Ivan N. Mefford

Liquid chromatographic analysis of catecholamines routine assay for regional brain mapping

Abstract A thoroughly tested and highly reliable catecholamine assay is described which routinely determines 20–100 picograms of norepinephrine and dopamine in small punches of brain tissue weighing 0.50 to 50 mg. The assay was designed for regional brain mapping. It employs liquid chromatography with electrochemical detection and involves a minimum of sample pretreatment. Its realistic performance is illustrated by typical experimental data. Modifications for larger or whole brain samples as well as details of construction and operation of this system are given.

Regional distribution of ascorbate in human brain

A comprehensive mapping of ascorbate distribution in human brain was carried out by liquid chromatography analysis. The data agree with earlier literature values where comparable and provide new information on several brain regions, including a detailed distribution in the thalamus. While ascorbate concentrations tend to be high in regions rich in catecholamines, there is no real correlation between the two.

THE USE OF LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION FOR THE DETERMINATION OF ADRENALINE AND OTHER BIOGENIC MONOAMINES IN THE CNS

High pressure liquid chromatography with electrochemical detection (LCEC) for the chemical-analytical determination of adrenaline and other biogenic monoamines in brain samples is briefly described and commented. This method has a reasonably good specificity and in routine use a sensitivity of about 20 pg (0.1 pmol) catecholamine per sample. Results from the use of this technique for studies of the adrenaline distribution in human post-mortem brains are presented. The data obtained suggest a distribution of adrenaline neurons in the human brain similar to that previously reported for rat brain, although the adrenaline systems seems to be more widely distributed in the human. Studies of the distribution of catecholamines in subcellular fractions in rat hypothalamus and pons-medulla using LCEC disclosed a similar distribution of all catecholamines (dopamine, noradrenaline and adrenaline); the main part (about 60%) being recovered in the nerve ending particle (synaptosomal) fraction. This is compatible with the view that adrenaline, like noradrenaline and dopamine, can serve as a neurotransmitter. Results from studies on the effect of tyrosine hydroxylase and dopamine-β-hydroxylase inhibition on catecholamine levels in rat brain, indicated a particular type of interaction between noradrenaline and adrenaline neurons in the pons-medulla. It is suggested that part of the noradrenaline released from the noradrenaline nerve terminals is taken up in nearby adrenaline nerve terminals where it can serve as precursor for adrenaline biosynthesis. The results indicated a similar situation in the striatum where released dopamine can be taken up in noradrenaline nerve terminals acting as precursor for noradrenaline synthesis. The functional significance of this type of interaction between catecholamine nerve terminals remains to be elucidated.

In vitro and in vivo depolarization coupled efflux of ascorbic acid in rat brain preparations

The depolarization-coupled efflux of endogenous ascorbate is demonstrated using rat synaptosome preparations and a rat cortical cup method. Ascorbate and catecholamines (monitored as a methodological control) were quantitated by high pressure liquid chromatography with electrochemical detection. In vitro potassium ion induces ascorbate efflux in a concentration-dependent, although calcium ion-dependent, manner. Veratridine also induces ascorbate efflux and its effect can be blocked by tetrodotoxin. In vivo, ascorbate efflux was likewise stimulated by increased potassium ion and by veratridine. In addition, electrical stimulation of medial lemniscus was accompanied by an increased efflux of ascorbate from somatosensory cortex. These results are intriguing in light of the recent evidence for the interaction of ascorbate with several neurotransmitter systems.

