O-methylated Metabolites Research Articles

Metabolism of norepinephrine during nerve stimulation in dog trachea.

We determined the relative importance of neuronal and extraneuronal uptake in the metabolism of norepinephrine (NE) released during electrical stimulation (ES) of isolated canine tracheal smooth muscle (TSM). Strips of TSM were labeled with L-[3H]NE (2 X 10(-7) M) and mounted for superfusion. Superfusate was collected continuously before, during, and after ES (15 V, 0.5 ms, 5 Hz). Measurements were made of [3H]NE and its metabolites in superfusate and in tissue. Neuronal uptake followed by metabolism was estimated by measuring the amount of 3,4-dihydroxyphenylglycol (DOPEG). Extraneuronal uptake was estimated by measuring O-methylated metabolites (OMM). ES caused large increases in the efflux of NE, DOPEG, and OMM from TSM. However, the overflow of OMM was six times greater than that of DOPEG. Cocaine (10(-5) M) abolished the increased efflux of DOPEG during ES and enhanced the overflow of NE and OMM. We conclude that extraneuronal uptake constitutes the primary metabolic pathway for NE released from adrenergic nerves innervating TSM.

Interference of labetalol metabolites in the determination of plasma catecholamines by HPLC with electrochemical detection

The alpha and beta adrenoceptor blocking drug labetalol is a potent antihypertensive agent in widespread clinical use. Its interference in the classical chemical estimations of urinary catecholamines and their metabolites has been the subject of several reports. Factitiously raised values have been noted in both the fluorimetric catecholamine assay, and the standard spectrophotometric procedure for total (free and conjugated) metadrenalines. To avoid such drug interference, modification of these methods is required in the estimation of catecholamines and their o-methylated metabolites. Alternatively, VMA estimations or plasma/urinary catecholamine measurements by radioenzymatic assay may be used in patients on labetalol. Although high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) methods for estimation of plasma catecholamines are now in widespread use, the interference of labetalol in this method has not been reported. We now report that significant direct interference of labetalol in the HPLC-ECD assay does indeed occur, and can yield spuriously raised adrenaline levels.

Uptake and metabolism of norepinephrine by endothelium of dog pulmonary artery.

The contribution of endothelium of dog pulmonary artery to the extraneuronal metabolism of norepinephrine was determined. Pulmonary artery was cut into helical strips; the endothelium was removed from half of the strips by gently stroking them with a wooden applicator stick. All strips were immersed in l-[3H]norepinephrine (2 X 10(-7) M) and mounted for superfusion. Superfusate was collected continuously before, during, and after electrical stimulation (10 V, 2 ms, 2 Hz). Column chromatography was used to separate [3H]norepinephrine and its radiolabeled metabolites in superfusate. Quantitation was by liquid scintillation spectrometry. Previous studies have established that 3,4-dihydroxyphenylglycol is of neuronal origin and that O-methylated metabolites are of extraneuronal origin. Since cocaine prevented neuronal uptake of norepinephrine, the reduction in metabolites of norepinephrine of extraneuronal origin in arteries with endothelium removed represented the contribution of endothelium to extraneuronal metabolism. O-Methylated metabolites were decreased from 3.50 to 2.17 X 10(3) dpm/2 ml of superfusate during basal conditions preceding electrical stimulation and from 11.16 to 6.94 X 10(3) dpm/2 ml of superfusate during electrical stimulation when endothelium had been removed. Decreases in extraneuronal metabolite production continued throughout the basal periods following stimulation. These studies suggest that in small pulmonary artery a substantial amount of the total norepinephrine that is released at the neuroeffector junction may be metabolized following uptake into endothelium.

