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Purpose: To assess biventricular functional reserve (FR), NT-proBNP levels and exercise performance, in relation to right ventricular volume in patients with pulmonary regurgitation (PR) after repair of tetralogy of Fallot (TOF) at young age., Methods: In 53 TOF patients (maximum age at repair 2.0 years, interval since repair 15 (5) years) without residual lesions except PR, biventricular FR (derived from magnetic resonance imaging with dobutamine stress), NT-proBNP levels, maximal workload, and peak oxygen uptake were assessed., Results: Mean right ventricular end-diastolic volume was 140(38) ml/m(2). Median pulmonary regurgitant fraction was 37% (range 0-57%). Biventricular systolic stress response was normal: mean (SD) ESV decreased (DeltaRVESV -17(8) ml/m(2), DeltaLVESV -11(5)), SV increased (DeltaRVSV +12(9) ml/m(2), DeltaLVSV +9(6)), FR was positive in all (RV-FR +11(5)%, LV-FR +13(6)). No serious adverse effects to dobutamine were encountered. NT-proBNP was increased in 2 patients. Median level was 10 pmol/L (range 2-42). NT-proBNP correlated with PR-percentage but not with right ventricular size. High-risk levels of NT-proBNP indicated a smaller RV-FR and a smaller decrease of biventricular ESV. Mean (SEM) VO2(max) was 96(3)%, mean Workload(max) 89(2)% of predicted., Conclusion: At mid to long term follow-up overall NT-proBNP levels are normal and biventricular functional reserve and exercise tolerance are well preserved in TOF repaired at young age, irrespective of RV volume. This questions the validity of isolated PR or RV volume criteria for pulmonary valve replacement in this group. Low-dose dobutamine stress testing is well tolerated and may be a useful additional tool for clinical decision making.

Background: Human 'new pressor protein' (NPP), related to coagulation beta-factor XIIa (beta-FXIIa), potently releases sympathoadrenal catecholamines in bioassay rats, with concurrent elevation of systolic and diastolic blood pressure (SBP/DBP) and heart rate (HR). Elevated plasma NPP/beta-FXIIa levels in hypertensive anephric pediatric patients on hemodialysis associated with fluid status and blood pressure changes were previously reported, suggesting that NPP/beta-FXIIa contributed to their hypertension., Objective: To investigate the mechanism of action of NPP/beta-FXIIa., Methods: Hemodynamic and sympathoadrenal responses to NPP (20 microL plasma equivalent/rat) or coagulation beta-FXIIa (300 ng/kg intravenously) were measured in rats treated with pentolinium (ganglion blockade [+GB]) and/or captopril (+CAP; angiotensin converting enzyme [ACE] inhibition)., Results: In controls not receiving GB or CAP (-GB-CAP), NPP/beta-FXIIa raised plasma epinephrine (E) sixfold, SBP/DBP by 14/8 mmHg and HR by 15 beats/min. With blockade of the cholinergic pathway to the sympathoadrenal system (+GB), basal E, norepinephrine (NE), SBP, DBP and HR all dropped. However NPP/beta-FXIIa remained capable of raising E 20-fold, NE fourfold, SBP/DBP by 27/11 mmHg and HR by 20 beats/min, suggesting that it acted through a 'noncholinergic' mechanism. With +CAP alone, NPP/beta-FXIIa raised plasma E 18-fold, NE threefold, SBP/ DBP by 29/8 mmHg and HR by 73 beats/min, implicating an ACE-regulated 'peptidergic' mechanism. Combining +GB with +CAP potentiated NPP/beta-FXIIa actions further by raising E 50-fold, NE sevenfold, SBP/DBP by 55/20 mmHg and HR by 87 beats/min, strengthening the efficacy of this alternate pathway., Conclusions: The cardiovascular effects of NPP/beta-FXIIa are considerably mediated by a noncholinergic (peptidergic) ACE-regulated mechanism for sympathoadrenal catecholamine release that is enhanced by +GB and/or +CAP. Under inflammatory procoagulant conditions, endogenously produced NPP/beta-FXIIa may interfere with the antihypertensive effects of ACE inhibition therapy.

Inhibition of renin with an active site inhibitor, aliskiren, lowers blood pressure (BP) in diabetic patients. Here, we studied the time course of the antihypertensive and antiproteinuric effect of renin inhibition in 15 patients with type 2 diabetes and elevated urinary albumin/creatinine ratios (UACRs) to check whether aliskiren can decrease proteinuria. After a 4-week washout of previous medications, patients received aliskiren and furosemide daily for 28 days followed by a 4-week withdrawal period. Twenty-four-hour BPs were measured at baseline throughout treatment and withdrawal periods. The UACR was significantly reduced after 2-4 days of treatment with another significant reduction after 28 days. Systolic blood pressure (SBP) was significantly lower after 7 days with no further reduction after 28 days. The BP returned toward baseline 3 days after withdrawal, whereas the UACR was still significantly reduced compared with baseline 12 days after withdrawal. Our study shows that aliskiren reduced 24 h SBP, and this was associated with a reduction in albuminuria in type 2 diabetic patients.

