Species In Human Serum Research Articles

Systematic evaluation of Copper(II)-loaded immobilized metal affinity chromatography for selective enrichment of copper-binding species in human serum and plasma.

Copper is essential in a host of biological processes, and disruption of its homeostasis is associated with diseases including neurodegeneration and metabolic disorders. Extracellular copper shifts in its speciation between healthy and disease states, and identifying molecular components involved in these perturbations could widen the panel of biomarkers for copper status. While there have been exciting advances in approaches for studying the extracellular proteome with mass spectrometry-based methods, the typical workflows disrupt metal-protein interactions due to the lability of these bonds either during sample preparation or in gas-phase environments. We sought to develop and apply a workflow to enrich for and identify protein populations with copper-binding propensities in extracellular fluids using an immobilized metal affinity chromatography (IMAC) resin. The strategy was optimized using human serum to allow for maximum quantity and diversity of protein enrichment. Protein populations could be differentiated based on protein load on the resin, likely on account of differences in abundance and affinity. The enrichment workflow was applied to plasma samples from patients with Wilson's disease and protein IDs and differential abundancies relative to healthy subjects were compared to those yielded from a traditional proteomic workflow. While the IMAC workflow preserved differential abundance and protein ID information from the traditional workflow, it identified several additional proteins being differentially abundant including those involved in lipid metabolism, immune system, and antioxidant pathways. Our results suggest the potential for this IMAC workflow to identify new proteins as potential biomarkers in copper-associated disease states.

Bimetallic CuCo Prussian blue analogue nanocubes induced chemiluminescence of luminol under alkaline solution for uric acid detection in human serum

In this paper, bimetallic CuCo Prussian blue analogue (CuCo PBA) nanocubes, synthesized through a self-assembly method, were found to induce chemiluminescence (CL) of luminol-NaOH since the catalytic oxidation activity of CuCo PBA. The CL intensity of luminol-NaOH system was enhanced about 50 times with the addition of CuCo PBA. When uric acid (UA) was added to the new luminol-NaOH-CuCo PBA system, the CL signal could be reduced significantly. Based on these, an enzyme-free CL sensor was constructed for UA detection with a linear response range from 0.30 to 5.00 μM, and detection limit of 0.16 μM (S/N = 3). The other common species in human serum didn’t affect UA detection. Furthermore, this CL sensor was successfully applied to detect the UA in human serum samples with high sensitivity. This work not only discovered the enzyme-like properties of CuCo PBA, but also broadened its application in CL bioassays.

Profiling of lysophosphatidylethanolamine molecular species in human serum and in silico prediction of the binding site on albumin.

Lysophosphatidylethanolamine (LPE) is a major lysophospholipid produced by phospholipids and binds to human serum albumin (HSA). LPEs may play various roles in vivo depending on the differences in their acyl chains. However, only few reports have been published on the biological functions of LPEs. Hence, we determined the exact relative abundance of the major LPEs in the serum of healthy participants (n= 8) using liquid chromatography-tandem mass spectrometry. Consequently, LPE 18:2 (24.1 ± 5.2%) was found to be the most abundant in serum. To understand the distribution of LPEs, the serum separated via gel-filtration high-performance liquid chromatography was subjected to quantitative measurement. LPEs were more observed in the albumin fraction than the lipoprotein fraction. We also performed a fluorescence displacement assay and an in silico molecular docking experiment using AutoDock to confirm the affinity and binding sites of the LPEs on HSA. The binding affinities of the LPEs for drug sites 1 and 2 on HSA were relatively low, with Ki values of approximately 11 and 3.8μM, respectively. AutoDock analysis revealed the conformation of the LPEs bound to drug sites and the possibility of LPEs binding to other HSA sites. These findings could help to elucidate the biological and pathological functions of LPEs.

