Capillary-venous differences of free plasma catecholamines at rest and during graded exercise

Levels of free plasma catecholamines were simultaneously determined in 10 cyclists using capillary blood from one ear lobe and venous blood from one cubital vein. Catecholamine concentrations were higher in the ear lobe blood than in the venous blood at rest and during graded exercise. Average differences amounted to 1.7 nmol X 1(-1) (dopamine), 2.1 nmol X 1(-1) (noradrenaline) and 1.9 nmol X 1(-1) (adrenaline) at rest and increased only to 8.8 nmol X 1(-1) for noradrenaline during exercise. We assume that higher concentrations of dopamine and adrenaline in the capillary blood point to a significant neuronal release of these catecholamines, similar to noradrenaline. Catecholamine concentrations in capillary blood may better reflect sympathetic drive and delivery of catecholamines to the circulation than the concentrations in venous blood.     

A sensitive method for the determination of plasma catecholamines using liquid chromatography with electrochemical detection

Simple and sensitive methods for the determination of plasma catecholamines are of great interest since the level of catecholamines in plasma reflects the activity of the sympatho-adrenal system. In the present work a previously described procedure based on high pressure liquid chromatography with electrochemical detection has been adapted for assay of plasma catecholamines. This method permits simultaneous detection of noradrenaline, adrenaline and dopamine in concentrations down to 0.1 nmol/1 in less than one ml plasma.     

Simultaneous radioenzymatic determination of plasma and tissue adrenaline,noradrenaline and dopamine within the femtomole range

A radiometric-enzymatic assay for measuring simultaneously femtomole quantities of adrenaline, noradrenaline and dopamine has been developed. The three catecholamines are first converted to their O-methylated analogues by catechol-O-methyltransferase in the presence of S-adenosyl-methionine-³H and thereafter extracted following addition of sodium tetraphenylborate. This extraction, together with an improved quick chromatographic separation and the oxidation of the adrenaline and noradrenaline derivatives to vanillin, yields an extremely high sensitivity and specificity of the method.The present assay allows the determination of adrenaline, noradrenaline and dopamine in tissue samples with a protein content of 100 μg or less and in plasma volumes of 20 – 100 μl. The amine content of 40 – 50 samples can be determined in two days by one person.Due to the high sensitivity achieved, this method promises to be a valid alternative to the gas chromatography-mass spectrometry technique.     

Effect of a fall of blood pressure on the release of catecholamines in the hypothalamus

The posterior hypothalamus of anaesthetized cats was superfused through a push-pull cannula and the release of endogenous catecholamines was determined in the superfusate which was continuously collected in 15 min periods. Fall in blood pressure elicited by nitroprusside or bleeding led to an increased rate of release of noradrenaline, adrenaline and dopamine in the hypothalamus. Transection of the brain causal to hypothalamus greatly reduced the rate of resting release of the catecholamines and abolished the enhancing effects of bleeding and nitroprusside. Determination of the catecholamines in samples which were collected in 90 s periods suggested a different pattern of release of the three catecholamines. Further shortening of the collection period (10 s) showed that the fall in blood pressure immediately increased the release of dopamine, while the rates of release of noradrenaline and adrenaline were increased gradually. Hypotension did not influence the rates of release of the catecholamines in the anterior hypothalamus. It is concluded that dopamine, adrenaline and noradrenaline systems of the hypothalamus are involved in the regulation of the arterial blood pressure. The different patterns of release might indicate that dopamine exerts a different function from those of noradrenaline and adrenaline in the normalization of the blood pressure after acute hypotension.     

Changes in plasma free and sulfoconjugated catecholamines before and after acute physical exercise: experimental and clinical studies.

To elucidate whether sulfoconjugated catecholamines in plasma, especially dopamine, serve as a source of free catecholamines, we examined the change in afterload on the deconjugating activity of catecholamines in isolated Langendorff perfused rat hearts. Dopamine-sulfate was administered under ordinary or high-work-load conditions. Free dopamine in the effluent was increased by the high-work-load of the hearts, whereas conjugated dopamine showed an apparent decrease. These results indicate the possibility that deconjugation of sulfoconjugated catecholamines is accelerated by a high-work-load. To obtain further evidence in humans, we also examined the changes in the plasma levels of free and sulfoconjugated catecholamines in healthy volunteers before and after marathon running. Free dopamine increased 1.99-fold from the baseline value after exercise, whereas conjugated dopamine decreased by 12%. Similarly, the plasma levels of free noradrenaline and adrenaline increased after exercise to 2.45- and 1.51-fold their respective baseline values, while conjugated noradrenaline and adrenaline both decreased. These clinical results, as well as those of the experimental studies, suggest that the increase in plasma free catecholamines after exercise is due not only to increased release from the sympathoadrenal system but also to accelerated conversion from sulfoconjugated catecholamines in the plasma.     