Background: Right ventricular (RV) dysfunction is associated with increased morbidity and mortality in patients with chronic thromboembolic pulmonary hypertension (CTEPH) who undergo pulmonary endarterectomy (PEA). We studied whether plasma brain natriuretic peptide (BNP) levels can be used to identify RV dysfunction in CTEPH patients. Therefore, plasma BNP levels were studied in relation to cardiac remodeling and function as determined by cardiac magnetic resonance imaging (MRI)., Methods: Thirty-eight patients with CTEPH (55 +/- 15 years), and ten healthy controls (46 +/- 15 years) were studied. The BNP was determined by an immunoradiometric assay., Results: The CTEPH patients had a mean pulmonary artery pressure of 49 +/- 13 mm Hg, cardiac index 2.1 +/- 0.7 l x min(-1) x m(-2), and pulmonary vascular resistance of 867 +/- 432 dynes x s x cm(-5). In CTEPH patients, compared with controls, right ventricular (RV) remodeling was demonstrated. In the patients, BNP was increased and correlated (all p < 0.0001; Spearman rank test) with MRI parameters of RV remodeling and function: end diastolic (r = 0.71) and end systolic (r = 0.74) volumes, RV mass (r = 0.68), leftward ventricular septal bowing (r = -0.80) and ejection fraction (EF; r = -0.81). By receiver operating curve analysis, BNP levels of 11.5 picomole (pmol)/L and 48.5 pmol/L, respectively, detected RV dysfunction as defined by RVEF less than 0.45 and less than 0.30, respectively, with high sensitivity and specificity. Hemodynamically, BNP levels greater than 48.5 pmol/L identified the most severely affected patients., Conclusions: In CTEPH patients, BNP levels correlate with RV remodeling and can be used to identify RV dysfunction. Future studies are warranted on the role of BNP to identify "high risk" CTEPH patients and its relation to postoperative hemodynamic outcome, RV failure, and mortality.

Orthostatic intolerance (OI) syndromes are frequent and share symptoms like dizziness and orthostatic syncope. Their pathophysiology however seems to be different. The aim of our work was to evaluate autonomic and hemodynamic behaviour in patients with familial amyloidotic polyneuropathy and neurally mediated syncope in supine position and after acute orthostatic passive stress. We studied 12 patients with autonomic failure (group A), 12 patients with neurally mediated syncope (group B) and 16 aged matched normal controls (group C), in supine position and during the first 10 min of head-up tilt test (HUTT). Beat-by-beat blood pressure and heart rate were continuously monitored and digitised at 500 Hz. The baroreceptor alfa-index gain (vagal reflex-BRG), high frequency of RR variability (HFRR, vagal tonus) and low frequency of systolic arterial pressure variability (LFSAP, sympathetic tone) were calculated. Catecholamines, plasma brain (BNP) and atrial natriuretic (ANP) peptides were also measured. Hemodynamic data were derived and calculated by the non-invasive modelflow method. During supine position, cardiac output (CO) and stroke volume (SV) were similar in all groups. Mean arterial pressure (MAP) and BNP were higher in group A. Noradrenaline (NOR), BRG, HFRR and LFSAP were extremely low in this group. BRG and adrenaline (ADR) were higher in group B than in controls. Within the first 10 min of HUTT, there was a huge drop of CO, SV and MAP in group A, maintenance of very low levels of neurohormones and lack of autonomic function. HR, LFSAP and ADR had a higher rise at HUTT in group B compared with controls (p<0.01) but a significant decrease of BRG was noted (p<0.05). ANP or BNP did not change with tilt in any group. Different orthostatic intolerance syndromes may show important hormonal, autonomic and hemodynamic differences during supine rest and enhanced after passive orthostatism.

Reduction of nephrotic range albuminuria is associated with markedly improved renal and cardiovascular outcome in patients with diabetic nephropathy. Aldosterone has been suggested to play a role in the progression of diabetic nephropathy. We therefore aimed to evaluate the short-term effect of aldosterone antagonism with spironolactone on nephrotic range albuminuria and blood pressure in diabetic nephropathy. Twenty Caucasian patients with diabetic nephropathy and nephrotic range albuminuria (>2500 mg/24 h) despite recommended antihypertensive treatment completed this double-masked, randomized crossover trial. Patients were treated in random order with spironolactone 25 mg once daily and matched placebo for 2 months, on top of ongoing antihypertensive treatment, including an angiotensin-converting enzyme inhibitor or an angiotensin II receptor blocker in maximally recommended doses. Median (range) number of antihypertensive drugs was 3 (2-5). After each treatment period, albuminuria, 24-h ambulatory blood pressure, and glomerular filtration rate (GFR) were determined. Spironolactone on top of recommended renoprotective treatment induced a 32% (95% confidence interval (CI): 21-42%) reduction in albuminuria from (geometric mean (95% CI)) 3718 (2910-4749) mg/24 h on placebo treatment (P<0.001). There was a significant reduction in 24-h blood pressure of 6 (2-10)/4 (2-6) mm Hg and day blood pressure of 7 (3-12)/5 (3-7) mm Hg (P<0.01), whereas night blood pressure remained unchanged. Spironolactone induced an insignificant reversible reduction in GFR of 3 ml/min/1.73 m2 from 64 (27) ml/min/1.73 m2. No patients were excluded due to adverse events. Our results suggest that spironolactone treatment on top of recommended renoprotective treatment including maximal renin-angiotensin system blockade may offer additional renoprotection in patients with diabetic nephropathy and nephrotic range albuminuria.

Background: Atrial fibrillation is common in chronic heart failure. Long-term restoration of sinus rhythm is generally unsuccessful. It may be speculated that higher heart rates are unfavorable, since this may lead to tachycardiomyopathy, but there are no data which have examined this., Methods and Results: Seventy-seven patients with atrial fibrillation and advanced chronic heart failure, age 70 +/- 7 years, left-ventricular ejection fraction 0.23 +/- 0.08, 61% with ischemic etiology were included. Patients were dichotomized according to the median heart rate (80 bpm) at inclusion (39 patients with "low" heart rate and 38 patients with "high" heart rate). At baseline, both patient groups were remarkably comparable. After a mean follow-up of 3.3 +/- 0.9 years, mortality was comparable (62% versus 55%, p = non-significant). An independent relation was found between lower heart rate and survival, in addition to absence of hypertension, digoxin use, and higher N-ANP, dopamine, and renin levels., Conclusion: In the present analysis, patients with atrial fibrillation and advanced chronic heart failure with higher heart rates are comparable to those with lower heart rates. Not higher heart rates at baseline but, on the contrary, lower heart rates seem associated with a worse outcome.