Bacterial O-Methylation of Chloroguaiacols: Effect of Substrate Concentration, Cell Density, and Growth Conditions

O-methylation of chloroguaiacols has been examined in a number of gram-positive and gram-negative bacteria to elucidate the effects of substrate concentration, growth conditions, and cell density. Substrate concentrations between 0.1 and 20.0 mg liter were used, and it was found that (i) yields of the O-methylated products were significantly higher at the lowest concentrations and (ii) rates of O-methylation were not linear functions of concentration. With 3,4,5-trichloroguaiacol, the nature of the metabolites also changed with concentration. During growth with a range of substrates, O-methylation of chloroguaiacols also took place. With vanillate, however, de-O-methylation occurred: the chlorocatechol formed from 4,5,6-trichloroguaiacol was successively O-methylated to 3,4,5-trichloroguaiacol and 3,4,5-trichloroveratrole, whereas that produced from 4,5-dichloroguaiacol was degraded without O-methylation. Effective O-methylation in nonproliferating suspensions occurred at cell densities as low as 10 cells ml, although both the yields and the rates were lower than in more dense cultures. By using disk assays, it was shown that, compared with their precursors, all of the O-methylated metabolites were virtually nontoxic to the strains examined. It is therefore proposed that O-methylation functions as a detoxification mechanism for cells exposed to chloroguaiacols and chlorophenols. In detail, significant differences were observed in the response of gram-positive and gram-negative cell strains to chloroguaiacols. It is concluded that bacterial O-methylation is to be expected in the natural environment subjected to discharge of chloroguaiacols.

Simultaneous assay of 3,4-dihydroxyphenylalanine, catecholamines and O-methylated metabolites in human plasma using high-performance liquid chromatography

We devised a procedure for the simultaneous determination of 3,4-dihydroxyphenylalanine, catecholamines and O-methylated metabolites using a reversed-phase liquid chromatographic system. Detection is achieved by an electrochemical detector and a fluorescence detector connected in series. Sample preparation is kept to a minimum, and involves precipitation of proteins with trichloroacetic acid and perchloric acid, and subsequent neutralization, thus omitting the commonly adopted adsorption step. Chromatographic peaks were identified on the basis of retention behaviour and the ratio of responses at several oxidation potentials. The method was applied to the quantitative determination of 3,4-dihydroxyphenylalanine, catecholamines and O-methylated metabolites in human plasma.

Effect of Prazosin on the Efflux of <sup>3</sup>H-Norepinephrine and Metabolites from the Intima and Adventitia of the Rabbit Ear Artery

The spontaneous and stimulation-induced (SI) effluxes of 3H-norepinephrine (3H-NE) and its metabolites from the intimal and adventitial surfaces of perfused segments of rabbit ear arteries were determined; vessels were previously incubated with 3H-NE (0.6 microM). The total SI adventitial efflux of 3H was approximately 10-fold greater than the intimal efflux, contained a higher percentage of unchanged 3H-NE (48 vs. 12%) and a lower percentage of O-methylated metabolites (17 vs. 55%); there was little difference between the percentages of deaminated catechols (35 vs. 31%). Prazosin, at a concentration (0.24 microM) which prevented the arteries constricting during stimulation, had little effect on the composition of the SI effluxes; however, it caused 2- to 3-fold increases in the effluxes of 3H-NE and its metabolites into the lumen during the period of stimulation. This effect is attributed to the failure of the vessel wall to thicken during stimulation, thus facilitating diffusion of 3H-NE and its intraneuronally formed metabolites across the media. Prazosin decreased the percentage of unchanged 3H-NE and increased that of the deaminated catechols in the spontaneous efflux; these effects are attributed to a direct effect of prazosin on the intra-neuronal metabolism of 3H-NE.

Determination of fenoldopam (SK&F 82526) and its metabolites in human plasma and urine by high-performance liquid chromatography with electrochemical detection

