A direct rapid radio enzymatic assay for measuring simultaneously picogram quantities of norepinephrine, epinephrine and dopamine by a combination of high pressure liquid chromatography has been developed, and the relationship of changes in cerebrospinal fluid norepinephrine concentrations during haloperidol treatment to changes in psychosis ratings of 12 schizophrenic patients has been investigated.
Twenty micro liters of cerebrospinal fluid are incubated with catechol-O-methyltransferase and S-adenosyl-L-[methyl-3H] methionine to convert the three catecholamines to their O-methylated analogues. These derivatives are extracted with diethyl ether containing tetraphenylborate at pH 8.2 followed by re-extraction with 0. 1 N HCl and then isolated by high pressure liquid chromatography using mBondapak Ci¹? column and 0. 17 M acetic acid-6% methanol-0. 02 M Na2SO4 as solvent. Manual collection of each fraction is started two seconds after in appeared mark.
The high pressure liquid chromatography fractions of norepinephine (NE) and epinephrine (E) derivatives are oxidized to vanillin followed by extracting the vanillin with toluene, and dopamine (DA) derivative fraction is extracted by toluene/isoamylalcohol and then. re-extracted by 0. 1 N HCl. Owing to a higher
recovery (NE, 75.6±0.9%; E,76±0.8%; DA, 70±0.4% and incorporation rate of radioactivity (NE, 163±11.4; E, 125±10.5; DA,~ 112±3.9 cpm), together with quick
separation of the methylated derivatives within 8 min by high pressure liquid
chromatography, the method yielded extremely high sensitivity (NE, 0.4 pg; E, 0.3 pg; DA, 1 pg), accuracy, reproducibility and specificity of the differential assay, and thus enabled us to complete the differential analysis of 15 samples (30 tubes) in a day. In case of norepinephrine only, more than 35 samples can be assayed in a day.
It was shown by this method that haloperidol induced decreases in norepinephrine and that the decreases correlated significantly with psychosis ratings, suggesting that cerebrospinal fluid norepinephrine can be used for evaluation of neuroleptics treatment of schizophrenia.
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