Indomethacin prevents increased catecholamine turnover in rat brain following systemic endotoxin challenge

1. Key features of the acute phase response to infection are replicated by systemic administrations of lipopolysaccharide and may be mediated via the production of lymphokines and cytokines, including interleukin-1. Inhibition of prostaglandin synthesis may attenuate certain features of the acute phase response. 2. In the present study, the effects of systemic administration of the lipopolysaccharide (LPS, 250 micrograms/rat) and interleukin-1 (IL-1, 10 micrograms/rat) on catecholamine metabolism in different brain regions were compared and the effects of indomethacin, a cyclooxygenase inhibitor was determined. 3. The ratio of metabolite to parent amine was used as an index of turnover of catecholamines. 4. In hypothalamus, both epinephrine and norepinephrine concentrations were decreased and their major metabolite, 3-methoxy,4-hydroxyphenylglycol (MHPG), was elevated at 4, 8 and 24 hr following LPS. The major metabolite of dopamine (homovanillic acid, HVA) was increased at 8 hours in striatum, hypothalamus and medulla. LPS increased dopamine turnover at 8 and 24 hr and norepinephrine turnover at 4, 8 and 24 hr. 5. In all regions examined, IL-1 produced effects similar to LPS on amine and metabolite contents and norepinephrine and dopamine turnover. 6. Significantly, co-administration of a single dose of indomethacin (50 mg/kg) completely blocked LPS-induced changes in hypothalamic catecholamines and metabolites and the increase in turnover at 4 and 8 hr. Furthermore, the effects of IL-1 on hypothalamic MHPG content and norepinephrine turnover were also blocked by indomethacin, although the effects of IL-1 on regional catecholamines and HVA content and turnover were either not modified or partially antagonized by indomethacin. 7. The present results suggest that in the rat, activation of noradrenergic, dopaminergic and epinephrine-containing neurons in hypothalamus, as well as dopaminergic neurons in other regions is associated with the acute phase response to endotoxin and that synthesis of prostaglandins plays a pivotal role in catecholamine responses in all brain regions examined.

Determination of reduced glutathione in guinea pig and rat tissue by HPLC with electrochemical detection.

Abstract A method using HPLC with electrochemical detection has been developed for the determination of GSH in tissue. The method is based upon the separation of GSH from other components by cation exchange chromatography coupled with the electrochemical oxidation of GSH to the corresponding disulfide. Detection limits of ca. 5 × 10−12 moles GSH were established and the method was used to measure GSH content of rat and guinea pig brain, liver and synaptosome preparations.

Epinephrine localization in human brain stem.

A study was undertaken to determine the presence and possible localization of epinephrine (E) in the human brain stem. Three brain stems were analyzed, although detailed mapping was performed only on the third. E was found to be highly localized in the reticular formation and the aqueductal gray. The highest concentrations were found in the region of the floor of the fourth ventricle. Norepinephrine (NE) and dopamine (DA) determinations were included in the analyses. These catecholamines were also found to be localized in the same regions. The relative E concentrations ranged from about 1-10% of the total catecholamine content.

Epinephrine distribution in human brain.

The distribution of epinephrine (E) in human brain has been analyzed by a previously described [11] liquid chromatographic method. E, though detectable in most areas sampled, was found to be highly localized in those areas previously known to contain highest norepinephrine (NE) concentrations [4]. The regions found to be highest in E concentration were the ventromedial, dorsomedial, supraoptic and paraventricular nuclei of the hypothalamus.

Evidence for the presence of PNMT-containing cell bodies in the hypothalamus

The presence of intrinsic neurons in the hypothalamus containing the enzyme phenylethanolamine N-methyltransferase (PNMT), was investigated by selective destruction of the local cell bodies with kainic acid (KA), a potent excitotoxin. The unilateral stereotaxic injection of 2 micrograms of KA into the lateral hypothalamus produced a 30-35% decrease in the hypothalamic PNMT activity in the lesioned side compared with the contralateral uninjected side. The difference was statistically significant for up to 4 days after the lesion. The unilateral lesion did not have a unilateral effect on epinephrine or norepinephrine content as there was a significant reduction on both sides (50% for epinephrine and 30% for norepinephrine compared with saline injected). These data suggest the presence of intrinsic cell bodies in the lateral hypothalamus, containing the epinephrine-forming enzyme but lacking epinephrine storage sites. Our results support the previous hypothesis of the dissociation between epinephrine present in the hypothalamus and hypothalamic PNMT activity.