Establishment and application of high performance liquid chromatography-inductively coupled plasma mass spectrometry method for the determination of iodine species in serum

To establish a method for the determination of iodine species in human serum by inductively coupled plasma mass spectrometry(ICP-MS). Total iodine was determined by ICP-MS in helium mode after dilution of serum with tetramethylammonium hydroxide solution. The serum was added to methanol solution to precipitate the protein, and the supernatant was separated by liquid chromatography(LC) followed by ICP-MS for the determination of inorganic iodine. The limit of detection(LOD) of serum I~- by ICP-MS was 0.17 μg/L, the limit of quantification(LOQ) was 0.57 μg/L, and the linear correlation coefficient R~2=0.9998; the LOD of serum IO_3~- was 0.16 μg/L, the LOQ was 0.55 μg/L, and the linear correlation coefficient R~2=0.9998.The I~- recoveries were 96.2%-104.5% for the total serum iodine assay and 93.7%-98.6% for the inorganic iodine assay. Analysis of the iodine species of the actual serum samples showed that the I~- content was 2.6-12.2 μg/L, the organic iodine content was 45.3-66.0 μg/L, and the serum samples were essentially free of IO_3~-. A convenient, efficient and accurate method for the determination of serum iodine species was established using high performance liquid chromatography tandem with inductively coupled plasma mass spectrometry.

Kinetics of interaction of Cr(VI) and Cr(III) with serum constituents and detection of Cr species in human serum at physiological concentration levels

A novel analytical method, based on monolithic convective interaction media (CIM) chromatography coupled to UV and inductively coupled plasma mass spectrometry (ICP-MS) detectors, was developed to investigate the kinetics of interactions of Cr(VI) and Cr(III) with serum constituents, and perform Cr speciation at physiological concentration levels. Cr(VI) was separated from Cr-transferrin (Cr-Tf) and Cr-albumin (Cr-HSA) on a CIM diethylamino (DEAE) column using linear gradient elution from 100% buffer A (50 mM Tris-HCl + 10 mM NaHCO3, pH 7.4) to 60% buffer B (buffer A + 2 M NH4Cl) in 10 min at a flow rate of 1 mL min−1. Good column recovery of separated Cr species (close to 100%) and satisfactory method repeatability (RSDs below 8%) was obtained. Kinetics of interaction of Cr(VI) and Cr(III) with serum constituents were followed after human serum was doubly spiked with enriched 50Cr(VI) and 53Cr(III) solutions, and speciation analysis applied from 5 min up to 48 h after spiking. The results showed that in serum 53Cr(III) rapidly interacted with Tf, while 50Cr(VI) reduction was slow. 48 h after spiking, more than 90% of added 53Cr(III) was bound to Tf and the remaining 10% associated with HSA. About 20% of spiked 50Cr(VI) was still present in serum, while the resulting 50Cr(III) was bound predominantly to Tf. High sensitivity of the developed speciation procedure enabled detection of Cr-Tf complex as the main Cr species in human serum at physiological concentration levels. To the best of our knowledge, Cr-Tf concentrations in serum of unexposed individuals have not been reported yet.

Characterization of galactosyl and lactosyl sulfatide species in human serum by MALDI-TOF mass spectrometry.

Background Sulfatides are found in a variety of tissues and serum lipoproteins. Sulfatide is a molecular species composed of various sphingoid bases, fatty acids and sugar chains; therefore, rapid analysis of the qualitative structure is important in clinical assessment. Methods In this study, sulfatide-rich fractions were isolated from serum lipids, and the sulfatide species were analysed by negative ion mode using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results Sulfatide species identified in human serum included two different sugar chains, eight sphingoid molecules and various fatty acid side chains including hydroxy fatty acids. In total, 64 galactosyl sulfatides (SM4s) and 49 lactosyl sulfatides (SM3) were identified. Quantitatively, the amount of SM3 was less than 1% of the amount of SM4s. The fatty acids of SM4s of healthy serum ( n = 8) were predominantly C16:0 and a hydroxylation C16:0 (C16:0h), followed by very long chain fatty acids (VLCFAs) predominant species, and SM3 was a major component of VLCFAs. Conclusion This present study described a simple method of human serum sulfatide analysis using MALDI-TOF MS. This method is suitable for clinical laboratories and is likely to increase the understanding of the roles of sulfatide species in both physiological and disease states.

