Organic Ion-associate Phase Extraction/Back-microextraction for the Preconcentration and Determination of Lithium Using 2,2,6,6-Tetramethyl-3,5-heptanedione by Liquid Electrode Plasma Atomic Emission Spectrometry and GF-AAS in Environmental Water.

We developed an ion-associate phase (IAP)-extraction/acid back-extraction system for the preconcentration and atomic spectrometric determination of lithium trace amounts in water. The chelating reagent for lithium also works as a constituent of the extraction phase. The lithium in a 10 mL sample solution was converted through a chelate complex reaction with 2,2,6,6-tetramethyl-3,5-heptanedione (HDPM). The addition of a benzyldimethyltetradecylammonium ion caused the formation of IAP suspension in the solution. Centrifugation of the solution led to the isolation of a liquid organic phase and the lithium complex was extracted as the upper phase from the centrifuge tube. After the aqueous phase was removed, lithium was back-extracted with a 400 µL nitric acid solution from the IAP. The acid phase was measured using liquid-electrode-plasma atomic-emission-spectrometry (LEP-AES) or graphite-furnace atomic-absorption spectroscopy (GF-AAS). The detection limits were 0.02 mg/L for LEP-AES and 0.02 µg/L for GF-AAS. This system was applied to the determination of environmental water. The HDPM in the organic phase was reusable.

Structural insights into the catalytic reaction trigger and inhibition of D-3-hydroxybutyrate dehydrogenase.

D-3-Hydroxybutyrate dehydrogenase catalyzes the reversible conversion of acetoacetate and D-3-hydroxybutyrate. These ketone bodies are both energy-storage forms of acetyl-CoA. In order to clarify the structural mechanisms of the catalytic reaction with the cognate substrate D-3-hydroxybutyrate and of the inhibition of the reaction by inhibitors, the enzyme from Alcaligenes faecalis has been analyzed by X-ray crystallography in liganded states with the substrate and with two types of inhibitor: malonate and methylmalonate. In each subunit of the tetrameric enzyme, the substrate is trapped on the nicotinamide plane of the bound NAD(+). An OMIT map definitively shows that the bound ligand is D-3-hydroxybutyrate and not acetoacetate. The two carboxylate O atoms form four hydrogen bonds to four conserved amino-acid residues. The methyl group is accommodated in the nearby hydrophobic pocket so that the formation of a hydrogen bond from the OH group of the substrate to the hydroxy group of Tyr155 at the active centre is facilitated. In this geometry, the H atom attached to the C(3) atom of the substrate in the sp(3) configuration is positioned at a distance of 3.1 Šfrom the nicotinamide C(4) atom in the direction normal to the plane. In addition, the donor-acceptor relationship of the hydrogen bonds suggests that the Tyr155 OH group is allowed to ionize by the two donations from the Ser142 OH group and the ribose OH group. A comparison of the protein structures with and without ligands indicates that the Gln196 residue of the small movable domain participates in the formation of additional hydrogen bonds. It is likely that this situation can facilitate H-atom movements as the trigger of the catalytic reaction. In the complexes with inhibitors, however, their principal carboxylate groups interact with the enzyme in a similar way, while the interactions of other groups are changed. The crucial determinant for inhibition is that the inhibitors have no active H atom at C(3). A second determinant is the Tyr155 OH group, which is perturbed by the inhibitors to donate its H atom for hydrogen-bond formation, losing its nucleophilicity.

Highly sensitive elemental analysis for Cd and Pb by liquid electrode plasma atomic emission spectrometry with quartz glass chip and sample flow.

