Relationships between platelet and plasma monoamine oxidase,plasma dopamine-B-hydroxylase,and urinary 3-methoxy-4-hydroxy phenylglycol

The activity of dopamine-B-hydroxylase in blood has recently been demonstrated to be under genetic control and to correlate closely with urinary catecholamine excretion. The results of the present study did not demonstrate any relationship between a major catecholamine metabolite in urine, 3-methoxy-4-hydroxy phenylglycol, and dopamine-B-hydroxylase activity in plasma, monoamine oxidase activity in platelets, or monoamine oxidase activity in plasma. Differences in the origin of urinary catecholamines and urinary 3-methoxy-4-hydroxy phenyglycol may be responsible for these divergent results.     

Assay of urinary free and conjugated 3-methoxy-4-hydroxyphenyleneglycol by high-performance liquid chromatography with amperometric detection

The aim of this study was to develop an analytical method for free and conjugated 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) in urine. After hydrolysis of the conjugated forms, the urinary MHPG was purified by solid-phase extraction on anion exchanger and eluted with a water-methanol (1:1, v/v) mixture. After addition of ethyl acetate to the eluate and back-extraction into acetic acid, the aqueous phase was separated on a C₁₈ column by HPLC and detected amperometrically. The results obtained from forty healthy human subjects were compared with the literature values. The precision and accuracy of the assay were studied using 4-methoxy-3-hydroxyphenylethyleneglycol (iso-MHPG) as internal standard.     

Urinary 3-methoxy-4-hydroxyphenylglycol determination using reversed-phase chromatography with amperometric detection

A reversed-phase liquid chromatographic method with amperometric detection has been developed for the determination of urinary 3-methoxy-4-hydroxyphenylglycol. Before and after enzymatic deconjugation, it was purified by an extraction procedure and rapidly quantified under isocratic conditions. The 24-h excretion profile in normal human subjects (eight males and seven females) was determined; our results are consistent with those arrived at in a number of other studies. The present method is highly sensitive and selective; in addition, a good degree of precision is assured by use of 4-methoxy-3-hydroxyphenylglycol as internal standard.     

Improved high-performance liquid chromatographic method for the routine determination of unconjugated 3-methoxy-4-hydroxyphenylethyleneglycol in human plasma using solid-phase extraction and electrochemical detection

An improved semi-automated high-performance liquid chromatographic method is described for the routine determination of unconjugated 3-methoxy-4-hydroxyphenylethyleneglycol in plasma. The 3-ethoxy analogue of the compound is used as an internal standard. The method is based on purification of 0.5-ml plasma samples with phenyl-type reversed-phase extraction columns, reversed-phase separation with an acetate—citrate—methanol mobile phase with an octadecyl-bonded column, and dual-electrode coulometric detection with oxidation at +0.44 V and reduction at −0.25 V. The precision and accuracy of the assay are satisfactory: the lower limit of reliable detection corresponds to a plasma concentration of 1.5 nM. The validity of the determination is demonstrated by an 18% mean increase in plasma levels of 3-methoxy-4-hydroxyphenylethyleneglycol during physical exercise (duration 16 min, n = 13) and a 50% mean reduction in plasma levels induced by a single dose of the monoamine oxidase inhibitor, moclobemide (n = 8). The method is suitable for routine use in pharmacological and physiological experiments.     

Detection of 3-methoxy-4-hydroxyphenylglycol in rabbit skeletal muscle microdialysate

A high-performance liquid chromatography with electrochemical detection (HPLC-ED) method is described for determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in microdialysate from the skeletal muscle interstitial space. Using a microdialysis technique, we sampled 30 microl dialysate from the skeletal muscle interstitial space and injected dialysate directly into HPLC-ED system. The control MHPG concentration of dialysate was 213+/-18 pg/ml. The MHPG concentrations were reduced by entacapone (catechol-O-methyltransferase inhibitor, COMT), augmented by local infusion of dihydroxyphenylglycol. This system offers a new possibility for simple, rapid monitoring of MHPG as an index of COMT activity in skeletal muscle.     

Determination of 3-methoxy-4-hydroxyphenylglycol in urine by high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of 3-methoxy-4-hydroxyphenylglycol (MHPG) in urine. After incubation with glusulase, free MHPG is extracted into ethyl acetate and further isolated by a combination of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The addition of amperometric detection provides increased sensitivity to a highly specific assay.     

