Paul Hjemdahl

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

Abstract 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.

The sympathetic response to euglycaemic hyperinsulinaemia

SummarySympathetic nervous system activation by insulin has been suggested as a mechanism explaining the association between insulin resistance and hypertension. We further examined the effect of insulin by direct microneurographic muscle and skin nerve sympathetic activity recordings during euglycaemic insulin clamps in healthy subjects. The mean plasma insulin level was elevated from 5.3±0.7 to 92.2±2.2 mU/l in seven subjects during a 90-min one-step clamp. In six other subjects plasma insulin was further raised from 85.7±4.0 mU/l to 747±53 mU/l between 45–90 min (two-step clamp). Four of the latter subjects received a sham clamp with NaCl infusions only on a second recording session. At the low dose of insulin muscle nerve sympathetic activity increased from a resting level of 22.7±5.0 bursts per min to 27.7±5.0 bursts per min at 15 min (p<0.05). The increases in muscle nerve sympathetic activity were significant (p<0.001; ANOVA) throughout insulin infusion, with a slight further increase (from 29.2±1.6 to 32.3±1.9 bursts per min) at the supraphysiological insulin concentration. During sham clamps muscle nerve sympathetic activity did not increase. Both insulin clamps induced minor, but significant, increases in forearm venous plasma noradrenaline concentrations. Skin nerve sympathetic activity (n=3) did not change during insulin infusions. Heart rate increased slightly but significantly (p<0.005), during the insulin clamps. Blood pressure was not notably affected. In conclusion, hyperinsulinaemia was associated with increased vasoconstrictor nerve activity to skeletal muscle and with no change of sympathetic outflow to skin.

Co-release of neuropeptide Y and catecholamines during physical exercise in man

Venous plasma levels of neuropeptide Y-like immunoreactivity (with chromatographic properties of synthetic neuropeptide Y) increased in parallel with catecholamines, heart rate and blood pressure during graded physical exercise in man. The plasma levels of neuropeptide Y correlated better with the levels of noradrenaline than adrenaline, suggesting release of a neural origin. Taken together with previous results, this suggests that neuropeptide Y is released together with noradrenaline upon sympathetic activation during physiological conditions in man. Determinations of plasma neuropeptide Y may therefore be valuable in the assessment of sympathetic nerve activity.

Platelet-Leukocyte Cross Talk in Whole Blood

Abstract—Thrombosis and inflammation involve complex platelet-leukocyte interaction, the details of which are not fully elucidated. Therefore, we investigated cross talk between platelets and leukocytes in whole blood, under the following physiological conditions: at 37°C, with normal calcium concentrations, and with shear force. Platelet P-selectin and leukocyte CD11b expression were used to monitor platelet and leukocyte activation, respectively, and platelet-leukocyte aggregation (PLA) was analyzed. The leukocyte-specific agonist N-formyl-methionyl-leucyl-phenylalanine (10−6 mol/L) increased P-selectin–positive platelets from 2.5±0.1% to 5.1±0.6% (P <0.05). The increase was inhibited by either the platelet-activating factor (PAF) antagonist SR27417, the superoxide anion scavenger superoxide dismutase, the 5-lipoxygenase inhibitor Zileuton, or the 5-lipoxygenase–activating protein inhibitor MK-886, suggesting the involvement of PAF, superoxide anion, and 5-lipoxygenase products in leukocyte-induced platelet activation. The platelet-specific agonist collagen (1 &mgr;g/mL) increased leukocyte CD11b expression from 2.94±0.52 to 3.81±0.58 (P <0.05); this was not inhibited by the thromboxane A2 receptor antagonist ICI 192.605 or the PAF antagonist SR27417. Platelet P-selectin expression induced by N-formyl-methionyl-leucyl-phenylalanine and leukocyte CD11b expression induced by collagen could be suppressed by glycoprotein IIb/IIIa blockade or P-selectin blockade. This study documents platelet-leukocyte cross talk under conditions that mimic a physiological state and suggests that this involves multiple mediators and mechanisms. Furthermore, new evidence of integrin and selectin involvement in intracellular and intercellular signaling during platelet-leukocyte cross talk is provided.

Norepinephrine-induced human platelet activation in vivo is only partly counteracted by aspirin.