Fenoldopam [6-chloro-2,3,4,5-tetrahydro-1-(4-hydroxyphenyl)-1H-3-benzazepine-7,8-diol] is a potent renal vasodilator that is currently undergoing Phase II clinical trials. Quantitative analytical methods, based on high-performance liquid chromatography with electrochemical detection (HPLC3—ED) after ethyl acetate extraction from plasma or urine were developed for the determination of fenoldopam and its identified metabolites in biological media. The lower limit of quantitation for fenoldopam in plasma was 50 pg/ml. In assays for fenoldopam glucuronide(s) and fenoldopam conjugates, urine was treated with β-glucoronidase and Glusulase, respectively, and the liberated fenoldopam was quantified by HPLC-ED. A novel assay by dual-electrode (in series) HPLC-ED was developed for the 8-sulfate of fenoldopam. In this method, the 8-sulfate was oxidized to the o-quinone at the first electrode and quantitated at the second electrode after reduction to the cathechol. A similar dual-electrode HPLC-ED method was used for 7- and 8-O-methyl fenoldopam. Conjugates of the O-methyl metabolites were determined by HPLC—ED after hydrolysis to O-methyl fenoldopam. These methods have been used to study the kinetics and metabolism of fenoldopam in healthy volunteers. The methods are specific, sensitive, reproducible, and linear over a wide range of concentrations. Precision of the analyses, expressed as coefficients of variation, were less than 10% for all analyses.

Effect of ovarian steroids on the metabolism of noradrenaline in rabbit uterus.

The metabolism of (-)-3H-noradrenaline was examined in uterine slices from ovariectomized rabbits which were either untreated or treated with 17 beta-oestradiol, alone or in combination with progesterone. 17 beta-oestradiol caused uterine enlargement which was not accompanied by a change in the formation per g of O-methylated metabolites (3H-NMN, 3H-VMA, 3H-MOPEG). Accumulation of unchanged 3H-noradrenaline and the formation of deaminated catechols (3H-DOMA and 3H-DOPEG) were decreased per g tissue, but increased per uterine horn. Progesterone produced further enlargement of the oestrogen-dominated uteri which was accompanied by (a) a decrease in deaminated catechol formation and (b) an increase in 3H-NMN formation per unit mass of tissue. In all uteri (control and hormone-treated), cocaine inhibited the formation of deaminated catechols, but not that of the O-methylated metabolites. It is suggested, therefore, that, per unit of uterine mass, the neuronal deamination of (-)-3H-noradrenaline is decreased by 17 beta-oestradiol and further decreased by progesterone, and that these changes reflect failure of the intraneuronal deaminating system in the whole uterus to increase in proportion to the increase in uterine mass. Since other agents which decreased the deamination of (-)-3H-noradrenaline (cocaine and nialamide) did not affect 3H-NMN formation in oestrogen-dominated uteri, it is suggested that stimulation of 3H-NMN formation represents a direct effect of progesterone on the extraneuronal O-methylation of noradrenaline.

Non-radiochemical procedure for the measurement of O-methylation of the stereoisomers of isoprenaline

A non-radiochemical procedure has been developed which permits the separation and measurement of isoprenaline (ISO) and the O-methylated metabolite, 3-methoxyisoprenaline (MeOISO). This methodology employs alumina chromatography and toluene solvent extraction to separate the catecholamine, ISO, from the O-methylated derivative, MeOISO. High-performance liquid chromatography with electrochemical detection has been used to quantify these compounds. The biological application of this procedure includes the evaluation of O-methylation of the stereoisomers of ISO by intact tissues.

Platelet phenol sulfotransferase and erythrocyte catechol-O-methyltransferase activities: correlation with methyldopa metabolism.

Methyldopa is metabolized by sulfate conjugation catalyzed by phenol sulfotransferase (PST), O-methylation catalyzed by catechol-O-methyltransferase (COMT), and decarboxylation catalyzed by aromatic L-amino acid decarboxylase. These experiments were performed to determine whether individual variations in red blood cell (RBC) COMT and platelet PST activities might reflect variations in the metabolism of methyldopa in man. Methyldopa, 3.5 mg/kg, was taken orally by 28 subjects. Blood samples were obtained from these subjects for the assay of platelet PST and RBC COMT activities, and a 24-hr urine sample was collected for the measurement of methyldopa and its major metabolites. Human platelets contain two independently regulated forms of PST. One form is thermolabile (TL), and the other is thermostable (TS). Methyldopa and alpha-methyldopamine are substrates for the TL but not for the TS form of PST. The results of the experiment showed significant correlations between TL platelet PST activity and the proportion of alpha-methyldopamine excreted as a sulfate conjugate, and between RBC COMT activity and the proportion of methyldopa excreted as an O-methyl metabolite. There was no significant correlation, however, between TL platelet PST activity, and the proportion of methyldopa itself excreted as a sulfate conjugate. These results are compatible with the conclusion that differences among subjects in drug metabolizing enzyme activities are one factor responsible for wide individual variations in methyldopa metabolism in man.

