Plasma free and sulfoconjugated catecholamines during sustained exercise

Previous research established a relationship between circulating sulfoconjugated norepinephrine (NE-SO4) and oxygen consumption at various exercise intensities. In this study, the stability of the NE-SO4 response was examined during sustained exercise at a constant relative intensity. Seven trained men bicycled at 78 +/- 3% of their maximal O2 consumption for 28 min and then rested on the ergometer for a comparable duration. After a 30-min rest, plasma samples were collected through an indwelling catheter at 7-min intervals during the exercise and recovery periods. Free NE and epinephrine increased sixfold during exercise. These changes were accompanied by increases in sulfoconjugated catecholamines, but only NE-SO4 achieved statistical significance (rest, 712 +/- 602; exercise, 1,329 +/- 1,163 pg/ml). This occurred at three collection periods (14, 21, and 28 min). Approximately 35, 52, and 95% of NE, epinephrine, and dopamine, respectively, existed as sulfoconjugated during exercise. Subject variation was present in the sulfoconjugated catecholamine response that could not be attributed to corresponding differences in circulating free catecholamine release. These findings implicate blood flow as a factor in the sulfoconjugation of NE, but not epinephrine or dopamine.     

Plasma free and sulfoconjugated catecholamine responses to varying exercise intensity

Plasma free catecholamines rise during exercise, but sulfoconjugated catecholamines reportedly fall. This study examined the relationship between exercise intensity and circulating levels of sulfoconjugated norepinephrine, epinephrine, and dopamine. Seven exercise-trained men biked at approximately 30, 60, and 90% of their individual maximal oxygen consumption (VO2max) for 8 min. The 90% VO2max period resulted in significantly increased plasma free norepinephrine (rest, 219 +/- 85; exercise, 2,738 +/- 1,149 pg/ml; P less than or equal to 0.01) and epinephrine (rest, 49 +/- 49; exercise, 555 +/- 516 pg/ml; P less than or equal to 0.05). These changes were accompanied by consistent increases in sulfoconjugated norepinephrine at both the 60% (rest, 852 +/- 292; exercise, 1,431 +/- 639; P less than or equal to 0.05) and 90% (rest, 859 +/- 311; exercise, 2,223 +/- 1,015; P less than or equal to 0.05) VO2max periods. Plasma sulfoconjugated epinephrine and dopamine displayed erratic changes at the three exercise intensities. These findings suggest that sulfoconjugated norepinephrine rises during high-intensity exercise.     

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.     

Effect of eating bananas on plasma free and sulfate-conjugated catecholamines

The effect of eating bananas, a known and rich source of biogenic amines, on plasma free and sulfate-conjugated catecholamines was examined in normal human volunteers. Before ingestion conjugated norepinephrine (NE) and epinephrine (E) comprised 60% and 84% of the total concentration respectively. Free dopamine (DA) was undetectable. Although there was no change in free NE and E levels after eating bananas, free DA became measurable in some subjects. In contrast, conjugated NE and particularly DA, increased markedly in all subjects. More detailed study in 2 subjects indicated that this increase may persist for 8 hours after consumption. Available evidence suggests that sulfate conjugation of free catecholamines in banana largely takes place in the gut. It is important therefore to consider dietary influences when the physiological role of conjugated catecholamines. and perhaps free dopamine, is being assessed.     

Plasma free and sulphated catecholamines after ultra-long exercise and recovery

We investigated the early and late effects of two types of ultra-long exercise on sympatho-adrenal and dopaminergic activity. With this aim both free and sulphoconjugated plasma catecholamines (CA), noradrenaline (NA), adrenaline (A), and dopamine (DA) were determined in two groups of athletes immediately after completion of 24-h running or a 10-h triathlon and on recovery during the next 1-3 days. Both races stimulated the sympathetic activity, but differences were observed in the CA pattern: the 24-h run induced a marked elevation of free and sulphoconjugated NA (+175% and +180%, respectively) but failed to alter significantly A and DA levels. The triathlon challenge increased the three conjugated CA (NA sulphate +350%; A sulphate +110%; DA sulphate +270%) and to a lesser extent free CA (NA +45%; A +30%). On the first post-exercise morning, a sustained intense noradrenergic activity was still present in the 24 h-runners, as evidenced by the large increase in free and sulphated NA levels (+140% and +100%, respectively). Such a prolonged activity was also indicated after completion of the triathlon, by the increase of NA sulphate (+140%) observed on the 1st recovery day. However, after the triathlon there was a decreased release of A from the adrenal medulla for several days. These data show that both types of ultralong exercise are able to induce for several hours a sustained sympathetic activation during the test and in the recovery period. Furthermore, the study shows that plasma conjugated CA may provide delayed and cumulative indexes of sympathetic activation, complementary to the instantaneous markers such as free CA.     

