Edna K. Gordon

Catecholamine metabolism in affective disorders: I.: Normetanephrine and VMA excretion in depressed patients treated with imipramine

INTRODUCTION DATA deriving both from studies in man and laboratory animals suggest that the antidepressant actions of imipramine may be mediated through its effect on the metabolism of catecholamines. l-14 Such a mechanism of action of imipramine would be compatible with the hypothesis that some depressions may be associated with an absolute or relative functional deficiency of catecholamines, especially norepinephrine, at specific brain receptor sites, whereas elations may be associated with an excess of such amines.1~2J~4J5J6 Clinical studies relevant to the catecholamine hypothesis of affective disorders are limited, however, and it is not possible either to confirm definitively or to reject this hypothesis on the basis of data currently available.l~2~4,16-21 In a previous study of depressed patients, it was found that imipramine (like the monoamine oxidase inhibitors) also decreased the excretion of 3-methoxy-4-hydroxymandelic acid (vanillylmandelic acid) (VMA), the major urinary metabolite of norepinephrine.2 Most VMA is believed to derive from norepinephrine which has been synthesized in the nerve cell, and deaminated intracellularly by mitochondrial monoamine oxidase and thereby inactivated without necessarily having exerted a physiological effect.22123 The decrease in VMA excretion during treatment with imipramine, a drug which does not chemically inhibit monoamine oxidase, therefore suggested the possibility that imipramine by a mechanism of action other than chemical inhibition of monoamine oxidase might also decrease the intracellular deamination of norepinephrine and thereby increase the norepinephrine available for use at receptor sites .2 This reasoning led to the prediction that

Central Norepinephrine Metabolism in Affective Illness: MHPG in the Cerebrospinal Fluid

Concentrations of the norepinephrine metabolite 3-methoxy-4-hydroxyphenyl glycol in cerebrospinal fluid were measured by a gas chromatographic method in 34 patients with affective illness and in 44 controls. Concentrations of this metabolite in spinal fluid were significantly lower in depressed patients than in controls or manic patients. These low values may occur secondary to depressive phenomena such as reduced psychomotor activity, or they may reflect a primary change in norepinephrine metabolism in depressive illness.

Amine Metabolites in Human Cerebrospinal Fluid: Effects of Cord Transection and Spinal Fluid Block

Patients with spinal cord transection had normal concentrations of 5-hydroxyindoleacetic acid and low concentrations of 3-methoxy-4-hydroxyphenyl glycol in lumbar cerebrospinal fluid. The presence or absence of spinal fluid block in these patients did not affect concentrations of either amine metabolite. However, the concentration of homovanillic acid was lower in patients with spinal fluid block than in those without block. The results suggest that the spinal cord contributes to concentrations of 3-methoxy-4-hydroxyphenyl glycol and possibly 5-hydroxyindoleacetic acid, but contributes little to that of homovanillic acid in the lumbar spinal fluid of man.

Norepinephrine metabolism in the central nervous system of man: studies using 3-methoxy-4-hydroxyphenylethylene glycol levels in cerebrospinal fluid.

—Levels of 3‐methoxy‐4‐hydroxyphenylethylene glycol (MHPG), a major metabolite of norepinephrine, were measured in human CSF by gas‐liquid chromatography. MHPG concentrations were similar in both ventricular and lumbar CSF samples; about 30 per cent of the MHPG from either source occurred as the sulphate conjugate. There was relatively little entry of intravenously infused [14C]MHPG into lumbar spinal fluid. Both α‐methylparatyrosine, an inhibitor of tyrosine hydroxylase, and fusaric acid, an inhibitor of dopamine‐β‐hydroxylase, significantly diminished MHPG values. On the other hand, doses of l‐DOPA or probenecid, sufficient to substantially elevate CSF levels of the dopamine metabolite, homovanillic acid, failed to alter the spinal fluid content of MHPG. CSF concentrations of MHPG in patients with Parkinsons disease or the other central nervous system disorders studied did not differ significantly from control levels. The results suggest that MHPG values in CSF may provide an index to norepinephrine metabolism in the central nervous system of man.

Simultaneous assay by mass fragmentography of vanillyl mandelic acid, homovanillic acid, and 3-methoxy-4-hydroxy-phenethylene glycol in cerebrospinal fluid and urine.