Speciation Analysis of Arsenic Compounds by HPLC-ICP-MS: Application for Human Serum and Urine.

A high-performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS) as an elemental specific detector was used for the speciation analysis of arsenic compounds in urine and serum samples from Vietnam. Five arsenic species including arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and arsenobetaine (AsB) were studied. A gradient elution of ammonium carbonate ((NH4)2CO3), ethylenediaminetetraacetic acid disodium salt (Na2EDTA), and methanol at pH 9.0 utilizing Hamilton PRP-X100 strong anion-exchange column allowed the chromatographic separation of five arsenic species. In this study, urine and serum samples were prepared by dilution in solvent and protein precipitation by trichloroacetic acid, respectively. The extraction efficiency was greater than 91% for urine matrix, and recoveries from spiked samples were in the range of 94–139% for the arsenic species in human serum. The method limit of detection (MDL) and limit of quantification (MQL), which were calculated by signal to noise ratio, were found to be 0.3–1.5 and 1.0–5.0 ng·mL−1, respectively. The concentration of arsenic species in 17 pairs of urine and serum samples from Vietnam was also quantified and evaluated. The major species of arsenic in the urine and serum samples were AsB and DMA.

Identification of Ganglioside GM3 Molecular Species in Human Serum Associated with Risk Factors of Metabolic Syndrome

Serum GM3 molecular species were quantified in 125 Japanese residents using tandem mass spectrometry multiple reaction monitoring. Individuals were categorized by the presence or absence of metabolic disease risk factors including visceral fat accumulation, hyperglycemia and dyslipidemia. A total of 23 GM3 molecular species were measured, of these, eight were found to be significantly elevated in individuals with visceral fat accumulation and metabolic disease, defined as the presence of hyperglycemia and dyslipidemia. All of the GM3 molecular species were composed of the sphingoid base sphingosine (d18:1 (Δ4)) and, interestingly, six of the eight elevated GM3 molecular species contained a hydroxylated ceramide moiety. The hydroxylated GM3 species were, in order of decreasing abundance, d18:1-h24:0 ≈ d18:1-h24:1 > d18:1-h22:0 » d18:1-h20:0 > d18:1-h21:0 > d18:1-h18:1. Univariate and multiple linear regression analyses were conducted using a number of clinical health variables associated with obesity, type 2 diabetes, metabolic disease, atherosclerosis and hypertension. GM3(d18:1-h24:1) was identified as the best candidate for metabolic screening, proving to be significantly correlated with intima-media thickness, used for the detection of atherosclerotic disease in humans, and a number of metabolic disease risk factors including autotaxin, LDL-c and homeostatic model assessment insulin resistance (HOMA-IR).

Synthesis, characterization, and preclinical validation of a PET radiopharmaceutical for interrogating Aβ (β-amyloid) plaques in Alzheimer's disease.