Background: Controversy exists about the indication of thrombolytic therapy in the subgroup of hemodynamically stable patients with acute pulmonary embolism (PE) and right ventricular dysfunction. Brain natriuretic peptide (BNP) is excreted from the cardiac ventricles in response to cardiomyocyte stretch and can be measured with an easy-to-perform blood test., Objective: The objective of this study was to determine the predictive value of elevated BNP levels for early recurrent venous thromboembolism with or without fatal outcome in hemodynamically stable patients with acute PE. In addition, we assessed the potential clinical consequences of initiating thrombolytic therapy based on the BNP levels alone., Methods: A nested case-control study was performed within the framework of a large randomized-controlled trial totalling 2213 hemodynamically stable patients with confirmed acute, symptomatic PE. Ninety patients experienced a fatal or non-fatal recurrent venous thromboembolism during the first 3 months of follow-up (cases); Two hundred and ninety-seven patients with uneventful follow-up served as controls. Blood for BNP levels was obtained at referral and assayed in a central laboratory., Results: Cases had significantly higher mean baseline BNP levels (P = 0.0002). The odds ratio (OR) for every logarithmic (log) unit increase in BNP concentration was 2.4 (95% CI: 1.5-3.7). A BNP cut-off level of 1.25 pmol L(-1) [the optimal point on the receiver-operating characteristic (ROC) curve] was associated with a sensitivity and specificity of 60% and 62%, respectively. In theory, for every patient correctly receiving thrombolytic therapy at this cut-off, 16 patients will receive this therapy unnecessarily., Conclusions: Brain natriuretic peptide level at presentation is significantly associated with early (fatal) recurrent venous thromboembolism in hemodynamically stable patients with acute PE. However, this relationship appears clinically insufficient to guide the initiation of thrombolytic therapy.

Introduction: The beneficial effects of ACE inhibitors are generally ascribed to blockade of neurohormonal activation. However, especially in chronic heart failure (CHF) patients plasma angiotensin II and aldosterone levels can be elevated despite ACE inhibition, the so-called ACE escape. In the present study, we aimed to identify the frequency and determinants of ACE escape in CHF patients., Methods: We studied 99 stable chronic heart failure patients (NYHA class III and IV, 66% ischemic etiology) receiving long-term therapy with ACE inhibitors. In all patients, cardiac, renal, and neurohormonal parameters were measured. ACE escape was defined as plasma angiotensin level > or = 16 pmol/L., Results: Mean (+/- SD) left ventricular ejection fraction of our 99 patients (79 men and 20 women, age 69 +/- 12 years) was 28 +/- 10%. In addition to an ACE inhibitor, 93% of patients received diuretics, 71% a beta-blocker, and 49% spironolactone. None of the patients used an angiotensin receptor blocker. In our population, 45% of the patients had an angiotensin II plasma concentration higher than 16 pmol/L (median concentration was 14.1 pmol/L). Spironolactone use was an independent predictor of elevated plasma angiotensin II levels. Furthermore, spironolactone users had significantly higher plasma active renin protein and aldosterone levels. Plasma angiotensin II concentration was positively correlated to active renin, plasma angiotensin I and plasma aldosterone. No correlation was found between plasma angiotensin II levels and serum ACE activity, dose of ACE inhibitor, or duration of use., Conclusion: In a group of severe chronic heart failure patients, 45% had elevated plasma angiotensin II levels independent of serum ACE activity despite long-term ACE inhibitor use. Although a causal link could not be proven, an association was found between spironolactone use and active renin protein, angiotensin II and aldosterone levels, suggesting that escape from ACE is mainly caused by a feedback mechanism.

Background: Aldosterone has been suggested to play a role in the initiation and progression of diabetic nephropathy. Currently recommended treatment with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers [renin-angiotensin system (RAS) blockade] does not suppress circulating aldosterone sufficiently. We therefore aimed to evaluate the short-term effect of aldosterone antagonism with spironolactone on albuminuria and blood pressure in diabetic nephropathy., Methods: Twenty Caucasian type 1 diabetic patients with persistent macroalbuminuria despite antihypertensive treatment, including RAS blockade, completed this double-masked, randomized cross-over trial. Patients were treated in random order with spironolactone 25 mg once daily and matched placebo for two months, respectively, on top of usual antihypertensive treatment. After each treatment period albuminuria, 24-hour blood pressure, and glomerular filtration rate (GFR) were determined., Results: Spironolactone on top of usual antihypertensive treatment induced a 30% (95% CI 17 to 41) reduction in albuminuria from [geometric mean (95% CI)] 831 (624 to 1106) mg/24-hour on placebo treatment (P < 0.001), and a reduction in fractional albumin clearance of 35% (20 to 46, P < 0.001). Twenty-four-hour blood pressure showed an insignificant reduction of [mean reduction (95% CI)] 8 (-1 to 17)/3 (-0.2 to 7) mm Hg (P < 0.10). There was an insignificant reversible reduction in GFR during treatment with spironolactone. On spironolactone treatment, one patient was excluded due to hyperkalemia (plasma potassium 5.7 mmol/L) and one due to orthostatic dizziness. Otherwise treatment was well tolerated., Conclusion: Our results suggest that spironolactone treatment on top of recommended antihypertensive treatment reduces blood pressure and may offer additional renoprotection in type 1 diabetic patients with diabetic nephropathy.

