Diurnal rhythm of 3-methoxy-4-hydroxyphenylglycol (MHPG): relationship between plasma and urinary levels

Plasma and urinary levels of MHPG were determined in six normal volunteers. Samples were obtained at 3-hour intervals for plasma and at 12-hour intervals for urine. Acrophase, amplitude and period were determined for plasma MHPG levels. A sinusoidal pattern was obtained for diurnal plasma MHPG with a peak at 15:00 hrs. +/- 46 min. Urinary MHPG, corrected for creatinine levels, correlated with both 9 AM plasma MHPG and with baseline plasma MHPG. Furthermore, the relationship between plasma and urinary MHPG was linear when the rhythm of urinary levels was assumed to lag 6.2 hours behind the plasma rhythm. It was concluded that free MHPG is evenly distributed in the total body space and that conjugated MHPG is largely restricted to the blood.     

Determination of 3-methoxy-4-hydroxyphenylethylene glycol in urine using reversed-phase liquid chromatography with column switching and electrochemical detection

A column-switching method was developed for the determination of total 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) in urine. This was performed by first treating samples with β-glucuronidase, followed by extraction with ethyl acetate. The reconstituted extracts with injected onto an HPLC system containing an amperometric detector and tandem Nucleosil C₁₈ and C₈ reversed-phase columns connected by a switching valve. The total analysis time for MHPG was 12 min. The limit of detection was 0.18 ng, or 9 μg/l for 20-μl injections of a 1.0-ml reconstituted extract prepared from 1.0 ml of urine. The linear range extended up to 80 mg/l. The within-day precision for a urine sample containing 170 μg/l total MHPG was ±6% and the day-to-day precision was ±15%. The average levels determined by this method for total MHPG in normal subjects showed good agreement with previous literature values. This approach could be modified for the determination of free MHPG by using only ethyl acetate extraction for sample pretreatment.     

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 Parkinson's 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.     

Reduction in human urinary MHPG excretion by guanethidine: urinary MHPG as index of sympathetic nervous activity.

The norepinephrine metabolites methoxyhydroxyphenyl glycol (MHPG) and vanillylmandelic acid (VMA) were measured in the urine of hypertensive subjects before and during adminstration of guanethidine, a peripheral sympatholytic agent which does not cross the blood-brain barrier or deplete adrenal catecholamines. Dosages of guanethidine (1.2 mg/kg/day) sufficient to cause at least a 20 torr reduction in standing systolic blood pressure caused a mean 63% (maximum of 68%) reduction in urinary MHPG excretion (p=0.01) while only causing a mean 37% (maximum of 44%) reduction (p<0.005) in excretion of VMA. These results indicate that MHPG in human urine, as in lower animals, is predominantly the product of peripheral sympathetic nervous system, rather than central nervous system nonrepinephrine metabolism. Urinary MHPG is more sensitive to specific sympatholytic therapy than is urinary VMA, and may be a useful index of sympathetic nervous activity.     

Urinary excretion of catecholamines in the night monkey (Aotus trivirgatus) (Primates, Cebidae)

A seasonal variation in the urinary catecholamines output has been demonstrated in two simians kept under constant ambient conditions : the nocturnal Aotus and the diurnal Sa?miri sciureus. In Aotus, catecholamines output (NA + A), in spring, is higher than in other Primates including man and even more so in winter. Cold exposure increases the NA + A excretion in Aotus as it does in squirrel monkey and rat but the A output is particularly prominent in Sa?miri. Fasting does not alter significantly the catecholamines excretion. Associated fasting and cold exposure do not modify the adrenosympathetic response observed in Aotus in cold conditions alone, but depresses the sympathetic activity and greatly enhance the adrenomedullary excretion in squirrel monkey, as it is the case in rat. Associated fasting and cold represents a highly stressful situation for squirrel monkey but not for night monkey. Catecholamines metabolites (MN, NMN, DOPAC, HVA, VMA and MHPG) are found in urine of both species, DOPAC and VMA being predominant in Aotus but DOPAC and MHPG in Sa?miri. The proportions of conjugated forms vary according to the metabolite : DOPAC and VMA are mainly under free form but NMN, MN and MHPG are mostly conjugated in both species. The daily output of pooled adrenergic metabolites (expressed as ng/mg creatinine) is higher in Aotus than in Sa?miri and man. Both monkey species display a high adrenosympathetic activity which does not correlate with their resting metabolic rate.     

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.     

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.     

Monoamines and their metabolites in the amphibian (Ambystoma tigrinum) brain: Quantitative changes during metamorphosis and captivity

• 1.1. Monoamine neurotransmitters (epinephrine, norepinephrine, dopamine, serotonin and some of their metabolites (DOPEG, MHPG, DOPAC, 5-HIAA) were measured by HPLC in extracts from telencephalon (TEL) and diencephalon-midbrain (DM) before, during and at the end of metamorphosis.
• 2.2. During metamorphosis MHPG increased and 5-HIAA decreased in TEL and DM while DOPEG decreased only in DM.
• 3.3. Monoamine levels were greater in the TEL and a larger increase in MHPG occurred there.
• 4.4. Captivity without metamorphosis also caused a significant depression of 5-HIAA in TEL and depression of DOPEG, MHPG and DOPAC in DM.

