Yi-Ming Liu

Neurotoxic Effects and Biomarkers of Lead Exposure: A Review

Lead, a systemic toxicant affecting virtually every organ system, primarily affects the central nervous system, particularly the developing brain. Consequently, children are at a greater risk than adults of suffering from the neurotoxic effects of lead. To date, no safe lead-exposure threshold has been identified. The ability of lead to pass through the blood-brain barrier is due in large part to its ability to substitute for calcium ions. Within the brain, lead-induced damage in the prefrontal cerebral cortex, hippocampus, and cerebellum can lead to a variety of neurologic disorders. At the molecular level, lead interferes with the regulatory action of calcium on cell functions and disrupts many intracellular biological activities. Experimental studies have also shown that lead exposure may have genotoxic effects, especially in the brain, bone marrow, liver, and lung cells. Knowledge of the neurotoxicology of lead has advanced in recent decades due to new information on its toxic mechanisms and cellular specificity. This paper presents an overview, updated to January 2009, of the neurotoxic effects of lead with regard to children, adults, and experimental animals at both cellular and molecular levels, and discusses the biomarkers of lead exposure that are useful for risk assessment in the field of environmental health.

Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: characterization and application for enzymatic inhibition assays.

Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe3O4-SiO2) possessed three dimensional core-shell structures with an average diameter of ~20 nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25 emu·g(-1). The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The Km and the Vmax values (0.02 mM, 6.40 U·mg(-1) enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29 mM, 3.16 U·mg(-1) enzyme). Furthermore, at an elevated temperature of 70 °C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds.

Chemiluminescence resonance energy transfer-based detection for microchip electrophoresis.

Since the channels in micro- and nanofluidic devices are extremely small, a sensitive detection is required following microchip electrophoresis (MCE). This work describes a highly sensitive and yet universal detection scheme based on chemiluminescence resonance energy transfer (CRET) for MCE. It was found that an efficient CRET occurred between a luminol donor and a CdTe quantum dot (QD) acceptor in the luminol-NaBrO-QD system and that it was sensitively suppressed by the presence of certain organic compounds of biological interest including biogenic amines and thiols, amino acids, organic acids, and steroids. These findings allowed developing sensitive MCE-CL assays for the tested compounds. The proposed MCE-CL methods showed desired analytical figures of merit such as a wide concentration range of linear response. Detection limits obtained were approximately 10(-9) M for biogenic amines including dopamine and epinephrine and approximately 10(-8) M for biogenic thiols (e.g., glutathione and acetylcysteine), organic acids (i.e., ascorbic acid and uric acid), estrogens, and native amino acids. These were 10-1000 times more sensitive than those of previously reported MCE-based methods with chemiluminescence, electrochemical, or laser-induced fluorescence detection for quantifying corresponding compounds. To evaluate the applicability of the present MCE-CL method for analyzing real biological samples, it was used to determine amino acids in individual human red blood cells. Nine amino acids, including Lys, Ser, Ala, Glu, Trp, etc., were detected. The contents ranged from 3 to 31 amol/cell. The assay proved to be simple, quick, reproducible, and very sensitive.

Integrated microfluidic system with chemiluminescence detection for single cell analysis after intracellular labeling.

This work describes the first application of microchip electrophoresis with chemiluminescence detection (MCE-CL) in single cell analysis. Human red blood cells were assayed to determine intracellular content of glutathione (GSH). Intracellular GSH was first labeled by incubating cells with diazo-luminol, and then individual cells were injected, in-line lysed, and MCE separated. CL detection was based on the oxidation reaction of luminol-labeled GSH with NaBrO. The MCE-CL assay had a linear calibration curve over a range from 0.2-90 amol GSH injected with a correlation coefficient of 0.9991 and a detection limit of 50 zmol or 3.6 x 10(-9) M (S/N = 3). The average content of GSH in individual human red blood cells was found 64.9 amol (n = 17). Compared with the MCE methods with laser induced fluorescence detection (LIF) reported so far for single cell analysis, the present MCE-CL assay of GSH is simple and about 100 times more sensitive.

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K3[Fe(CN)6]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 x 10(-4) M luminol added to the CE running buffer and 1.0 x 10(-4) M K3[Fe(CN)6] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD]=3.5%, n=11) and a detection limit of 3.5 x 10(-7) M UA (signal/noise ratio [S/N]=3). A linear calibration curve ranging from 6.0 x 10(-7) to 3.0 x 10(-5) M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.

Quantification of biogenic amines by microchip electrophoresis with chemiluminescence detection

A highly sensitive microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of biogenic amines including agmatine (Agm), epinephrine (E), dopamine (DA), tyramine, and histamine in human urine samples. To achieve a high assay sensitivity, the targeted analytes were pre-column labeled by a CL tagging reagent, N-(4-aminobutyl)-N-ethylisoluminol (ABEI). ABEI-tagged biogenic amines after MCE separation reacted with hydrogen peroxide in the presence of horseradish peroxidase (HRP), producing CL emission. Since no CL reagent was added to the running buffer, the background of the CL detection was extremely low, resulting in a significant improvement in detection sensitivity. Detection limits (S/N=3) were in the range from 5.9x10(-8) to 7.7x10(-8) M for the biogenic amines tested, which were at least 10 times lower than those of the MCE-CL methods previously reported. Separation of a urine sample on a 7 cm glass/poly(dimethylsiloxane) (PDMS) microchip channel was completed within 3 min. Analysis of human urine samples found that the levels of Agm, E and DA were in the ranges of 2.61x10(-7) to 4.30x10(-7) M, 0.81x10(-7) to 1.12x10(-7) M, and 8.76x10(-7) to 11.21x10(-7) M (n=4), respectively.