This paper describes the development of a highly sensitive liquid-electrode plasma atomic emission spectrometry (LEP-AES) by combination of quartz glass chip and sample flow system. LEP-AES is an ultracompact elemental analysis method, in which the electroconductive sample solution is put into a microfluidic channel whose center is made narrower (∼100 µm in width). When high voltage pulses (1500 V) are applied at both ends of the channel, the sample evaporates locally at the narrow part and generates plasma. By the emission from the plasma, elemental concentration is analyzed. In this paper, the limits of detection (LODs) were investigated in various conditions of accumulation time, material of the chip, and the sample flow. It was found that the long accumulation using the quartz chip with sample flow was effective to improve LOD. Authors suggested that this was because bubbles remaining after each plasma pulse were removed from the narrow channel by sample flow, resulting in highly reproducible plasma generation, to enable a high accumulation effect. Finally, LODs were calculated from a calibration curve, to be 0.52 µg/L for Cd and 19.0 µg/L for Pb at optimized condition. Sub-ppb level LOD was achieved for Cd.

Basic performance of an enzymatic method for glycated albumin and reference range determination.

BACKGROUND: Glycated albumin (GA) is a medium-term glycemic control marker of diabetes and may be more sensitive to changes in plasma glucose than hemoglobin A1c. We studied where and how many fructosyl groups bind to albumin, and which glycation sites are measured by the enzymatic method for GA. We also studied the basic performance of the enzymatic method for GA. METHODS: Glycated albumin was measured using an enzymatic method (Lucica®GA-L, Asahi Kasei Pharma) on a biochemical autoanalyzer. Molecular weights of purified GA and nonglycated albumin were measured by a mass spectrometry system. Two hundred one healthy volunteers with normal results of oral glucose tolerance testing were recruited to determine the reference range in Americans. RESULTS: The present method measured only glycated amino acids from albumin in serum protein. We estimate that the number of glycated amino acids measured by this method was approximately two per molecule of albumin. The general performance (sensitivity, specificity, reproducibility, linearity, interference) of the method was good. The reference range of GA% in Americans with normal glucose tolerance was determined to be 11.9-15.8% (mean ± 2 standard deviations). Significant differences were not observed between the sexes; however, race differences were observed (higher levels in blacks relative to whites). CONCLUSIONS: The method was specific for measuring glycated amino acids in albumin and had good basic performance characteristics. The reference range in Americans was 11.9-15.8%. This method may be a useful indicator for diabetes control.

Determination of cadmium in water samples by liquid electrode plasma atomic emission spectrometry after solid phase extraction using a mini cartridge packed with chelate resin immobilizing carboxymethylated pentaethylenehexamine.

Solid phase extraction using a mini cartridge packed with 22 mg of chelate resin immobilizing carboxymethylated pentaethylenehexamine was successfully utilized for separation/preconcentration of cadmium in water samples prior to liquid electrode plasma atomic emission spectrometric (LEP-AES) determination. The combined method with the extraction and LEP-AES was applicable to the determination of cadmium in the certified reference materials (EU-L-1 wastewater and ES-L-1 groundwater); the detection limit was 0.20 microg in 200 mL of sample solution (500-fold preconcentration).

Structure of D-3-hydroxybutyrate dehydrogenase prepared in the presence of the substrate D-3-hydroxybutyrate and NAD+.

D-3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis catalyzes the reversible conversion between D-3-hydroxybutyrate and acetoacetate. The enzyme was crystallized in the presence of the substrate D-3-hydroxybutyrate and the cofactor NAD(+) at the optimum pH for the catalytic reaction. The structure, which was solved by X-ray crystallography, is isomorphous to that of the complex with the substrate analogue acetate. The product as well as the substrate molecule are accommodated well in the catalytic site. Their binding geometries suggest that the reversible reactions occur by shuttle movements of a hydrogen negative ion from the C3 atom of the substrate to the C4 atom of NAD(+) and from the C4 atom of NADH to the C3 atom of the product. The reaction might be further coupled to the withdrawal of a proton from the hydroxyl group of the substrate by the ionized Tyr155 residue. These structural features strongly support the previously proposed reaction mechanism of D-3-hydroxybutyrate dehydrogenase, which was based on the acetate-bound complex structure.

Structures of Arthrobacter globiformis urate oxidase-ligand complexes.