     

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.     

Effect of L-threo-3,4-dihydroxyphenylserine(L-threo-DOPS) on brain and serum MHPG levels in mice: evidence for NE formation in CNS

Effects of L-threo-DOPS on brain and serum concentrations of 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), a major metabolite of 1-norepinephrine(NE) were studied in mice. An intraperitoneal(i.p.) injection of L-threo-DOPS markedly increased both serum and brain MHPG levels in mice. This increase in the brain was dose-dependent at doses up to 800 mg/kg, and lasted for 4 h or more. Though the increase in serum total-MHPG was 3-4 times greater than that in brain MHPG, the decline was rapid as compared with the case of brain MHPG. The L-threo-DOPS-induced increase in MHPG was inhibited by i.p. pretreatment with benserazide, a peripheral decarboxylase inhibitor, in both serum and brain. This inhibition in the brain, however, was observed at about 20 times higher doses of benserazide than that in serum. On the contrary, an intracerebroventricular(i.c.v.) injection of benserazide inhibited the increase in brain MHPG to about the same degree as that in serum MHPG. These results suggest that the L-threo-DOPS-induced increase in brain MHPG is not likely to originate in peripheral organs including the brain capillary, and that L-threo-DOPS can be converted to NE by aromatic L-amino acid decarboxylase(AADC) in the brain parenchyma.     

Effects of naloxone-precipitated withdrawal after a single dose of morphine on catecholamine concentrations in guinea-pig brain

The effect of naloxone-precipitated withdrawal after acute morphine was studied on the concentrations of noradrenaline (NA), 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and on the metabolite/parent amine ratios MHPG/NA, DOPAC/DA and HVA/DA, in eight regions of the guineapig brain. Guinea-pigs were treated with a single dose of morphine sulphate (15 mg/kg s.c.) or saline (control) and 2h later with naloxone hydrochloride (15 mg/kg s.c.) to precipitate withdrawal. The animals were decapitated at 0.5 h or 1 h after naloxone injections and their brains analysed for monoamine concentrations by HPLC-ECD. At 0.5 h after naloxone-precipitated withdrawal NA and MHPG levels, and the MHPG/NA ratio, were increased in the hypothalamus, and the NA levels were increased in the hypothalamus, medulla/pons and cortex 1 h after naloxone. Naloxoneprecipitated withdrawal also produced increased DA metabolism in the cortex, midbrain and medulla 0.5 h later, and in the cortex, hypothalamus and striatum 1 h later. Hence naloxone-precipitated withdrawal from acute morphine treatment produced a complex pattern of increased synthesis and metabolism of NA and DA which varied over time and with the brain region examined.     

THE BIOSYNTHESIS OF 3-METHOXY-4-HYDROXYPHENYLGLYCOL SULPHATE BY LIVER AND BRAIN

—3-Methoxy-4-hydroxyphenylglycol (MHPG) formed a sulphate conjugate when incubated with ATP, Mg²⁺ ions, Na₂³⁵SO₄ and the high-speed supernatant preparations of rabbit or rat brain. The same reactions could be catalysed by similar enzyme preparations from liver. The sulphated product was separated and identified by paper chromatography. On acid hydrolysis, it released both Na₂³⁵SO₄ and the free glycol. The measurement of this labelled sulphate was used as a specific assay procedure for determining the overall sulphoconjugatory process. The pH optimum of the reaction is 7.8. For rabbit brain, the K_m for Na₂SO₄ determined for the activating system is 3.6 × 10⁻⁴m , and that for MHPG for the sulphotransferase reaction is 1.05 × 10⁻⁴m . The specific enzyme activity, expressed as nmol ³⁵SO₄ incorporated/h/mg protein for a 30-min assay is as follows: rat brain, 2.8; rabbit brain, 1.6; rat liver, 33.4and rabbit liver, 15.0. Dithiothreitol at 3 mm concentration had no significant effect on the sulphation of MHPG in all these preparations.     

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.     

Determination of urinary 4-hydroxy-3-methoxyphenylethylene glycol in man by high performance liquid chromatography with electrochemical detection

A reverse phase high performance liquid chromatographic method for determining levels of 4-hydroxy-3-methoxyphenylethylene glycol (MHPG) in human urine that is virtually free of all interfering peaks has been developed. After addition of a homologous internal standard, enzymatic hydrolysis is performed. Samples are then placed onto columns containing AG1-X8, and the MHPG is collected in a phosphate buffer wash of the column. After ethyl acetate extraction and evaporation of the organic solvent, the dry residue is redissolved in mobile phase, and injected onto a reverse phase column. Results obtained with this assay were almost identical (101±5.6%, mean±SD, n=6) with those obtained using a gas chromatography - mass spectrometry (GCMS) assay.     

