A rapid radioenzymatic assay for dopamine and N-acetyldopamine.

Dopamine (DA) was measured in various tissue extracts as [³H]methoxy-N-acetyldopamine after incubation with two partially purified enzymes, catechol-O-methyl transferase (EC 2.1.1.1) and N-acetyltransferase (EC 2.3.1.5), in the presence of [³H]adenosylmethionine and acetyl-CoA. This product can be separated quantitatively from labeled products of norepinephrine and epinephrine by solvent extraction. N-Acetyl-DA can be assayed by omitting the acetylating system from the incubation mixture. The procedure is rapid, convenient for processing large numbers of samples, and has a sensitivity of approximately 0.1 pmol. It has been used to measure DA in ganglia and in individual neurons from gastropod mollusks.     

Simultaneous single isotope radioenzymatic assay of plasma norepinephrine,epinephrine and dopamine

Modification of the original single isotope radioenzymatic assay of Passon and Peuler (1) permits the direct and simultaneous analysis of norepinephrine, epinephrine and dopamine in plasma samples of 50 μl or less. Plasma or cerebrospinal fluid without prior extraction of catecholamines or deproteinization is added directly into a mixture of 100 μl. This catechol-O-methyl-transferase-catalyzed assay is sensitive to 1 pg (20 pg/ml of plasma) for norepinephrine and epinephrine and 6 pg (120 pg/ml) for dopamine. A rapid thin layer chromatographic separation of the three ³H-methylcatecholamines contributes to the excellent specificity of the differential assay of the three catecholamines. The differential analysis of 15–20 plasma samples can be completed easily within one day. A total assay which omits the chromatographic step and, thus, measures norepinephrine plus epinephrine at the same sensitivity can be completed in 20 samples in one-half a working day.     

Plasma catecholamines in resting rainbow trout,Salmo gairdneri Richardson,by high pressure liquid chromatography*

Plasma norepinephrine and epinephrine from cannulated trout were measured by high pressure liquid chromatography with electrochemical detection. The catecholamines were extracted with acid-washed alumina using a microfilter assembly which permitted analysis of small volumes of plasma. Mean (± S.D.) values for plasma norepinephrine and epinephrine were 1.83 (0.97) pmol ml^?1 and 8.95 (4.94) pmol ml^?1, respectively. These values are compared with catecholamine levels from other vertebrate species.     

A radioenzymatic assay for catecholamines and dihydroxyphenylacetic acid.

Picogram quantities of the catecholamines, dopamine, norepinephrine, and epinephrine, and the dopamine metabolite, dihydroxyphenylacetic acid, can be measured in tissue or plasma samples utilizing a rapid radioenzymatic procedure. The catechols are converted to their ³H-methylated derivatives (3-methoxytyramine, normetanephrine, metanephrine and homovanillic acid, respectively) by the enzyme catechol-O-methyltransferase with ³H-S-adenosylmethionine serving as the ³H-methyl donor. Following the enzymatic reaction, unreacted ³H-S-Adenosylmethionine is removed by precipitation and the reaction products are separated by thin layer chromatography on silica plates. The areas corresponding to the ³H-methylated derivatives are scraped into scintillation vials, eluted with aqueous buffer, extracted into nonpolar scintillation cocktail, and counted by liquid scintillation spectrometry. Using the standard assay procedure described here, over 100 tubes can be assayed in a single day with a sensitivity of 15–25 pg for all compounds measured. With the application of additional procedures, as little as 1 pg norepinephrine and epinephrine and 5–10 pg dopamine and dihydroxyphenylacetic acid can be quantified in a single sample.     

Catecholamines in fetal pig plasma and the response to acute hypoxia and chronic fetal decapitation

Summary Dopamine, norepinephrine and epinephrine were measured by radioenzymatic assay in blood plasma samples drawn from the umbilical arteries of 30 anaesthetised Landrace pig fetuses. Just prior to term, the concentrations of dopamine (0.46±0.14 ng·ml^–1) and norepinephrine (1.74±0.60 ng·mg^–1) were lower than earlier in gestation, whereas epinephrine concentrations at term (0.80±0.31 ng·ml^–1) were similar to those at mid-gestation, intervening stages of gestation having higher levels of plasma epinephrine. Fetal hypoxia was induced by clamping the umbilical cord for 2 min and the catecholamines determined in arterial blood samples immediately thereafter, then again 3 min after removal of the clamp. Inconsistent effects of cord clamping on catecholamine levels were seen at 55 days, but thereafter, in all but one instance, the hormone levels were increased. Fetuses near term tended to respond less than fetuses at 75 and 96 days gestation (term=114±1 day). Catecholamines were also present in the circulation of fetuses decapitated at 42 days gestation and studied at 109±1 days. The average concentrations of dopamine (1.12±0.27 ng·ml^–1) and norepinephrine (8.23±3.04 ng·ml^–1) were greater than in intact fetuses, the plasma epinephrine levels being comparable to, or slightly higher than, those in intact fetuses. The results demonstrate that catecholamines are present in the circulation of the intact and decapitated pig fetus and that the actual concentrations and the type of response to umbilical cord clamping are dependent on gestation age.     