Catecholamine biosynthesis in vitro and in vivo in the chromaffin tissue of the Atlantic cod, Gadus morhua

1. The biosynthesis of [3H]catecholamines from [3H]L-tyrosine in the intact chromaffin tissue of cod posterior cardinal veins was studied in vitro and in vivo at 10 degrees C. 2. The tritiated products dopamine, noradrenaline and adrenaline were separated from the [3H]tyrosine by paper chromatography of tissue extracts and the radioactivity of 1 cm strips of the chromatogram was determined by liquid scintillation spectrophotometry. DOPA could never be demonstrated in the tissue extracts from any of the experiments performed. 3. The content of [3H]noradrenaline in pieces of the cardinal veins incubated in vitro was found to increase rapidly. The tissue content of dopamine and adrenaline remained at lower levels which were reached during the first few hours of the incubation. A similar pattern could be demonstrated in the chromaffin tissue in vivo after infusion of [3H]tyrosine, but the total content of the [3H]catecholamines was lower than in the in vitro experiments. 4. The results are consistent with the view that the methylation of noradrenaline is the rate-limiting step in the biosynthesis of adrenaline in cod chromaffin tissue.     

Measurement of catecholamines in biological fluids by high-performance liquid chromatography : A comparison of fluorimetric with electrochemical detection

An improved method for the determination of catecholamines in biological fluids, by reversed-phase high-performance liquid chromatography (HPLC) with fluorimetric detection is presented. The pH titration previously employed in the alumina extraction was abandoned in favour of the use of a molar excess of pH 8.5 Tris—HCl buffer. A novel lyophilisation step serves to concentrate the catechols and by reconstituting in mobile phase, chromatography disturbances are minimised. The addition of 2 mM octanesulphonic acid to a citrate—phosphate mobile phase at pH 6.0 gave optimal resolution and sensitivity.That HPLC separation can improve the specificity of the trihydroxyindole reaction, to the extent of providing a reliable analytical method, has been demonstrated and validated by the technique of HPLC with electrochemical detection. A correlation coefficient of 0.98 was obtained between the two techniques as applied to the measurement of urinary catecholamines. The HPLC—fluorimetric method was sensitive enough to measure 0.1 ng/ml of noradrenaline or adrenaline at a signal-to-noise ratio of 2.0. Application of the method to the quantitative determination of catecholamines in human urine, plasma and rat brain homogenates is demonstrated.     

Analysis of catecholamines and related substances using porous graphitic carbon as separation media in liquid chromatography-tandem mass spectrometry

Capillary porous graphitic carbon (PGC) columns have been utilized for separation of several catecholamines and related compounds (i.e. L-tyrosine, L-DOPA, 3-O-methyl-DOPA, dopamine, 3,4-dihydroxy-phenyl-acetic acid (DOPAC), homovanillic acid, noradrenaline, vanillomandelic acid and adrenaline) on-line with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The use of a mobile phase without ion-pairing agents and with high content of organic modifier facilitated the coupling to the selective and sensitive mass spectrometric detection. Minimum detectable sample concentration (MDC sample) for noradrenaline, dopamine and L-tyrosine in a standard solution was estimated to 3, 10 and 30 nM, respectively (3 S/N corresponds to MDQ for L-tyrosine of approximately 8 x 10(-14)mol). The developed strategy was applied for analysis of brain tissue, i.e. a substantia nigra (ns) sample.     

Pulmonary extraction of dopamine in the conscious dog

Thus far non-respiratory pulmonary functions have mainly been studied in isolated lungs or in intact anaesthetized animals. Therefore, we studied pulmonary handling of vasoactive hormones in the conscious dog. Dopamine, adrenaline and noradrenaline (3 nmol/ml) were injected together with cardiogreen (4 mg/ml) into the caval vein during 5, 10 and 15 s in three separate experiments. Indicator-dilution curves were derived from samples originating from the pulmonary artery and the aorta. During a single passage of the lung dopamine was removed to the greatest extent, noradrenaline somewhat less, and adrenaline not at all, irrespective of the injection duration. In particular, the extraction of dopamine, which was not emphasized in literature, was a remarkable finding.     