Is the platelet phenolsulfotransferase involved in the sulfoconjugation of plasma catecholamines?

Catecholamines are predominantly present in the sulfoconjugated forms in human plasma. Phenolsulfotransferase (EC 2.8.2.1), which catalyses the sulfation of phenolic compounds, is widely distributed in human tissues. In blood, a phenolsulfotransferase, more specific for catecholamine sulfation is found exclusively in platelets. Free and sulfoconjugated catecholamines were measured in plasma and platelets of healthy volunteers and compared with those present in patients with uremia or pheochromocytoma to determine the ability of platelet phenolsulfotransferase to sulfurylate plasma catecholamines. In patients with pheochromocytoma, the rise in free and sulfoconjugated plasma catecholamines is accompanied by a simultaneous rise of these molecules in platelets. In uremia, where the level of plasma catecholamines is normal, the rise in the sulfoconjugates is not accompanied by a concomitant increase in either free or sulfoconjugated catecholamines in platelets. Platelet phenolsulfotransferase activity remains unchanged in pheochromocytoma and uremia. These data indicate that the platelet phenolsulfotransferase is involved in the sulfation of the catecholamines present in platelets, but its contribution, if any, to the high level of sulfoconjugated catecholamines found in plasma is negligible. This assertion is confirmed by our observations in thrombocytopenic patients. Indeed, despite the very low number of platelets and the absence of plasma phenolsulfotransferase activity, thrombocytopenic patients have normal plasma levels of free and sulfoconjugated catecholamines.     

Plasma free and sulfate conjugated catecholamine levels during acute physiological stimulation in man

The responses of plasma free and sulfate-conjugated catecholamines to acute physiological stimulation was examined in normal male subjects. Catecholamines were measured with a sensitive radioenzymatic assay incorporating simultaneous hydrolysis of sulfate conjugates and O-methylation of free norepinephrine and epinephrine. Following 20 minutes recumbency after venepuncture 30 +/- 3% of norepinephrine and 16 +/- 5% of epinephrine was in thr free form. Free catecholamines generally increased during standing, cold immersion and isometric handgrip, but sulfates did not change. Bicycle ergometry markedly increased free catecholamines which rapidly returned to basal levels at the end of exercise. In contrast, sulfated norepinephrine decreased substantially with exercise in all subjects but returned to basal levels 3 minutes after stopping exercise. Epinephrine sulfate varied considerably between subjects but showed a similar, although smaller, fall with exercise. Thus, during physiological stimulation, which caused increases in free norepinephrine and epinephrine levels in plasma, the only consistent change in sulfated catecholamines was a marked fall in norepinephrine sulfate after bicycle exercise. This may indicate saturation of sulfotransferase activity, substrate inhibition or impaired tissue conjugation.     

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.     

Assay of free and conjugated catecholamines by high-performance liquid chromatography with electrochemical detection

A rapid and simple method for the analysis of free and conjugated catecholamines in body tissues and fluids is described. The free catecholamines were isolated by standard alumina procedures before and after hydrolysis of the conjugated compounds to free compounds by heating the samples in perchloric acid. Free catecholamines were then separated by high-performance liquid chromatography and detected by electrochemical detection. Conjugated compound was the difference between the total and free amount in each sample. This method was utilized to measure free and conjugated norepinephrine, epinephrine, and dopamine in human urine and rat adrenal gland, and to measure free and conjugated dopamine in rat whole brain and kidney.     

Cardiac Responses to Thermal,Physical, and Emotional Stress

We have studied the effect of a short period of exposure to the intense heat of a sauna bath on the electrocardiogram and plasma catecholamine, free fatty acid, and triglyceride concentrations in 17 subjects with apparently normal hearts and 18 persons with coronary heart disease. Similar observations were made on 11 of the 17 normal subjects and on 7 of the persons with coronary heart disease in response to exercise.Exposure to heat was associated with an increase in plasma adrenaline with no change in noradrenaline, free fatty acid, or triglyceride concentrations. Exercise was associated with the expected increase in both plasma noradrenaline and adrenaline concentrations. A heart rate up to 180 beats/min was observed in response to both heat and exercise. Apart from the ST-T changes inherent to sinus tachycardia, ST-T segment abnormalities were frequent in response to heat in both the subjects with normal and abnormal hearts, but little change occurred in the ST-T configuration when the subjects were exercised to produce comparable heart rates. Ectopic beats, sometimes numerous and multifocal, were observed in some subjects of both groups in response to heat, but not to exercise. It seems likely that the net unbalanced adrenaline component of the increased plasma catecholamine concentrations (which is also seen in certain emotional stress situations) is predominantly responsible for ischaemic-like manifestations of the electrocardiogram in susceptible subjects. The observations provide further validation for previously reported studies that it is the increased plasma noradrenaline in response to emotional stress that is associated with the release of free fatty acids and ultimate hypertriglyceridaemia, of probable importance in the aetiology of atheroma.     