Human nonshivering thermogenesis

Metabolic responses, skin temperatures and changes in heart rate and blood pressure were measured in a control group and in “polar swimmers” after infusion of different doses of epinephrine, norepinephrine and isoprenaline. In controls the highest infusion dose of isoprenaline (0.1 μg min⁻¹ kg⁻¹) increased metabolic rate in normal humans by 36%, while the highest infusion doses of epinephrine and norepinephrine (0.45 μg min⁻¹ kg⁻¹) increased metabolic rate by 24%, only. In “polar swimmers” the epinephrine thermogenesis was potentiated significantly, reaching about 45% of the basal metabolic rate. The norepinephrine and isoprenaline thermogenesis were not different from that of the control group. It is concluded that in humans the epinephrine thermogenesis is probably located in muscles and in the white fat (Simonsen et al., 1992), and may be the principal mechanism of metabolic adaptation to cold. It was calculated that the increased capacity of epinephrine thermogenesis in cold exposed “polar swimmers” could theoretically shift the survival limit downwards to lower environmental temperatures by about 5°C.     

Detection of endogenous salsolinol in neonatal rat tissue by a radioenzymatic—thin-layer chromatographic assay

A sensitive radioenzymatic—thin-layer chromatographic assay for the quantitative analysis of the tetrahydroisoquinoline alkaloid, salsolinol, in plasma and neonatal rat tissue is described. The assay involves the enzymatic O-methylation of salsolinol by catechol-O-methyltransferase in presence of [³H] S-adenosylmethionine, and subsequent separation by thin-layer chromatography of the resultant [³H] O-methyl-salsolinol from the O-methylated derivatives of dopamine, epinephrine and norepinephrine. The method allows the detection of as little as 100 pg salsolinol per g tissue, and the accurate quantitation of as little as 100 pg/ml plasma and 500 pg/g tissue. This assay permitted the detection of trace amounts of endogenous salsolinol in neonatal rat tissue (< 500 pg/g tissue).     

CATECHOLAMINES IN FETAL AND NEWBORN RAT BRAIN

The levels of dopamine and norepinephrine were determined in the brains of fetal and newborn rats by means of a sensitive, radiometric-enzymatic assay. Catecholamines were converted to their 3-O-methylated derivatives in the presence of catechol-O-methyl transferase (EC 2.1.1.1) and [³ H-methyl]S-adenosylmethionine; and the [³H]-derivatives were isolated by selective extraction. The assay had a sensitivity for dopamine and norepinephrine of 100 picograms and was linear to at least 30 nanograms of catecholamines. Both amines were present at 15 days of gestation and increased 15-fold in content during the last week of gestation. The regional distribution of these neurotransmitters in the brain of the newborn rat correlated with the distribution of their biosynthetic enzymes. An investigation of the effects of reserpine, pheniprazine, α-methyl-para-tyrosine, diethyldithiocarbamate and l -DOPA on the levels of dopamine and norepinephrine in the brains of the 18-day gestational fetus indicated that the levels of these neurotransmitters are under controls similar to those known to occur in the brain of the adult rat.     

A radioimmunoassay for epinephrine and norepinephrine in tissues and plasma

An I¹²⁵ radioimmunoassay (RIA) has been developed for the measurement of plasma and tissue epinephrine (E) and norepinephrine (NE). The assay utilizes an antibody which specifically binds metanephrine. E and NE are detected by conversion to metanephrine with the enzymes catechol-0-methyltransferase and phenylethanol-amine-N-methyltransferase. The assay is very specific and will allow the measurement of E and NE in less than 500 μl of normal human plasma. E and NE concentrations were determined by both the RIA and a radioenzymatic assay in canine, human and rat biologic samples. The correlation coefficients between the two assays were .962 for E and .956 for NE. The RIA is sensitive, specific, precise and significantly less costly and time consuming than present radioenzymatic methods.     