Endogenous Noradrenaline and Dopamine in Nerve Terminals of the Hippocampus: Differences in Levels and Release Kinetics

The presence and release of endogenous catecholamines in rat and guinea pig hippocampal nerve terminals was studied by fluorimetric HPLC analysis. In isolated nerve terminals (synaptosomes) the levels and breakdown of endogenous catecholamines were determined and the release process was characterized with respect to its kinetics and Ca2+ and ATP dependence. Endogenous noradrenaline and dopamine, but not adrenaline, were detected in isolated hippocampal nerve terminals. For dopamine both the levels and the amounts released were more than 100-fold lower than those for noradrenaline. In suspension, released endogenous catecholamines were rapidly broken down. This could effectively be blocked by monoamine oxidase inhibitors, Ca(2+)-free conditions, and glutathione. The release of both noradrenaline and dopamine was highly Ca2+ and ATP dependent. Marked differences were observed in the kinetics of release between the two catecholamines. Noradrenaline showed an initial burst of release within 10 s after K+ depolarization. The release of noradrenaline was terminated after approximately 3 min of K+ depolarization. In contrast, dopamine release was more gradual, without an initial burst and without clear termination of release within 5 min. It is concluded that both catecholamines are present in nerve terminals in the rat hippocampus and that their release from (isolated) nerve terminals is exocytotic. The characteristics of noradrenaline release show several similarities with those of other classical transmitters, whereas dopamine release characteristics resemble those of neuropeptide release in the hippocampus but not those of dopamine release in other brain areas. It is hypothesized that in the hippocampus dopamine is released from large, dense-cored vesicles, probably colocalized with neuropeptides.     

Catecholamines in sow graafian follicles at proestrus and at diestrus

Endogenous dopamine, noradrenaline, and adrenaline were detected in the sow graafian follicular wall and in the follicular fluid. Noradrenaline represented the highest level and adrenaline the lowest. Dopamine and noradrenaline concentrations found in the follicular fluid were lower at early proestrus than at mid-diestrus, whereas adrenaline levels in the fluid did not differ at either stage of the estrous cycle. The sow follicular wall contained less dopamine, noradrenaline and adrenaline at early proestrus than at mid-diestrus. Concomitantly, a decrement of [3H]-dopamine and [3H]-noradrenaline uptake, and of dopamine-beta-hydroxylase activity was detected at early proestrus compared to levels detected at mid-diestrus. The findings in sow graafian follicles show the existence of relationships between hormonal status, dopamine, noradrenaline and adrenaline endogenous levels and uptake, and dopamine-beta-hydroxylase activity. Possible links between estradiol, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels during the pig estrous cycle and ovarian catecholamines are discussed, as is a plausible involvement of these neurotransmitters in the contractile activity of the theca layer and the processes of follicular rupture and ovulation.     

Automated analysis of urinary catecholamines by high-performance liquid chromatography and on-line sample pretreatment

A simple and automated solid-phase extraction for the selective and quantitative HPLC analysis of free catecholamines in urine is described. The urinary catecholamines react with diphenylboric acid, giving a complex at pH 8.5 which is strongly retained on a PLRP-S cartridge; elution is accomplished with the same mobile phase used for HPLC analysis. Separation is performed by ion-pair reversed-phase HPLC, with sodium heptanesulphate as counter-ion, and a totally end-capped C₁₈ analytical column. Quantitation is achieved with an electrochemical detector. A Spark Holland Prospekt system controls the on-line solid-phase extraction, preconcentration and direct elution to the LC column. Chromatography run-time is 10 min and the total time to process one urine sample is ca. 12 min.     