Enzymatic deconjugation of catecholamines in human and rat plasma and red blood cell lysate

We have developed a method for enzymatic hydrolysis of both sulfated and glucuronidated catecholamines in plasma and red blood cell lysate. Hydrolysis occurs in the course of the radioenzymatic assay for catecholamines. In human plasma, catecholamines are conjugated almost entirely with sulfate while, in rat plasma, glucuronides are the main conjugates of epinephrine and dopamine but not norepinephrine. Rat plasma contains less percent conjugated catecholamine than human plasma. Human red blood cell lysate contains less conjugated catecholamine than plasma, whereas free E in lysate exceeds that of plasma and free NE has same level both in plasma and lysate. This method is useful in detecting total (free + sulfated + glucuronidated) catecholamines and the nature of conjugated catecholamines.     

Free and conjugated plasma catecholamines, DOPA and 3-O-methyldopa in humans and in various animal species

The aim of the present study was to determine the extent to which plasma catecholamines are conjugated in different animals compared to man and how widespread is the presence of dihydroxyphenylalanine (DOPA) and 3-methoxy-4-hydroxyphenylalanine (3-OMD) in plasma among the different animal species. Free and conjugated norepinephrine, epinephrine, and dopamine were measured in plasma in humans and in several animal species (dog, rat, Gunn rat, cat, rabbit, guinea pig, African green monkey, young pig, calf, and one American black bear) using HPLC with electrochemical detection. The same technique was used to measure free and conjugated DOPA and 3-OMD in plasma of man, dog, rat, Gunn rat, calf, and American black bear. Human plasma contains the highest concentration of total (free and conjugated) catecholamines (46.1 pmole/ml), while low concentrations (below 15 pmole/ml) were observed in unstressed rats, calves, cats, and young pigs. In man, 95.3% of total plasma catecholamines were conjugated. The extent to which plasma catecholamines were conjugated varied greatly between animal species. The conjugated fraction expressed as percentages of the total catecholamines is lowest in the young pig (4.7%) and highest in the bear (100%). Conjugated dopamine was present in the plasma of all species, varying between 3% of the total catecholamine pool in young pig to 90% in dog. Conjugated norepinephrine was also present in plasma of all species except in unstressed rats with access to food. Conjugated epinephrine was detected only in cat and rat. Free DOPA and 3-OMD were present in plasma of all tested species with especially high levels of 3-OMD being present in dog. Conjugated DOPA and 3-OMD were not consistently found in any species. Our results indicate that man, dog, bear, and African green monkey are particularly good catecholamine conjugators and that young pig, guinea pig, rabbit, and calf are poor conjugators.     

Free, glucuronide, and sulfate catecholamines in the rat: effect of hypoxia

The formation and excretion of conjugated catecholamines (CA) was studied in conscious rats after sympathetic stimulation by hypoxia (5.5-6% O2, 4 h). Hypoxia induced a rapid and intense increase of free epinephrine (E, X 12) and norepinephrine (NE, X 6) but only a limited enhancement of free dopamine (DA, X 2). Sulfate conjugates of E and NE had kinetics similar to the free forms, while glucuronides were only moderately and lately altered. In contrast to free and sulfated DA, DA glucuronide, the major plasma conjugate, was decreased (-25%). This result suggests that DA glucuronide, unlike other CA conjugates, is not related to detoxication but might supply a CA precursor. Urinary conjugates badly reflected plasma conjugates. In normoxic controls, CA conjugates prevailed in the plasma, whereas the free amines prevailed in the urine. Hypoxia increased mainly the excretion of E and NE glucuronide but not of the free amines. Urinary DA, free or conjugated, was decreased (-25%), a result in keeping with plasma DA glucuronide only. The poor relations between plasma and urine catecholamines pinpoint the importance of the kidney in CA handling.     

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.     

