Damodar D. Godse

Rat Brain and Plasma Norepinephrine Glycol Metabolites Determined by Gas Chromatography‐Mass Fragmentography

Abstract: A gas chromatographic‐mass fragmentographic (GC‐MF) procedure is described for the simultaneous quantitation of 3,4‐dihydroxyphenyl‐ethyleneglycol (DHPG) and 3‐methoxy‐4‐hydroxyphenylethyleneglycol (MHPG) in brain tissue and plasma. DHPG and MHPG were assayed as their respective acetyl‐trifluoroacyl esters, using [2H2]DHPG and [2H3]MHPG as internal standards. Assay sensitivities of at least 1 ng per sample were attainable for the quantitation of free glycols, whereas for determination of total DHPG, assay sensitivity was 2.5 ng. Whole rat brain total (99.2 ±4.11 ng/g) and free (13.0 ± 1.14 ng/g) DHPG concentrations were similar to respective total (86.0 ± 3.70 ng/g) and free (12.3 ± 0.41 ng/g) MHPG levels. Total DHPG concentrations exceeded total MHPG levels in hypothalamus (3.0:1), midbrain (1.4:1), pons plus medulla (1.3:1), and hippocampus (1.5:1), whereas in other brain regions the levels of these metabolites were similar. In plasma, however, total DHPG levels were only 20% as high as MHPG concentrations. In mouse brain, DHPG and MHPG occurred almost entirely in free form (>90%), but total DHPG levels were only 50% as high as respective MHPG concentrations. These results emphasize the substantial formation of DHPG compared with MHPG in rat and mouse brain and suggest that DHPG formation and eMux may be of equal or greater importance than MHPG in the metabolic clearance of CNS norepinephrine in some species.

A gas chromatographic-mass spectrometric (GC-MS) assay for 3-methoxy-4-hydroxyphenethyleneglycol and vanilmandelic acid in human serum

Procedure for the quantification of 3-methoxy-4-hydroxyphenethyleneglycol (MHPG) and vanilmandelic acid (VMA) in human serum are described. MHPG was selectively acetylated then determined as its 4-acetyl-di-trifluoroacetyl derivative and VMA as its di-pentabluoropropionyl-methyl ester. Deuterium-labeled MHPG and VMA were used as internal standards. Each of these metabolities and the resprective internal standards was recorded by double ion monitoring respectively, and the ratios were determined for specificity. Assay sensitivities of 1.0 ng/ml for MHPG and 2.0 ng/ml for VMA were achieved using 0.5 ml of serum. Total and free MHPG concentrations in human serum were determined to be 16.5 ng/ml +/- 4.4 (S.D.) and 4.6 ng/ml +/- 1.0 respectively from 10 normal male subjects. Free VMA concentrations were 7.0 ng/ml +/- 1.5; thus the ratio of MHPG/VMA was calculated to be 2.4 +/- 0.7 in these subjects. Of these two major normal metabolites of norepinephrine, MHPG is regarded to be largely derived from the central nervous system while VMA is from the periphery. The procedures are highly specific as well as simple and sensitive enough to permit simultaneous measurement of these two metabolites in a series of multiple samples from an individual.

Induction of rage in rats by central injection of 6-hydroxydopamine.

Abstract Intracisternal injection of 300 μg of 6-hydroxydopamine in male rats elicited a syndrome of hyperreactivity or hyperemotionality, i.e., rage, similar to that previously reported after septal or ventromedial hypothalamic (VMH) lesions. Specifically, rats showed increased resistance to capture as well as increased number and magnitude of startle responses compared to vehicle injected or normal controls. As with septal but not VMH lesions, this rage subsided with repeated testings (handling). These findings are discussed with regard to the possible importance of brain neurotransmitters in the expression of behaviors.

Brain noradrenergic neuronal activity affects 3,4-dihydroxyphenylethyleneglycol (DHPG) levels

Abstract Brain regional DHPG levels were determined following pharmacological manipulations that are known to alter brain noradrenergic neuronal activity. In rats given the α-adrenergic antagonist yohimbine (1, 5 and 10 mg/kg, i.p.) 2 h prior to sacrifice, there was a dose-dependent increase in cortical, midbrain, pons + medulla, hypothalamic and spinal total DHPG and MHPG concentrations. In contrast, cortical and spinal total DHPG and MHPG concentrations were markedly decreased 2 h following the α-adrenergic agonist, clonidine (10 and 250 μg/kg, i.p.). These findings indicate that rat brain DHPG formation is also sensitive to changes in brain noradrenergic neuronal impulse flow.

Dependence of brain tryptamine formation on tryptophan availability.