Ligand fishing from Dioscorea nipponica extract using human serum albumin functionalized magnetic nanoparticles

Dioscorea nipponica and the preparations made from it have been used for long to prevent and treat coronary heart disease in traditional Chinese medicine. A group of steroidal saponins present in the plant are believed to be the active ingredients. It has been a challenge to study the individual saponins separately due to the similarities in their chemical and physical properties. In this work, human serum albumin (HSA) functionalized magnetic nanoparticles (MNPs) were used to isolate and identify saponin ligands that bind to HSA from D. nipponica extract. Electrospray ionization mass spectrometry (ESI-MS) was used for compound identification and semi-quantification. Three saponins, i.e. dioscin, gracillin, and pseudo-protodioscin showed affinity to HSA-MNPs and thus isolated effectively from the extract. The other two saponins detected in the extract (i.e. protodioscin and 26-O-β-D-glucopyranosyl-3β,20α,26-triol-25(R)-Δ(5,22)-dienofurostan-3-O-α-L-rhamnopyranosyl (1→2)-[α-L-rhamnopyranosyl (1→4)]-β-D-glucopyranoside) exhibited no affinity at all. Among the three saponins fished out, dioscin bound to HSA much stronger than gracillin and pseudo-protodioscin did. The results indicated that affinity interaction between HSA immobilized on MNPs and small molecule compounds were highly dependent on chemical structures and, potentially, medicinal usefulness. The present work demonstrates a facile and effective way to isolate and identify ligands of receptors from medicinal plants.

A novel capillary electrophoresis method for the determination of D-serine in neural samples

A capillary electrophoresis method has been developed for the determination of d-serine in neural samples. d/l-serine was tagged with naphthalene-2,3-dicarboxaldehyde (CBI-d/l-Ser), and the separation of CBI-d/l-Ser enantiomer was achieved by using a dual chiral selector system consisting of beta-cyclodextrin (beta-CD) and chiral micelles formed by sodium deoxycholate (SDC). No resolution was observed when either beta-CD or SDC was used alone. Moreover, the combined use of beta-CD with achiral micelles of sodium dodecylsulfate (SDS) exhibited no resolving effect. With laser induced fluorescence detection, the limit of detection was 3.0x10(-8)M Ser. Under the separation conditions selected, no other amino acids co-eluted with l-/d-Ser enantiomers. Using the present method, d-Ser level in Aplysia ganglia homogenates was found to vary significantly from animal to animal. Interestingly, d-Ser was not detected in single neurons isolated from Aplysia ganglia.

Extraction of aflatoxins from liquid foodstuff samples with polydopamine-coated superparamagnetic nanoparticles for HPLC-MS/MS analysis.

A facile magnetic solid phase extraction (MSPE) of aflatoxins (AFs) from liquid samples was developed using polydopamine-coated magnetic nanoparticles (PD-MNPs) as the adsorbent. PD-MNPs were prepared from amine-terminated MNPs and dopamine via an in situ oxidative self-polymerization approach. Under the selected MSPE conditions, extraction yields ranging from 59.3% for AF G2 to 89.0% for AF B1 were obtained with good repeatability. Coupled with HPLC-MS/MS quantification, the MSPE procedure serves not only for sample cleanup but also for AFs enrichment that is highly desired for trace analysis. The proposed MSPE-HPLC-MS/MS method had a linear calibration curve in the concentration range from 0.00600 to 3.00 ng/mL aflatoxin and limits of detection of 0.0012 ng/mL for AF B1, AF B2, and AF G1, and 0.0031 ng/mL for AF G2.

A Nonenzymatic Chemiluminescent Reaction Enabling Chemiluminescence Resonance Energy Transfer to Quantum Dots

Resonance energy transfer (RET)-based measurement approaches are very useful for innovative studies such as that of protein-protein interactions in living cells with spatial and /or temporal resolution [1]. RET involves non-radiative (dipole-dipole) energy transfer between a donor and an acceptor that are in close proximity (normally <10 nm). RET that occurs between two fluorophores is known as fluorescence RET (FRET), whereas RET that occurs between a light-emitting donor enzyme (e.g. luciferase) and a fluorophore is known as bioluminescence RET (BRET). Numerous publications have been seen on FRET being used in various areas such as structural elucidation of biological macromolecules, their interactions, in vitro assays, in vivo monitoring, and signal transduction in living cells [2, 3]. BRET is also well documented as a technique useful for these studies [4, 5]. Major disadvantages of BRET include the requirement of at least one carefully designed protein fusion and the low emission intensity that compromises the spatial and /or temporal resolutions in BRET measurements. Chemiluminescence RET (CRET) involves nonradiative transfer of energy from a chemiluminescent (CL) donor (instead of a bioluminescent enzyme donor as in BRET) to a fluorophore acceptor [6]. CRET occurs by the oxidation of a CL compound that then excites the fluorescent acceptor. Since no external light source is used for excitation in CRET approaches, nonspecific signals caused by external light excitation as often observed in FRET measurements can be minimized. Compared with BRET, a CRET-based approach involves no protein fusion. Both the CL donor and fluorescent acceptor can be conjugated to antibodies, promising a widespread application. However, little study has been so far reported on CRET [6-8]. A major difficulty is to identify an effective CL donor or reaction that can excite a fluorescent acceptor by energy transfer. In all of the previous CRET works reported, the luminol-H2O2 CL reaction catalyzed by horseradish peroxidase (HRP) was used. Unfortunately, involving an exogenous enzyme (i.e. HRP) limits the applicability of the CRET system. In many cases, it complicates the assay by, for example, disturbing the biological interactions under study.