BackgroundPET radiopharmaceuticals capable of imaging β-amyloid (Aβ) plaque burden in the brain could offer highly valuable diagnostic tools for clinical studies of Alzheimer’s disease. To further supplement existing armamentarium of FDA-approved agents as well as those under development, and to correlate multiphoton-imaging data reported earlier, herein, we describe preclinical validation of a PET tracer.MethodsA novel PET radiopharmaceutical (18F-7B) was synthesized and characterized. To assess its affinity for Aβ, binding assays with Aβ1-42 fibrils, Alzheimer’s disease (AD) homogenates, and autoradiography studies and their IHC correlations were performed. For assessing its overall pharmacokinetic profiles in general and its ability to cross the blood-brain barrier (BBB) in particular, biodistribution studies in normal mice were performed. Finally, for evaluating potential for 18F-7B to serve as a targeted Aβ probe, the microPET/CT imaging was performed in age-matched amyloid precursor protein/presenilin-1 (APP/PS1) mice and wild-type (WT) counterparts.ResultsThe radiotracer 18F-7B shows saturable binding to autopsy-confirmed AD homogenates (Kd = 17.7 nM) and Aβ1-42 fibrils (Kd = 61 nM). Preliminary autoradiography studies show binding of 18F-7B to cortical Aβ plaques in autopsy-confirmed AD tissue sections, inhibition of that binding by unlabeled counterpart 7A-indicating specificity, and a good correlation of tracer binding with Aβ immunostaining. The agent indicates high initial penetration into brains (7.23 ± 0.47%ID/g; 5 min) of normal mice, thus indicating a 5-min/120-min brain uptake clearance ratio of 4.7, a benchmark value (>4) consistent with the ability of agents to traverse the BBB to enable PET brain imaging. Additionally, 18F-7B demonstrates the presence of parental species in human serum. Preliminary microPET/CT imaging demonstrates significantly higher retention of 18F-7B in brains of transgenic mice compared with their WT counterparts, consistent with expected binding of the radiotracer to Aβ plaques, present in APP/PS1 mice, compared with their age-matched WT counterparts lacking those Aβ aggregates.ConclusionsThese data offer a platform scaffold conducive to further optimization for developing new PET tracers to study Aβ pathophysiology in vitro and in vivo.

Simultaneous identification and comparison of pathogenic Brucella species in human serum and blood samples by means of multiplex PCR and confirmation of the results by PCR-RFLP.

Simultaneous identification and comparison of pathogenic Brucella species in human serum and blood samples by means of multiplex PCR and confirmation of the results by PCR-RFLP.

Development, validation, and application of an ultra-performance liquid chromatography–sector field inductively coupled plasma mass spectrometry method for simultaneous determination of six organotin compounds in human serum

Organotin compounds (OTCs) are heavily employed by industry for a wide variety of applications, including the production of plastics and as biocides. Reports of environmental prevalence, differential toxicity between OTCs, and poorly characterized human exposure have fueled the demand for sensitive, selective speciation methods. The objective of this investigation was to develop and validate a rapid, sensitive, and selective analytical method for the simultaneous determination of a suite of organotin compounds, including butyl (mono-, di-, and tri-substituted) and phenyl (mono-, di-, and tri-substituted) species in human serum. The analytical method utilized ultra-performance liquid chromatography (UPLC) coupled with sector field inductively coupled plasma mass spectrometry (SF-ICP-MS). The small (sub-2µm) particle size of the UPLC column stationary phase and the sensitivity of the SF-ICP-MS enabled separation and sensitive determination of the analyte suite with a runtime of approximately 3min. Validation activities included demonstration of method linearity over the concentration range of approximately 0.250–13.661ngmL−1, depending on the species; intraday precision of less than 21%, interday precision of less than 18%, intraday accuracy of −5.3% to 19%, and interday accuracy of −14% to 15% for all species; specificity, and matrix impact. In addition, sensitivity, and analyte stability under different storage scenarios were evaluated. Analyte stability was found to be limited for most species in freezer, refrigerator, and freeze–thaw conditions. The validated method was then applied for the determination of the OTCs in human serum samples from women participating in the Snart-Foraeldre/MiljØ (Soon-Parents/Environment) Study. The concentration of each OTC ranged from below the experimental limit of quantitation to 10.929ngtin (Sn)mL−1 serum. Speciation values were confirmed by a total Sn analysis.

New method for the speciation of ruthenium-based chemotherapeutics in human serum by conjoint liquid chromatography on affinity and anion-exchange monolithic disks