A Sensitive and Reliable Assay for Dopamine (β-Hydroxylase in Tissue

A new assay procedure for dopamine β-hydroxylase (DBH) in tissue extracts is described. Solubilized DBH was adsorbed from crude extracts on Concanavalin A-Sepharose (Con A-Sepharose), resulting in enrichment of the enzyme as well as removal of endogenous catecholamines and inhibitory substances. The enzymatic assay was carried out with DBH still adsorbed to Con A-Sepharose. The adsorption of the DBH to Con A-Sepharose offers three advantages over previous assay procedures. (1) Because of removal of the endogenous inhibitory substances, a single Cu²⁺ concentration can be used for the determination of DBH activity, regardless of the tissue dilution or inhibitor content of the analysed sample. Using this procedure, the optimal Cu²⁺ concentration for DBH of bovine adrenal gland extracts was 3 μM and for rat brain 10 μM. (2) Because of removal of endogenous catecholamines, dopamine, the main physiological substrate of DBH in noradrenergic neurons, can be used for the assay. The enzymatic reaction product, noradrenaline, was determined by high performance liquid chromatography and electrochemical detection (hplc-ec). This procedure resulted in an approx. 10-fold increase in sensitivity of the assay compared with other procedures, e.g., the radioenzymatic assay. (3) Direct determination of the immediate product of the enzymatic reaction (noradrenaline) permits kinetic analysis. It was found that the Michaelis constants for the substrate (dopamine) and co-factor (ascorbic acid) (2 mM and 0.65 mM, respectively) determined in bovine adrenal tissue extracts by the described procedure were identical with the values for the purified DBH preparation.     

Determination of plasma catecholamines by high performance liquid chromatography with electrochemical detection: comparison with a radioenzymatic method.

High performance liquid chromatography with electrochemical detection has been compared to a radioenzymatic method for the determination of plasma catecholamines. With the use of an internal standard highly accurate determinations of plasma noradrenaline, adrenaline and dopamine were performed on 0.2–2 ml plasma with the chromatographic method. The radioenzymatic method required only 3 × 50 μl plasma. A comparison of noradrenaline and adrenaline concentrations measured by the two methods in a set of nine plasma samples showed an excellent agreement between the methods (r=0.993 and 0.994, respectively). Advantages and disadvantages with the two methods are discussed.     

Flow-injection chemiluminescence determination of catecholamines based on their enhancing effects on the luminol-potassium periodate system.

A rapid and sensitive chemiluminescence (CL) method using flow injection analysis is described for the determination of four catecholamines, dopamine, adrenaline, isoprenaline and noradrenaline, based on their greatly enhancing effects on the CL reaction of luminol-potassium periodate in basic solutions. The optimized chemical conditions for the chemiluminescence reaction were 1.0 x 10(-4) mol/L luminol and 1.0 x 10(-5) mol/L potassium periodate in 0.2 mol/L sodium hydroxide (NaOH). Under the optimized conditions, the calibration graphs relating the CL signal intensity (peak height) to the concentration of the analytes were curvilinear and they were suitable for determining dopamine, adrenaline, isoprenaline, and noradrenaline in the range 0.1-10 ng/mL, 0.1-100 ng/mL, 1-100 ng/mL and 5-50 ng/mL, respectively, with the relative standard deviations of 0.8-1.7%. The detection limits of the method are 0.02 ng/mL for dopamine, 0.01 ng/mL for adrenaline, 0.1 ng/mL for isoprenaline and 2.0 ng/mL for noradrenaline. The sampling frequency was calculated to be about 60/h. The selectivity of the method was good, because a series of common ions or excipients, such as K(+), Ba(2+), CO(3)(2-), NO(3)(-), SO(4)(2-), PO(4)(3-), sodium citrate, sodium bisulphite, oxidate dopamine, starch, lactose, carbamide and gelatin, could not produce interference when their concentrations were 1000-fold than those of dopamine. The present method was successfully applied to the determination of the four catecholamines in pharmaceutical injections.     

Arrhythmia in patients with mitral valve prolapse syndrome and the status of the sympathetic nervous system]

The study aimed at evaluating a possible relationship between the adrenergic system tone determined with the excretion of catecholamines with the urine and an incidence of the ventricular arrhythmias in patients with the mitral valve prolapse. The study included 20 patients (13 women and 7 men aged between 20 and 50 years; mean = 31.6 years) with the mitral valve prolapse syndrome diagnosed with the aid of the patients' history, physical examinations and echocardiography. Echocardiograms have shown anterior mitral leaflet prolapse in 7 patients, posterior mitral leaflet prolapse in 8 patients, and both mitral leaflets prolapse in the remaining 5 patients. Daily excretion of adrenaline and noradrenaline was measured with Van Euler and Lishajko's fluorimetric technique. Cardiac arrhythmias were determined with a 24-hour ECG monitoring and classified according to Lown. Premature ventricular contractions of class I were seen in 1 patient, of class II in 5, class III in 1, class IV in 2, and class V in 3 patients. Holter monitoring technique did not show the arrhythmias in 8 patients. Daily adrenaline and noradrenaline excretion with the urine was within the normal values (3.2-30.8 ug and 0.2-16.2 ug, respectively) in all examined patients. Daily urine noradrenaline was higher in patients with serious ventricular arrhythmias (Lown's class V) than mean values in the whole examined group.     

