Fluorometric determination of 2-oxoadipic acid, a common metabolite of tryptophan and lysine, by high-performance liquid chromatography with pre-chemical derivatization

2-Oxoadipic acid, a key metabolite of tryptophan and lysine, reacted with 1,2-diamino-4,5-methylenebenzene in an acidic solution to produce a fluorescent derivative. The reaction product was separated using a Tosoh ODS-80Ts column with 20 mmol/L of KH?PO?-K?HPO? buffer (pH 7.0) containing 26% methanol at a flow rate 0.8 mL/min. The excitation wavelength of detection was 367 nm, and the emission wavelength was 446 nm. The limit of quantification was 1 pmol per injection, sufficiently sensitive for the determination of 2-oxoadipic acid in human and experimental animal urine.     

Analysis of monoamines and their metabolites by high performance liquid chromatography with coulometric electrochemical detection

High performance liquid chromatography with coulometric electrochemical detection has been used to achieve simultaneous determination of norepinephrine, epinephrine, 5-hydroxytryptophan, normetanephrine, dopamine, metanephrine, 3,4-dihydroxyphenylacetic acid, N-acetyldopamine, tyramine, tryptophan, 5-hydroxyindoleacetic acid, 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine, homovanillic acid, tyrosine, p-octopamine, N-acetyl-p-octopamine, and p-synephrine. The procedure has been applied to study monoamine degradation in the insect brain and to demonstrate that N-acetylation rather than oxidative deamination is the primary route of monoamine catabolism in insects.     

Simultaneous measurement of dopamine and its metabolites, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and tryptophan in brain tissue using liquid chromatography and electrochemical detection

An assay has been developed for brain tryptophan using reverse-phase liquid chromatography with electrochemical detection. The method simultaneously assays dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). The method does not require elution from ion exchange resins. After deproteinization and centrifugation samples are injected directly onto the chromatographic column. It was found that small changes in mobile phase pH markedly influenced the retention time of tryptophan while elution of the indoleamines and catecholamines did not change. The assay of these endogenous compounds in a single injection proved not expedient but inexpensive. Values obtained using alumina and ion exchange resins yielded comparable values.     

Rapid Determination of Norepinephrine, Dopamine, Serotonin, Their Precursor Amino Acids, and Related Metabolites in Discrete Brain Areas of Mice Within Ten Minutes by HPLC with Electrochemical Detection

A rapid and simple chromatographic procedure using HPLC with electrochemical detection is described for simultaneous determination of the substrates from precursor amino acids to metabolites related to synthesis and metabolism of three monoamine neurotransmitters--norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT, serotonin)--in discrete brain areas of the mouse. Under the present instrumental and mobile phase conditions, the procedure permits simultaneous determination of three monoamines (NE, DA, and 5-HT), two precursor amino acids (tyrosine and tryptophan), and four respective metabolites (3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid) within 10 min in one chromatographic run. By varying column temperature, this procedure also permits simultaneous determination of 10-14 monoamine-related substrates including the nine substrates described above within 15-21 min. The validity of the present procedure is demonstrated by analyzing the effect of an alpha 2-adrenergic agonist (clonidine) and an alpha 2-antagonist (yohimbine) in mouse hypothalamus.     

Distribution of 5-hydroxytryptamine and indolealkylamine metabolites in the American cockroach, Periplaneta americana L

A procedure is described for simultaneous estimation of tryptophan (TP), 5-hydroxytryptophan (5-OHTP), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), N-acetyl 5-hydroxytryptamine (NA5-HT) and N-acetyldopamine (NADA) using high performance liquid chromatography with coulometric electrochemical detection. The procedure has been used to determine the distribution of these compounds in the central nervous system of the American cockroach, Periplaneta americana. The ratio of TP:5-HT is greatest in the cerebral ganglia (6.5) with lesser ratios evident in the thoracic ganglia (15.5-18.9) and abdominal ganglia (9.6-11.2). Relatively low concentrations of 5-OHTP and NA5-HT were observed in the cerebral ganglia whereas 5-HIAA was not detected. Incubation of ganglia resulted in increased concentrations of NA5-HT. Reserpine reduced levels of 5-HT and NADA whereas probenecid caused a marked reduction in TP and slight elevation of NADA levels. No MAO activity was detected in the central nervous system.     