The enzyme urate oxidase catalyzes the conversion of uric acid to 5-hydroxyisourate, one of the steps in the ureide pathway. Arthrobacter globiformis urate oxidase (AgUOX) was crystallized and structures of crystals soaked in the substrate uric acid, the inhibitor 8-azaxanthin and allantoin have been determined at 1.9-2.2 A resolution. The biological unit is a homotetramer and two homotetramers comprise the asymmetric crystallographic unit. Each subunit contains two T-fold domains of betabetaalphaalphabetabeta topology, which are usually found in purine- and pterin-binding enzymes. The uric acid substrate is bound tightly to the enzyme by interactions with Arg180, Leu222 and Gln223 from one subunit and with Thr67 and Asp68 of the neighbouring subunit in the tetramer. In the other crystal structures, lithium borate, 8-azaxanthin and allantoate are bound to the enzyme in a similar manner as uric acid. Based on these AgUOX structures, the enzymatic reaction mechanism of UOX has been proposed.

The structures of Alcaligenes faecalis D-3-hydroxybutyrate dehydrogenase before and after NAD+ and acetate binding suggest a dynamical reaction mechanism as a member of the SDR family.

D-3-Hydroxybutyrate dehydrogenase, which catalyzes the reversible reaction between D-3-hydroxybutyrate and acetoacetate, has been classified into the short-chain dehydrogenase/reductase family and is a useful marker in the assay of diabetes mellitus and/or ketoacidosis. The enzyme from Alcaligenes faecalis was crystallized in the apo form and in the holo form with acetate as a substrate analogue. The crystal structures of both forms were determined at 2.2 angstroms resolution. The enzyme is a tetramer composed of four subunits assembled with noncrystallographic 222 point symmetry. Each subunit has two domains. The principal domain adopts the Rossmann fold essential for nucleotide binding, which is a common feature of the SDR family. NAD+ is bound in a large cleft in the domain. The pyrophosphate group of NAD+ is covered by the small additional domain, which is supported by two extended arms allowing domain movement. In the catalytic site, a water molecule is trapped by the catalytic Tyr155 and Ser142 residues in the vicinity of the bound NAD+ and acetate. The substrate analogue acetate is bound above the nicotinamide plane. A substrate (D-3-hydroxybutylate) bound model can reasonably be constructed by adding two C atoms into the void space between the water O atom and the methyl group of the acetate, suggesting a substrate-bound state before enzymatic reaction occurs. Based on these structural features, a reaction mechanism has been proposed.

The structures of pyruvate oxidase from Aerococcus viridans with cofactors and with a reaction intermediate reveal the flexibility of the active-site tunnel for catalysis.

The crystal structures of pyruvate oxidase from Aerococcus viridans (AvPOX) complexed with flavin adenine dinucleotide (FAD), with FAD and thiamine diphosphate (ThDP) and with FAD and the 2-acetyl-ThDP intermediate (AcThDP) have been determined at 1.6, 1.8 and 1.9 A resolution, respectively. Each subunit of the homotetrameric AvPOX enzyme consists of three domains, as observed in other ThDP-dependent enzymes. FAD is bound within one subunit in the elongated conformation and with the flavin moiety being planar in the oxidized form, while ThDP is bound in a conserved V-conformation at the subunit-subunit interface. The structures reveal flexible regions in the active-site tunnel which may undergo conformational changes to allow the entrance of the substrates and the exit of the reaction products. Of particular interest is the role of Lys478, the side chain of which may be bent or extended depending on the stage of catalysis. The structures also provide insight into the routes for electron transfer to FAD and the involvement of active-site residues in the catalysis of pyruvate to its products.

Structural and glycation site changes of albumin in diabetic patient with very high glycated albumin.