Dopamine-selective response in membrane potential by homooxacalix[3]arene triether host incorporated in PVC liquid membrane.

Selectivities of membrane potential changes for catecholamines and inorganic cations were investigated with lipophilic derivatives of calix[6]arene and related hosts incorporated in poly(vinyl chloride) (PVC) matrix liquid membranes. Homooxacalix[3]arene triether displayed an excellent selectivity for dopamine against other catecholamines (adrenaline, noradrenaline) and also against inorganic cations (K+, Na+).     

Catecholamine levels in whole brain of stressed and control domestic and wild rats (Rattus Norvegicus)

Whole brain catecholamine (dopamine, nonadrenaline, adrenaline) levels were measured in control and electric footshocked Wild and domestic (Sprague Dawley and Long Evans) rats. No significant differences were found among the three strains of rats for combined total catecholamine content, or for combined total catecholamines between the control and footshocked groups. Significant differences were found for the total of each catecholamine taken separately, dopamine being present at three times the level of noradrenaline and ten times the level of adrenaline. No significant differences were found for dopamine in both control and footshocked animals among all three rat strains. Noradrenaline was significantly higher in the control domestic rats compared to the control Wild rats, and in the footshocked domestic rats compared to the footshocked Wild rats. No differences in noradrenaline levels were found between Sprague Dawley and Long Evans rats, but noradrenaline increased significantly in the latter following footshock. Adrenaline was significantly highest in the Sprague Dawley controls and lowest in the Wild controls. Footshocking resulted in almost identical levels of adrenaline in the domestic strains and an increase in the F₁ Wild strain.     

A reliable and sensitive method for the simultaneous determination of dopamine, noradrenaline, 5-hydroxytryptamine and 5-hydroxy-indolacetic acid in small brain samples

A sensitive and reliable fluorometric method for the simultaneous determination of dopamine, noradrenaline, 5-hydroxytryptamine and 5-hydroxy-indol acetic acid in small samples of brain tissues is described. The procedure is based on solvent extraction; catecholamines are oxidized by the Chang's method and 5-hydroxytryptamine and 5-hydroxy-indol acetic acid determined by reaction with o-phthalaldehyde, alpha-methyl-p-tyrosine causes a negligible interference with the procedure. Results of determination of these amines in different brain areas are reported.     

Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase). Evidence that the feedback inhibitors adrenaline and noradrenaline are coordinated to iron

Tyrosine 3-monooxygenase (tyrosine hydroxylase) is a non-heme iron, tetrahydropterin-dependent enzyme which catalyzes the rate-limiting step in the biosynthesis of catecholamines. The highly purified bovine adrenal enzyme contains an unusual blue-green chromophore with lambda max at around 700 nm (epsilon = 1.3 (mM subunit enzyme)-1 cm-1). On excitation at 605.2 nm, resonance-enhanced Raman vibrations are observed at 454, 494, 527, 604, 635, 835, 1130, 1271, 1320, 1426, and 1476 cm-1. The excitation profiles of the modes of 1276 and 1476 cm-1 (from 488 to 620 nm) follow the contour of the 700 nm absorption band. The vibrations observed strongly indicate the presence of a bidentate catecholamine-Fe(III) complex in the enzyme as isolated which gives rise to the characteristic charge-transfer transitions. This is further supported by the release of 0.11 +/- 0.04 mol of noradrenaline and 0.25 +/- 0.06 mol of adrenaline per mol of enzyme subunit on denaturation of the enzyme. The energies of the catecholate to Fe(III) charge-transfer transitions indicate a mixture of histidines and carboxylate(s) coordinated to the iron center in tyrosine hydroxylase. At neutral pH, the enzymatic activity was inhibited more than 50% by 10 microM dopamine, noradrenaline, and adrenaline. The high affinity of the catecholamines to the nonphosphorylated form of tyrosine hydroxylase may have significance in vivo since catecholamines are potent feedback inhibitors of the enzyme.