Thomas K. Green

Separation and sensitive detection of D-amino acids in biological matrices

The increase in our understanding of D-amino acid function and distribution in mammals is in many ways a result of the initial development of sensitive enantioselective separation strategies that allow for quantification in real biological samples. This article reviews progress on the development of chiral selective separation and detection of D-amino acids including enzyme-based microbiosensors, GC/MS, HPLC/fluorescence, HPLC/MS-MS, cEKC/fluorescence, and MEKC/fluorescence. Only methods capable of analyzing D-isomers in biological matrices are given here and significant effort is made to highlight approaches that offer speed, resolution, high sensitivity, and versatility.

Sampling glutamate and GABA with microdialysis: suggestions on how to get the dialysis membrane closer to the synapse

Microdialysis is currently optimized to sample the extrasynaptic pool. As such, the technique has facilitated discovery of ischemia-induced excitotoxic glutamate overflow (Benveniste H, Drejer J, Schousboe A, Diemer NH, 1987, Regional cerebral glucose phosphorylation and blood flow after insertion of a microdialysis fiber through the dorsal hippocampus in the rat. J. Neurochem., 49, 729-734) and adenosinergic sleep drive (Porkka-Heiskanen T, Strecker RE, Thakkar M, Bjorkum AA, Greene RW, McCarley RW, 1997, Adenosine: a mediator of the sleep-inducing effects of prolonged wakefulness. Science, 276 (5316), 1265-1268); and is proving essential for clinical monitoring of glutamate and cellular metabolites in stroke and head trauma (Sarrafzadeh AS, Sakowitz OW, Kiening KL, Benndorf G, Lanksch WR, Unterberg AW. Bedside microdialysis: a tool to monitor cerebral metabolism in subarachnoid hemorrhage patients? Crit. Care Med. 2002, 30 (5): 1062-1070). Study of the origin of extrasynaptic glutamate sampled with microdialysis has advanced understanding of extrasynaptic signal processing (Baker DA, Xi ZX, Shen H, Swanson CJ, Kalivas PW. The origin and neuronal function of in vivo nonsynaptic glutamate. J. Neurosci. 2002, 22 (20): 9134-9141; Baker DA, McFarland K, Lake RW, Shen H, Tang XC, Toda S, Kalivas PW, 2003, Neuroadaptations in cystine-glutamate exchange underlie cocaine relapse. Nat. Neurosci., 6, 743-749) in the CNS. Microdialysis studies furthermore demonstrate that synaptic pools of some neurotransmitters spill into the extrasynaptic space. For this reason, microdialysis has provided a window into the synaptic pool that has significantly advanced understanding of neurotransmitter control of behavior (Tanda G, Pontieri FE, Di Chiara G, 1997, Cannabinoid and heroin activation of mesolimbic dopamine transmission by a common mu1 opioid receptor mechanism. Science, 276, 2048-2050). Nonetheless, ability to sample synaptic pools of neurotransmitters is limited. Here we summarize evidence that microdialysis often fails to sample synaptic pools of neurotransmitters, such as glutamate and GABA because of rapid clearance and limited diffusion of these neurotransmitters from the synapse. Moreover, we consider means to move the dialysis membrane closer to the synapse to facilitate sampling of the synaptic pool of these neurotransmitters by minimizing tissue trauma, decreasing probe size and increasing temporal resolution.

Simultaneous efflux of endogenous D-ser and L-glu from single acute hippocampus slices during oxygen glucose deprivation.

D‐serine and L‐glutamate play crucial roles in excitotoxicity through N‐methyl‐D‐aspartate receptor coactivation, but little is known about the temporal profile of efflux during cerebral ischemia. We utilized a newly designed brain slice microperfusion device coupled offline to capillary electrophoresis laser‐induced fluorescence to monitor dynamic efflux of endogenous D‐ser and L‐glu in response to oxygen glucose deprivation (OGD) in single acute hippocampus slices. Efflux profiles with 2‐min temporal resolution in response to 24‐min OGD show that efflux of D‐ser slightly precedes efflux of L‐glu by one 2‐min sampling interval. Thus both coagonists are available to activate NMDA receptors by the time when glu is released. The magnitude of D‐ser efflux relative to baseline values is, however, less than that for L‐glu. Peak efflux during OGD, expressed as pre‐OGD baseline values, was as follows: D‐ser 254% ± 24%, L‐glu 1,675% ± 259%, L‐asp 519% ± 128%, and L‐thr 313% ± 33%. L‐glutamine efflux was shown to decrease significantly in response to OGD. The microperfusion/CE‐LIF approach shows several promising attributes for studying endogenous chemical efflux from single, acute brain slices.