Background: The purpose of this study was to evaluate the renoprotective effect as reflected by short-term changes in albuminuria of ultrahigh doses of irbesartan in type 2 diabetic patients with microalbuminuria., Methods: This double-masked randomized crossover trial included 52 (41 males) hypertensive type 2 diabetic patients with microalbuminuria on ongoing antihypertensive medication. At inclusion, previous antihypertensive treatment was discontinued and replaced with bendroflumethiazide, 5 mg once daily, for the entire study. Following 2 months wash-out (baseline), patients were treated randomly with irbesartan 300, 600, and 900 mg once daily, each dose for 2 months. End points evaluated at the end of each study period included urinary albumin excretion rate (UAE) (mean of three 24-hour collections), 24-hour ambulatory blood pressure, and glomerular filtration rate (GFR) [chromium 51 ethylenediaminetetraacetic acid (51Cr-EDTA)]., Results: Baseline values were: 24-hour UAE [geometric mean (95% CI)] 134 (103 to 170) mg/24 hours, ambulatory blood pressure [mean (SD)] 140 (10)/77 (7) mm Hg, and GFR 103 (19) mL/min/1.73 m2. All doses of irbesartan significantly reduced UAE, ambulatory blood pressure, and GFR from baseline. Reductions in UAE from baseline were 52% (46% to 57%), 49% (43% to 54%), and 59% (54% to 63%) with increasing doses of irbesartan (P < 0.01). UAE was reduced significantly more by irbesartan 900 mg compared with lower doses with an additional reduction in UAE of 15% (2% to 26%) by irbesartan 900 mg compared with 300 mg (P = 0.02). The greater reduction in albuminuria by irbesartan 900 vs. 300 mg was more pronounced in patients with UAE during irbesartan 300 mg above vs. below the median [31% (18% to 42%) vs. -9% (-25% to 6%), respectively (P < 0.05)]. With increasing doses systolic ambulatory blood pressure was reduced from baseline by 8 (4 to 12), 9 (5 to 13), and 9 (5 to13) mm Hg, and diastolic ambulatory blood pressure by 6 (4 to 7), 7 (6 to 9), and 7 (6 to 9) mm Hg (NS between doses)., Conclusion: Ultrahigh dosing of irbesartan (900 mg once daily) is generally safe and offers additional renoprotection independent of changes in systemic blood pressure and GFR in comparison to the currently recommended dose of 300 mg.

Background: Brain natriuretic peptide (BNP) levels have been used to assess clinical status and predict prognosis of patients with chronic heart failure (CHF). However, BNP levels can only be measured in specialized laboratories which has hampered its use in daily clinical practice. We compared a new, rapid, BNP assay with a conventional BNP measurement and evaluated the applicability to current practice by comparing it with standard clinical parameters., Methods: BNP levels were determined in 78 stable CHF patients and 20 controls. The severity of CHF was assessed by determination of New York Heart Association functional class (NYHA), left ventricular ejection fraction (LVEF) and peak oxygen consumption (peak VO(2)), and these parameters were compared to BNP levels., Results: Overall, rapid BNP assessment was highly correlated with the conventional BNP assay (r=0.95, P<0.0001). In the higher ranges (>200 pmol/l), however, correlation was less accurate, and tended to overestimate. BNP levels also strongly correlated with both NYHA class, LVEF and peak VO(2) (all P<0.001). A cut-off value for BNP of 20 pmol/l yielded a sensitivity of 91% and a specificity of 92% to detect the presence of left ventricular systolic dysfunction., Conclusions: Rapid measurement of BNP levels is comparable to conventional BNP measurement and strongly correlated to clinical tests that are currently used to stratify CHF patients. Wider use of this method may yield a reduction of costly and time-consuming clinical tests and may reduce the medical burden of CHF.

Objective: To investigate the role of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and -27 as possible mediators of the actions of "new pressor protein" (NPP), which is related to coagulation factor XIIa, on blood pressure (systolic and diastolic blood pressure) and heart rate, and on the release of adrenal medullary catecholamines., Methods: Adult male Wistar bioassay rats (n=8 to 18 per group) were anesthetized with inactin (100 mg/kg intraperitoneally), ganglion-blocked with pentolinium (19.2 mg/kg subcutaneously) and treated with captopril (2.5 mg/kg intravenously). Human NPP was injected at 20 L plasma equivalent per approximately 300 g of rat intravenously, and both PACAPs were injected at 10 g/kg intravenously. The systolic and diastolic blood pressure and heart rate responses to all of these agonists were determined using a MacLab/8 system. Arterial plasma adrenaline and noradrenaline were determined by high performance liquid chromatography with fluorimetric detection. Responses to NPP, the PACAPs and a specific PACAP antagonist were compared to assess the PACAPs as potential mediators of the cardiovascular effects of NPP., Results: PACAP-38 mimicked the effects of NPP on systolic and diastolic blood pressure and heart rate more closely than did PACAP-27. Generally, NPP raised diastolic blood pressure and heart rate, and especially plasma adrenaline and noradrenaline, more impressively in degree and duration than that achieved by the PACAPs. The antagonism of PACAP receptors (PAC-1) significantly reduced the cardiovascular effects of NPP by 30% to 50%., Conclusions: PACAP-38, especially, may qualify as a potential mediator of the cardiovascular and sympathoadrenal effects of NPP but incomplete inhibition of NPP activity by PAC-1 receptor antagonism and the observed differences all suggest that PACAP is not the only peptide involved. Such peptidic mediation of the effects of NPP may explain the potentiation of NPP by captopril and why NPP remains effective after cholinergic blockade. These data suggest that PACAP is involved in a novel axis between NPP, cardiac function and blood pressure that resists angiotensin-converting enzyme inhibition. Any endogenous production of NPP could raise clinically relevant issues pertaining to therapy with ACE inhibitors.

Objective: To investigate the major cardiovascular effects of human plasma "new pressor protein" (NPP) and how the adrenal medulla contributes to these effects., Methods: NPP was injected into bioassay rats intravenously, and the effects on blood pressure and cardiac function were investigated. Acute adrenal medullectomy (2MDX), alpha- and beta-adrenergic blockade and plasma catecholamine levels were also used to evaluate the role of the sympathoadrenal system in mediating the NPP effects., Results: NPP significantly raised systolic blood pressure (SBP) and mean arterial pressure but not diastolic blood pressure (DBP), with no significant change in total peripheral resistance. Heart rate, cardiac output and stroke volume rose by 16%, 53% and 36%, respectively. Plasma catecholamines increased massively, notably adrenaline, raising the adrenaline to noradrenaline ratio from about 4:1 to 18:1. 2MDX attenuated the increments of SBP and heart rate by more than 90% and more than 70%, respectively, implicating the adrenal medulla. Beta-adrenergic blockade (propranolol) potentiated the NPP-induced increase of SBP and DBP, but not that of heart rate. Combined alpha- and beta-adrenergic blockade (phentolamine and propranolol) blocked the rise in SBP, DBP and heart rate., Conclusions: NPP's hypertensive action is attributable mainly to increases in systolic blood pressure, heart rate and cardiac output (an increase in heart rate and stroke volume) with massive release of adrenal medullary catecholamines. Such effects suggest a novel axis between coagulation factor XII and the sympathoadrenal system, the cardiovascular effects of which are controlled by combined alpha- and beta-adrenergic blockade, but not by angiotensin-converting enzyme inhibition. Clinical relevance depends on whether NPP is formed in vivo in thrombotic states.