Crystalline adenylate kinase from carp muscle.

1. The concentration of adenylate kinase in carp muscle is about 0.3 mg/g. An improved isolation procedure makes use of a dilute solution of the substrates, ATP and AMP, to elute the enzyme from a phosphocellulose column in overall yields of 60% before crystallization. By the hexokinase--pH-stat assay the specific activity is 3550 units/mg. The preparation has been found to be essentially homogeneous by dodecylsulfate gel electrophoresis, isoelectrofocusing and gel filtration. 2. The molecular weight has been determined to be 22000 by several methods. The absorbance of a 1% solution at 280 nm is 6.9 and the isoelectric point by electrofocusing is pH 5.9. 3. The crystals of carp adenylate kinase have the space group P4-1-22 or P4-3-22. 4. The amino acid composition has been determined. There is no tryptophan, no cystine. There is one amino acid residue each of cysteine and histidine which are at or close to the catalytic center. 5. Several peptides derived by tryptic hydrolysis have been isolated and identified with corresponding peptides of porcine adenylate kinase. Consideration is given to histidine and cysteine being a part of the active site.     

Surfactant ion-pair high-performance liquid chromatography of tryptophan and some of its metabolites in biological fluids

A rapid, sensitive assay for tryptophan and some of its metabolites in urine, plasma and saliva has been developed using sodium dodecylsulphate as a pairing ion in a surfactant ion-pair high-performance liquid chromatography technique. The method is highly selective for tryptophan which is separated from its main indoleamine metabolites, 5-hydroxytryptophan, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, and from kynurenine. The usefulness of the assay has been demonstrated in plasma level and urinary excretion studies of orally administered tryptophan.     

METABOLISM OF A TRYPTOPHAN LOAD IN THE HYPOTHALAMUS AND OTHER BRAIN REGIONS

Abstract— Results confirm previous findings that after injecting rats with 50mg/kg tryptophan the percentage increase of 5-hydroxytryptamine metabolism (as shown by 5-hydroxyindolylacetic acid changes) is particularly small in the hypothalamus. However, 15–30 min after tryptophan injection (when brain 5-hydroxytryptamine changes were maximal) percentage 5-hydroxytryptamine increases in the hypothalamus and in the rest of the brain were comparable. The small 5-hydroxyindolylacetic acid changes in the hypothalamus are consistent with a long 5-hydroxytryptamine turnover time therein as indicated by experiments using pargyline or probenecid and by the relatively small increases of 5-hydroxytryptamine after injecting tryptophan into tranylcypromine treated rats. When 5-hydroxytryptamine synthesis was partially inhibited by p -chlorophenylalanine and tryptophan was injected, there was a large percentage rise of hypothalamic 5-hydroxytryptamine but the concentration found in rats given neither drug was not attained and 5-hydroxyindolylacetic acid showed little change. Elsewhere in the brain 5-hydroxytryptamine attained concentrations comparable to those in rats given neither drug and 5-hydroxyindolylacetic acid rose considerably. Results are discussed in relation to the contributions made to brain 5-hydroxytryptamine turnover by functional and non-functional metabolism.     

Day/night differences in pineal indoles in the adult pigeon (Columba livia)

Day/night differences in concentrations of 5-hydroxy and 5-methoxy indole metabolites in the pineal gland of the pigeon are described. A simultaneous determination of 5-hydroxytryptamine (serotonin), 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, N-acetyl-5-hydroxytryptamine (N-acetyl serotonin), 5-methoxyindoleacetic acid, 5-methoxytryptophol, tryptophan, indoleacetic acid and melatonin was accomplished using a recently developed procedure employing high-performance liquid chromatography with electrochemical detection. As in mammalian species, an inverse relationship was observed between N-acetylated indoles and serotonin and its acid metabolites. Melatonin and N-acetyl serotonin were increased approximately three-fold at night to concentrations of 0.730 and 1.79 ng/pineal respectively. Daytime serotonin values were 44.9 +/- 13.0 ng/pineal and decreased to 12.3 +/- 6.5 ng/pineal during the dark phase.     

Evidence for the role of brain biogenic amines in depressed motor activity seen in chemically thyroidectomized rats.