A sensitive and specific method is described for the simultaneous determination of vanillyl mandelic acid (VMA), homovanillic acid (HVA) and 3-methoxy-4-hydroxy-phenyl ethylene glycol (MHPG) in cerebrospinal fluid (CSF) and urine, using gas chromatography-mass spectroscopy. The catecholamine metabolites, each labelled with three deuterium atoms on the methyl group in the 3 positions, are used as internal standards. The procedure, quantified by measuring the relative amounts of the endogenous protium (H) and the added deuterated (D) form of a major fragment of the trifluoroacetyl derivative of each compound, offers the advantages of determining, in a single sample, small amounts of these O-methylated metabolites of the catecholamines. VMA, which hitherto has not been positively identified and quantified in CSF was easily determined by this method.

Conjugated 3,4 dihydroxy phenyl acetic acid (DOPAC) in human and monkey cerebrospinal fluid and rat brain and the effects of probenecid treatment.

Abstract Gas chromatography-mass spectrometry (GC-MS) was used to measure 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in cerebrospinal fluid from humans and monkeys and in rat caudate nuclei. DOPAC was found to be present mainly in conjugated form. In human lumbar CSF the average concentration of total DOPAC before probenecid treatment was 1.48 ± 0.31 ng/ml; after probenecid it increased to 15.06 ± 3.17 ng/ml. This increase was mainly due to conjugated DOPAC but increases in free DOPAC also occurred. There is a relatively greater accumulation of DOPAC than of HVA, suggesting that in human CSF conjugated DOPAC may have a faster turnover rate than HVA. In monkey, ventricular CSF contained higher concentrations of DOPAC and HVA than did lumbar CSF. In rat brain, treatment with probenecid caused increases in DOPAC, HVA and their conjugates. These results suggest that DOPAC is conjugated in brain and that both compounds are removed from brain and CSF by a probenecid-sensitive acid transport system in the same manner as is HVA.

The circadian variation of catecholamine metabolism in the subhuman primate.

Cerebrospinal fluid (CSF) was removed continuously in 2- or 3-h aliquots from the lateral and fourth cerebral ventricles of chronic chair restrained rhesus monkeys. Under conditions of 12 h light (06.00-18.00 h) and 12 h darkness (18.00-06.00 h) the concentrations of norepinephrine (NE) were found to describe a circadian pattern, with maximal concentrations occurring during the light hours and minimal concentrations occurring during the dark hours. The patterns were generally coincident with the circadian patterns of brain temperature and body activity. When assayed for 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) and 3-methoxy-4-hydroxymandelic acid (VMA), samples of CSF collected over 3-4 days demonstrated no reproducible pattern of change. Fluctuation in the concentration of MHPG did not correspond in direction or magnitude to changes in the concentration of VMA. These random fluctuations may in part be accounted for by the influx of the metabolites from peripheral sources to the brain and CSF, and by the relatively slow movement of these metabolites as they diffuse from brain parenchyma to the CSF.