Automated mass spectrometric analysis of urinary free catecholamines using on-line solid phase extraction

Analysis of catecholamines (epinephrine, norepinephrine and dopamine) in plasma and urine is used for diagnosis and treatment of catecholamine-producing tumors. Current analytical techniques for catecholamine quantification are laborious, time-consuming and technically demanding. Our aim was to develop an automated on-line solid phase extraction method coupled to high performance liquid chromatography–tandem mass spectrometry (XLC–MS/MS) for the quantification of free catecholamines in urine. Five microlitre urine equivalent was pre-purified by automated on-line solid phase extraction, using phenylboronic acid complexation. Reversed phase (pentafluorophenylpropyl column) chromatography was applied. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Urinary reference intervals were set in 24-h urine collections of 120 healthy subjects. XLC–MS/MS was compared with liquid chromatography with electrochemical detection (HPLC–ECD). Total run-time was 14 min. Intra- and inter-assay analytical variations were <10%. Linearity was excellent (R² > 0.99). Quantification limits were 1.47 nmol/L, 15.8 nmol/L and 11.7 nmol/L for epinephrine, norepinephrine and dopamine, respectively. XLC–MS/MS correlated well with HPLC–ECD (correlation coefficient >0.98). Reference intervals were 1–10 μmol/mol, 10–50 μmol/mol and 60–225 μmol/mol creatinine for epinephrine, norepinephrine and dopamine, respectively. Advantages of the XLC–MS/MS catecholamine method include its high analytical performance by selective PBA affinity and high specificity and sensitivity by unique MS/MS fragmentation.     

Bactericidal Activity of Catecholamine Copper Complexes

Washed or growing E. coli cells are killed by epinephrine, norepinephrine or dopamine in the presence of non lethal concentrations of Cu(II). Killing is enhanced by anoxia and by sublethal Concentrations of H₂O₁. The rate of killing is proportional to the rate of catecholamine oxidation. The copper epinephrine complex binds to E. coli cells, induces membrane damage and depletion of the cellular ATP pool. The cells may be partially protected by SOD or catalase but not by OH radical scavengers. Addition of H²O₂ to cells which were sensitized by preincubation with the epinephrine-copper complex, causes rapid killing and DNA degradation. Sensitized cells are not protected by BSA.     

Assay of catecholamines in human plasma: Studies of a single isotope radioenzymatic procedure

The sensitive specific radioenzymatic procedure for determination of catecholamines originally described from our laboratory by Coyle and Henry (1) has been optimized for use in assay of human plasma levels of dopamine, norepinephrine and epinephrine. Dopamine and the total of norepinephrine and epinephrine are assayed by 0-methylation while norepinephrine is determined by N-methylation. Epinephrine is calculated from the difference between the 0-methylation and N-methylation procedures. In a group of 13 normal subjects, plasma levels of epinephrine were found to be 67 ± 9.2 pg/ml, norepinephrine 208 ± 16.9 pg/ml and dopamine 33 ± 8.1 pg/ml. Dopamine determinations are of low reliability because of relatively high blanks and necessary corrections.     

Induction of phosphoenolpyruvate carboxykinase by sympathetic agents in primary cultures of adult rat hepatocytes

Phosphoenolpyruvate carboxykinase was studied in primary adult rat hepatocyte cultures maintained for 48 h. Between 48 h and 52 h norepinephrine (10^?5 mol/l) and epinephrine (10^?6 mol/l) in the presence of dexamethasone (10^?8 mol/l) and insulin (10^?9 mol/l) increased the enzyme activity about fourfold. This increase was prevented by cycloheximide. The induction by norepinephrine and epinephrine could be inhibited almost completely by the β-blocking agent propranolol, while that by glucagon remained unaffected. The concentration dependence of enzyme induction may indicate that epinephrine might act as a circulating hormone, while norepinephrine might be operative as a neurotransmitter requiring higher local concentrations. The results are in line with the proposal that hepatic nerves might directly control gene expression.     