A new approach to biochemical evaluation of brain dopamine metabolism

1. Dopaminergic neurotransmission in brain is receiving increased attention because of its known involvement in Parkinson's disease and new methods for the treatment of this disorder and because of hypotheses relating several psychiatric disorders to abnormalities in brain dopaminergic systems. 2. Chemical assessment of brain dopamine metabolism has been attempted by measuring levels of its major metabolite, homovanillic acid (HVA), in cerebrospinal fluid, plasma, or urine. Because HVA is derived in part from dopamine formed in noradrenergic neurons, plasma levels and urinary excretion rates of HVA do not adequately reflect solely metabolism of brain dopamine. 3. Using debrisoquin, the peripheral contributions of HVA to plasma or urinary HVA can be diminished, but the extent of residual HVA formation in noradrenergic neurons is unknown. By measuring the levels of methoxy-hydroxyphenylglycol (MHPG) in plasma or of urinary norepinephrine metabolites (total MHPG in monkeys; the sum of total MHPG and vanillyl mandelic acid (VMA) in humans) along with HVA, it is possible to estimate the degree of impairment by debrisoquin of HVA formation from noradrenergic neuronal dopamine and thereby better assess brain dopamine metabolism. 4. This method was applied to a monkey before and after destruction of the nigrostriatal pathway by the administration of MPTP.     

The metabolism and excretion of methylglucamine (N-methyl-D-glucamine) in the rat

Methylglucamine is a commonly used cation in radiocontrast media. The present study sheds light on its fate in the rat. When administered intraperitoneally, 93% of the compound was excreted unchanged in the urine in 24 hr. When administered orally, about 15% of the dose was found in the urine, about 40% in the faeces and 20% in expired air in 24 hr. When administered orally to rats whose gut flora had been depleted by treatment with neomycin sulphate, 19% was excreted in the urine, 69% in the faeces and 3% in expired air in 72 hr. This indicated that the gut flora played a role in the degradation of the compound and its eventual loss as expired carbon dioxide.     

Sulphate can induce differential expression of thioglucoside glucohydrolases (myrosinases)

Thioglucoside glucohydrolase (EC 3.2.3.1; myrosinase) hydrolyses glucosinolates and thereby liberates glucose and sulphur and nitrogen compounds. To examine the hypothesis that the myrosinase-glucosinolate system is influenced by environmental factors, the effect of sulphate on the expression of myrosinases was examined. On examining different plant organs at various stages, it was observed that sulphate induces a differential expression of myrosinase polypeptides in plants ofSinapis alba L. (white mustard). Specific myrosinase polypeptides, dependent on sulphate in the growth medium, were detected on immunoblots. Without sulphate a maximum of three polypeptides was detected in buds, two in cotyledons and one in stems and roots. In plants cultured on medium with sulphate up to four polypeptides could be observed in cotyledons, five polypeptides in buds, two in stems and one in roots. Expression of myrosinases was, in general, high in plants cultured on a medium supplemented with sulphate. In floweringS. alba plants, sulphate-starved plants showed a higher expression of myrosinase in cotyledons and stems compared to plants fed with sulphate. Sulphate-fed plants had a high expression in inflorescences and roots. The organ- and time-specific induction of the myrosinase expression is discussed in relation to sulphate metabolism and availability of sulphate under normal conditions of cultivation and in relation to protection of Brassicaceae species. This is the first evidence for a specific induction of individual myrosinase proteins.     

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.     

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.     

The metabolism of labelled hexadecyl sulphate salts in the rat, dog and human.

The metabolic fate of [1-14-C]hexadecylsulphate and hexadecyl[35-S]sulphate, administered intravenously as the sodium and trimethylammonium salt to dogs and orally as the erythromycin salt to dogs, rats and humans, was studied. Studies with rats indicated that the compounds were well absorbed and rapidly excreted in the urine. However, after oral administration of the 14-C-and 35-S-labelled hexadecyl sulphate erythromycin salt to dogs, considerable amounts of radioactivity were excreted in the faeces as unmetabolized hexadecyl sulphate. Studies with two humans showed that orally administered erythromycin salt of [1-14C]hexadecyl sulphate was well absorbed in one person but poorly absorbed in the other. Radioactive metabolites in urine were separated by t.l.c. in two solvent systems. The main metabolite of hexadecyl sulphate in the dog, rat and human was identified as the sulphate ester of 4-hydroxybutyric acid. In addition, psi-[14-C]butyrolactone as a minor metabolic product of [1-14-C]hexadecyl sulphate was also isolated from the urine of rat, dog and man. However, there was still another metabolite in dog urine, which comprised about 20% of the total urinary radioactivity and carried both 14-C and 35-S labels. This metabolite was absent from rat urine. The metabolite in dog urine was isolated and subsequently identified by t.l.c. and g.l.c. and by isotope-dilution experiments as the sulphate ester of glycollic acid. Small amounts (about 5% of the total recovered radioactivity in excreta) of labelled glycollic acid sulphate were also found in human urine after ingestion of erythromycin [1-14-C]hexadecyl sulphate.