Alexandra Pinter

MD¹ Beatrix Mersich

Andrea Laszlo

MD, PhD² Krisztina Kadar

MD¹ Mark Kollai
email: kollai@elet2.sote.hu

Alexandra Pinter¹

Adaptation of baroreflex function to increased carotid artery stiffening in patients with transposition of great arteries

INTRODUCTION

Transposition of great arteries (TGA) is a congenital heart defect in which the aorta arises from the right ventricle and the pulmonary artery from the left ventricle. TGA is the consequence of abnormal aortico-pulmonary septal development. Although these TGApatients after surgical correction have good long-term prognosis, they also have considerable late morbidity and mortality caused, in part, by rhythm disturbances [START_REF] Konstantinov | Atrial switch operation: past, present and future[END_REF][START_REF] Agnetti | Longterm outcome after Senning operation for transposition of the great arteries[END_REF][START_REF] Moons | Long term outcome up to 30 years after the Mustard or Senning operation: a nationwide multicentre study in Belgium[END_REF]. The mechanism of these rhythm disturbances is not clear.

In animal models experimentally induced defects in aortico-pulmonary septation was found to be associated with impaired large artery elastogenesis [START_REF] Rosenquist | Origin and propagation of elastogenesis in the developing cardiovascular system[END_REF][START_REF] Hutson | Neural crest and cardiovascular development: a 20-year perspective[END_REF]. In line with this observation we have recently demonstrated that the carotid artery was markedly stiffer in TGA patients than in age matched controls [START_REF] Mersich | Transposition of great arteries is associated with increased carotid artery stiffness[END_REF]. Stiffening of large elastic arteries, in which high pressure baroreceptors are embedded, has been shown to be associated with reduced cardiovagal baroreflex sensitivity [START_REF] Bonyhay | Relation between baroreflex sensitivity and carotid artery elasticity in healthy humans[END_REF][START_REF] Visontai | Impaired baroreflex function during pregnancy is associated with stiffening of the carotid artery[END_REF][START_REF] Lipman | Mental stress response, arterial stiffness, and baroreflex sensitivity in healthy aging[END_REF][START_REF] Monahan | Ageassociated changes in cardiovagal baroreflex sensitivity are related to central arterial compliance[END_REF][START_REF] Lage | Relationship of arterial compliance to baroreflex function in hypertensive patients[END_REF][START_REF] Monahan | Central arterial compliance is associated with age-and habitual exercise-related differences in cardiovagal baroreflex sensitivity[END_REF][START_REF] Hunt | Quantification of mechanical and neural components of vagal baroreflex in humans[END_REF]. Impaired baroreflex function shifts cardiac autonomic balance towards sympathetic dominance, promoting the occurrence of arrhythmic events [START_REF] Eckberg | Human baroreflexes in health and disease[END_REF].

Reduced baroreflex sensitivity has been established as independent predictor of mortality in heart failure and after myocardial infarction [START_REF] Larovere | Baroreflex sensitivity and heart-rate variability in prediction of total cardiac mortality after myocardial infarction: ATRAMI (Autonomic Tone and Reflexes After Myocardial Infarction) Investigators[END_REF][START_REF] Mortara | Arterial baroreflex modulation of heart rate in chronic heart failure: clinical and hemodynamic correlates and prognostic implications[END_REF]. Baroreflex function has never been studied in congenital heart disease, including TGA.

In this study, we tested the hypothesis that increased carotid artery stiffness in TGA patients was associated with reduced baroreflex sensitivity and altered cardiovagal autonomic function in the post-operational state.

MATERIALS AND METHODS

Subjects

Thirty-two TGA patients (23 male) aged 9 to 19 years were recruited from the Gottsegen Cardiology Institute, Budapest. Patients had an atrial switch operation (Senning-procedure), which was performed between the ages 6 months to 3 years; average time after repair was 12 ± 3 years. The Senning-operation creates a tunnel between the atria, redirecting oxygen rich blood to the right ventricle and aorta and the oxygen-poor blood to the left ventricle and pulmonary artery. Exclusion criteria included permanent pacing, atrial fibrillation or > 2 ectopic beats per min during data acquisition, clinical instability within preceding 2 months, hypertension and diabetes mellitus. Surgical data were obtained from operative notes. All patients were in clinical status Class І as defined according the New York Heart Association functional classifications and none of the children was taking any medications. Thirty-two age-matched healthy control subjects were also studied. All subjects gave written informed consent to participate in the study, which was approved by the Ethical Committee of the Semmelweis University, Budapest, Hungary.