Quantitative determination of secondary metabolites in Cladina stellaris and other lichens by micellar electrokinetic chromatography.

The quantitative determination of usnic acid (UA), perlatolic acid (PA), and atranorin (AT) in Alaska lichens by micellar electrokinetic chromatography (MEKC) is reported. The background electrolyte (BGE) included sodium docecyl sulfate (SDS), and beta-cyclodextrin (beta-CD) in a high-pH borate buffer. The presence of beta-CD in the buffer significantly decreases peak width, especially for UA, as it decreases migration time for both UA and PA. Linear calibration curves for UA, PA, and AT were established using an internal standard of benzoic acid (BA). Concentration limits of detection (cLODs) are 2.5, 2.2 and 2.0microg/mL (S/N 3) for UA, PA, and AT, respectively. Dry samples of lichen were extracted at room temperature with acetone for 24h in the presence of BA as internal standard. Recoveries of UA from spiked samples ranged from 92 to 98%. Amounts of UA and PA in the lichen samples ranged from 0.28 to 1.7% dry weight and 0.02 to 0.23%, respectively.

Separation of sulfoalkylated cyclodextrins with hydrophilic interaction liquid chromatography

Determination of the charged state distributions of partially- and fully-substituted sulfoalkylated cyclodextrins was achieved using hydrophilic interaction liquid chromatography (HILIC). HILIC analysis of a spiked sample of the partially sulfopropylated cyclodextrins was achieved using a gradient to baseline resolve the charged states from -1 to -14. The fully-substituted CDs yielded a major peak with some trace impurities and the partially-substituted sulfopropylated cyclodextrins showed a wide range of charge states present in the mixture. Small changes in the structure of the cyclodextrins have a significant impact on the retention times of the various types of cyclodextrins investigated.

Solubilization of Hexafluorobenzene by the Micellar Aromatic Core Formed from Aggregation of Amphiphilic (2,3-O-Dibenzyl-6-O-sulfobutyl) Cyclodextrins

Aggregation colloids that possess an aromatic pseudophase in an aqueous system could provide new avenues of research including micellar catalysis, aqueous remediation, and emulsion polymerization studies. The apparent aggregation of two macrocyclic surfactants, hexakis (2,3-O-dibenzyl-6-O-sulfobutyl) cyclomaltohexaose (DBSBA) and heptakis (2,3-O-dibenzyl-6-O-sulfobutyl) cyclomaltoheptaose (DBSBB), was investigated using diffusion ordered nuclear magnetic resonance (NMR) spectroscopy (DOSY), conductivity, and pyrene fluorescence techniques. These amphiphiles were found to possess near spherical symmetry at critical micelle concentrations of approximately 0.1 mM in all techniques used to study the phenomenon. Aggregation of both surfactants was found to be entropically driven at low temperatures but enthalpically driven at higher temperatures. The calculated compensation temperatures of DBSBA and DBSBB were determined to be 317 and 307 K, respectively. These surfactants contain a high percentage of aromatic moieties in their structures, which affects the thermodynamics of aggregation and their interior micellar environment. The proposed aromatic micellar core was tested using hexafluorobenzene (HFB) as a molecular probe in (19)F NMR experiments. (19)F NMR relaxation and chemical shift studies found the HFB quantitatively partitioned into the micellar interiors. Global regression analysis found that HFB interaction with DBSBA micelles possessed at least two association constants, differing by an order of magnitude, the largest being in excess of 8300 M(-1). DBSBB micellar interactions with HFB were found to be weaker, although in excess of 1100 M(-1), with a subsequent association constant of similar magnitude. Benzyl substituents of DBSBB are required for solubilization of HFB. Heteronuclear Overhauser effect spectroscopy (HOESY, (19)F-(1)H) of the DBSBB:HFB complex revealed strong interaction of HFB with benzyl substituents but not the cyclodextrin cavity.