Relationship between plasma and cerebrospinal fluid norepinephrine and dopamine metabolites in a nonhuman primate.

Major and minor pathways of metabolism in the mammalian CNS result in the formation of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) and normetanephrine (NMN) from norepinephrine (NE), and homovanillic acid (HVA) and 3-methoxytyramine (3-MT) from dopamine (DA), respectively. The correlational relationships between HVA and 3-MT and between MHPG and NMN in primate CSF and plasma have not been described. These relationships may help to elucidate the usefulness of CSF and plasma metabolites as indices of CNS NE and DA activity. In addition, because NMN is unlikely to cross the blood-brain barrier. CSF NMN concentrations would not be confounded by contributions from plasma, which is a major issue with CSF MHPG. We have obtained repeated samples of plasma and CSF from drug-naive male squirrel monkeys and have measured the concentrations of MHPG, HVA, NMN, and 3-MT to define their correlational relationships. For the NE metabolites, significant correlations were obtained for CSF MHPG and NMN (r = 0.806, p less than 0.001), plasma MHPG and CSF NMN (r = 0.753, p less than 0.001), and plasma and CSF MHPG (r = 0.776, p less than 0.001). These results suggest that CSF and plasma MHPG and CSF NMN may reflect gross changes in whole brain steady-state noradrenergic metabolism. Only a single significant relationship was demonstrated for the DA metabolites, with CSF 3-MT correlating with plasma HVA (r = 0.301, p less than 0.025). The results for the DA metabolites probably reflect regional differences in steady-state brain dopaminergic metabolism.

Liquid chromatographic assay for cerebrospinal fluid normetanephrine.

A method for quantitation of normetanephrine in human cerebrospinal fluid is described. An amine-specific reagent, sulfosuccinimidyl propionate, is used to obtain the lipid soluble N-propionyl derivative of normetanephrine, which can be separated and quantitated in presence of other biogenic amines by liquid chromatography with electrochemical detection. The method is reproducible, linear, and precise at the relatively low concentrations of unconjugated normetanephrine occurring in human cerebrospinal fluid. Hospitalized, drug-free, alcoholic patients were found to have cerebrospinal fluid unconjugated normetanephrine concentrations in the 0.5-1.5 nanomolar range. The practical limit of sensitivity for the method is about 0.025 pmole per ml of CSF.

Effects of clonidine on central and peripheral nerve tone in primary hypertension.

To define the mechanisms whereby clonidine lowers blood pressure, we measured cerebrospinal fluid and plasma levels of norepinephrine, normetanephrine, epinephrine, dopamine, and the dopamine metabolite homovanillic acid in 10 primary hypertensive subjects before and after 3 months of clonidine treatment (mean dose, 0.68 mg/day). Catecholamines were measured by radioenzymatic methods. Cerebrospinal fluid and plasma sampling was performed after subjects had fasted and remained supine overnight, and plasma sampling was repeated 2 hours later, after subjects had ambulated. Supine and upright blood pressure fell, as might be expected. Cerebrospinal fluid levels of norepinephrine and normetanephrine fell significantly, but dopamine and homovanillic acid levels were unchanged. Plasma norepinephrine, normetanephrine, and epinephrine levels decreased 30 to 50%, and supine dopamine levels also fell. The percent fall in supine blood pressure was related to the fall of cerebrospinal fluid and plasma norepinephrine. There were also positive relationships between the decreases of plasma norepinephrine and of normetanephrine and dopamine. The cerebrospinal fluid/plasma norepinephrine ratio was unaffected by clonidine, suggesting that the drug lowered both pools equally. Our findings indicate that clonidine decreases both central and peripheral norepinephrine activity. The dopaminergic activity of cerebrospinal fluid was unaffected by clonidine, and though plasma dopamine levels tended to be lower after treatment, mean plasma prolactin level, an index of dopaminergic activity, was also unchanged. The fall in plasma epinephrine level is probably related to diminished sympathetic adrenomedullary stimulation and is unlikely to contribute to clonidine's antihypertensive action. These results also suggest that measurement of normetanephrine in cerebrospinal fluid and plasma provides a good index of norepinephrine activity.

Extractive acylation and mass spectrometric assay of 3-methoxytyramine, normetanephrine, and metanephrine in cerebrospinal fluid.