An important step in pharmacological characterisation of a candidate drug is the study of the drugs interactions with serum proteins. In the present work, conjoint liquid chromatography (CLC) was used for separation of ruthenium (Ru)-based drug candidates in human serum. CIM Protein G and CIM DEAE disks were assembled together in a single housing forming a CLC monolithic column. By applying isocratic elution with Tris–HCl–NaHCO3 buffer (pH 7.4) in the first min, followed by gradient elution with 1molL−1 NH4Cl (pH 7.4) in the next 9min, immunoglobulins (IgG) were retained by the Protein G disk enabling subsequent separation of unbound Ru species from Ru species bound to human serum transferrin (Tf) and albumin (HSA) on the CIM DEAE disk. Finally, elution with acetic acid (AcOH) in the next 3min allowed separation of Ru species associated with IgG. Protein elution was followed on-line with UV detection at 278nm, while the separated Ru species were quantified by post-column isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS). The instrumental set-up enabled fast two-dimensional separation by affinity and ion-exchange modes to be carried out in a single chromatographic run. Two Ru-based chemotherapeutics: a newly synthesised compound chlorido(η6-p-cymene)(nalidixicato-κ2O,O)Ru(II) (1) and (H2im)[trans-Ru(III)Cl4(Him)2] (2; KP418), which is currently undergoing preclinical studies, were investigated. The CLC procedure applied is sensitive with low limit of detection (LOD) (0.027μgRumL−1 for (1)) and good method repeatability (RSD±3.5%). The experimental data revealed that it enables investigation of the kinetics of interaction of positively charged and neutral complexes of metallodrugs with serum proteins as well as the distribution of metallodrug species in human serum. However, negatively charged metallic complexes co-eluted with Tf and HSA and thus hindered their speciation analysis. An example of successful application of the kinetic studies on the CLC column is (1), a neutral Ru complex that hydrolyses to a positively charged species. For comparison, speciation data obtained for serum samples spiked with cisplatin are also shown.

Outcome of a public consultation on the Draft Scientific Opinion of the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) on Dietary Reference Values for selenium

EFSA Supporting PublicationsVolume 11, Issue 10 670E Technical reportOpen Access Outcome of a public consultation on the Draft Scientific Opinion of the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) on Dietary Reference Values for selenium European Food Safety Authority (EFSA), European Food Safety Authority (EFSA)Search for more papers by this author European Food Safety Authority (EFSA), European Food Safety Authority (EFSA)Search for more papers by this author First published: 10 October 2014 https://doi.org/10.2903/sp.efsa.2014.EN-670 Published date: 10 October 2014 Question number: EFSA-Q-2014-00469 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References Combs GF, Jr., Jackson MI, Watts JC, Johnson LK, Zeng H, Idso J, Schomburg L, Hoeg A, Hoefig CS, Chiang EC, Waters DJ, Davis CD and Milner JA, 2012. Differential responses to selenomethionine supplementation by sex and genotype in healthy adults. British Journal of Nutrition, 107, 1514– 1525. Dernovics M, Giusti P and Lobinski R, 2007. ICP-MS-assisted nanoHPLC-electrospray Q/time-of-flight MS/MS selenopeptide mapping in Brazil nuts. Journal of Analytical Atomic Spectrometry, 22, 41– 50. Duffield AJ, Thomson CD, Hill KE and Williams S, 1999. An estimation of selenium requirements for New Zealanders. American Journal of Clinical Nutrition, 70, 896– 903. EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies), 2010. Scientific Opinion on principles for deriving and applying Dietary Reference Values. 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Symposium on ‘Geographical and geological influences on nutrition’: Factors controlling the distribution of selenium in the environment and their impact on health and nutrition. Proceedings of the Nutrition Society, 69, 119– 132. Kokarnig S, Tsirigotaki A, Wiesenhofer T, Lackner V, Francesconi KA, Pergantis SA and Kuehnelt D, 2014. Concurrent quantitative HPLC-mass spectrometry profiling of small selenium species in human serum and urine after ingestion of selenium supplements. Journal of Trace Elements in Medicine and Biology. Kryukov GV, Castellano S, Novoselov SV, Lobanov AV, Zehtab O, Guigo R and Gladyshev VN, 2003. Characterization of mammalian selenoproteomes. Science, 300, 1439– 1443. Li Q, Li XZ, Wang T, Zhou LW, Feng HQ, Gao L, Pei JR, Lin C and Jiang CX, 2013. Selenoprotein P and Yunnan endemic sudden cardiac death–an ecological study. Biological Trace Element Research, 151, 14– 17. Loui A, Raab A, Braetter P, Obladen M and de Braetter VN, 2008. Selenium status in term and preterm infants during the first months of life. European Journal of Clinical Nutrition, 62, 349– 355. Michaelis M, Gralla O, Behrends T, Scharpf M, Endermann T, Rijntjes E, Pietschmann N, Hollenbach B and Schomburg L, 2014. Selenoprotein P in seminal fluid is a novel biomarker of sperm quality. Biochemical and Biophysical Research Communications, 443, 905– 910. Persson-Moschos M, Alfthan G and Akesson B, 1998. Plasma selenoprotein P levels of healthy males in different selenium status after oral supplementation with different forms of selenium. European Journal of Clinical Nutrition, 52, 363– 367. Rayman MP, Infante HG and Sargent M, 2008. Food-chain selenium and human health: spotlight on speciation. British Journal of Nutrition, 100, 238– 253. Rayman MP, Searle E, Kelly L, Johnsen S, Bodman-Smith K, Bath SC, Mao J and Redman CW, 2014. Effect of selenium on markers of risk of pre-eclampsia in UK pregnant women: a randomised, controlled pilot trial. British Journal of Nutrition, 1– 13. Xia Y, Hill KE, Li P, Xu J, Zhou D, Motley AK, Wang L, Byrne DW and Burk RF, 2010. Optimization of selenoprotein P and other plasma selenium biomarkers for the assessment of the selenium nutritional requirement: a placebo-controlled, double-blind study of selenomethionine supplementation in selenium-deficient Chinese subjects. American Journal of Clinical Nutrition, 92, 525– 531. Volume11, Issue10October 2014670E ReferencesRelatedInformation

