxidation of antioxidant compounds without affecting sugars and citric acid commonly contained in foodstuffs and the capability to assay -SH bearing antioxidants (unlike FRAP). Other similar ET-based antioxidant assays that we have developed or modified for phenolics are the Fe(III)− and Ce(IV)−reducing capacity methods.

It would be desirable to establish and standardize methods that can measure the total antioxidant capacity level directly from vegetable extracts containing phenolics. Antioxidant capacity assays may be broadly classified as electron transfer (ET)− and hydrogen atom transfer (HAT)−based assays. The majority of HAT assays are kineticsbased, and involve a competitive reaction scheme in which antioxidant and substrate compete for peroxyl radicals thermally generated through the decomposition of azo compounds. ET−based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes colour when reduced. ET assays include the ABTS/TEAC, CUPRAC, DPPH, Folin-Ciocalteu and FRAP methods, each using different chromogenic redox reagents with different standard potentials. This review intends to offer a critical evaluation of existing antioxidant assays applied to phenolics, and reports the development by our research group of a simple and low-cost antioxidant capacity assay for dietary polyphenols, vitamins C and E, and human serum antioxidants, utilizing the copper(II)neocuproine reagent as the chromogenic oxidizing agent, which we haved named the CUPRAC (cupric ion reducing antioxidant capacity) method. This method offers distinct advantages over other ET−based assays, namely the selection of working pH at Molecules 2007, 12 1497 physiological pH (as opposed to the Folin and FRAP methods, which work at alkaline and acidic pHs, respectively), applicability to both hydrophilic and lipophilic antioxidants (unlike Folin and DPPH), completion of the redox reactions for most common flavonoids (unlike FRAP), selective oxidation of antioxidant compounds without affecting sugars and citric acid commonly contained in foodstuffs and the capability to assay -SH bearing antioxidants (unlike FRAP). Other similar ET-based antioxidant assays that we have developed or modified for phenolics are the Fe(III)− and Ce(IV)−reducing capacity methods.

A novel sensitive fluorimetric method was investigated for the assay of fexofenadine hydrochloride (FEX) using silver nanoparticles (NPs) as a fluorescence probe. The NPs, which were prepared by chemical reduction of silver nitrate with sodium borohydride (reducing agent) in aqueous solution (without organic stabilizers) were water soluble, stable and had narrow emission band. The addition of drug to NPs solution caused considerable quenching of the emission band of silver NPs, which was likely due to the complexation of the drug to silver NPs. Under the optimum conditions, the quenched fluorescence (FL) intensity was linear with the concentration of FEX in the range of 1 · 10 À7 to 2.5 · 10 À5 mol L À1 (0.9985) with a detection limit of 1.2 · 10 À8 mol L À1 . The quenching mechanism of the studied drug on the emission band of silver NPs was explained by Stern-Volmer law. The developed method was applied to FEX determination in a pharmaceutical formulation (allegra tablets) and biological fluids (human serum and urine).

Certified reference materials (CRMs) enable the meaningful comparison of measurement results over time and place. When CRMs are used to calibrate or verify the performance of a measurement system, results produced by that system can be related through the CRM to well-defined, stable, and globally accessible reference(s). Properly done, this directly establishes the metrological traceability of the results. However, achieving the meaningful comparison of results from measurement systems calibrated and/or verified with different CRMs requires that the different materials truly deliver the same measurand, that is, are "the same" within stated uncertainty except for differences in the level of the analyte of interest. We here detail experimental and data analysis techniques for establishing and demonstrating the comparability of materials. We focus on (1) establishing a uniform interpretation of the common forms of CRM uncertainty statements, (2) estimating consistent measurement system response uncertainties from sometimes inconsistent experimental designs, (3) using "errors-in-variables" analysis methods to evaluate comparability studies and novel graphical tools for communicating results of the evaluation to reviewing authorities and potential CRM customers, and (4) augmenting established comparability studies with new materials using measurements provided by the certifying institution. These experimental and data analytic tools were developed in support of the Joint Committee for Traceability in Laboratory Medicine's efforts to enhance the reliability of clinical laboratory measurements and are illustrated with potassium and cholesterol measurands of clinical relevance; however, these tools can be applied to any group of materials that deliver the same nominal measurand with stated value and uncertainty.