The influence of inhibition of catechol-O-methyl transferase or of monoamine oxidase on the extraneuronal metabolism of 3H-(-)-noradrenaline in the rat heart.

The extraneuronal metabolism of 3H-(-)-noradrenaline (1 nmol/l) was determined in rat hearts obtained from reserpine-pretreated animals (in the presence of 30 mumol/l cocaine). Inhibition of monoamine oxidase (MAO) (by pretreatment of the animals with pargyline) increased the formation of O-methylated metabolites by nearly that amount by which the formation of deaminated metabolites declined; hence, catechol-O-methyl transferase (COMT) seemed to be able to nearly fully compensate for the loss of MAO activity. However, when COMT was inhibited (by the presence of either 1 or 10 mumol/l U-O521), the increase in the formation of deaminated metabolites was smaller than the decrease in the formation of O-methylated metabolites; hence, MAO seemed to be unable to fully compensate for the loss of COMT activity. These results are discussed with regard to the hypothesis that the two extraneuronal enzymes co-exist in one compartment. As inhibition of COMT causes a much greater increase in the steady-state tissue/medium ratio for 3H-(-)-noradrenaline than does inhibition of MAO, it is suggested that it is this increase in the intracellular concentration of 3H-(-)-noradrenaline which - by promoting an efflux of the unchanged amine that is proportional to the tissue/medium ratio - actually decreases the net removal of 3H-(-)-noradrenaline from the perfusion fluid. The results are compatible with (but no evidence for) the hypothesis that the two enzymes co-exist in the same extraneuronal compartment.

Regioselective O-methylation of tetrahydropapaveroline and tetrahydroxyberbine in vivo in rat brain

Enzymatic O-methylation is a primary pathway for the metabolism of catecholamines in mammals and of isoquinoline alkaloids in plants. This report describes the differential O-methylation patterns of the racemates and enantiomers of two catecholamine-derived alkaloids, tetrahydropapaveroline (THP) and 2,3,10,11-tetrahydroxyberbine (THB), in the brain of the rat. One hour after intracerebroventricular administration of a specific isomeric form of each alkaloid, the O-methylated metabolites were isolated from the rat brain and subsequently quantified using high performance liquid chromatography. The isomeric form of THP or THB which was administered markedly influenced the pattern of O-methylation. The racemate and R-(+)-enantiomer of THP were mono- O-methylated predominantly at the 7 and 3′ positions, while the S-(−)-enantiomer of THP was mono- O-methylated to an essentially equal degree at the 6, 7 and 3′ positions. Minimal mono- O-methylation at the 4′ position was detectable only with the racemate and (−)-enantiomer of THP. The racemate and enantiomers of THB were mono- O-methylated predominantly at the 2 and 11 positions and to a lesser extent at the 3 and 10 positions. Although minimal with the R-(+)-enantiomer, the 3 and the 10- O-methylation pathways were enhanced significantly with the S-(−)-enantiomer of THB. These results demonstrate that both enantiomers of THP and THB are O-methylated in vivo in rat brain and that the chiral centers of these alkaloids influence the position of O-methylation, thereby dictating the relative amounts of specific products formed.

Enzymatic O-methylation of the dopamine agonist dipropyl-5,6-dihydroxyaminotetralin: isolation and structure elucidation of the O-methylated metabolite.