Effects of oral clonidine on plasma 3-methoxy-4-hydroxyphenethyleneglycol (MHPG) in man: Preliminary report

Repeated (N=15) administration of clonidine (0,1,5 μg/kg,p.o.) to three normotensive male subjects resulted in significant decreases in plasma free 3-methoxy-4-hydroxyphenethyleneglycol (MHPG) at three hours for both the 1 μg/kg dose (p < .05) and the 5 μg/kg dose (p < .01) when compared to concentrations following placebo. The mean decrement in plasma free MHPG following a 5 μg/kg dose was 36%. Systolic blood pressure fell a mean of 17 mmHg after 1 μg/kg and 37 mmHg after 5 μg/kg of clonidine. The application of a clonidine challenge test to assess noradrenergic receptor sensitivity $\text{in}$$\text{vivo}$ is discussed.     

6-(Levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl groups as novel base-labile groups for 5'-hydroxy protection in solid-phase oligonucleotide synthesis

The 6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl (LMMoNBz) and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl (LMMpNBz) groups were developed as novel base-labile groups for 5'-hydroxy protection in solid-phase oligonucleotide synthesis. A comparative study of the utility of LMMoNBz, LMMpNBz, and 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) groups is described.     

Use of peptidyl-4-methoxy-2-naphthylamides to assay plasmin

Several tripeptide amides of 4-methoxy-2-naphthylamide were synthesized and tested as possible substrates for human plasmin. These peptides contained arginine or lysine as the carboxyl terminus. One such amide, benzyloxycarbonyl-Ala-Ala-Lys-4-methoxy-2-naphthylamine (CBZ-Ala-Ala-Lys-MNA), was found to be a better substrate for plasmin than for other mammalian serine proteases. Measuring the release of 4-methoxy-2-naphthylamine fluorometrically or by colorimetry after coupling with fast blue B dye provided convenient assays for as little as 0.05 CTA unit of plasmin. K_m and k_cat values obtained were 0.90 m and 0.7 sec⁻¹, respectively. The assays were simple, sensitive, versatile, and specific.     

Analysis of urinary 3-methoxy-4-hydroxyphenylglycol by high-performance liquid chromatography and electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection for the quantitation of total 3-methoxy-4-hydroxyphenylglycol (MHPG) in human urine is described. Existing methods for deconjugation and extraction have been optimized. The present method is simpler than existing methods with a high precision. Urinary MHPG is deconjugated enzymatically and subsequently extracted with ethyl acetate. The organic layer is extracted with acetic acid and a sample of the aqueous layer is injected into a reversed-phase column. In one run 90 samples can be processed. The critical parameters of deconjugation, extraction and chromatography are described. Data for reproducibility and selectivity are presented.     

Metabolism and properties of 3-methoxy-4-hydroxyphenylpyruvate; a metabolite of dihydroxyphenylalanine

The pathway of 3,4-dihydroxyphenylalanine undergoing metabolism via transamination and subsequent oxidative rearrangement to 2,4,5-trihydroxyphenylacetate was investigated. 3-Methoxytyrosine does not pursue an identical course, since its corresponding keto acid is not subject to action of p-hydroxyphenylpyruvate hydroxylase. The radiochemical synthesis of 3-methoxy-4-hydroxy[carboxy-¹⁴C]pyruvate was accomplished. This metabolite was used to demonstrate that this keto acid does not proceed through oxidative rearrangement both in vitro and in vivo. The keto acid was found to be a competitive inhibitor of the hydroxylase and can help account for some of the metabolites observed in the urine of patients treated with 3,4-dihyroxyphenylalanine.     

Characteristics of enzymes forming 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) in brain

The subcellular localization and character of the enzymes forming 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) were determined in rat brain. The aldehyde derivative of normetanephrine was produced in situ by monoamine oxidase, and two forms of aldehyde reductase were shown to metabolize the aldehyde to MOPEG. One form of the enzyme was found to have a low affinity for NADH and a higher affinity for NADPH as a cofactor, and was shown to be inhibited by pentobarbital and by high concentrations of 5-hydroxyindoleacetic acid. This enzyme form was localized primarily in the cytosol. The second aldehyde reductase had a high affinity for both NADH and NADPH, and was not inhibited to a great extent by either pentobarbital or 5-hydroxyindoleacetic acid. This second enzyme form was localized primarily in the mitochondrial fraction. The relative contribution of the two enzyme forms to MOPEG formation in homogenates was estimated, using the various inhibitors and cofactors.     

