Acidic Catecholamine Metabolites Research Articles

Single-Molecule Sensing of Acidic Catecholamine Metabolites Using a Programmable Nanopore.

Acidic catecholamine metabolites, which could serve as diagnostic markers for many diseases, demonstrate an importance of accurate sensing. However, they share a highly similar chemical structure, which is a challenge in the design of sensing strategies. A nanopore may be engineered to sense these metabolites in a single molecule manner. To achieve this, a recently developed programmable nano-reactor for stochastic sensing (PNRSS) technique adapted with a phenylboronic acid (PBA) adaptor was applied. Three acidic catecholamine metabolites, including 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxymandelic acid (DHMA) and 3-methoxy-4-hydroxymandetic acid (VMA) were investigated by PNRSS. Specifically, DHMA, which contains an α-hydroxycarboxylate moiety and an adjacent cis-hydroxyl groups on its benzene ring, reports two binding modes simultaneously resolvable by PNRSS. Assisted with the high resolution of PNRSS, direct regulation of these two binding modes by pH can also be observed. A custom machine learning algorithm was also developed to achieve automatic event classification.

Voltammetric Behavior of Acidic Catecholamine Metabolites in Presence of Cationic Surfactants

The voltammetric behavior of two acidic catecholamine metabolites, vanillylmandelic acid (VMA) and homovanillic acid (HVA) in the presence of cationic, anionic, or nonionic surfactants was monitored at the disposable screen-printed electrodes. In basic medium above their pKa values, the cationic surfactant cetylpyridinium chloride (CPC) enhanced the oxidation peak current based on electrostatic and hydrophobic interactions with the ionized carboxylic group and stabilization of some of the electrochemical reaction intermediates. Under the optimum measuring conditions at pH 9.5, calibration graphs were linear within HVA concentration range from 0.25 to 2.5 × 10−6 mol l−1 and LOD 0.021 × 10−6 mol l−1. Simultaneous assay of HVA in the presence of VMA within the submicromolar concentration range can be performed with acceptable average recovery of 106.0 ± 3.4%. This preliminary study represents the base for simultaneous quantification of both compounds in of urine samples for Neuroblastoma diagnosis.

Thin-layer chromatography--an image-processing method for the determination of acidic catecholamine metabolites.

A sensitive and convenient method for acidic catecholamine metabolites (including homovanillic acid, vanillylmandelic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylacetic acid) determination was developed based on thin-layer chromatography and image-processing analysis. The metabolites were separated without a prederivatization step using reversed phase RP-18W high-performance plates. The mobile phase composition, detection, and quantification conditions were systematically investigated through several trials. The reaction with 2,2-diphenyl-1-picrylhydrazyl radical allowed specific detection of acidic catecholamine metabolites with a high sensitivity and a wide linear range. The limit of detection and the limit of quantification were in the range of 13-103 and 18-120 ng/spot, respectively, in all cases. Mean recoveries determined were in the range 95-106% for all of the investigated compounds. The proposed method allowed rapid simultaneous determination of acidic catecholamine metabolites from spiked human urine sample.

Using Chemical Derivatization to Determine the Human Urinary Acidic Catecholamine Metabolites Quantitatively by Liquid Chromatography/Electrospray Ionization‐Tandem Mass Spectrometry

AbstractBased on a 4,6‐dimethoxy‐2‐(methylsulfonyl)pyrimidine (DMMSP) labeling reagent, a sensitive method to determine acidic catecholamine metabolites homovanillic acid (HVA), vanillymandelic acid (VMA), 3,4‐dihydroxymandelic acid (DOMA) and 3,4‐dihydroxyphenylacetic acid (DOPAC) has been developed using simple chemical derivatization with liquid chromatography/electrospray ionization‐tandem mass spectrometry (LC/ESI‐MS/MS). Acidic catecholamine metabolites were converted to stable 4,6‐dimethoxypyrimidinyl (DMP) derivatives by one‐step derivatization. Because of bulky DMP group(s) introduced into the structures of these metabolites, subsequent analysis by LC/ESI‐MS/MS in positive ionization with multiple reaction monitoring (MRM) allowed specific detection of DMP derivatives of these metabolites with a wide linear relationship range. The limits of detection were in the range of 1–5 ng/mL. The mean recoveries determined were 100.2% –104.4%. Both relative standard deviation and error for intra‐ and inter‐day measurements were less than 10% for acidic catecholamine metabolites. This strategy enabled accurate and specific determination of acidic catecholamine metabolites in human urine.