Background: Semicarbazide sensitive amine-oxidase (SSAO) is an adhesion molecule and thought to play a role in endothelial cell dysfunction (ECD). SSAO has never been associated with markers of ECD. Pre-eclampsia (PE) is, like the early stages of atherosclerosis, characterised by ECD. SSAO could contribute to ECD in PE., Methods: Plasma samples were obtained in 14 pre-eclamptic patients and 14 matched controls. In these SSAO-activity, von Willebrand factor (vWF) levels and ED1 fibronectin levels were determined. Placental tissue was collected of 12 pre-eclamptic pregnancies, 8 preterm deliveries and 12 term controls. In these samples, SSAO activity was assessed. In a subset of these placentas, immunohistochemical staining was performed for SSAO, ICAM-1 and VCAM-1., Results: Plasma SSAO activity was not significantly different between pre-eclamptic subjects and controls. VWF and ED1 fibronectin were both significantly increased in the pre-eclamptic subjects. There was no correlation between SSAO activity and vWF or ED1 fibronectin levels. Placental SSAO activity was not different between pre-eclamptic pregnancies, preterm deliveries and term controls. There was strong staining of SSAO in vascular smooth muscle cells (VSM) and moderate staining of trophoblast in all three groups. Endothelial cell expression of SSAO was only seen in term controls and the placentas of pre-eclamptic patients. There was no association between the expression of SSAO, ICAM-1 or VCAM-1 in the placentas of pre-eclamptic patients., Conclusion: SSAO expression and activity are not related to markers of ECD., (Copyright 2002 Elsevier Science B.V.)

Children aged 0-3 yr were stratified for age and randomized to receive either continuous morphine (CM, 10 microg x kg(-1) x h(-1)) with three-hourly placebo boluses or intermittent morphine (IM, 30 microg x kg(-1) every 3 h) with a placebo infusion for postoperative analgesia. Plasma concentrations of epinephrine, norepinephrine, insulin, glucose and lactate were measured before and at the end of surgery and 6, 12 and 24 h after surgery. Pain was assessed with validated pain scales [the COMFORT scale and a visual analogue scale (VAS)] with the availability of additional morphine doses. Minor differences occurred between the randomized treatment groups, the oldest IM group (aged 1-3 yr) having a higher blood glucose concentration (P=0.003), mean arterial pressure (P=0.02) and COMFORT score (P=0.02) than the CM group. In the neonates, preoperative plasma concentrations of norepinephrine (P=0.01) and lactate (P<0.001) were significantly higher, while the postoperative plasma concentrations of epinephrine were significantly lower (P<0.001) and plasma concentrations of insulin significantly higher (P<0.005) than in the older age groups. Postoperative pain scores (P<0.003) and morphine consumption (P<0.001) were significantly lower in the neonates than in the older age groups. Our results show that continuous infusion of morphine does not provide any major advantages over intermittent morphine boluses for postoperative analgesia in neonates and infants.

Background: To date, the use of beta-blockers in treating patients with chronic heart failure gains support, this since several large clinical trials reported reduced mortality after chronic beta-blockade. Part of these beneficial effects may result from inhibition of deleterious neurohormone activation that accompanies progression of chronic heart failure. The present study evaluates whether this neurohormone inhibition is preserved after chronic beta-blockade., Methods: In a retrospective analysis the neurohormonal profiles of patients with moderate to severe chronic heart failure were studied from three treatment subgroups: (1) Without beta-blockers or ACE-inhibitors (n=15), (2) without beta-blockers, with ACE-inhibitors (n=324), (3) with beta-blockers and ACE-inhibitors (n=31). Patients were on beta-blockers for an average period of 3.8 years. Plasma samples were obtained under controlled conditions., Results: Despite uneven group sizes, the groups were well matched for clinical characteristics. Plasma renin levels were significantly lower in patients treated adjunctively with beta-blockers. Plasma aldosterone and endothelin-I levels also tended to be lower after chronic beta-blockade, however, this did not reach statistical significance., Conclusions: Chronic adjunctive beta-blocker treatment shows significantly lower plasma renin levels when compared to single ACE-inhibition. This persistent reduction of plasma neurohormone activation may concomitantly reduce the chance of neurohormones to escape from inhibition.

The relatively new technique of microdialysis provides new possibilities for investigating in vivo the functioning of the sympathetic nervous system. The small sample volumes obtained, however, are a great challenge for analytical chemists. We report here a HPLC method for measuring in one run both natural and synthetic catecholamines [dopamine, (nor)epinephrine, alpha-methylnorepinephrine, isoproterenol and epinine] and the intraneuronal metabolite 3,4-dihydroxyphenylglycol in small microdialysis samples after derivatization with the fluorogenic agent 1,2-diphenylethylenediamine. No prior clean-up step is necessary. N-Ethylmaleimide is necessary for preventing an inhibitory action on derivatization occurring in in vivo microdialysis samples. The method can handle large numbers of samples, is sensitive (on-column detection limits 30 to 200 fg) and reproducible (RSD 1 to 7%). Recovery characteristics of the commercial microdialysis probe used (CMA/20) were extensively investigated both in vitro and in vivo at various perfusion rates; for practical purposes a rate of 2 microl/min and sampling at 10-min intervals was found to be workable and to give good and reproducible recoveries (50 to 70%).

