C. LeRoy Blank

Dual electrochemical detector for liquid chromatography

Abstract Selectivity, sensitivity, and wide dynamic range are all displayed by the dual electrochemical detector for use with liquid chromatography. Exhibiting a linear range of about 105, it is, at the same time, capable of sensing sub-picomole quantities of certain species. Moreover, only those compounds which display electroactivity (oxidation or reduction) at the chosen potential(s) are detected. Two distinctive modes of operation are available with the dual detector. For compounds which overlap chromatographically, but have differing electrochemical formal potentials, the system offers an instrumental separation. For routine investigations, the system provides significant time reductions without causing proportionate increases in cost.

A novel, inexpensive, and sensitive method for analysis of tyrosine hydroxylase activity in tissue samples

Abstract Tyrosine hydroxylase activity in whole mouse brains was measured in v vitro . The L-dihydroxyphenylaline (L-DOPA) formed by the enzyme was quantitated by liquid chromatography with electrochemical detection (LCEC). An investigation of the incubation factors (added Fe+2, DOPA decarboxylase inhibitor concentration, substrate concentration, amount of tissue, time of incubation) is reported. Under optimal conditions the activity was found to be 15.1 ± 0.6 (S.E.M.) nmol DOPA formed/hr./g. tissue.

LEVELS OF NOREPINEPHRINE AND DOPAMINE IN MOUSE BRAIN REGIONS FOLLOWING MICROWAVE INACTIVATION‐RAPID POST‐MORTEM DEGRADATION OF STRIATAL DOPAMINE IN DECAPITATED ANIMALS

—The effects of 2 methods of killing on norepinephrine and dopamine in mouse brain regions were examined. One method utilized decapitation, while the other method utilized heating with microwave irradiation concentrated on the head. The norepinephrine and dopamine contents of the cerebellum, medulla‐pons, midbrain, diencephalon, hippocampus, corpus striatum, and cerebral cortex were determined by methods using liquid chromatography with electrochemical detection. Dopamine content in striatum was also quantitated by the method of gas chromatography with mass fragmentography. A significantly lower value for decapitated animals, as compared to the microwave heated group, was found only for dopamine exclusively in the striatum.

Simultaneous determination of norepinephrine, dopamine, and serotonin in brain tissue by high-pressure liquid chromatography with electrochemical detection

Abstract High-pressure liquid chromatography with electrochemical detection (l.c.e.c.) is utilized in a procedure for the simultaneous determination of three of the most important neurotransmitters in nervous tissue samples. The high degree of selectivity and sensitivity (subpicomole limits for each component) makes this technique directly applicable to extremely small samples. After optimal conditions had been established, the procedure was employed in the determination of all three components in whole mouse brains, seven separate mouse brain parts, and diurnal variations of whole mouse brains. The technique outlined does not require sample splitting, tissue pooling, component derivatization, or coupled enzymatic reactions to achieve the desired results. Twenty individual samples may be accommodated per day.

5,5′‐Dihydroxy‐4,4′‐Bitryptamine: A Potentially Aberrant, Neurotoxic Metabolite of Serotonin

Abstract: Previous investigators have detected unknown oxidized forms of 5‐hydroxytryptamine (5‐HT) in the CSF of Alzheimers disease (AD) patients. Furthermore, an unidentified autoxidation product of this neurotransmitter is an inhibitor of acetylcholinesterase (AChE), an enzyme compromised in the Alzheimer brain. In this study it is demonstrated that the major product of autoxidation of 5‐HT is 5,5′‐dihydroxy‐4,4′‐bitryptamine (DHBT). Central administration of DHBT to mice at a dose of 40 μg (free base) evokes profound behavioral responses, which persist until the animals die (∼24 h). One hour after central administration of DHBT, the levels of norepinephrine, dopamine, 5‐HT, and acetylcholine and their metabolites in whole brain are greatly elevated. Disturbances to the catecholaminergic and serotonergic systems were still evident shortly before the death of animals. DHBT is also shown to be a noncompetitive inhibitor of AChE in vitro. These observations suggest that if DHBT is formed as an aberrant metabolite of 5‐HT in the human brain, it could potentially be neurotoxic and contribute to the neuronal degeneration and other neurochemical and neurobiochemical changes associated with AD or perhaps other neurodegenerative diseases.