Abstract— The dependence of rat brain tryptamine (TA) formation on tryptophan availability was investigated. Rat brain TA was found to accumulate linearly with respect to time over 90min following pargyline (75 mg/kg. i.p.). The in vivo rate of brain TA appearance was estimated to be 0.1 nmol/g/h. After tryptophan loading the accumulation of brain TA and 5‐HT in pargyline‐pretreated rats was linearly related to both brain and plasma tryptophan concentrations. Low doses of tryptophan (5–10 mg/kg. i.p.) produced statistically significant increments in brain TA formation. The findings of this study indicate that the in vivo formation of TA in rat brain is directly dependent upon brain and plasma tryptophan concentrations.

Brain tryptamine in rats by a new gas chromatography-mass fragmentographic method

Abstract A simplified assay for tryptamine (TRYP) by gas chromatographymass fragmentography (GC-MF) is described. TRYP was analyzed as its N-acetyl-1-trifluoroacetyl derivative. Acetylation of TRYP was carried out at basic pH using acetic anhydride dissolved in a diethyl ether vehicle. N-acetyl-TRYP was extracted into the diethyl ether from a basic aqueous phase during the acetylation reaction. TRYP-D2 was employed as an internal standard. TRYP was quantitatively assayed in human urine and rat brain using multiple ion detection GC-MF. Assay sensitivities of 500 pg could be achieved by single ion and 5 ng by double ion GC-MF. In rats treated with pargyline and pargyline plus tryptophan, brain TRYP was found to be 35.6 ± 4.9 (mean ± SE) and 133 ± 3.8 ng/g, respectively. However, TRYP was not detected in normal rat brain at an assay sensitivity of 500 pg.

Consummatory behaviors of hypothalamic hyperphagic rats after central injection of 6-hydroxydopamine ☆

Abstract Two experimenrs examined the ability of medial hypothalamic lesions to produce overeating, weight gain and finickiness toward palatable and unpalatable solutions in rats with persisting decrements in brain norepinephrine and dopamine due to central injection of 6-hydroxydopamine. In both experiments, such disruption of central catecholamine systems did not significantly reduce overeating and weight gain subsequent to hypothalamic injury. However, acceptance of quinine solutions was increased following 6-hydroxydopamine treatment in both lesioned and non-lesioned rats. The relative importance of brain catecholamines for the expression of different consummatory behaviors is discussed.

Gas chromatography--mass fragmentographic determination of indole-3-acetic acid in rat brain.

Abstract— A gas chromatography‐mass fragmentographic procedure is described for the quantitation of indole‐3‐acetic acid (IAA) in rat brain. IAA was determined as its IV‐1‐pentafluoropropionyl‐methyl ester. Deuterium labeled IAA was employed as an internal standard. Assay sensitivities of at least 500 pg and 2.5 ng could be achieved by single and double ion monitoring respectively. Whole brain IAA concentrations were determined to be 11.6 + 0.75 ng/g (n = 6). IAA concentrations in brain areas ranged from 10 to 64 ng/g with highest levels in the striatum. Rat brain IAA accumulated curvilinearly with respect to time over a 90 min interval following probenecid (200 mg/kg, i.p.). The efflux of IAA from brain appears mediated in part through a probenecid‐sensitive active efflex process.

Rat Brain Norepinephrine Metabolism: Substantial Clearance Through 3,4-Dihydroxyphenylethyleneglycol Formation

Abstract: To assess whether the metabolic clearance of rat brain norepinephrine (NE) through 3,4‐dihydroxyphenylethyleneglycol (DHPG) formation is quantitatively comparable or greater than through 3‐methoxy‐4‐hydroxyphenylethyleneglycol (MHPG) production, we studied the accumulation rates of conjugated DHPG and MHPG following probenecid administration in whole brain as well as in several brain regions. Administration of increasing doses of probenecid (100–500 mg/kg, i.p.) 1.5 h before sacrifice produced a dose‐dependent increase of conjugated DHPG and MHPG levels. The maximum increment of these conjugated metabolites occurred at a dose of 300 mg/kg or higher. During the first hour following probenecid administration (300 mg/kg, i.p.), rat brain conjugated DHPG and MHPG levels accumulated linearly at a rate of 646 and 319 pmol/g/h, respectively. With the probenecid technique, the estimated appearance rates of conjugated DHPG significantly exceeded those of conjugated MHPG in hypothalamus, midbrain, brainstem, hippocampus, and cerebral cortex. These results clearly indicate that under resting conditions, formation and efflux of conjugated DHPG is the major route of metabolic clearance of rat brain NE.

Determination of picogram levels of brain serotonin by a simplified liquid chromatographic electrochemical detection assay.

A simplified assay is described for measurement of picogram amounts of serotonin (5-HT) by high pressure liquid chromatography with electrochemical detection (LCEC). The procedure involves pre-purification of brain 5-HT by adsorption on Amberlite CG-50. Serotonin is subsequently resolved and detected by LCEC on Zipax SCX resin. The present method gives working sensitivities of at least 100 pg, tissue recoveries of 95% and very low interassay variability (coefficient of variation = 3%). Determination of rat brain area 5-HT by this LCEC method is described and compared to other high sensitivity methods.