The (+)-enantiomer of the potent dopamine agonist dipropyl-5,6-dihydroxyaminotetralin (DP-5,6-diOH-ATN) is metabolized in vitro by catechol-O-methyltransferase (COMT) to one O-methyl derivative. This compound was isolated from the incubation mixture by reversed-phase HPLC, after the incubation mixture had been purified over Sephadex G 10 columns and the excess parent catecholamine had been removed with alumina. One of the possible O-methyl metabolites, dipropyl-5-hydroxy-6-methoxyaminotetralin, was synthesized from dipropyl-5,6-dimethoxyaminotetralin by a regioselective mono-demethylation with iodotrimethylsilane. Comparison of chromatographic and spectral data of the products from incubation and synthesis showed that different isomers are formed; i,e. DP-5,6-diOH-ATN is exclusively meta-O-methylated by COMT in vitro to dipropyl-5-methoxy-6-hydroxyaminotetralin.

Norepinephrine content and disposition in large and small pulmonary artery of dog.

Comparisons were made between large and small pulmonary arteries in 1) the content of norepinephrine (NE) and 2) the disposition of released NE. Endogenous NE was measured by liquid chromatography with electrochemical detection. The disposition of NE was studied using superfused strips of arteries previously incubated in l-[3H]NE. Superfusate was collected continuously before, during, and after electrical stimulation. 3,4-Dihydroxyphenylglycol (DOPEG) was shown to be of neuronal, and O-methylated metabolites to be of extraneuronal, origin. Thus extraneuronal uptake was estimated directly by measuring the O-methylated metabolites in the superfusate, and neuronal uptake followed by metabolism was estimated by measuring [3H]DOPEG. The magnitude of the neuronal uptake fraction entering vesicles for reuse was estimated from the compensatory increases in [3H]NE and O-methylated metabolites when neuronal uptake was blocked. The predominant route of disposition for NE was neuronal uptake in the small arteries (41% of released NE) but was extraneuronal uptake or NE overflow in the large vessels (35 and 37% of released NE, respectively). Recapture of NE by storage vesicles was greater in small than in large vessels (22 and 7%, respectively, of released NE).

Determination of urinary normetanephrine and metanephrine by radial-compression liquid chromatography and electrochemical detection.

A procedure has been developed for determining the O-methylated catecholamine metabolites, normetanephrine and metanephrine, in urine by use of radial-compression liquid chromatography followed by electrochemical detection. Normetanephrine and metanephrine are isolated from hydrolyzed urine by ion-exchange on small, commercially available, disposable columns and preconcentrated by solvent extraction. They are then separated by reversed-phase ion-pair chromatography, with use of a radial compression cartridge and radial compression module, and quantified with 3-methoxy-4-hydroxybenzylamine as internal standard. Normetanephrine, metanephrine, and the internal standard are separated from interfering peaks in about 15 min. The method is applicable to the relatively low amounts of normetanephrine (100-600 micrograms/24 h) and metanephrine (50-400 micrograms/24 h) found in normal subjects and patients with depressive disorders or hypertension. Within-day CVs ranged from 1.1 to 2.2% for normetanephrine and 1.2 to 6.9% for metanephrine; the corresponding between-day CVs were 4.9 and 5.7% over these ranges.

Placental monoamine oxidase content and inhibition: Effect of enzyme inhibition on maternofetal transfer of noradrenaline (norepinephrine) in the human placenta in vitro

The effect of monoamine oxidase (MAO) inhibition on maternofetal transfer of noradrenaline (NA) has been investigated in vitro using dual perfusion of isolated human placental lobules. As a first step, placental MAO content and its sensitivity to inhibition by pargyline were assessed in incubation studies of homogenates as well as during perfusion, taking rat liver as reference. Our results show that the human placenta, though it contains as great an enzyme activity as rat liver, was less sensitive to inhibition by pargyline than the latter. MAO inhibition by pargyline significantly reduced the NA clearance from maternal to fetal circulation. Thus the proportion of unmetabolized NA radioactivity in fetal venous samples decreased significantly after pargyline treatment. A concomitant rise in the proportion of mainly O-methylated metabolites was also observed. We speculate that the apparent activation of catechol-O-methyl transferase pathway, observed in our studies on MAO inhibition, may play an important role in limiting NA transfer towards the fetus in toxaemic pregnancies associated with the reduction in placental MAO.

Urinary catecholamine metabolites and effects of clonidine in patients with alcohol amnestic disorder.