Abstract— The effects of exposure to an antithyroid drug, methimazole, on brain tyrosine hydroxylase and tryptophan hydroxylase activity, as well as the levels of norepinephrine, dopamine, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid have been investigated in maturing brain. Daily treatment of neonatal rats with methimazole for 30 days induced chemical thyroidectomy as evidenced by significant impairment of body and brain growth. The activities or brain tyrosine hydroxylase and tryptophan hydroxylase and the levels of norepinephrine, dopamine and 5-hydroxytryptamine were markedly altered in a dose- and time-dependent manner in methimazole-treated rats. Conversely, the concentration of brain 5-hydroxyindoleacetic acid was elevated (46%) by methimazole administration. Treatment with the antithyroid drug failed to exert any significant effect on the endogenous levels of brain tryptophan, as well as on the activity of the deaminating enzyme, monoamine oxidase. Administration of triiodothyronine (25 or 100 μg/100 g) to hypothyroid rats for 30 days did not produce any appreciable effect upon the neurochemical parameters related to either norepinephrine or 5-hydroxytryptamine mctabolism. However, increasing the dose of triiodothyronine to 250 μg/100 g significantly elevated the levels of norepinephrine and 5-hydroxytryplamine as well as the activities of the two synthesizing enzymes, tyrosine hydroxylase and tryptophan hydroxylase. Brain 5-hydroxyindoleacetic acid levels were restored to normal values in thyroid hormone-deficient rats treated with this higher dose of triiodothyronine. Evidencc also was obtained to show that chemical thyroidectomy suppressed the spontancous locomotor activity in neonatal rats; the changes being apparent at 15 days of age. Our data support the view that thyroid hormone in neonatal life displays an important regulatory effect on the metabolism of norepinephrine, dopamine and 5-hydroxytryptamine. Since certain amines have been known to be implicated as the neurochemical substrates for behavioural arousal, it is conceivable that the observed hypoactivity in methimazolc-treated rats may, at least in part, be related to impaired maturation of norepinephrine and dopamine-synthesizing systems in brains of cretinous rats.     

Calcium-Dependent, Tetrodotoxin-Sensitive Stimulation of Cortical Serotonin Release After a Tryptophan Load

The effect of intraperitoneal administration of tryptophan (50, 100, or 200 mg/kg) on extracellular concentrations of tryptophan, serotonin (5-hydroxytryptamine, 5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) was studied in the cortex of freely moving rats by transcerebral dialysis. Rats were implanted with dialysis probes in the frontal cortex, and experiments were performed 24 h later. Tryptophan, 5-HT, and 5-HIAA were quantified in 20-min samples of dialysate by HPLC with electrochemical detection after separation on reverse-phase columns. Tryptophan administration resulted in a significant increase of tryptophan, 5-HT, and 5-HIAA levels in dialysates. The maximal increase of 5-HT and 5-HIAA output was approximately 150% over basal values. Perfusion with Ringer's solution containing tetrodotoxin (1 microM) reduced 5-HT output by 90% and prevented the increase of 5-HT and 5-HIAA content after 100 mg/kg of tryptophan. Similar results were obtained after perfusion with Ringer's solution without Ca2+. The results indicate that a tryptophan load stimulates the physiological release of 5-HT.     

REGIONAL BRAIN STUDY OF INDOLEAMINE METABOLISM IN THE RAT IN ACUTE HEPATIC FAILURE

Abstract— Tryptophan, 5-hydroxytryptamine and 5-hydroindoleacetic acid were found to be greatly increased in various parts of the brains of rats in acute hepatic failure following two stage hepatic devascularization. However, the increases in 5-hydroxytryptamine and 5-hydroxyindoleacetic acid varied by region and are not explicable solely in terms of increased concentrations of tryptophan. The results are discussed in terms of differences in the regional metabolism of 5-hydroxyindoleamines. Plasma free fatty acids, albumin, total tryptophan and free tryptophan were measured in plasma in hopes of elucidating the mechanism responsible for the cerebral elevation of tryptophan. Increased plasma free tryptophan appears sufficient to explain the rapid increase in brain tryptophan. The relationship between these results and recent observations in hepatic encephalopathy is discussed.     