BACKGROUND Epinephrine and mental stress may, via platelet stimulation, enhance the risk of thrombus formation. Norepinephrine is more likely than epinephrine to activate platelets in vivo because of higher levels in plasma but is less well studied in this respect. The antiplatelet drug of choice for patients with coronary artery disease, aspirin, may be less effective during sympathoadrenal activation. We therefore investigated platelet responses in vivo to exogenous norepinephrine with and without aspirin pretreatment. METHODS AND RESULTS Platelet aggregability in vivo was assessed in 11 healthy male subjects, by filtragometry ex vivo (which reflects platelet aggregability in vivo) and by measurements of plasma beta-thromboglobulin (beta-TG, which reflects platelet secretion). Norepinephrine infusions elevated venous plasma norepinephrine from 1.5 to 4 and 15 nmol/L, respectively, and enhanced platelet aggregability (filtragometry) concentration dependently (P < .001). Platelet secretion (beta-TG levels) increased during high-dose infusion (P < .01). Aspirin pretreatment (500 mg orally 12 hours earlier) reduced the excretion of 11-dehydrothromboxane B2 by 62 +/- 5% (P < .001) and attenuated platelet aggregability at rest (P < .05) but not the effect of norepinephrine infusion on platelet aggregability. Conversely, resting plasma beta-TG levels and the urinary excretion of high-molecular-weight beta-TG were not altered by aspirin pretreatment, whereas the norepinephrine-induced increase in plasma beta-TG was abolished. CONCLUSIONS Norepinephrine, at plasma levels easily attained during exercise, enhances platelet aggregability and platelet secretion in vivo in healthy humans. Aspirin may be less effective as an antithrombotic drug during sympathoadrenal activation in humans.

Evidence for Prothrombotic Effects of Exercise and Limited Protection by Aspirin

BACKGROUND Exercise may activate platelets and leukocytes and promote thrombosis. The effects of aspirin treatment on the prothrombotic effects of exercise have not been established. METHODS AND RESULTS A total of 15 healthy men performed exhaustive exercise without and with 1 week of pretreatment with aspirin (500 mg/day). Before and immediately after exercise, platelet aggregability ex vivo was measured by filtragometry, and venous blood samples were obtained. Whole-blood flow cytometry was used to determine platelet and leukocyte activation and platelet-leukocyte aggregates. Exercise increased platelet P-selectin expression, CD11b expression in neutrophils and lymphocytes, and platelet and leukocyte responses to thrombin, ADP, platelet activating factor, and N-formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro. Consistent with enhanced platelet and leukocyte activation, more circulating platelet-platelet and platelet-leukocyte aggregates were detected after exercise (P<0.001 for both). Filtragometry readings were shortened, and plasma soluble P-selectin and prothrombin fragment 1+2 were elevated. Aspirin markedly reduced the urinary excretion of 11-dehydrothromboxane B(2), decreased P-selectin expression in single platelets at rest (P<0.05), and inhibited fMLP-induced neutrophil CD11b expression, but it did not attenuate exercise-induced increases in platelet aggregability, platelet P-selectin expression, leukocyte CD11b expression, platelet-leukocyte aggregate formation, soluble P-selectin, or prothrombin fragment 1+2. CONCLUSIONS Exercise induced platelet and leukocyte activation and platelet-leukocyte aggregation in vivo, and it increased platelet and leukocyte responsiveness to in vitro stimulation. Aspirin treatment attenuated certain signs of platelet activity in vivo at rest and fMLP-induced neutrophil activation in vitro, but it did not attenuate the prothrombotic effects of exercise.

Fetal and maternal plasma catecholamine levels at elective cesarean section under general or epidural anesthesia versus vaginal delivery

Fetal and maternal plasma levels of catecholamines were measured at birth in 40 women with normal term pregnancies who underwent elective cesarean section. Twenty women were operated on under general anesthesia, and 20 under epidural anesthesia. For comparison, the same measurements were also made in 10 women who underwent vaginal delivery without signs of intrapartum fetal distress. Maternal venous levels of catecholamines were elevated in all three groups as compared to values in the resting adult. The highest levels were found in the vaginal delivery group (norepinephrine and epinephrine, 3.9 +/- 2.1 and 1.1 +/- 1.0 nmoles/L, respectively), and the lowest in the epidural cesarean section group. Fetal outcomes were similar in all three groups, as judged by Apgar scores and by measurements of umbilical arterial blood gases. In spite of that, neonates delivered vaginally showed a markedly higher sympathoadrenal activation (norepinephrine and epinephrine, 31.8 +/- 24.1 and 5.1 +/- 7.6 nmoles/L, respectively) than those born by elective cesarean section. In the latter group, however, it was found that the type of maternal anesthesia influenced fetal sympathoadrenal activation, since neonatal levels of catecholamines were higher in the epidural section group (norepinephrine and epinephrine, 9.5 +/- 6.4 and 4.0 +/- 4.5 nmoles/L, respectively) than in the general anesthesia group (norepinephrine and epinephrine, 3.2 +/- 2.7 and 1.0 +/- 1.4 nmoles/L, respectively). These results may have a certain clinical relevance since fetal sympathoadrenal activation is thought to promote extrauterine adaptation.

Effects of serotonin on platelet activation in whole blood.