Semicarbazide-sensitive amine oxidase (SSAO)-mediated deamination of methylamine and aminoacetone in vitro produces carbonyl compounds, such as formaldehyde and methylglyoxal, which have been proposed to be cytotoxic and may be responsible for some pathological conditions. An HPLC procedure was developed to assess different aldehydes, which were derivatized with 2,4-dinitrophenylhydrazine (DNPH). We have demonstrated in vivo deamination of methylamine and aminoacetone by examining the excretion of formaldehyde and methylglyoxal, respectively, in rats. Following chronic administration of methylamine, the urinary level of malondialdehyde (MDA), an end product of lipid peroxidation, was also found to be substantially increased. A selective SSAO inhibitor blocked the increase of MDA. The results support the idea that increased SSAO-mediated deamination of methylamine and aminoacetone can be a potential cytotoxic risk factor.

Atrial natriuretic peptide (ANP) has become an important parameter for assessing the condition of patients with cardiac disease. Recently, attention has also focused on N-terminal pro-atrial natriuretic peptide (NtproANP) in this context. NtproANP circulates in plasma in higher concentration, is more stable ex vivo, and may be a better parameter for cardiac function over time. We have evaluated a new commercially available radiommunoassay kit for NtproANP and compared results and method with those of ANP measurements. The NtproANP kit was found to be reliable and easy to use (no plasma extraction step is necessary), with good reproducibility (coefficients of variation 7-15%). Normal values in 15 healthy laboratory workers, 25 healthy elderly subjects and 25 patients with heart failure were 207 +/- 70, 368 +/- 134 and 1206 +/- 860 pmol/l, respectively, 8.3, 11.8 and 13.0 times higher, respectively, than corresponding ANP concentrations. NtproANP correlated well with ANP (r 0.64-0.78). We conclude that plasma NtproANP measurement may be a good alternative to plasma ANP measurement: technically, it is easier to perform, and NtproANP is more stable in plasma. Whether NtproANP is a better diagnostic and prognostic parameter than ANP remains to be further established.

We have evaluated a new commercially available ELISA kit for determination of plasma chromogranin A with respect to its usefulness in the diagnosis of neuroendocrine tumors, mainly pheochromocytoma. Serum and differently anticoagulated plasmas gave different chromogranin A concentrations. Control values (n = 21) were 18.9 +/- 5.8 units/l. Chromogranin A values > 30.4 units/l (mean + 2 S.D.) were considered elevated. In 22 patients suspected of (but found not to have) pheochromocytoma and in 24 patients with renovascular hypertension, 18% were found to have elevated chromogranin A concentrations. In renovascular hypertension chromogranin A correlated positively with serum creatinine; chromogranin A was strongly elevated especially in chronic renal failure. In 45 patients with pheochromocytoma, 13 (29%) had chromogranin A concentrations within the normal range, as had 3 out of 11 patients with neuroblastoma (27%). In 13 pheochromocytoma patients with elevated chromogranin A, measurements were repeated after surgical removal of the tumor; values then all fell within the normal range. We conclude that measurement of chromogranin A adds little to already existing methods for the diagnosis of pheochromocytoma.

We report here a simple and sensitive method for the measurement of semicarbazide-sensitive amine oxidase (SSAO) activity in human plasma. Benzaldehyde, generated during a 1-h incubation of plasma with benzylamine, is derivatized with the specific aldehyde reagent dimedone after prior deproteinization. Quantitation of the derivatization product is done by automated injection onto an isocratic high-performance liquid chromatographic system with fluorimetric detection. The assay shows good linearity and reproducibility (intra-assay C.V. 7%). Detection limit is 25 mU/l (= pmol/ml/min). In 51 healthy controls (age 49 +/- 13 yr, 20 males) the measured SSAO activity was 352 +/- 102 mU/l (mean +/- S.D.). A large number of samples (70-80) can easily be processed in one day by one technician.

The peripheral decarboxylase inhibitors benserazide and carbidopa, often administered in combination with L-dopa in the treatment of Parkinson's disease, are also very good inhibitors of semicarbazide-sensitive amine oxidase (SSAO). In untreated patients and in patients treated with L-dopa alone, plasma SSAO activity is normal. In patients treated with L-dopa plus benserazide or carbidopa (Madopar or Sinemnet), however, plasma SSAO activity is strongly inhibited, contrary to the paradoxical 3-fold increase in plasma aromatic-L-amino acid decarboxylase activity we reported previously. Single-dose and longitudinal studies show that the SSAO inhibition proceeds rapidly and increases even further to nearly complete inhibition after continued treatment, while aromatic-L-amino acid decarboxylase activity only transiently decreases after a single dose and increases slowly with continued treatment above pretreatment levels. Dialysis experiments confirm that the binding of benserazide to SSAO is irreversible, especially after chronic treatment. The lack of knowledge about the exact function of SSAO precludes definite conclusions about the effect of this chronic SSAO inhibition on patients. Careful follow-up studies of patients treated with Madopar or Sinemet might provide further information about the possible physiological role of SSAO.

We report a rapid breakdown of dopamine and especially of 3,4-dihydroxybenzylamine, the frequently-used internal standard in catecholamine determinations, in plasma of many but not all animal species. Species investigated were cow, sheep, goat, pig, horse, rabbit, dog, guinea pig, mouse, chicken, rat and man. In some species 3,4-dihydroxybenzylamine nearly completely disappeared at 4 degrees C within 15 min after addition to the plasma. Added dopamine, but not norepinephrine and epinephrine, also rapidly disappeared at 4 degrees C. Disappearance rates were increased at higher temperatures, and at 20 degrees C also norepinephrine showed some breakdown. The breakdown is caused by a semicarbazide-sensitive amine oxidase in the plasma, and can be completely blocked by the addition of the inhibitor semicarbazide. Measurement of plasma catecholamine concentrations in animal species can thus lead to erroneous results, especially when 3,4-dihydroxybenzylamine is used as an internal standard. Only when blood is collected in tubes containing an inhibitor of semicarbazide-sensitive amine oxidase like semicarbazide can reliable plasma catecholamine measurements be performed.