Concurrent quantitative HPLC–mass spectrometry profiling of small selenium species in human serum and urine after ingestion of selenium supplements

Selenium metabolic patterns in the human body originating from five distinct selenium dietary sources, selenate, selenite, selenomethionine (SeMet), methylselenocysteine (MeSeCys) and selenized yeast, were investigated by performing concurrent HPLC–mass spectrometric analysis of human serum and urine. Total selenium and selenium species time profiles were generated by sampling and analyzing serum and urine from volunteers treated with selenium supplements, up to 5 and 24h following ingestion, respectively. We found that an increase in total serum selenium levels, accompanied by elevated selenium urinary excretion, was the common pattern for all treatments, except for that of selenite supplementation. Selenosugar 1 was a universal serum metabolite in all treatments, indicating that ingested selenium is favorably metabolized to the sugar. Except for selenite and selenized yeast ingestion, these patterns were reflected in the urine time series of the different treatments. Selenosugar 1 was the major selenium species present in urine in all treatments except for the selenate treatment, accounting for about 80% of the identified excreted species within 24h of ingestion. Furthermore, the urinary metabolite trimethylselenonium ion (TMSe) was detected for the first time in human background serum by using HPLC coupled to elemental and molecular mass spectrometry. The concurrent monitoring of non-protein selenium species in both body fluids provides the relation between bioavailability and excretion of the individual ingested species and of their metabolic products, while the combined use of elemental and molecular mass spectrometry enables the accurate quantitation of structurally confirmed species. This successfully applied approach is anticipated to be a useful tool for more extensive future studies into human selenium metabolism.

Cobalt speciation assay for human serum, Part II. Method validation in a study of human volunteers ingesting cobalt(II) chloride dietary supplement for 90 days