Biogenic amines in cultured neuroblastoma and astrocytoma cells

The presence of biogenic amines in cultured cells of mouse neuroblastoma C-1300 (clone NB-2a) was suggested by fluorescence-microscope histochemistry. Incubation in media containing L-[¹⁴C]tyrosine and L-[¹⁴C]tryptophan for 24 h, followed by high-voltage electrophoresis, radiochromatogram scanning, and scintillation counting, confirmed the presence of [¹⁴C]dopamine, [¹⁴C]norepinephrine, [¹⁴C]epinephrine, [¹⁴C]serotonin, [¹⁴C]tyramine, and [¹⁴C]octopamine. Dopamine, norepinephrine, epinephrine, and serotonin were demonstrated spectrophotofluorometrically in concentrations, expressed as micrograms amine per milligram protein, of 1.19, 0.027, 0.038, and 0.148, respectively, for cells in a stationary growth phase. Fluorescence-microscope histochemistry also suggested the presence of biogenic amines in cultured astrocytoma cells (cell line C6). Spectrophotofluorometric assay of cells in a stationary growth phase demonstrated intracellular dopamine, norepinephrine, epinephrine, and serotonin in concentrations significantly lower than those of neuroblastoma cells.     

Development of a sensitive enzymeimmunoassay for oxytocin determination in bovine plasma

A highly sensitive and specific second antibody enzymeimmunoassay (EIA) on microtiterplates for oxytocin determination in bovine plasma using the biotin–streptavidin amplification system was developed. Biotin was coupled to oxytocin and used to bridge between streptavidin-peroxidase and the immobilized oxytocin antiserum in the competitive assay. The assay was carried out directly in 200 μl of bovine plasma. Oxytocin standards prepared in hormone-free plasma were used. The sensitivity of the assay was 0.25 pg/well which corresponded to 1.25 pg/ml plasma; the 50% relative binding was seen at 2.8 pg/well. Plasma volumes for the assay ranging from 50 to 200 μl did not influence the shape of the oxytocin standard curve; however a distinct drop in the OD₄₅₀ was observed with higher plasma volumes. The oxytocin antiserum used in the assay showed no significant cross-reaction with other octapeptides tested. The assay was compared with a radioimmunoassay (RIA) procedure employing prior solvent extraction of plasma samples. The oxytocin concentrations assayed by EIA and RIA in plasma samples obtained from four cows before, during and after milking were highly correlated and very similar (r=0.97). Hence the assay developed offers an attractive alternative to the RIA since no prior laborious plasma extraction is needed. Further, the assay has the distinct advantage of being non-radioactive in nature.     

Catecholamines and dopamine-β-hydroxylase activity during therapeutic abortion induced by sulprostone

To evaluate the changes in circulating norepinephrine (NE), epinephrine (E) and dopamine-β-hydroxylase (DBH) caused by an intravenous infusion of a derivate of PGE₂, sulprostone, in connection with legal termination of pregnancy, serial plasma samples were analyzed for six gravidae. Plasma catecholamines were measured by a sensitive radioenzymatic method (9,10) and DBH activities by a photometric assay (11). Intravenous infusion of sulprostone, in abortifacient doses as an intravenous infusion of 3–4 μg per minute for six to eight hours produced a decrease in circulating norepinephrine. No significant alteration was found in plasma epinephrine or dopamine-β-hydroxylase activity. The finding suggests an inhibitory effect of sulprostone on the release of norepinephrine from the adrenergic terminals without inhibition of the adrenal medulla.     

Solvent-extraction complex of uranium(VI) with cis,syn, cis-dicyclohexano-18-crown-6

The extraction of U(VI)with dicyclohexano-18-crown-6 (mixed isomers or isomer A) from HCl medium is effective and selective, and can be used for separating and analysing uranium and thorium. However, little is known of the properties of the extraction complex of uranium with crown-ether in organic phase. In this paper we report the preparation, characteristic and structure of the crystalline extraction complex IaUO₂Cl₂HClH₂O, Iabeing isomer A of dicyclohexano-18-crown-6.After extracting uranium(VI) from aqueous hydrochloric acid solution with Ia in 1,2-dichloroethane, the crystalline product of the extraction complex was prepared from the organic phase by diluting with a non-polar solvent at 25 °C. The content of uranium, crown-ether and HCl was determined. The IR spectrum of the crystals shows that the strong hydronium-crown ether/oxygen hydrogen bond absorption is found in the region 2300–2400 cm⁻¹. The chemical shift in the range 9–12 ppm was observed. The ¹H NMR signal of hydronium protons appears at 9.890 ppm. The results of assay correspond to the formula Ia₂·(H₃O⁺)₂·UO₂Cl₄²⁻.Crystal structure of the extraction complex has been determined by X-ray crystallography. Crystals are monoclinic, space group C_2/c (No. 15) a=32.464, b=10.203, c=21.616 Å, β=119.73° and Z=4. In the complex each of the two H₃O⁺ cations is anchored in the crown-ether cavity by three stronger hydrogen bonds (distances approximately 2.65 Å), whereas uranium forms UO₂Cl₄²⁻ with Cl⁻ as counterion about 8 Å away from the H₃O⁺.     