Blood pressure

Radial artery pressure was monitored continuously with an automated tonometric device (Colin CBM-7000, AD Instruments Ltd., Hastings, UK) for determination of BRS indices.

During data collection the servo-reset mechanism of the Colin apparatus was turned off to permit continuous data acquisition. Systolic and diastolic blood pressure values, measured on the brachial artery by an automatic microphonic sphygmomanometer built in the Colindevice, were used to calibrate the radial pressure pulse.

ECG and respiration

ECG was recorded continuously from the limb lead with the largest R wave. Respiration was recorded with an inductive system (Respitrace System, Ambulatory Monitoring Inc.).

Carotid ultrasonography

Diameter of the left common carotid artery and its pulsatile distension were measured with ultrasonography, the scanner was positioned 1.5 cm proximal to bifurcation. The ultrasound device consisted of a vessel wall echo-tracking system (Wall Track System, Pie Medical, Maastricht, The Netherlands) and has been described in detail before [START_REF] Hoeks | Assessment of the distensibility of superficial arteries[END_REF]. Carotid artery diameter was recorded in 5 epochs, each containing 4-8 distension pulses.

Carotid artery pressure

Carotid artery pressure was measured by applanation tonometry (SPT-301, Millar Instruments, Houston, Texas, USA), and the carotid pulse wave recording was calibrated by using diastolic and mean brachial pressure values measured by sphygmomanometry on the right brachial artery. Diastolic brachial pressure was assigned to the minimum value of the carotid pressure pulse wave and the mean pressure to its electrically averaged value [START_REF] Kelly | Non-invasive determination of aortic input impedance and external left ventricular power output: a validation and repeatability study of a new technique[END_REF]. The carotid tonometric pressure was used to calculate carotid artery elastic parameters.

Carotid artery elastic variables

The distensibility coefficient (DC) was calculated as 2*∆D(D*∆P), where D is the end diastolic diameter, ∆D is the change in diameter from end diastole to peak systole and ∆P is carotid pulse pressure. Stiffness index was expressed as ln (SP/DP) x D/∆D, where SP and DP are systolic and diastolic carotid pressure, respectively.

Baroreflex-sensitivity (BRS)

Spontaneous methods: The coupling between spontaneous fluctuations in heart rate and systolic pressure was determined by the sequence method and also by spectral analysis. The software (WinCPRS program, Absolute Aliens Oy, Finland) detected the ECG R-wave and computed RR-interval (RRI) and radial artery systolic blood pressure (SBP) time series and identified spontaneously occurring sequences in which SBP and RRI concurrently increased and decreased over three or more consecutive beats (BRS seq ).Minimal accepted change was 1 mmHg for SBP and 5 ms for RRI. Only sequences with a correlation coefficient > 0.85 were considered. To determine spectral indices, the signals were interpolated, resampled and their power spectra were determined using FFT-based methods. The low-frequency transfer function gain (LF gain ) was determined which expresses RRI and SBP cross-spectral magnitude in the frequency range of 0.05-0.15 Hz frequency band, where coherence is greater than 0.5.

Phenylephrine-method:

In 18 of our patients and in 18 control subjects bolus phenylephrine injections (3-4 µg/kg) were given iv., which caused 10-25 mmHg elevation in systolic blood pressure. Injections were repeated 3-4 times with approximately 5-10 minutes between each, until baseline conditions were re-established. RR-intervals were plotted against systolic blood pressure values, and the slope of the regression line was determined as BRS phe . Only regression lines that were statistically significant (p < 0.05) were accepted for analysis. The final slope was obtained by calculating the mean value of ≥ 2 measurements.

Heart rate variability (HRV)

Time and frequency domain measures of heart rate variability from 10 min recordings of RRintervals were calculated using the WinCPRS program. The following parameters were determined: the standard deviation of the RR-intervals (NNSD), the root mean square of successive differences (RMSSD), the percent successive of RR-intervals which differed more than 50 ms (pNN50) and low (0.05-0.15 Hz) and high frequency (0.15-0.4 Hz) power of RRinterval variability (LF and HF, respectively).