We report a reliable fluorimetric assay for the simultaneous determination of norepinephrine, epinephrine, dopamine and dobutamine in human plasma and urine, based on liquid-liquid extraction and derivatization with the fluorogenic agent 1,2-diphenylethylenediamine prior to chromatography. The method is sensitive (detection limit 0.3-0.8 pg injected) and reproducible (coefficients of variation 1-10%), and shows good accuracy (93-98%). The method should also be used when one only wants to measure the concentrations of the natural catecholamines, in order to avoid interference by metabolites of dobutamine and by the late-eluting dobutamine itself.

Effects of four doses of the alpha 2-receptor agonist clonidine (CLO) (0.25, 0.5, 1, and 2 micrograms/kg IV) and placebo were studied in seven healthy men who volunteered in a double-blind randomized design in order to delineate possible presynaptic and postsynaptic components in the mechanism of action of CLO. Blood pressure, heart rate, plasma noradrenaline (NOR), plasma 3-methoxy-4-hydroxyphenylglycol (MHPG), plasma growth hormone (GH), and subjective sedation were monitored for a period of 1 hr following infusion of CLO. NOR and MHPG were also analyzed in urine, collected at 1 and 4 hr after the infusions. Dose-dependent decrements were observed in systolic and diastolic blood pressure and plasma NOR levels, and dose-dependent increases in subjective sedation and plasma GH. CLO did not influence plasma MHPG levels, whereas only urinary MHPG excretion was reduced 4 hr after infusion of 2 micrograms/kg CLO. Because no obvious differences between dose-response relations of plasma NOR (believed to be a presynaptic and peripheral effect), blood pressure (believed to be mainly a central presynaptic and postsynaptic effect), and subjective sedation (believed to be a central and probably postsynaptic effect) were observed, our results do not provide simple parameters to discern the multiple mechanisms of action of CLO. However, at a dose of 0.5 micrograms/kg CLO (a dose lower than that generally used) clear effects on plasma NOR, blood pressure, and sedation, but not on plasma GH (a central postsynaptic effect) or urinary MHPG (a presynaptic effect), were observed. When using CLO as a challenge test in psychiatric disorders, a design with 0.5 micrograms/kg CLO, in addition to the traditional 2 micrograms/kg CLO, may provide more information to characterize discrete abnormalities in the noradrenergic system at the level of the brainstem, the pituitary, or the peripheral sympathetic nervous system.

Epinine (N-methyldopamine) is the pharmacologically active hydrolysis product of the prodrug ibopamine, which is currently being widely studied for the treatment of congestive heart failure. This paper reports a sensitive and reliable method for the simultaneous determination of free catecholamines and epinine in plasma and urine. The compounds are isolated from plasma or urine by a specific liquid-liquid extraction, derivatized with the selective fluorogenic agent 1,2-diphenylethylenediamine, and quantitated by high-performance liquid chromatography with gradient elution and fluorimetric detection. The limits of detection for the derivatized catecholamines and epinine are 0.3-0.6 pg of injected compound. Intra- and inter-assay coefficients of variation of all four compounds are good (1-8%), as are the accuracy and linearity. A method is also reported for the determination of total dopamine and epinine in plasma and urine based on the same principle. This method, in which deconjugation is accomplished by acid hydrolysis at 95 degrees C, also shows good sensitivity and reproducibility.

We report a sensitive and reliable assay for the determination of the urinary catecholamines norepinephrine, epinephrine and dopamine, based on selective extraction by a liquid-liquid extraction procedure, followed by selective derivatization with the fluorigenic agent 1,2-diphenylethylenediamine and quantification by high-performance liquid chromatography with fluorimetric detection. Comparison with a method using electrochemical detection shows that interference of an unknown compound, most probably N-methylepinephrine, which is an often-overlooked problem with methods using electrochemical detection and results in falsely high epinephrine concentrations, does not occur with the described fluorimetric method.

We report a reliable method for determining DOPA levels in plasma and cerebrospinal fluid. The method is based on complete conversion of DOPA to dopamine and quantification by HPLC-ECD of the dopamine formed. Lower limit of detection was 0.5 nmol/l. No differences in plasma DOPA levels were found between normal children (0-15 yr, n = 60), normal adults (n = 39) and patients with essential hypertension (n = 40) or Parkinson's disease (no DOPA therapy, n = 30). In normal individuals and in patients with essential hypertension venous plasma levels were higher than arterial levels (10.2 vs 9.3 nmol/l, p less than 0.001, V/A ratio 1.11 (SD 0.08), n = 15). Sympathetic stimuli (standing, tilting, bicycle exercise, tyramine) did not influence DOPA levels. In untreated depressed patients (n = 10) and in non-parkinsonian neurological patients (n = 12) cerebrospinal fluid levels of DOPA were 4.5 (SD 2.4) and 5.2 (SD 1.3) nmol/l respectively. A direct method for the measurement of DOPA by HPLC-ECD after deproteinization of plasma is also described and compared with the conversion method. Good agreement was found when plasma DOPA levels exceeded 0.25 mumol/l (y(conversion method) = 0.943x (direct method) + 0.118; n = 60; r = 0.985). The direct method, because of greater simplicity and the possibility of simultaneous measurement of the DOPA metabolite 3-O-methyldopa, is the method of choice with plasma samples from DOPA-treated patients. In non-DOPA treated individuals the conversion method is superior and has proved to be an accurate and sensitive method for the determination of DOPA levels in plasma and cerebrospinal fluid.

We report a sensitive fluorimetric method, in which catecholamines are concentrated from plasma by liquid-liquid extraction and derivatized with the selective fluorescent agent 1,2-diphenylethyl-enediamine prior to chromatography. Optimal conditions for extraction, derivatization and chromatography were investigated. With alpha-methylnorepinephrine as internal standard, the chromatographic separations are complete within 6 min. Limits of detection are 0.3 pg for norepinephrine and epinephrine and 0.5 pg for dopamine. Coefficients of variation are low (3-7%). Comparison of plasma catecholamine values determined with this method and with an established method with electrochemical detection (n = 135) shows good correlation (r = 0.94-1.00), and regression lines are close to lines of identity.