A differential pulse polarographic assay for O-methylation of catechols by catechol-O-methyltransferase

A new method is described for monitoring the enzyme-eatalyzed O-methylation of norepinephrine (or other catechol substrate) by catechol-O-methyltransferase. Norepinephrine and normetanephrine combine with bis-2-(ethylhexyl)hydrogen phosphate at pH 7.4 to form an ion pair which is quantitatively extracted with an immiscible organic solvent by an application of partition chromatography. The mixture of catechol substrate and O-methylated products are then simultaneously determined in 0.5 m H₂SO₄ solution by monitoring their oxidation at a carbon paste electrode by a differential pulse polarographic technique.     

Regulation of adenylate cyclase in cultured cardiocytes from neonatal rats by adenosine and other agonists

The presence of adenosine receptors coupled to adenylate cyclase in cultured cardiocytes from atria and ventricles from neonatal rats is demonstrated in these studies. N-Ethylcarboxamideadenosine (NECA), l-N⁶-phenylisopropyladenosine (PIA), and 2-chloroadenosine (2-cl-Ado) stimulated adenylate cyclase in a concentration-dependent manner in both cultured atrial and ventricular cells. The order of potency of stimulation was NECA > PIA > 2-cl-Ado. The stimulation of adenylate cyclase by NECA was enhanced by guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine in both these cells. Other agonists such as epinephrine, norepinephrine, dopamine, F^?, and forskolin were also able to stimulate adenylate cyclase, although the extent of stimulation by these agents was higher in ventricular than in atrial cells. The stimulation of adenylate cyclase by epinephrine and norepinephrine was inhibited by propranolol but not by phentolamine. On the other hand, phentolamine, propranolol, and haloperidol inhibited dopamine-stimulated adenylate cyclase activity to the same extent. Forskolin, at its maximal concentration, potentiated the stimulatory effect of epinephrine, norepinephrine, and dopamine on adenylate cyclase in both atrial and ventricular cardiocytes, but the interaction of NECA with epinephrine, norepinephrine, or dopamine was different in atrial and ventricular cells. The stimulation by an optimal concentration of NECA was additive with maximal stimulation by the catecholamines in atrial cells but not in ventricular cells. The data suggest the existence of adenosine “Ra” and catecholamine receptors in cultured atrial and ventricular cardiocytes. It can be postulated that adenosine in addition to its role as a potent vasodilator might regulate cardiac performance through its interaction with “Ra” receptors associated with adenylate cyclase. The difference in the mode of interaction of adenosine with catecholamines in atrial and ventricular cells suggests that the mechanism by which these agents activate adenylate cyclase may be different in these cells.     

Specific Selenium-Containing Macromolecules in the Marine Diatom Thalassiosira pseudonana

Thalassiosira pseudonana Husedt (Hasle and Heimdal) clone 3H was grown in axenic culture in artificial seawater medium containing 10⁻⁸ molar Na₂⁷⁵SeO₃. Biochemical distribution of radiolabeled Se was determined by solvent extraction techniques, gel filtration, and polyacrylamide gel electrophoresis. Of the total cellular Se, 51% was protein bound. Two soluble macromolecules of 21 and 29 kilodaltons contained ⁷⁵Se. These results are the first to provide evidence of specific Se-containing compounds in a photosynthetic organism. Glutathione peroxidase (GSH-Px) activity was measured in cell-free extracts and on nondenaturing polyacrylamide gels by a glutathione-reductase coupled assay. Two enzymes showing GSH-Px activity were present. One enzyme was active with H₂O₂ and tert-butyl hydroperoxide (tBOOH); consistent with known Sedependent GSH-Pxs, but the other enzyme was only active with tBOOH. Co-migration of the H₂O₂-active GSH-Px and ⁷⁵Se on nondenaturing polyacrylamide gels provides evidence that T. pseudonana contains a Sedependent GSH-Px. The molecular weight of one of the ⁷⁵Se-labeled macromolecules is identical with the weight of previously characterized GSH-PX subunits. We conclude that the obligate requirement for Se in Thalassiosira pseudonana is due in part to the presence of the selenoenzyme glutathione peroxidase.