Serotonin (5-hydrotryptamine, 5-HT) is a weak platelet agonist, which has not been evaluated fully with respect to platelet responses in whole blood. We thus re-evaluated platelet responses to 5-HT using three whole blood techniques: flow cytometry, impedance aggregometry and filtragometry. At concentrations up to 10–5 mol/l, 5-HT per se failed to induce platelet aggregation in whole blood, or to increase platelet fibrinogen binding or P-selectin expression. However, 5-HT potentiated platelet responses to low concentrations of adenosine diphosphate (ADP) or thrombin dose-dependently. 5-HT (10–7, 10–6 and 10–5 mol/l) increased ADP-induced platelet fibrinogen binding by 40, 59 and 79%, while P-selectin expression increased by 45, 64 and 95%, respectively (P < 0.05; n = 10). The enhancing effects of 5-HT were even more pronounced in thrombin-stimulated samples, as 5-HT at 10–8, 10–7, 10–6 and 10–5 mol/l increased fibrino gen binding by 56, 128, 212 and 260% (P < 0.05), and P-selectin expression by 31, 56, 89 and 109%, respectively (P < 0.01; n = 10). The response to 5-HT was inhibited dose-dependently by a highly selective 5-HT2A receptor antagonist (SR 46349), with almost complete inhibition at 10–6 mol/l. Impedance aggregometry showed a significant enhancement in 5 × 10–6 mol/l ADP-induced platelet aggregation caused by 5-HT at 8 × 10–8 mol/l (from 7.58 × 2.50 Ω to 9.02 × 2.43 Ω, P < 0.02, n = 12). Similarly, filtragometry readings, i.e. the time taken for platelet aggregates to occlude a microfilter, were shortened by 27% (P < 0.05, n = 9), reflecting increased platelet aggregability. Our data suggest that 5-HT per se does not activate platelets, but dose-dependently enhances platelet activation induced by ADP and, in particular, thrombin in whole blood. Thus, the idea that 5-HT is a ‘helper agonist’ is supported; this effect is mediated by 5-HT2A receptors.

Circulatory and sympatho-adrenal responses to stress in borderline and established hypertension.

Responses to mental stress [a colour word test (CWT), orthostatic testing (ORT) and a cold pressor test (CPT) were studied in 33 subjects with essential hypertension (EHT), 16 subjects with borderline hypertension (BHT) and 17 age and sex-matched normotensive controls (NT). Venous plasma noradrenaline (NA) was similar in all groups. CWT induced marked circulatory responses and metabolic activation with minor increases in NA. Circulatory and NA responses to ORT and CPT were similar in all groups. CWT elevated diastolic blood pressure more in BHT and tended to elevate HR more in EHT and BHT than in NT. Plasma adrenaline (ADR) tended to be higher in BHT and increased during all provocations in EHT and BHT but not in NT. Early hypertension appears to be associated with enhanced cardiovascular and sympatho-adrenal reactivity (resembling a hypothalamic defence reaction) which is revealed by mental stress, rather than stimuli such as ORT or CPT. Venous plasma NA has limitations in defining neurogenic alterations in hypertension since it reflects poorly sympathetic activity in the organs responsible for pressor responses to emotional stimuli. Plasma ADR is more valuable in this respect.

Fibrinolytic Variables and Cardiovascular Prognosisin Patients With Stable Angina Pectoris Treated With Verapamil or Metoprolol Results From the Angina Prognosis Study in Stockholm

BACKGROUND Disturbed fibrinolytic function may influence the progression of coronary atherosclerosis and contribute to thrombotic cardiovascular (CV) events. METHODS AND RESULTS In the Angina Prognosis Study in Stockholm (APSIS), patients with stable angina pectoris were studied prospectively during double-blind treatment with metoprolol or verapamil. Various measures of fibrinolytic function were studied in 631 (of 809) patients. During a median follow-up time of 3.2 years (2132 patient-years), 32 patients suffered a CV death, 21 had a nonfatal myocardial infarction (MI), and 77 underwent revascularization. Plasma levels of tissue plasminogen activator (TPA) activity and antigen (ag), plasminogen activator inhibitor (PAI-1) activity at test, and TPA responses to exercise were determined at baseline and after 1 months treatment and were related to subsequent fatal and nonfatal CV events. Univariate Cox regression analysis revealed that elevated levels of TPA-ag at rest (P < .05), high PAI-1 activity (P < .05), and low TPA-ag responses to exercise (P < .05) were associated with increased risk of subsequent CV death. After adjustment for baseline risk factors, TPA-ag independently predicted CV death or MI. In addition, PAI-1 activity independently predicted CV death or MI in male patients. Verapamil treatment was associated with a 10% decrease of TPA-ag levels and metoprolol treatment with a 2% increase (P < .001 for treatment difference). CONCLUSIONS Plasma TPA-ag levels at rest, and among male patients PAI-1 activity as well, independently predict subsequent CV death or MI in patients with stable angina pectoris.