An interesting electrochemical sensor has been constructed by the electrodeposition of palladium nanoclusters (Pd nano ) on poly(N-methylpyrrole) (PMPy) film-coated platinum (Pt) electrode. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS), and scanning electron microscopy were used to characterize the properties of the modified electrode. It was demonstrated that the electroactivity of the modified electrode depends strongly on the electrosynthesis conditions of the PMPy film and Pd nano . Moreover, the modified electrode exhibits strong electrocatalytic activity toward the oxidation of a mixture of dopamine (DA), ascorbic acid (AA), and uric acid (UA) with obvious reduction of overpotentials. The simultaneous analysis of this mixture at conventional (Pt, gold [Au], and glassy carbon) electrodes usually struggles. However, three well-resolved oxidation peaks for AA, DA, and UA with large peak separations allow this modified electrode to individually or simultaneously analyze AA, DA, and UA by using differential pulse voltammetry (DPV) with good stability, sensitivity, and selectivity. This sensor is also ideal for the simultaneous analysis of AA, UA and either of epinephrine (E), norepinephrine (NE) or L-DOPA. Additionally, the sensor shows strong electrocatalytic activity towards acetaminophen (ACOP) and other organic compounds. The calibration curves for AA, DA, and UA were obtained in the ranges

The current study reports the binding of curcumin (CUR) as the main pharmacologically active ingredient of turmeric and diacetylcurcumin (DAC) as a bioactive derivative of curcumin to human serum albumin (HSA) and bovine serum albumin (BSA). The apparent binding constants and number of substantive binding sites have been evaluated by fluorescence quenching method. The distance (r) between donor (HSA and BSA) and acceptor (CUR and DAC) was obtained on the basis of the Förster's theory of non-radiative energy transfer. The minor changes on the far-UV circular dichroism spectra resulted in partial changes in the calculated secondary structure contents of HSA and BSA. The negligible alteration in the secondary structure of both albumin proteins indicated that ligand-induced conformational changes are localized to the binding site and do not involve considerable changes in protein folding. The visible CD spectra indicated that the optical activity observed during the ligand binding due to induced-protein chirality. All of the achieved results suggested the important role of the phenolic OH group of CUR in the binding process.

A Glutathione (GSH) optical sensor with high sensitivity and exceptional selectivity was developed using one-step synthesized-upconversion nanoparticles (UCNPs, NaLuF4:Gd3+,Yb3+,Er3+) in conjunction with a Rhodamine B derivative (RBD). RBD was loaded on the surface of the UCNPs though non-covalent bonding to serve as an energy acceptor, while UCNPs served as energy donors. The sensor based on a coupling fluorescence resonance energy transfer (FRET) process at an excitation of 980 nm wavelength from UCNP to RBD with a ringopening process of RBD after the addition of GSH. The sensing probe exhibits a remarkable
limit of detection (LOD = 50 nM) for GSH through the enhancement of the fluorescence intensity of RBD at 592 nm, together with a concomitant reduction in the green band of the UCNPs. In addition, the sensing mechanism, characterization of UCNPs, and the selectivity of the fluorescence sensor toward GSH were discussed. The proposed sensor was evaluated on real human serum and urine samples and demonstrated as a highly selective and sensitive probe for the detection of GSH.

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 methanoldichloromethane (DCM) (5:1, v/v). The phenolic fraction was further derivatized with pentafluoropropionic acid anhydride (30 min at 70°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.

Gas chromatographic analysis of the fatty acids and sterols in the saponifiable and nonsaponifiable fractions of petroleum ether extracts of four kinds of Iraqi food plants was done. The major fatty acids of these plant materials are oleic in fruits of Zizyphus jujuba (nabk); linoleic, oleic, and palmitic in Allium cepa (onion) and AN&m sativum (garlic); and myristic in the seeds of Myristica fragrans (nutmeg). Sitosterol was the predominant sterol in all plants studied and the only one identified in onion and garlic. Stigmasterol was identified in nabk and campesterol and lanosterol were found in nutmeg. Desmosterol was identified as a minor component in nabk and nutmeg.

Group 13 cations exhibit an essentially similar chemical behavior in aqueous solution. Under physiological conditions these cations exist as metal complexes. They are known to bind tightly to human serum transferrin in the blood. Here, the numerous published studies on the interactions of Group 13 metals with transferrin are reviewed, particular attention being given to the comparative analysis of the binding constants and to the kinetics and mechanisms of metal ion uptake and release. The structural and functional information obtained on these metallotransferrins by advanced physicochemical methods, such as NMR spectroscopy, is presented in light of the recent crystal structures of ferric-and apotransferrin. The biological consequences of binding of aluminum(III), gallium(III) and indium(III) to transferrin are discussed in relation to the relevant roles played by these metal ions in pharmacology and toxicology. #