Optimized isocratic conditions for the simultaneous determination of serotonin precursors and metabolites by reversed-phase high-pressure liquid chromatography with electrochemical detection

We describe an analytical procedure for the simultaneous determination of 5-hydroxyindole derivatives using high-pressure liquid chromatography with electrochemical detection. The procedure clearly resolves 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxytryptophol, and 5-hydroxyindole-3-acetic acid. The C-18 extraction column methodology and high-pressure liquid chromatography-electrochemical detection parameters have been developed to provide a rapid, sensitive, and reproducible quantitative determination of these 5-hydroxyindoles with picogram sensitivity. Chromatograms obtained from the analysis of whole normal mouse brain by the present technique clearly resolve the 5-hydroxyindoles and appear to be uncomplicated by interfering substances.     

Simultaneous determination of urinary tryptophan, tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection

A high performance liquid chromatography method with ultraviolet and fluorimetric detection has been developed for the simultaneous determination of urinary creatinine (Cr), tryptophan (Trp) and three Trp-related metabolites including kynurenine (Kyn), kynurenic acid (Kyna) and 5-hydroxyindole-3-acetic acid (5-HIAA). Samples were pretreated by centrifugation after a freeze-thaw cycle to remove protein and other precipitates. Separation was achieved by an Agilent HC-C18 (2) analytical column and a gradient elution program with a constant flow rate 1mL/min at an ambient temperature. Total run time was 30 min. Cr, Kyn and Kyna were measured by a variable wavelength detector at wavelengths 258 nm, 365 nm and 344 nm respectively. Trp and 5-HIAA were measured by a fluorescence detector with an excitation wavelength of 295 nm and an emission wavelength of 340 nm. This allowed the determination of Kyn/Cr, Kyna/Cr, Trp/Cr and 5-HIAA/Cr concentration ratios in a single run on the same urine sample. Good linear responses were found with correlation coefficient (r)>0.999 for all analytes within the concentration range of physiological level. The limit of detection of the developed method was: Cr, 0.0002 g/L; Kyn, 0.1 μmol/L; Kyna, 0.04 μmol/L; Trp, 0.02 μmol/L and 5-HIAA, 0.01 μmol/L. Recoveries from spiked human urine were: Cr, 93.0-106.4%; Kyn, 97.9-106.9%; Kyna, 98.5-105.6%; Trp, 96.7-105.2% and 5-HIAA, 96.1-99.7%. CVs of repeatability and intermediate precision of all analytes were less than 5%. This method has been applied to the analysis of urine samples from normal subjects.     

Simultaneous Determination of 3,4-Dihydroxyphenylalanine, 5-Hydroxytryptophan, Dopamine, 4-Hydroxy-3-Methoxyphenylalanine, Norepinephrine, 3,4-Dihydroxyphenylacetic Acid, Homovanillic Acid, Serotonin, and 5-Hydroxyindoleacetic Acid in Rat Cerebrospinal Fluid and Brain by High-Performance Liquid Chromatography with Electrochemical Detection

A method using reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection for the simultaneous determination of tryptophan (TRP), 3,4-dihydroxyphenylalanine (DOPA), and their metabolites in whole brain, small-brain parts, and cerebrospinal fluid of rats has been developed. The sample preparation requires only homogenization in perchloric acid and centrifugation before injection onto the column. With a LiChrosorb RP-18 (10 micrometer) column and a mobile phase consisting of a phosphate (NaH2PO4, 0.1 M)-methanol mixture with octylsulfonate (2.6 x 10(-3) M) at pH 3.35 and 26 degrees C, the separation of DOPA, dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 4-hydroxy-3-methoxyphenylalanine, TRP, 5-hydroxytryptophan (5-HTP), serotonin, and 5-hydroxyindoleacetic acid was achieved. The method has been applied to study the effect of alpha-monofluoromethyldopa alone and in combination with L-DOPA or L-5-HTP, on the catechol and 5-OH indole levels in brain and CSF of the rat.     

A simplified procedure to determine tryptophan residues in proteins.