Protocol

Subjects were studied in the early afternoon under standardized conditions, in a quiet room at a comfortable temperature. All fasted at least two hours before testing and were asked to refrain from strenuous exercise or drinking alcohol or caffeine containing beverages for 24 hours before the study. Upon arrival at the investigation unit the subjects were equipped with measurement devices, and then rested in supine position for about 15 min until the absence of evident heart rate and mean blood pressure trends demonstrated that satisfactory baseline conditions had been achieved. The protocol began with the carotid measurements. Carotid artery tonometric pressure on the right side, and diameter on the left side were recorded simultaneously in 5-7 epochs, each containing 4-8 distension pulses, which recordings were used to determine carotid elastic parameters. Then patients were asked to synchronize their respiratory rate with a metronome beating at 0.25 Hz. RR-interval and radial artery pressure were recorded continuously for a 10-min period to determine spontaneous baroreflex indices, then BRS was also determined by the phenylephrine-method in selected patients.

Data analysis

Blood pressure-and ECG-recordings of 10 minutes duration were digitized and analyzed with the WinCPRS program using a sampling rate of 500 Hz and stored in a personal computer for subsequent off-line analysis. Data are expressed as mean ± 1SD. Differences in variables between controls and patients were analyzed by unpaired Student's t-test, or Mann-Whitney rank-sum for data failing test of normality. Relations between variables were investigated by univariate correlation analyses. Significance was accepted at p < 0.05. Statistical analysis was performed by the SigmaStat for Windows Version 2.03 (SPSS Inc.) program package.

RESULTS

Clinical characteristics and carotid artery variables of patients and controls are given in

Table1. Patients had lower BMI. Systolic pressure was higher in patients and diastolic pressure lower, resulting in higher pulse pressure amplitude but no change in mean arterial pressure. Carotid artery end-diastolic diameter was smaller and pulsatile distension was less in patients in spite of higher carotid pulse pressure. Elastic parameters indicated significant stiffening of the carotid artery, in support of our earlier observations. Carotid distensibility was decreasing with age in controls (r 2 = 0.45, p < 0.05) whereas it was not related to age in TGA patients. Carotid distensibility was inversely related to systolic blood pressure both in patients and controls (r 2 = 0.35, and r 2 = 0.31, respectively, p < 0.05 for both).

Spontaneous BRS-and HRV-index values were not statistically different in patients and in controls (Table 2). Also, spontaneous BRS indices were not related to carotid artery elastic parameters neither across all subjects, nor within the patient and control groups. BRS was determined by the phenylephrine method (Phe brs ) in 18 patients and in 18 control subjects.

The obtained values for patients (19.1 ± 8.6 ms/mmHg) tended to be lower than those in controls (24.8 ± 7.2ms/mmHg) but the difference did not reach statistical significance. In the control subjects Phe brs was significantly and directly related to carotid artery distensibility (r 2 = 0.53, p <0.05), as expected, whereas no such relationship existed in patients. When patients' data were inserted into the control Phe brs -carotid distensibility nomogram, 10 patients had higher Phe brs values than the upper 95 % confidence limits of control data (Fig. 1).

Spontaneous BRS and HRV indices were not related to age or systolic pressure neither in patients, nor in controls.

DISCUSSION

We compared carotid artery elasticity and baroreflex sensitivity indices in TGA patients after surgical correction and in age matched healthy control subjects, and found, that: 1) elastic variables indicated significant stiffening of the carotid artery in patients confirming earlier results; 2) reduced distensibility of the carotid artery, was not associated with reduced baroreflex sensitivity; 3) cardiovagal autonomic indices were not significantly different in patients and controls.

Baroreflex sensitivity and heart rate variability

BRS and HRV have been established as independent predictors of cardiovascular morbidity