Calcitonin gene-related peptide and adrenomedullin release in humans: effects of exercise and hypoxia

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are potent vasorelaxant peptides. This study examined exercise-induced changes in CGRP and AM levels in 12 healthy sea level natives at sea level (SL) and subsequently after 24 h (HA1) and 5 days (HA5) in high altitude hypoxia (4559 m). Plasma values of CGRP, AM, calcitonin, noradrenaline, adrenaline, lactate and heart rate were measured at rest and during maximal exercise (W(max)). On each study day, the dopamine D(2)-receptor antagonist, domperidone (30 mg; n=6), or no medication (n=6) was given 1 h before exercise. W(max) at SL, HA1 and HA5 increased CGRP and AM along with heart rate, lactate and catecholamines, whereas, calcitonin remained unchanged. The maximal CGRP levels at W(max) were significantly decreased at HA1 (74.3+/-6.1 pmol/l; p=0.002) and HA5 (69.6+/-6.0 pmol/l; p<0.001) compared to maximal CGRP at SL (85.1+/-4.9 pmol/l). A similar pattern was observed for lactate and the relation between CGRP and lactate release showed a close linear correlation (r(2)=0.63, P<0.0001). Domperidone produced a marked increase in noradrenaline at W(max), but had no affect on CGRP or AM. In conclusion, CGRP release during hypoxic exercise does not respond to domperidone-induced changes in circulating levels of noradrenaline, rather the release may be directly related to the production of lactate.     

Hormonal Neuroendocrine and Vasoconstrictor Peptide Responses of Ball Game and Cyclic Sport Elite Athletes by Treadmill Test

ObjectiveOur objective was to evaluate complex hormonal response in ball game and cyclic sport elite athletes through an incremental treadmill test, since, so far, variables in experimental procedures have often hampered comparisons of data.MethodsWe determined anthropometric data, heart rate, maximal oxygen uptake, workload, plasma levels of lactate, adrenaline, noradrenaline, dopamine, cortisol, angiontensinogen and endothelin in control (n = 6), soccer (n = 8), handball (n = 12), kayaking (n = 9) and triathlon (n = 9) groups based on a Bruce protocol through a maximal exercise type of spiroergometric test.ResultsWe obtained significant increases for adrenaline, 2.9- and 3.9-fold by comparing the normalized means for soccer players and kayakers and soccer players and triathletes after/before test, respectively. For noradrenaline, we observed an even stronger, three-time significant difference between each type of ball game and cyclic sport activity.ConclusionsExercise related adrenaline and noradrenaline changes were more pronounced than dopamine plasma level changes and revealed an opportunity to differentiate cyclic and ball game activities and control group upon these parameters. Normalization of concentration ratios of the monitored compounds by the corresponding maximal oxygen uptake reflected better the differences in the response level of adrenaline, noradrenaline, dopamine and cortisol.     

Dopamine 4-sulfate: effects on isolated perfused rat heart and role of atria

We studied the effects of sulfate conjugate of dopamine on the isolated perfused rat heart (Langendorff preparation). In the experimental group, we removed atria from half number of the hearts. In the hearts with intact atria, dopamine 4-sulfate significantly improved the DT (developed tension), +dT/dt max (maximal rate of contraction), -dT/dt max (maximum rate of relaxation) over baseline values. But when atria were removed, dopamine 4-sulfate had no effect on the mechanical functions of heart. We analysed the effluent perfusate for the free and conjugated catecholamines. In the control group (no drug), and when atria were excised, the free catecholamine levels were negligible. But when the atria were kept intact, the effluent contained significant amount of free dopamine (DA), and norepinephrine (NE). These data suggested that dopamine sulfate had no direct effect on the ventricular muscle of rat heart, but was converted within the atrial tissues into free catecholamines which might be responsible for the positive inotropic actions.     

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.     

Increased conjugation of catecholamines during normal pregnancy

Urinary elimination of conjugated catecholamines (adrenaline and noradrenaline) is increased during normal pregnancy (60.5 % in the normal ; 70 % in the pregnants).     

Ascorbic acid suppresses the deconjugation of noradrenaline but not dopamine in plasma

The characteristics of hydrolysis of sulfoconjugated noradrenaline (NA) and dopamine (DA) in plasma using sulfatase were investigated. Ascorbic acid has been used as an antioxidant during the hydrolysis of conjugated NA or DA. Hydrolysis of NA sulfates was considerably inhibited by adding ascorbic acid (0.5-10 mM), and slightly inhibited by adding dithiothreitol (1-10 mM). In contrast, the hydrolysis of DA sulfates was not affected after either ascorbic acid or DTT treatment. On the basis of these findings, the levels of NA sulfates previously reported are found to be markedly lower than the actual levels of NA sulfates in human plasma.