BACKGROUND: Glycated albumin (GA) has been utilized to monitor mid-term glycemic control, and reflects the status of blood glucose more rapidly and effectively than hemoglobin A(1c) (HbA(1c)). To examine the relationship between GA level and structural changes or glycation sites of albumin, we analyzed pre- and post-treatment samples from a diabetic patient with extraordinary increase of GA. METHOD: A female diabetic patient with poor glycemic control had a GA >94% and was treated with intensive insulin therapy to decrease blood glucose. We analyzed changes in fluorescence derived from tryptophan (Trp) and advanced glycation end product (AGE) of albumin isolated/purified from pre- and post-treatment samples. To determine the sites of glycation of albumin, samples were carboxymethylated and digested by Glu-C endoprotease, and peptides were analyzed using liquid chromatography/mass spectrometry. RESULTS: GA level decreased almost linearly and reflected the improved glycemic state well. Trp-related fluorescence of pre- and post-treated samples did not change while AGE-related fluorescence increased depending on GA level. Ten major glycation sites were detected in the pre-treatment sample, while 3 major glycation sites were detected in post-treated samples. CONCLUSIONS: GA level reflects the status of blood glucose more rapidly than HbA(1c). Since GA level was related to AGE-related fluorescence and number of glycation sites, it might be a good marker for not only glycemic control of diabetic patients but also structural and functional changes of albumin.

A method for designing conformationally restricted analogues based on allylic strain: synthesis of a novel class of noncompetitive NMDA receptor antagonists having the acrylamide structure.

A series of conformationally restricted analogues of milnacipran, a weak NMDA receptor antagonist, were designed by a method based on allylic strain. The conformational analysis study showed that the allylic-strain-based conformational restriction indeed occurred and that the affinity for the NMDA receptor was efficiently improved by the conformational restriction.

A systematic study of the hydride reduction of cyclopropyl ketones with structurally simplified substrates. highly stereoselective reductions of trans-substituted cyclopropyl ketones via the bisected s-cis conformation.

The stereoselective hydride reduction of the cis- and trans-substituted cyclopropyl ketones was systematically investigated using a series of structurally simplified substrates, trans-[tert-butyldiphenylsilyloxymethyl]cyclopropyl ketones 1a-e and trans-(benzyloxymethyl)cyclopropyl methyl ketone (2), and the corresponding cis congeners 3a,b,e and 4. The results showed that, not only in the reduction of the cis-substituted cyclopropyl ketones but also in that of the trans-substituted ketones, high stereoselectivity can be realized when the substrate has a bulky substituent on the cyclopropane ring, even though it is attached to the position trans to the acyl moiety. Ab initio calculations based on the density functional theory (DFT) of cyclopropyl ketones showed that (1) the bisected s-cis and s-trans conformers were the only two minimum energy conformers, while the s-cis conformer was more stable than the s-trans and (2) a bulky alkyl group in the acyl moiety and a cis substituent on the cyclopropane ring made the bisected s-cis conformer much more stable. On the basis of these calculations and experimental results, it is likely that the more stable the bisected s-cis conformer of the substrate, the more stereoselective the hydride reduction. Thus, the stereochemistry can be explained by hydride attack on the bisected s-cis conformation of the substrate from the less-hindered face. The predictability of the stereochemical results is predicated on the bisected s-cis transition-state model, which is very important from the viewpoint of synthetic organic chemistry.

Conformational analysis of the NMDA receptor antagonist (1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (PPDC) designed by a novel conformational restriction method based on the structural feature of cyclopropane ring.

(1S,2R)-1-phenyl-2-[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide (2b, PPDC), a new class of potent N-methyl-D-aspartic acid (NMDA) receptor antagonist, was designed based on a new method for restricting the conformation of compounds having a cyclopropane ring. The three-dimensional structures of PPDC obtained by the three different methods of X-ray crystallographic analysis, usual MM2-calculations in vacuum, and MM2 calculations based on the nuclear Overhauser effect (NOE) data in D2O are similar, which are in accord with that hypothesized. These results suggest that this conformational restriction method is particularly effective in designing novel biologically active molecules.