Acidic Catecholamine Metabolites and 5-Hydroxyindoleacetic Acid in Urine: The Influence of Diet

Concentrations of vanillylmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), vanillic acid (VA) and 5-hydroxyindoleacetic acid (5-HIAA) in urine from healthy subjects were determined by a high-performance liquid chromatography system with a mixed-mode (C18/anion-exchange) column and an 8-channel electrochemical detector, in order to study the influence of diet, diurnal variation and age. The urinary excretion of 5-HIAA increased significantly after eating banana, pineapple, tomato, kiwi fruit and walnut. An increase in the urinary excretion of DOPAC and HVA after eating banana and that of VA after taking vanilla was also noted. The urinary excretion of VMA was not significantly influenced by any of the foods examined. The urinary excretion of 5-HIAA in the first-morning urine increased beyond the upper limit of the reference value when banana was taken at 2000 h the previous day. The excretion of all metabolites in the second-morning urine in the fasting state was within respective reference ranges. Diurnal variation of the excretion of VMA, DOPAC, HVA and 5-HIAA in urine was relatively small, but that of VA was large. The concentrations (mmol/mol creatinine) of VMA, DOPAC, HVA, 5-HIAA and VA in the first-morning urine from healthy subjects increased from 7 days after birth to 1 year and then decreased to adult levels at 13 years of age.

A new solid-phase extraction method for human urinary 3-methoxy-4-hydroxyphenylethyleneglycol

A new solid-phase extraction procedure for urinary 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) was established. Sep-Pak Diol cartridge was used, because MHPG is a neutral and alcoholic compound. Aqueous samples were adsorbed to the cartridge, then MHPG was eluted by the addition of ethyl acetate. After the eluate was evaporated, the residuum was dissolved with HPLC mobile phase and injected into HPLC. The extraction procedure was highly specific to MHPG, and none of other acidic catecholamine metabolites, such as vanillylmandelic acid (VMA), homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), was extracted. The recovery of MHPG using this method was over 90% and higher than those using previously described methods such as liquid-liquid extraction with ethyl acetate. Of the three vanillyl alcohol isomers, isovanillyl alcohol was the most suitable as an internal standard for the correction of column-to-column variation of the recovery. Human urinary unconjugated MHPG extracted by the new procedure could be measured by HPLC with a fluorescence detection. Further complicated derivatization and more sensitive detection systems, such as GC-MS and HPLC-electrochemical detector (ECD), were not needed due to high selectivity and high recovery of the extraction procedure. In addition, the urinary total (conjugated plus unconjugated) MHPG content could also be determined by the same procedure after an enzymatic hydrolysis of conjugated MHPG. The newly developed extraction procedure was simple, rapid and highly specific, and might be applicable to the analysis of MHPG in various body fluids.

The addition of dopamine determination to the measurement of acidic catecholamine metabolites in urine screening for neuroblastoma

Introduction: Urinary dopamine (DA) is well-known as a useful marker in the detection and follow-up of neuroblastomas, especially advanced cases. We have established a new method based on the simultaneous measurement of urinary DA and acidic catecholamine metabolites using high performance liquid chromatography (HPLC). The method described here has been applied in a regular neuroblastoma screening at age 6 months and in a second pilot screening at age 14 moths in Sapporo. Methods: Urine samples were taken from either dried specimens on filter paper or acidified 24-h urine specimens collected at a hospital. The analytical column used was an Inertsil ODS-2. N-octanesulfonate salt as an ion-pair reagent was added in the mobile phase. A coulometric detector with dual electrodes was utilized so that dopamine and other acidic catecholamine metabolites could be monitored on each electrode separately. Results: DA, vanillylmandelic acid (VMA) and homovanillic acid (HVA) were eluted at 9.9, 3.5 and 13.5 min, respectively. Samples were injected into the column every 15.5 min. Urinary DA was detectable between the range of 1.5 ng/ml and 5 μg/ml. Of 41809 infants who tested negative or did not participate in the first neuroblastoma screening and who received screening at age 14 months, we detected two neuroblastoma cases. Urinary DA levels were within normal limits in both. Conclusion: Measurement of urinary DA in addition to VMA and HVA was described. The method could be useful to reduce the likelihood of missing a patient who may develop neuroblastoma, even advanced or dopaminergic cases, though a pilot study using this assay could not prove the possibility yet.

High-performance liquid chromatographic determination of catecholamine metabolites and 5-hydroxyindoleacetic acid in human urine using a mixed-mode column and an eight-channel electrode electrochemical detector.