A new analytical method for determining cobalt (Co) species in human serum by size exclusion chromatography with inductively coupled plasma mass spectrometry (SEC-ICP-MS) was applied to serum samples collected from 12 human volunteers who participated in a Co(II) chloride supplement study involving ingestion of 1 mg Co/day for up to 90 consecutive days. The study protocol included determination of serum total Co by acid digestion followed by ICP-MS. Co speciation assay measurements were conducted for up to 13 time points per individual spanning from one to two weeks before dosing began to two weeks after dosing ceased. The Co speciation assay showed good recovery >91% relative to total Co measurements. Undiluted serum demonstrated uniform fractions of large molecular Co defined as Co bound to albumin and other proteins >50 kDa at 96% and the residual as small molecular Co defined as free Co(II) and <1 kDa Co-complexes for individual serum Co concentrations up to 146 μg/L. There were no dose-related changes in Co distribution. Analysis of the same serum samples with tenfold dilution in 0.1 M acetic acid led to a lower fraction of large molecular Co at 87%, with the difference between diluted and undiluted measurements being 8.4%. The difference noted between undiluted and diluted large molecular Co may be attributed to Co release from albumin. Data demonstrated that large molecular Co was the predominant Co species in both undiluted and diluted human serum over a broad range of in vivo Co concentrations, reflecting high albumin–Co binding capacity. These data validate the Co speciation assay and may be employed in understanding further the toxicokinetics and dose-response relationships for Co species.

Quantitative determination of small selenium species in human serum by HPLC/ICPMS following a protein-removal, pre-concentration procedure

Protein precipitation was incorporated into a sample preparation method for the quantitative determination of small "non-protein" selenium species in human serum by high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC/ICPMS). The advantages of cleaner matrix and concomitant concentration of the small compounds result in quantification limits in the native serum at the sub-micrograms Se per litre level. Spiking experiments with methyl 2-acetamido-2-deoxy-1-seleno-β-D-galactopyranoside (selenosugar 1), trimethylselenonium ion, selenomethionine, methylselenocysteine (MeSeCys) and selenate yielded recoveries from 73% to 103%. Selenite had a low recovery (44%), possibly owing to protein binding. The validated method was applied to serum samples from two volunteers before and after ingestion of a selenium food supplement. HPLC/ICPMS analysis showed, besides ingested selenate, the presence of selenosugar 1 and trace amounts of MeSeCys and methyl 2-amino-2-deoxy-1-seleno-β-D-galactopyranoside, which have not been reported in human serum before.

Profiling Cys34 Adducts of Human Serum Albumin by Fixed-Step Selected Reaction Monitoring

A method is described for profiling putative adducts (or other unknown covalent modifications) at the Cys34 locus of human serum albumin (HSA), which represents the preferred reaction site for small electrophilic species in human serum. By comparing profiles of putative HSA-Cys34 adducts across populations of interest it is theoretically possible to explore environmental causes of degenerative diseases and cancer caused by both exogenous and endogenous chemicals. We report a novel application of selected-reaction-monitoring (SRM) mass spectrometry, termed fixed-step SRM (FS-SRM), that allows detection of essentially all HSA-Cys34 modifications over a specified range of mass increases (added masses). After tryptic digestion, HSA-Cys34 adducts are contained in the third largest peptide (T3), which contains 21 amino acids and an average mass of 2433.87 Da. The FS-SRM method does not require that exact masses of T3 adducts be known in advance but rather uses a theoretical list of T3-adduct m/z values separated by a fixed increment of 1.5. In terms of added masses, each triply charged parent ion represents a bin of ±2.3 Da between 9.1 Da and 351.1 Da. Synthetic T3 adducts were used to optimize FS-SRM and to establish screening rules based upon selected b- and y-series fragment ions. An isotopically labeled T3 adduct is added to protein digests to facilitate quantification of putative adducts. We used FS-SRM to generate putative adduct profiles from six archived specimens of HSA that had been pooled by gender, race, and smoking status. An average of 66 putative adduct hits (out of a possible 77) were detected in these samples. Putative adducts covered a wide range of concentrations, were most abundant in the mass range below 100 Da, and were more abundant in smokers than in nonsmokers. With minor modifications, the FS-SRM methodology can be applied to other nucleophilic sites and proteins.