and mortality. There are only a handful of studies, however, in which these indices were determined in congenital heart disease. Some of those results indicated reduced BRS and HRV in various forms of congenital heart disease, both before and after surgical interventions [START_REF] Davos | Global impairment of cardiac autonomic nervous activity late after Fontan operation[END_REF][START_REF] Ohuchi | Severely impaired cardiac autonomic nervous activity after the Fontan operation[END_REF][START_REF] Mcleod | Reduced heart rate variability following repair of tetralogy of Fallot[END_REF][START_REF] Davos | Global impairment of cardiac autonomic nervous activity late after repair of tetralogy of Fallot[END_REF][START_REF] Ouchi | Abnormal cardiac autonomic nervous activity after right ventricular outflow tract reconstruction[END_REF][START_REF] Heragu | Heart rate variability in healthy children and in those with congenital heart disease both before and after operation[END_REF]. In patients late after the Fontan operation and also in patients after repair of tetralogy of Fallot (ToF) autonomic nervous control of the heart was found to be markedly deranged, with reduced BRS and HRV [START_REF] Davos | Global impairment of cardiac autonomic nervous activity late after Fontan operation[END_REF][START_REF] Ohuchi | Severely impaired cardiac autonomic nervous activity after the Fontan operation[END_REF][START_REF] Mcleod | Reduced heart rate variability following repair of tetralogy of Fallot[END_REF][START_REF] Davos | Global impairment of cardiac autonomic nervous activity late after repair of tetralogy of Fallot[END_REF]. The reduction was related to previous surgical interventions, their timing and underlying right and left sided hemodynamics [START_REF] Davos | Global impairment of cardiac autonomic nervous activity late after repair of tetralogy of Fallot[END_REF]. In another study on ToF patients, however, only 43 % of patients had lower HRV values [START_REF] Mcleod | Reduced heart rate variability following repair of tetralogy of Fallot[END_REF]. In a study on patients with atrial (ASD) or ventricular septal defects (VSD), or with right ventricular outflow tract reconstruction (RVOTR), BRS and HRV were lower then control, with the reduction, however, being less in ADS and VSD than in RVOTR. The reduction in BRS and HRV was greater immediately after surgery, but recovered partially later on. HRV and BRS correlated inversely with the number of surgical procedures and correlated directly with the follow up duration after RVOTR [START_REF] Ouchi | Abnormal cardiac autonomic nervous activity after right ventricular outflow tract reconstruction[END_REF]. BRS and HRV, however, has never been studied in patients with TGA.

Our present data are indicate no significant differences in cardiovagal autonomic indices between TGA patients and control subjects. This finding was unexpected, considering the unavoidable damage to cardiac autonomic nerves during surgery. Cardiopulmonary nerves, which pass along the posteromedial surface of the superior vena cava and the right atrium, form plexuses, which contain both vagal and sympathetic cardiac efferent fibres, together with afferents of cardio-pulmonary origin. Consequently, removal of pericardium overlying the right atrium during correction surgery of TGA must cause mechanical damage to cardiac autonomic innervation. Additional damage may also occur due to ischemia of nerves and/or of sinoatrial node. Indeed in two studies, when autonomic indices were compared before and immediately after corrective surgery, the postoperative values were markedly reduced [START_REF] Ouchi | Abnormal cardiac autonomic nervous activity after right ventricular outflow tract reconstruction[END_REF][START_REF] Heragu | Heart rate variability in healthy children and in those with congenital heart disease both before and after operation[END_REF]. Our present data therefore, may indicate complete recovery or reinnervation of the sinoatrial node late after repair in TGA patients. In the rat model, re-innervation in the baroreceptor region was observed a few months after sino-aortic denervation [START_REF] Guo-Jun | Morphological evidence of reinnervation of the baroreceptive regions in sinoaorticdenervated rats[END_REF]. The question arises as to what might explain the difference in the extent of autonomic recovery between this study and earlier reports. Two factors may be considered: 1) Difference in time after repair. Autonomic indices were found to recover with time after corrective surgery; in the study of Ouchi et al. BRS significantly correlated with the follow-up duration after RVOTR [START_REF] Ouchi | Abnormal cardiac autonomic nervous activity after right ventricular outflow tract reconstruction[END_REF]. Difference in time after repair, however, seems an unlikely explanation in our case, because HRV and BRS were found to be lower even 26 years after repair of TOF [START_REF] Davos | Global impairment of cardiac autonomic nervous activity late after repair of tetralogy of Fallot[END_REF], while in the present study the mean (± SD) time after repair was only 12 ± 3 years. 2) Difference in the complexity of surgery. In the complex forms of congenital heart disease reduction in HRV and BRS was inversely related to the number of surgical procedures. In the atrial switch type of repair the corrective surgery of TGA is limited to the right atrium and does not involve the ventricular septum, the RVOT, or the pulmonary artery. It seems possible that the more extensive surgical damage to cardiac tissues and to areas where cardiopulmonary baroreceptors may be located, can result in long term impairment of baroreflex function.

Lack of relation between carotid artery distensibility and baroreflex sensitivity

Previous investigations have demonstrated that BRS is significantly related to carotid artery distensibility. A positive association between elastic properties of the carotid artery and BRS has been reported in healthy volunteers, pregnant women and hypertensive patients [START_REF] Bonyhay | Relation between baroreflex sensitivity and carotid artery elasticity in healthy humans[END_REF][START_REF] Visontai | Impaired baroreflex function during pregnancy is associated with stiffening of the carotid artery[END_REF][START_REF] Lage | Relationship of arterial compliance to baroreflex function in hypertensive patients[END_REF].