An HPLC system for the simultaneous determination of acidic catecholamine metabolites, related compounds and 5-hydroxyindoleacetic acid (5-HIAA) in human urine was developed. A mixed-mode (C18/anion-exchange) column with isocratic elution using citrate buffer and an eight-channel electrochemical detector were used. Vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenyllactic acid (vanillactic acid, VLA), homovanillic acid (HVA), vanillic acid (VA) and 5-HIAA in urine were determined simultaneously. Detection limits and inter (n = 5) and intra-assay (n = 5) coefficients of variation were satisfactory. The mean of analytical recoveries (n = 3, +/- C.V. (%)) were between 97 +/- 3.2 (VMA) and 105 +/- 4.8 (VA). Correlations between the analytical results for VMA, HVA and 5-HIAA obtained by an established method and the present method were satisfactory. The mean +/- 2 S.D. of the excretion rates of VMA, DOPAC, VLA, HVA, 5-HIAA and VA in urine from healthy adult volunteers were 0.61-4.36, 0.13-1.02, 0-0.35, 0.67-6.55, 0.50-5.14 and 0-0.55 mg/g creatinine, respectively.

High Performance Liquid Chromatographic Determination of Acidic Catecholamine Metabolites in Human Urine Using Postcolumn Fluorescence Derivatization with dl-1,2-Diphenylethylenediamine

dl-1,2-Diphenylethylenediamine was found to react sensitively in an alkaline solution with 3,4-dihydroxymandelic acid as well as other catechol compounds and 4-hydroxy-3-methoxyphenyl compounds to yield fluorescent products. By using this reaction, a postcolumn fluorescence derivatization high performance liquid chromatography of the acidic metabolites of catecholamines (3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, vanillylmandelic acid and homovanillic acid) in human urine was established. These compounds were separated on an anion-exchanger column (TSK-gel DEAE-2SW) and subjected to derivatization involving coulometric oxidation followed by a fluorescence reaction with the named reagent. The detection limits (S/N=3) for the acidic metabolites in urine were 15–30 fmol in a 100-μl injection volume.

Conjugated versus “Free” acidic metabolites of catecholamines in random urine samples: Significance for the diagnosis of neuroblastome

Conjugated versus “Free” acidic metabolites of catecholamines in random urine samples: Significance for the diagnosis of neuroblastome

Conjugated versus "free" acidic metabolites of catecholamines in random urine samples: significance for the diagnosis of neuroblastoma.

Random urine samples were obtained from 31 patients with neuroblastoma (newborn to 8 yr of age) and from 100 children without this tumor (newborn to 10 yr). The urine samples were studied for the presence of sulfate and glucuronide conjugates of homovanillic (HVA), dihydroxyphenylacetic, vanilmandelic, and vanillactic acids. The urinary concentrations of these acids were determined by capillary gas-chromatography before and after enzymatic treatment with glucuronidase and sulfatase. Concentrations of the "free" fraction and "total" urinary content of these acids were determined using the results from untreated and treated urines respectively. Age-related reference values were established for children without neuroblastoma. Fractions of the total content of urinary HVA (18-39%) and dihydroxyphenylacetic acid (36-66%) were excreted as glucuronides and/or sulfates by the control group, with the highest conjugated fractions found in the urine of young infants (0-3 months). Vanilmandelic was excreted mainly as "free" acid (unconjugated), whereas vanillactic acid was undetectable in almost all control samples. Patients with neuroblastoma also excreted a fraction of these acids as glucuronide and/or sulfate conjugates, (25% of urinary HVA, 39% of dihydroxyphenylacetic acid and 45% of vanillactic acid) whereas vanilmandelic acid was excreted only as "free" in controls. Determination of "total" rather than "free" urinary HVA was diagnostic in one neuroblastoma patient with borderline "free" HVA levels, whereas determination of "free" or "total" dihydroxyphenylacetic acid and vanillactic acid did not improve the diagnostic sensitivity in the cases examined. We conclude that it may be clinically useful to determine "total" urinary HVA in patients with borderline "free" HVA levels who are suspected of having neuroblastoma.

Simultaneous determination of the major acidic metabolites of catecholamines and serotonin in urine by liquid chromatography with electrochemical detection after a one-step sample clean-up on sephadex G-10; Influence of vanilla and banana ingestion

A simple method is described for the simultaneous determination of vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HIAA) and homovanillic acid (HVA) in urine. The compounds are isolated by a one-step sample clean-up on Sephadex G-10, separated by ion-pair reversed-phase liquid chromatography and detected electrochemically. A single analysis is completed within 65 min. Sample clean-up did not cause losses of the compounds of interest. The detection limits in urine were 0.4, 0.8, 1.0 and 1.6μmol/l for VMA, DOPAC, 5-HIAA and HVA, respectively. 3,4-Dihydroxymandelic acid and vanillic acid (VA) were also detectable, but, under the chromatographic conditions used, they were not resolved from interfering components. VA and 5-HIAA could be analysed separately in the Sephadex G-10 eluate if more restrictive sampling conditions were used. Ingestion of bananas caused an increase of VMA, DOPAC, 5-HIAA and HVA in 24-h urine. After ingestion of vanilla an increased excretion of VA was observed, while the excretion of VMA, DOPAC and HVA was unaffected.