Sympatho-adrenal activation induced by stress contributes to the development of pathological states such as hypertension and anxiety disorders. The Stroop Color Word Test (CWT) is evaluated as a test for the study of stress-induced sympathetic effects, on the basis of psychological, physiological and biochemical responses. The CWT induced increases in plasma and urinary adrenaline, heart rate, respiration rate, electrodermal activity, electromyography, feelings of anxiety, and decreased finger pulse amplitude.

DL-threo-3,4-Dihydroxyphenylserine (DOPS) is increasingly being investigated for treatment of disorders involving defects of the sympathetic nervous system, such as Parkinson's disease, Shy-Drager syndrome and congenital dopamine-beta-hydroxylase deficiency. Whilst L-DOPS is converted by aromatic L-amino acid decarboxylase into natural norepinephrine in vitro, D-DOPS inhibits this process. There are no data on the interaction between D- and L-DOPS in vivo because a reliable method for the measurement of the D- and L-enantiomers in plasma and urine is lacking. We describe here such a method based on reversed-phase chromatography after derivatization with o-phthaldialdehyde and N-acetyl-L-cysteine. Good separation was achieved with this procedure (resolution factor 2.33). Two simple and sensitive methods are also presented for total D,L-DOPS estimation, based on reversed-phase chromatography with electrochemical detection after either deproteinization (DP) or liquid-liquid extraction (LE) as sample preparation steps. The two methods gave identical results (regression line DOPS (DP) = 1.026 DOPS (LE) + 33.28; r = 0.997; n = 52). Excellent agreement was found between the sum of the D- and L-DOPS concentrations and the measured total D,L-DOPS concentration (regression line DOPS (D + L) = 0.955 DOPS (total, LE) + 116.65; r = 0.992; n = 100).

In a double blind, crossover study 6 h infusions of adrenaline (15 ng/kg/min; 1 ng = 5.458 pmol), noradrenaline (30 ng/kg/min; 1 ng = 5.911 pmol), and a 5% dextrose solution (5.4 ml/h), were given to ten healthy volunteers in random order 2 weeks apart. By means of intra-arterial ambulatory monitoring the haemodynamic effects were followed for 18 h after the infusions were stopped. Adrenaline, but not noradrenaline, caused a delayed and protracted pressor effect. Over the total postinfusion period systolic and diastolic arterial pressure were 6 (SEM 2)% and 7 (2)%, respectively, higher than after dextrose infusion (ANOVA, p less than 0.001). Thus, "stress" levels of adrenaline (230 pg/ml) for 6 h cause a delayed and protracted pressor effect. These findings are strong support for the adrenaline-hypertension hypothesis in man.

A patient with severe orthostatic hypotension due to dopamine-beta-hydroxylase deficiency was treated with the unnatural aminoacid D,L-threo-3,4-dihydroxyphenylserine (DOPS) in the hope that it would serve as a substrate of aromatic-L-aminoacid decarboxylase to produce (-)-noradrenaline. With a dose of 500 mg twice daily by mouth, blood pressure rose gradually from 100/55 to 145/85 mm Hg, and orthostatic hypotension disappeared. After 4 months' treatment the patient is free of symptoms and able to live a normal life. DOPS switched on the production of noradrenaline and reduced the excessive production of dopamine. During treatment plasma noradrenaline rose normally after standing and after infusion of tyramine, a biogenic amine that liberates stored neurotransmitter from sympathetic nerve terminals. These data demonstrate that in congenital dopamine-beta-hydroxylase deficiency dopamine instead of noradrenaline is released as the sympathetic neurotransmitter but that the integrity of the sympathetic neuron is otherwise intact.

A woman was referred with severe orthostatic hypotension at the age of 21. Ptosis, skeletal muscle hypotonia, and recurrent hypoglycaemia had been noticed in early childhood. There was noradrenergic denervation and adrenomedullary failure but baroreflex afferents, cholinergic innervation, and adrenocortical function were intact. Noradrenaline and adrenaline were undetectable in plasma, urine, and cerebrospinal fluid (CSF), but dopamine was 7-fold to 12-fold normal in plasma, 4-fold normal in urine, and 20-fold normal in CSF. Measurements of catecholamine metabolites showed further evidence for impairment of noradrenaline and adrenaline biosynthesis due to deficient dopamine-beta-hydroxylation. Dopamine-beta-hydroxylase was undetectable in plasma and CSF. Physiological and pharmacological stimuli of sympathetic neurotransmitter release caused increases in plasma dopamine rather than plasma noradrenaline.

Aromatic-L-aminoacid (dopa) decarboxylase (ALAAD) was determined in human plasma by its ability to form dopamine from the substrate 3,4-dihydroxyphenylalanine in the presence of pyridoxal-5-phosphate as cofactor. Dopamine formed was quantitated by high performance liquid chromatography with electrochemical detection. A preincubation step of plasma with the cofactor and dithioerythritol was necessary to obtain optimal reaction conditions. The assay method showed good linearity and reproducibility. The inhibition pattern of the therapeutically used peripheral dopa decarboxylase inhibitors, carbidopa and benserazide, was studied and appeared to be dependent on whether the inhibitor was added before or after the preincubation step. Mean levels in 40 control subjects, in 40 patients with essential hypertension and in 15 patients with phaeochromocytoma, were 34.6 (SD 12.1), 28.5 (SD 10.9) and 34.7 (SD 18.4) mU/l respectively. In the patients with essential hypertension the enzyme level decreased with age (p less than 0.05). Very high levels were found in plasma of two patients with metastatic phaeochromocytoma and in two patients with untreated neuroblastoma, but not in two patients with neuroblastoma after chemotherapy. The method described can be used for measuring uninhibited ALAAD activity in patients treated with benserazide, as well as for measuring total, i.e. the sum of inhibited and uninhibited, ALAAD activity in patients treated with carbidopa.