Seven normotensive patients with alcohol amnestic disorder were treated with 2 micrograms/kg clonidine (C) three times daily for 1 wk. Four patients received 12 micrograms/kg/day during the subsequent week; three developed hypotensive symptoms at this dose and remained on 6 micrograms/kg/day. During a predrug placebo period and after 60 hr on each dose of C, urinary excretion rates of catecholamine metabolites were determined. C, 6 micrograms/kg/day, reduced the ratio of norepinephrine (NE) metabolites (mumol/24 hr) to normetanephrine (NM), vanillylmandelic acid (VMA), and 3-methoxy-4-hydroxyphenyl glycol (MHPG). The excretion of metanephrine (M) was not reduced significantly. The ratio M/NM and M/(VMA + MHPG) increased, indicating Cs effects are primarily noradrenergic. Reduction in NM/(VMA + MHPG) indicates disproportionate lowering of the O-methylated metabolite of NE compared to its deaminated metabolites, consistent with C inhibition of NE release. Patients with the highest predrug NM excretion had the greatest decrements with C. The dopamine metabolites 3-methoxytyramine and homovanillic acid were not decreased by C. C-induced reductions in the ratio NM/(VMA + MHPG), an index of NE release, correlated (n = 7) with reductions in supine systolic blood pressure, mean arterial pressure, and salivary flow rate.

Metabolism of norepinephrine in vitro by dog pulmonary arterial endothelium

The importance of endothelial cells in the removal of norepinephrine from synaptic clefts in dog pulmonary artery was studied. Strips of artery cut helically were denuded of endothelium by gently stroking the intimal surface with a wooden applicator stick or were studied with endothelium intact. All strips were prelabeled in L-[3H]norepinephrine (2 X 10(-7) M) and mounted for superfusion. Superfusate was collected continuously before, during, and after electrical stimulation (10 V, 2 ms, 2 Hz). Measurements were made of the amounts of [3H]norepinephrine and its metabolites in superfusate and in tissue. These studies have established that 3,4-dihydroxyphenylglycol is of neuronal origin and O-methylated metabolites are of extraneuronal origin. The formation of extraneuronal metabolites in smooth muscle and endothelium was examined by either blocking uptake of norepinephrine into each, in turn, or by combining blockade of uptakes with the elimination of access of norepinephrine to endothelial tissue by completely removing it. Electrical stimulation elicited the overflow of large amounts of norepinephrine, 3,4-dihydroxyphenylglycol, and O-methylated metabolites into superfusate in strips with intact endothelium; in strips denuded of endothelium there were striking decreases in the amounts of O-methylated metabolites produced. These studies show that pulmonary arterial endothelium participates in the extraneuronal metabolism of norepinephrine released by sympathetic nerve stimulation.

Fused silica capillary gas chromatography/negative chemical ionization mass spectrometry for determination of catecholamines and their O-methylated metabolites

Fused silica capillary gas chromatography/negative chemical ionization mass spectrometry for determination of catecholamines and their O-methylated metabolites

Flow dependence of norepinephrine extraction by isolated perfused rat lungs.

Disposition of perfused norepinephrine (NE) was studied in isolated rat lungs at several perfusate flows, including that occurring in vivo. Lungs were ventilated with 95% O2-5% CO2 and perfused at 37 degrees C with recirculating medium initially containing 90 nM [14C]NE. In agreement with previous studies the fraction of NE removed in a single pass through the pulmonary vasculature was 0.30 +/- 0.03 by lungs perfused at low flow (10 ml/min; 7.2 ml.min-1.g lung-1). This extraction ratio decreased with increasing flow, so that in lungs perfused at 45 ml/min (31.8 ml.min-1.g lung-1) the extraction ratio was 0.09 +/- 0.01. The concentration of NE metabolites in the perfusate reservoir increased with time of perfusion. The rate of appearance of deaminated and O-methylated metabolites in the recirculating perfusion medium was not markedly influenced by flow. NE that accumulated in lung was metabolized before reentry into the perfusion medium. These results indicate that extraction of NE by rat lung is markedly dependent on flow and suggest that removal of NE by lung is not highly efficient at normal organ perfusion rates.