Problems and progresses in speciation of Al in human serum: An overview

Aluminium (Al) is associated with many clinical disorders in renal patients. Al accumulation in brain has also been related to the neurodegenerative processes in Alzheimer's disease. In order to better understand Al transport in the human body, it is necessary to identify and quantify chemical species in which Al is present in body fluids and tissues. Among a variety of biological samples, Al speciation was the most frequently investigated in human serum. Improvements were made in the development of analytical techniques for the determination of the amount and composition of high molecular mass Al (HMM-Al) and low molecular mass Al (LMM-Al) species in human serum. However, due to the complex chemistry of Al in serum, its low total concentration and the high risk of contamination, speciation of Al in biological samples is still a difficult task for analytical chemists. In this work, problems related to speciation of Al in human serum are critically discussed. An overview of the progress that was made by the use of different analytical procedures, in order to propose analytical protocols for reliable speciation of Al in serum at low ng mL(-1) concentration range, is presented.

Stability of the conjugated species of environmental phenols and parabens in human serum

In humans, the metabolism of environmental phenols may include the formation of conjugated species (e.g., glucuronides and sulfates), but the free species—not the conjugated forms—are considered biologically active. Therefore, information on the concentration of these free species in blood or urine could be helpful for risk assessment. Because conjugates could hydrolyze to their corresponding free forms during collection, handling, and storage of biological specimens, information on the temporal stability of the conjugates is of interest. Previously, we reported the temporal stability of urinary conjugates of several environmental phenols, but data on the stability of phenols' conjugated species in serum, albeit critical if concentrations of free and conjugated species are compared, are largely unknown. In the present study, we investigate the stability of the conjugates of four phenols—bisphenol A, benzophenone-3, triclosan, and 2,5-dichlorophenol—and two parabens—methyl paraben and propyl paraben—in 16 human serum samples for 30 days at above-freezing temperature storage conditions (4 °C, room temperature, and 37 °C). These conditions reflect the worst-case scenarios that could occur during the short-term storage of biological samples before their long-term storage at controlled subfreezing temperatures. We found that the percentage of the conjugated species of the four detected compounds (2,5-dichlorophenol, triclosan, and methyl and propyl parabens) in these serum specimens even when stored at 37 °C for at least 30 days did not vary significantly. These preliminary data suggest that the phenols' serum conjugates appear to be more stable than their corresponding urinary conjugates, some of which started to hydrolyze within 24 h under similar storage conditions. The reported stability of these conjugated species in human serum also suggests that the free species are unlikely to have resulted from the hydrolysis of their corresponding conjugates. This information could be important for interpreting the low concentrations of free phenol species detected in serum samples of nonoccupationally exposed populations. To our knowledge, this is the first study to report on the stability of conjugated species in serum, and as such requires replication.

Electrospray ionisation with selected reaction monitoring for the determination of Mn‐citrate, Fe‐citrate, Cu‐citrate and Zn‐citrate

Citrate complexes of Mn and Fe, and potentially those of Cu and Zn, are considered as important low molecular mass species in human serum and cerebrospinal fluid (CSF). For example, Mn is supposed to enter the brain under excess exposure as Mn-citrate leading to neurotoxic effects. Mn-citrate has been characterised in human CSF using chromatography and electrophoresis online with inductively coupled plasma mass spectrometry, but not yet with molecular mass spectrometry. Therefore, this study explores the potential of electrospray ionisation (ESI) with selected reaction monitoring (SRM) for the detection of metal-citrate complexes, in particular Mn-citrate. The collision-induced dissociation of precursor ions with various metal:citrate stoichiometries was studied for Mn-citrate, Fe-citrate, Cu-citrate and Zn-citrate. High selectivity was achieved for Mn(II)-citrate even in respect to Fe(III)-citrate which forms isobaric precursor ions. The limit of detection for Mn-citrate was estimated to be around 250 microg L(-1) (referring to the total Mn content in the standard) using flow injection. The sensitivity was sufficient for the determination of Mn-citrate in standard solutions and in an extract of an Mn-citrate-containing supplement. An improved ESI source design is expected to reduce the limits of detection significantly. The developed ESI-SRM method has the potential to provide complementary data for the quality control of current separation methods for metal citrates using element-selective detection, with application to biomedical samples and further matrices.