7-S-Glutathionyl-tryptamine-4,5-dione: A possible aberrant metabolite of serotonin

Tryptamine-4,5-dione (Compound 1) is an in vitro oxidation product of 5-hydroxytryptamine (5-HT). Recent evidence has suggested that aberrant oxidations of 5-HT occur in the central nervous system of individuals with Alzheimers disease (AD). In the event that Compound 1 is formed as a result of oxidation of 5-HT within serotonergic nerve terminals or axons, it would be expected to be rapidly conjugated by intraneuronal glutathione (GSH) to give 7-S-glutathionyl-tryptamine-4,5-dione (Compound 2). When injected into the brains of laboratory mice, Compound 2 was lethal (LD50 = 21 micrograms) and evoked hyperactivity for the first 30 min following drug administration. Particularly during this hyperactive phase Compound 2 caused a statistically significant decrease in whole brain levels of norepinephrine and 5-HT. Levels of dopamine were also decreased while whole brain concentrations of its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were increased significantly. In the presence of GSH, NADPH and ascorbic acid, Compound 2 redox cycled in reactions that catalyzed the oxidation of these cellular reductants by molecular oxygen and formed H2O2 as a byproduct. Compound 2 also reacted with molar excesses of GSH to form more structurally complex glutathionyl conjugates. Several of these conjugates have been isolated and their structures determined using spectroscopic methods. It is conceivable that one or more of these conjugates might serve as analytical markers in a search for evidence in support of the hypothesis that aberrant oxidations of 5-HT occur in the Alzheimer brain. The redox cycling properties of Compound 2 and its facile reactions with cellular nucleophiles such as GSH may represent mechanisms that contribute to the toxicity of this drug.

Glassy carbon pre-column for direct determination of acetylcholine and choline in biological samples using liquid chromatography with electrochemical detection

The determination of acetylcholine and choline has been quite successfully accomplished using liquid chromatography with electrochemical detection following the original reports of Potter et al. [J. Neurochem., 41 (1984) 188]. A post-column reactor containing acetylcholinesterase and choline oxidase allows conversion of the desired species into hydrogen peroxide, an electrochemically active substance. However, the direct injection of tissue homogenates and other biological samples into such a system exhibits quite large solvent fronts and unidentified peaks. Using a pre-column packed with glassy carbon particles, we were able to dramatically decrease the size of the solvent front for such injections and tentatively identify the unknown peaks to be caused, at least in part, by common catecholamines. The glassy carbon pre-column, in addition to increasing the selectivity of the results, allowed the required chromatographic time per sample to be decreased from 20 to 10 min.

Utilizing the Urinary 5-HTOL/5-HIAA Ratio to Determine Ethanol Origin in Civil Aviation Accident Victims

Specimens from fatal aviation accident victims are submitted to the FAA Civil Aerospace Medical Institute for toxicological analysis. During toxicological evaluations, ethanol analysis is performed on all cases. Care must be taken when interpreting a positive ethanol result due to the potential for postmortem ethanol formation. Several indicators of postmortem ethanol formation exist; however, none are completely reliable. The consumption of ethanol has been shown to alter the concentration of two major serotonin metabolites, 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA). While the 5-HTOL/5-HIAA ratio is normally very low, previous studies using living subjects have demonstrated that the urinary 5-HTOL/5-HIAA ratio is significantly elevated for 11-19 h after acute ethanol ingestion. Recently, our laboratory developed and validated an analytical method for the simultaneous determination of both 5-HTOL and 5-HIAA in forensic urine samples using a simple liquid/liquid extraction and LC/MS/MS and LC/MS/MS/MS. In this previous work a 15 pmol/nmol serotonin metabolite ratio cutoff was established in postmortem urine, below which it could be conclusively determined that no recent antemortem ethanol consumption had occurred. In the current study this newly validated analytical method was applied to five ethanol-positive aviation fatalities where the origin of the ethanol present could not previously be conclusively determined. In four of the five cases examined the detected ethanol was demonstrated to be present due to postmortem microbial formation, and not consumption, even though some indication of ethanol consumption may have been present.

Synthesis and Neurotoxic Potential of Racemic and Chiral Dihydroxytetrahydroquinoline Derivatives

Abstract The synthesis and preliminary neurotoxic investigation of (±), (+) and (−)-3-amino-6,7-dihydroxy-1,2,3,4-tetrahydroquinoline, (±)-3-amino-6,8-dihydroxy-1,2,3,4-tetrahydroquinoline and (±)-3-aminomethyl-6,7-dihydroxy-1,2,3,4-tetrahydroquinoline are described. While exhibiting relatively no dopamine and only moderate norepinephrine depletions, these compounds elicit serotonin depletions equal to those provided by the well-known serotonergic neurotoxin 5,7-dihydroxytryptamine in whole mouse brain.

Binding characteristics of various neurochemicals to glassy carbon

The determination of acetylcholine and choline using liquid chromatography with electrochemical detection using the approach of Potter et al. [J. Neurochem., 41 (1983) 188] normally requires isolation of the desired species before an analysis of tissue samples can be undertaken due to coeluting interferences afforded by other neutrochemicals. We have recently shown that this problem can be overcome by the use of a glassy carbon precolumn to effectively trap the interfering species [Ikarashi et al., J. Chromatogr., 575 (1992) 29]. We now report on the nature and mechanism of this adsorption onto glassy carbon for norepinephrine, dopamine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid. For both the acidic and basic compounds which comprise this group, distinct Langmuir adsorption processes appear to be involved for both the neutral and ionic forms of the individual compounds. Using various data fitting approaches, we have attempted to derive appropriate adsorption constants for the two forms of each compound. Theoretical predictions employing these derived constants provided results which match reasonably well with the observed adsorption data in most cases.