Simultaneous determination of noradrenaline and 3-methoxy-4-hydroxyphenylethyleneglycol sulfate in discrete brain regions of the rat

A fluorometric method for measuring both noradrenaline (NA) and 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (MHPG-SO₄) in the rat brain was studied. The MHPG-SO₄ assay was improved in regard to separation and sensitivity to the point where 2–3 ng of the compound can be detected. The simultaneous assay of NA and MHPG-SO₄ from the same sample was also attained by dividing the H₂SO₄ extract from the brain tissue into two portions. The method is so sensitive and accurate that it permits determination of both NA and MHPG-SO₄ in brain samples as small as the hypothalamus of the rat.     

3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in human cerebrospinal fluid : Storage and measurement by reversed-phase high-performance liquid chromatography and coulometric detection using 3-methoxy-4-hydroxyphenyllactic acid as an internal standard

To simultaneously measure 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5HIAA), and homovanillic acid (HVA) in human cerebrospinal fluid (CSF), we used an acetonitrile protein precipitation, reversed-phase high-perforamance liquid chromatography with coulometric detection, and 3-methoxy-4-hydroxyphenyllactic acid (MHPLA) as an internal standard for all three metabolites. MHPG, 5HIAA, HVA, and MHPLA were stable for one month when stored in CSF at −70°C. Three determinations were made in triplicate for each of seven subjects over a 30-day storage period and the coefficients of variation within subject for these determinations ranged from 0.075 to 0.165 for MHPG, 0.045 to 0.148 for 5HIAA and 0.053 to 0.181 for HVA. Means and standard deviations fo CSF concentrations were 10.7 ± 3.0 ng/ml for MHPG, 22.4 ± 9.9 ng/ml for 5HIAA, and 39.9 ± 21.4 ng/ml for HVA. This method provides simple sample preparation, sensitivity, and cost advantages, as well as simultaneous extraction and quantitation of MHPG, 5HIAA, and HVA using an internal standard.     

Plasma 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) are insensitive indicators of alpha 2-adrenoceptor mediated regulation of norepinephrine release in healthy human volunteers.

The usefulness of the plasma concentrations of two major metabolites of norepinephrine (NE), 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG), as indicators of neuronal NE release was investigated. The potent alpha 2-adrenoceptor agonist, dexmedetomidine, induced only about 15% maximal reductions in the metabolite concentrations, in spite of almost total inhibition of neuronal NE release, as evidenced by 90% reductions in plasma NE concentrations. Similarly, administration of the alpha 2-adrenoceptor antagonist atipamezole was followed by only small increases in plasma DHPG and no change in MHPG levels, in spite of almost six-fold, albeit short-lasting, increases in plasma NE. In contrast, a single dose of the reversible monoamine oxidase type A (MAO-A) inhibitor moclobemide reduced plasma DHPG levels by 78% and MHPG levels by 51%. It is concluded that the plasma concentrations of DHPG and MHPG are largely determined by intraneuronal, MAO-A-dependent metabolism of NE, and do not accurately reflect acute alterations in neuronal NE release. The concentration of NE in venous plasma is clearly a more sensitive indicator of alpha 2-adrenoceptor-mediated regulation of NE release.     

A semiautomated electron capture gas chromatographic assay of 3-methoxy-4-hydroxyphenylethyleneglycol in urine

A semiautomated method for the assay of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) in urine has been developed. The method incorporates a new efficient (95%) extraction procedure combined with an automated gas chromatographic system. This system (consisting of a pulsed electron capture detector and an automatic sample injector which valves the sample between two columns) will accept continuous, unattended, sequential sample input. The limit of sensitivity of the trifluoroacetic anhydride derivative of pure MHPG is 3 pg. Analysis of samples from the same 24-h urine (n = 21) over a 2-month period resulted in a mean of 794 ng/ml with a coefficient of variation of 3.5%. MHPG levels from 12 control males and 9 females with no history of psychiatric disorder, were found to contain 1605 and 1034 μg/24 h, respectively.     