A procedure is described to determine tryptophan residues in proteins using a tryptophan reagent, 2-hydroxy-5-nitrobenzyl bromide. The method involves the treatment of the unfolded protein with the reagent in 9 m urea at acid pH; incubation of the mixture at room temperature for 2 hr and the removal of the excess reagent by centrifugation and gel filtration. The amount of tryptophan in a protein is determined from the optical density of the labeled protein at 280 and 410 nm, and from the known optical density of 1 mg/ml of the protein at 280 nm and of the reagent at 280 and 410 nm. The efficacy of the method was tested with eight proteins whose tryptophan content is known.     

Simultaneous measurement of dolasetron and its major metabolite, MDL 74,156, in human plasma and urine

A selective and sensitive analytical method for the simultaneous measurement of dolasetron (I) and its major metabolite, MDL 74,156 (II), in human plasma and urine samples has been developed using a structural analogue, MDL 101,858, as internal standard (I.S.). The compounds were extracted from plasma and urine using solvent extraction after the addition of the I.S. Chromatographic separation was carried out on a reversed-phase HPLC column and detection and quantification was by fluorescence with excitation and emission wavelengths of 285 and 345 nm, respectively. Linear responses were obtained over concentration ranges of 5 to 1000 pmol/ml for plasma samples and 20 to 1000 pmol/ml for urine samples with correlation coefficients for the calibration curves exceeding 0.999 in all cases. Intra-day and inter-day reproducibility yielded limits of quantification of 10 pmol/ml for I and 5 pmol/ml for II in plasma and 50 pmol/ml for I and II in urine. The method has been applied to the simultaneous analysis of both compounds in plasma and urine samples coming from clinical pharmacokinetic studies.     

PROPERTIES OF PARTIALLY PURIFIED PIG BRAIN STEM TRYPTOPHAN HYDROXYLASE

—Tryptophan hydroxylase form pig brain has been purified using a method which involved sonic disintegration of a whole homogenate, ammonium sulphate fractionation, hydroxylapatite fractionation, column chromatography on Sephadex G-100 or G-200 and finally electrophoresis on poly-acrylamide gel. The enzyme was stabilized during purification by tryptophan and dithiothreitol. The partially purified enzyme has a molecular weight of 55,000-60,000 as measured by gel-filtration. The K_m of the soluble partially purified enzyme was 0-4 mm , which differed significantly from that of the particulate enzyme (0·02mm ). Enzyme activity was not stimulated by ferrous ion. However, it was inhibited by the chelating agents 8-hydroxyquinoline, O-phenanthroline and EDTA. In contrast to dopamine, high concentration of tryptophan (10 mm ), 5-hydroxytryptamine, tryptamine and tyramine at 0-5 mm concentration did not inhibit the enzyme in the presence of dimethyltetrahydropterin (DMPH4). A number of monoamine oxidase inhibitors, phenelzine, pheniprazine and chlorgyline at 1 mm strongly inhibit the formation of 5-hydroxytryptamine. Evidence is presented for the presence of an endogenous inhibitor of tryptophan hydroxylase.     

Effect of orally administered l-tryptophan on serotonin,melatonin, and the innate immune response in the rat

To assess the effects of external administration of L-tryptophan on the synthesis of serotonin and melatonin as well as on the immune function of Wistar rats, 300 mg of the amino acid were administered through an oral cannula either during daylight (08:00) or at night (20:00) for 5 days. Brain, plasma, and peritoneal macrophage samples were collected 4 h after the administration. The accumulation of 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition was used to measure the rate of tryptophan hydroxylation in vivo. Circulating melatonin levels were determined by radioimmunoassay, and the phagocytic activity of macrophages was measured by counting, under oil-immersion phase-contrast microscopy, the number of particles ingested. The results showed a diurnal increase (p < 0.05) in the brain 5-HTP, serotonin (5-hydroxytryptamine, 5-HT), and 5-hydroxyindolacetic acid (5-HIAA) of the animals which had received tryptophan at 08:00 and were killed 4 h later. In the animals which received tryptophan during the dark period, the 5-HT declined but the 5-HT/5-HIAA ratio remained unchanged. There was also a significant increase (p < 0.05) in nocturnal circulating melatonin levels and in the innate immune response of the peritoneal macrophages in the animals which had received tryptophan at 20:00. The results indicated that the synthesis of serotonin and melatonin, as well as the innate immune response, can be modulated by oral ingestion of tryptophan.