The circadian variation in dopamine metabolism in the subhuman primate

DOPAMINE (DAI containing neuronal systems in the CNS of man now appear to contribute to the regulation of extrapyramidal motor function (POIRER rr ul.. 1975: KLAWANS er ul.. 1976) as well as of affective state (SNYDER er ul.. 1974: GOODWIN er ul.. 1975). There is also some evidence to suggest that central catecholaminergic pathways participate in the control of such rhythmic phenomena as body temperature (HELLON. 1975). sleep-walking cycles (JOUVET. 1972). and the release of certain anterior pituitary hormones (COPPOLA. 1971; VAN LOON. 1973). These latter observations prompted this attempt to measure circadian fluctuations in cerebral DA metabolism in the primate. Male rhesus monkeys (MUCQCQ I?1&rru). weighing 5 4 . 5 kg were adapted to a primate restraining chair in a well brntilated sound-attenuated chamber in which lights were kept on from 0600 to 1800h and turned o f f from 1800 to 0600 h. Under ketamine hydrochloride (Parke Davis. Detroit. MI) and sodium pentobarbital (Abbott. N. Chicago. IL) anesthesia. cannula were implanted stereotaxically into the right lateral or 4th cerebral ventricles. The cannulae were secured to the skull with stainless steel screws and dental cement. Polyethylene tubing connected to the ventricular cannulae. passed through a small hole in the roof of the chamber to the pumping and collection apparatus. Ventricular fluid was withdrawn continuously 10.4 ml: h) from the cannula and collected in 2 h aliquots. Cerebrospinal fluid remained at room temperature in the collecting tubing for approx 1 h until i t could be refrigerated (4 C). Fractions from each 14-h period were collected in the morning and frozen (-20 C) until assaysed by the gas chromatographic-mass spectroscopy for homovanillic acid (3-methoxy-4-hydroxy-phenyl-acetic acid. HVA) ~GORDOX er ul.. 1974). the principal metabolite of DA in primates (GORDON er ul.. 1975. 1976). Data for this study was obtained from analysis of lateral ventricular fluid samples for 3 contiguous 24-h periods on 4 animals. and rrom fourth ventricular fluid samples for 3 contiguous 24-h periods on one animal. Although HVA concentrations are lower in the fourth. than in the lateral ventricle (GORDON vr ul.. 1075). the pattern of HVA concentration in both \entricks was essentially the same: results obtained from all animals were thus analyzed together. and plotted to compensate for dead space in the collection system. When analyzed as to variation about a daily mean, a graph of HVA concentration describes a circadian pattern as illustrated in Fig. I . A two factor analysis of variance with repeated measures was performed. Individual monkels comprised the non-repeated factor and time of day

Studies of uptake of 1-norepinephrine-14C

During a constant slow infusion, l-norepinephrine-14C accumulates in the tissues. The rate of accumulation from the circulation depends upon the relative blood flow to each tissue and the ability of the tissue to extract the delivered compound. The efficiency of extraction from the circulation seems to be related to the norepinephrine content of the tissue which, in turn, reflects sympathetic nerve density. The heart, which has a very high relative blood flow and a high norepinephrine concentration, accumulates the labeled catecholamine most rapidly. The seminal vesicle, which has a high norepinephrine concentration but a low blood flow, extracts norepinephrine efficiently so that its rate of norepinephrine-14C accumulation is about the same as that of the intestine, which has a high blood flow but a low norepinephrine concentration. After a long infusion period, there is a steady state in which the ratio of the specific activities of norepinephrine in the tissue and urine can be used to estimate the proportion of the catecholamine in the tissue derived from the circulation. Only 20 per cent of the norepinephrine in the heart appears to be derived from the circulation. In other tissues less than 10 per cent comes from the circulation. Thus synthesis appears to be the main source of tissue catecholamine, with uptake from the circulation playing only a minor role in maintaining stores of this catecholamine.

Origins of catecholamine metabolites in monkey cerebrospinal fluid.

THE CONCENTRATION of HVA present in CSF drawn from the lateral ventricles of experimental animals and man is much higher than that present in fluid drawn from the lumbar subarachnoid space (GULDBERG et al., 1966; GORDON & OLIVER, 1971; CHASE et al., 1973). Since the HVA found in lumbar CSF has been shown to come from the brain rather than from the spinal cord, (CURZON et al., 1971; POST et a[., 1973; YOUNG et a/., 1973), the concentration of lumbar HVA can be considered the result of this metabolite entering the CSF from the periventricular brain structures less the amount removed by a probenecid-sensitive active transport system during passage of the fluid from the cerebral ventricles to the lumbar subarachnoid space. In contrast to HVA (GORDON & OLIVER, 1971; CHASE et al., 1973) MHPG is present in lateral ventricular and lumbar CSF of man in roughly equal concentrations. In rat brain where MHPG exists primarily as the acidic sulfate conjugate it, like other organic acids, will accumulate in response to probenecid administration (MEEK & NEFF, 1972). The majority of MHPG in human CSF is present as the free, neutral compound (GORDON & OLIVER, 1971; GORDON rt al., 1973; WILK et al., 1971; BERTILLSON, 1973) and little if any elevation in total MHPG is seen in lumbar CSF following probenecid treatment in man (GORDON et al., 1973). The recent development of a sensitive gas chromatography-mass fragrnentography technique for the simultaneous determination of HVA, MHPG and VMA (GORM)N et a/., 1974) in the same sample of CSF, has made possible comparison of the levels of these metabolites in the small vol of CSF that can be obtained from the lateral, third and fourth ventricles and lumbar subarachnoid space of monkeys.