The chemical analysis of 3-methoxytyramine, normetanephrine, and metanephrine in tissues, blood, and cerebrospinal fluid is complicated by the low levels in which they occur and the amphoteric nature of the functional groups, which hampers their isolation from aqueous media. In the present report, we describe a practical and simple method for the routine isolation and derivatization of 3-methoxytyramine, normetanephrine, and metanephrine in high yield from aqueous samples like cerebrospinal fluid. The metabolites are simultaneously derivatized with pentafluoropropionic anhydride and extracted into an organic solvent. After further treatment with pentafluoropropionic anhydride under anhydrous conditions, the samples are ready for GC/MS analysis. The method is able to exploit the theoretical maximal sensitivity available in the electron capture negative-ion chemical ionization mode without any apparent losses during extraction and derivatization, giving limits of detection in the low picogram range. Mean levels of free 3-methoxytyramine, normetanephrine, and metanephrine in human cerebrospinal fluid were 3.77, 5.20, and 0.40 pmol/ml. Assay of pools of squirrel monkey, human, and canine cerebrospinal fluid with and without previous enzymatic hydrolysis demonstrated that the three metabolites occur predominantly in a conjugated form.

Measurement of tritiated norepinephrine metabolism in intact rat brain.

A procedure for the study of NE metabolism in the intact rat brain is described. The method involves ventriculocisternal perfusion of the adult male rat with artificial CSF containing [3H]NE. Radioactivity in the perfusate associated with NE and its metabolites 3,4-dihydroxymandelic acid (DOMA), 3,4-dihydroxyphenylethyleneglycol (DHPG), 3-methoxy-4-hydroxymandelic acid (VMA), 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), and normetanephrine (NMN) is separated using high-performance liquid chromatography (HPLC). After 80 min the radioactivity in the perfusate reaches an apparent steady-state. Analysis of the steady-state samples shows higher activity in the fractions corresponding to DHPG and MHPG than in those corresponding to DOMA and VMA, confirming glycol formation as the major pathway of NE metabolism in rat brain. Pretreatment with an MAO inhibitor (tranylcypromine) results in a marked decrease in the deaminated metabolites DHPG and MHPG and a concurrent increase in NMN. The results indicate this to be a sensitive procedure for the in vivo determination of changes in NE metabolism.

The relationships of free to conjugated normetanephrine in plasma and spinal fluid of hypertensive patients.

The concentrations of free and total normetanephrine (NMN) were determined in the plasma of normotensives and patients with primary hypertension and pheochromocytoma. NMN values were measured in the cerebrospinal fluid (CSF) of patients. Free and conjugated NMN, the latter after acid hydrolysis, were assayed using S-adenosylmethionine in the presence of phenylethanolamine-N-methyltransferase to form labeled metanephrine. The conjugates of NMN were present in plasma as sulfates principally, as they were also liberated with arylsulfatase. Free and conjugated NMN levels were 117 +/- 10 and 1417 +/- 109 ng/liter, respectively in plasma of normotensives. The mean ratio of the content of conjugated to free NMN was 14.9 +/- 1.8 (mean +/- SEM). The contents of free and conjugated NMN were 155 +/- 33 and 1670 +/- 320 ng/liter in primary hypertensives, respectively, and the ratio of conjugated to free NMN was 18.5 +/- 3.3. These values did not differ significantly from those in normotensives. The contents of free and total NMN in the plasma of patients with pheochromocytoma were 50- to 60-fold greater than values in normotensive and primary hypertensives. The mean ratio of conjugated to free NMN in the plasma of patients with pheochromocytoma was similar to those in normotensives and primary hypertensives. The contents of free and conjugated NMN in the CSF of patients with pheochromocytoma exceeded those in primary hypertensives (P less than 0.01 and P less than 0.001). Further, the ratio of conjugated to free NMN in CSF was increased in patients with pheochromocytoma (33.9 +/- 8.1) compared to that primary hypertensives (8.3 +/- 2.3; P less than 0.001). The measurement of NMN in plasma and CSF may help characterize sympathetic nerve tone in patients with primary hypertension to elucidate the pathophysiology of the elevated blood pressure.

Central and regional normetadrenaline in evaluation of neurogenic aspects of hypertension: aid to diagnosis of phaeochromocytoma.

1. Free normetadrenaline concentrations in plasma of patients with phaeochromocytoma were elevated 20-1000-fold as compared with values in primary hypertensive subjects. 2. Spinal fluid normetadrenaline was 1.8-16% of the simultaneous plasma values in patients with phaeochromocytoma, whereas levels in spinal fluid exceeded those of plasma in primary hypertension. 3. Spinal fluid noradrenaline was 7.9-58% of simultaneous values of plasma noradrenaline in patients with phaeochromocytoma, whereas values in plasma and cerebrospinal fluid were similar in primary hypertension. 4. Plasma noradrenaline and normetadrenaline levels were both elevated in veins from regions subsequently found to harbour chromaffin tumours. 5. The excessive amounts of normetadrenaline produced in phaeochromocytoma enters plasma but not spinal fluid and can be used for diagnosis and localization of phaeochromocytoma.