Simultaneous determination of noradrenaline and 3-methoxy-4-hydroxyphenylglycol in plasma with high-performance liquid chromatography using electrochemical detection

We herein report the simultaneous determination of the levels of noradrenaline (NA), and 3-methoxy-4-hydroxyphenylglycol (MHPG), a major metabolite of NA. The sample was subjected to a Sep Pak C18 cartridge prior to the NA and MHPG assay by high-performance liquid chromatography with an electrochemical detector. The results correlated well with the established methods. The average percentage of recovery was 91.2 and 98.7% for NA and MHPG, respectively. The intraassay coefficients of variation were 3.7 and 4.6% for NA and MHPG. The interassay coefficients of variation were 3.5 and 7.5% for NA and MHPG, respectively.     

Liquid chromatographic determination of free and conjugated 3-methoxy-4-hydroxyphenylethylene glycol with wide-ranging substances related to monoamine metabolism

A simple and sensitive procedure was developed for the simultaneous determination of substances metabolically related to monoamine transmitters including 3-methoxy-4-hydroxy-phenylethylene glycol (MOPEG) in dissected brain regions of rats using high-performance liquid chromatography combined with electrochemical detection. The tissue sample was homogenized in HCl solution. The homogenate was divided into two portions, of which one was used for the assay of MOPEG after enzymatic hydrolysis with sulfatase. A butanol extraction process was performed on the remaining portion to obtain the sample of monoamine transmitters, precursor amino acids, and acidic metabolites. The monoamines and precursor amino acids were finally recovered in HCl solution, while the acidic metabolites shifted into the alkaline buffer from the organic layer. The basic and neutral substances were separated with a 0.1 M sodium citrate/citric acid buffer system (pH 4.0) containing 1% tetrahydrofuran, and the acidic ones with 0.075 M sodium citrate/citric acid buffer (pH 3.5) containing 1% tetrahydrofuran, 10% methanol, and 12% acetic acid. The steady-state concentrations of three monoamine transmitters (noradrenaline, dopamine, and 5-hydroxytryptamine) were determined together with their precursors and metabolites. Changes in the concentrations of these substances were examined for various drugs, of which the effects had been previously confirmed. The changes reflected putative drug effects and demonstrated that the procedure was applicable to the regional determination of monoamines and their metabolically related substances.     

Organ selective induction of cytochrome P-448 isozymes in the rat by 2-methoxy-4-aminoazobenzene and 3-methylcholanthrene

Male Sprague Dawley rats were injected intraperitoneally with 2-methoxy-4-amino-azobenzene (2-MeO-AAB) or 3-methylcholanthrene (MC), and then the expression of microsomal cytochrome P-450 isozymes in liver and extrahepatic tissues was investigated by means of immunological methods and a bacterial mutation test. The results of protein A-enzyme-linked immunosorbent assaying and immunoblotting using anti-rat cytochrome P-448 monoclonal antibodies showed that MC induced at least two microsomal cytochrome P-448 isozymes, a high spin form (cytochrome P-448H) and a low spin form (cytochrome P-448L), in liver, but that it induced only cytochrome P-448L in extrahepatic tissues such as lung, kidney, small intestine, and colon. The results also indicated that, in contrast to MC, 2-MeO-AAB selectively induced microsomal cytochrome P-448H in liver but did not induce any cytochrome P-448 isozymes in extrahepatic tissues. The activities of 9,000 X g supernatants from the individual organs, as to the mutagenic conversion of 3 aromatic amines (3-amino-1-methyl-5H-pyrido(4,3-b)indole, 2-amino-6-methyldipyrido(1,2-a: 3',2'-d)-imidazole and 3-methoxy-4-aminoazobenzene), toward Salmonella typhimurium TA 98 bacteria were dependent upon the quantity and/or quality of the microsomal cytochrome P-448 isozymes in the organs.     

Interaction of antidepressants with clonidine on rat brain total 3-methoxy-4-hydroxyphenylglycol.

The effects of some antidepressants on brain total 3-methoxy-4-hydroxyphenylglycol (MHPG) were studied in the rat. Desipramine decreased and mianserine increased the brain total MHPG concentration while the other antidepressants had no effect. They were also the only antidepressants that attenuated the lowering action of clonidine on brain total MHPG, but possibly through different mechanisms. Two tertiary amine tricyclic antidepressants, amitriptyline and imipramine, appeared to enhance the lowering action of clonidine on brain total MHPG. The results suggest that antidepressants are heterogeneous in their action on brain noradrenergic mechanisms in the rat.