Acidic catecholamine metabolites: substrates for human phenolsulphotransferase M

Acidic catecholamine metabolites: substrates for human phenolsulphotransferase M

Determination of acidic catecholamine metabolites in plasma and cerebrospinal fluid using gas chromatography—negative-ion mass spectrometry

A method for the assay of acidic catecholamine metabolites in biological fluids using capillary gas chromatography—electron-capture negative-ion mass spectrometry is described. The method combines acetylation of phenolic hydroxy groups in buffered aqueous solution followed by pentafluorobenzyl ester formation and acetylation of aliphatic hydroxy groups under anhydrous conditions. The resulting per-O-acetyl carboxypentafluorobenzyl esters provided excellent negative-ion mass spectra with intense and diagnostic anions. The sensitivity of the analysis using electron-capture negative-ion mass spectrometry exceeds that using electron-impact mass spectrometry by two to three orders of magnitude. Analysis of acidic catecholamine metabolites in human lumbar cerebrospinal fluid and plasma were performed with good precision (σ rel < 5%) at the low nanomoles per litre level.

"Total" acidic metabolites of catecholamines in urine as determined by hydrolysis with hydriodic acid and liquid chromatography: application to patients with neuroblastoma and melanoma.

We describe a method for determining the "total" excretion of acidic metabolites of catecholamines by measuring 3,4-dihydroxyphenylacetic acid (DOPAC) formed by hydriodic acid hydrolysis of 4-hydroxy-3-methoxyphenylacetic acid (HVA), 4-hydroxy-3-methoxymandelic acid (VMA), and their conjugates. The DOPAC in the diluted hydrolysate is measured directly by liquid chromatography with electrochemical detection. Normal values, expressed in relation to excretion of creatinine, vary as a function of age. For healthy subjects, the mean DOPAC value after hydrolysis was 1.15 times that for unconjugated HVA, VMA, and DOPAC combined. Preliminary results for patients with neuroblastoma and melanoma indicate the potential usefulness of the method for diagnosis and prognosis of patients with neural crest tumors that produce dopa or catecholamines.

Clinical pharmacokinetics of probenecid.

A review of the clinical applications and of the disposition of probenecid in man, including drug interactions, is presented. Probenecid is the classical competitive inhibitor of organic acid transport in the kidney and other organs. There are 2 primary clinical uses for probenecid: as a uricosuric agent in the treatment of chronic gout and as an adjunct to enhance blood levels of antibiotics (such as penicillins and cephalosporins). Adsorption of probenecid is essentially complete following oral administration. The drug is extensively metabolised by glucuronide conjugation and by oxidation of the alkyl side chains; oxidation of the aromatic ring does not occur. The half-life of probenecid in plasma (4 to 12 hours) is dose-dependent. Renal excretion is the major route of elimination of the metabolites; excretion of the parent drug is minimal and is dependent on urinary pH. Probenecid and its oxidised metabolites are extensively bound to plasma proteins, mainly to albumin. Tissue concentrations (based on animal studies) are generally lower than plasma concentrations. Most of the drug-drug interactions involving probenecid are due to an effect on the kidney-block of transport of acidic drugs. Similarly probenecid affects the tubular secretion of a number of acidic endogenous substances by the kidney. Probenecid is also involved in the block of transport of acidic metabolites of catecholamines, for example homovanillic and hydroxyindoleacetic acids, in the brain. There are a number of analytical procedures for the assay of probenecid. These are based on spectrophotometry, spectrofluorometry, gas and liquid chromatography and radioimmunoassay.

Mass spectrometry of dimethylthiophosphinates of aromatic hydroxy compounds.

The mass spectra of 20 differently substituted dimethylthiophosphinic esters of aromatic hydroxy compounds are presented. Fragmentation routes were investigated using high resolution mass measurements, decoupled metastable determinations and deuterium labelling. All compounds exhibited abundant molecular ions and typical phosphorus-containing ions. Characteristic elimination processes strongly dependent upon the respective type of substitution were observed. Due to their high stability, their great ease of formation and their good gas chromatographic properties these new types of derivatives are of special interest for establishing gas chromatography mass spectrometry profiles of acidic catecholamine metabolites.