Age associated changes in BRS were found to be related to central arterial compliance [START_REF] Monahan | Ageassociated changes in cardiovagal baroreflex sensitivity are related to central arterial compliance[END_REF].

Regular exercise in sedentary older subjects increased both cardiovagal BRS and carotid artery compliance and the two events were strongly and positively related [START_REF] Monahan | Central arterial compliance is associated with age-and habitual exercise-related differences in cardiovagal baroreflex sensitivity[END_REF]. In healthy volunteers approximately 50% variance in BRS was explained by differences in carotid artery distensibility [START_REF] Bonyhay | Relation between baroreflex sensitivity and carotid artery elasticity in healthy humans[END_REF][START_REF] Monahan | Ageassociated changes in cardiovagal baroreflex sensitivity are related to central arterial compliance[END_REF][START_REF] Hunt | Quantification of mechanical and neural components of vagal baroreflex in humans[END_REF]. It might be argued that the relatively low number of patients prevented the difference in baroreflex sensitivity determined by phenylephrine in patients vs. controls to reach statistical significance. On the other hand, the complete lack of relation between baroreflex sensitivity and carotid artery distensibility in patients indicate that baroreflex sensitivity is determined -at least in part -by different mechanisms in patients vs.

controls.

Although the ability of barosensory vessels to transduce arterial pressure changes into vessel wall stretch is a key mechanisms in baroreflex function, recent data emphesized the important role of neural function, which encompasses baroreceptor output, afferent neural conduction, central integration, efferent autonomic outflow and sinoatrial node responsiveness [START_REF] Hunt | Quantification of mechanical and neural components of vagal baroreflex in humans[END_REF]. In one study carotid artery distensibility was found to decrease from early childhood, with a concomitant increase in BRS until puberty. This finding indicates that in spite of unremitting vascular stiffening, maturation of neural function predominantly determines baroreflex gain during this period of life [START_REF] Lenard | Maturation of cardiovagal autonomic function from childhood to young adult age[END_REF]. In another study vascular and neural deficits were both shown to contribute to age related declines in cardiovagal BRS, whereas short term exercise attenuated this decline primarily by maintaining neural vagal control [START_REF] Hunt | Does reduced vascular stiffening fully explain preserved cardiovagal baroreflex function in older, physically active men?[END_REF]. These observations indicate that neural plasticity might play an important role in determining cardiovascular autonomic regulation. Neural plasticity might explain our present findings, as well. We hypothesize that the reduction of baroreceptor input, due to congenital vascular stiffening, might be compensated by central autonomic adaptation, maintaining BRS at close to normal level.

Limitations

Due to the cross-sectional design of the study, we could not examine direct influence of surgical procedures on either arterial stiffness or on cardiac autonomic control. Also, we were not able to establish whether the normal HRV and BRS values that we observed in our TGApatients were the result of gradual improvement after a postoperative decline, and whether carotid artery stiffening was the result of gradual postoperative derangement in vessel wall distensibility. The number of patients in whom BRS was determined by the phenylephrine method is relatively low, because many of our patients were reluctant to undergo the phenylephrine procedure.

We measured carotid arterial diameter in the common carotid artery, ~ 1cm below the carotid bulb housing the carotid sinus baroreceptors. It is difficult to measure the diameter of the carotid bulb because its shape is oval and the opposing walls are not parallel. Measurement of common carotid arterial compliance may not be identical to that of the adjacent carotid bulb or the aortic arch. Nonetheless, it is likely that this measurement is more representative of local vascular wall properties than measurements made from more peripheral vessels. We did not assess diameter and BP changes in the aorta, which is also richly innervated with arterial baroreceptors. Because the stimulus to these 2 receptor regions is directionally similar during phenylephrine administration and both vessels demonstrate similar mechanical and hemodynamic properties, this should not represent a significant confound in the present study.

We cannot exclude this possibility, however. NNSD -standard deviation of RR-intervals; RMSSD -root mean square of successive RRinterval differences; pNN50 -percent of RR-intervals, which differ more than 50 ms; HFhigh (0.15-0.4) frequency power of RR-interval variability; LF -low (0.05-0.15) frequency power of RR-interval variability; BRS seq -baroreflex sensitivity determined by the sequence method; LF gain -cross-spectral transfer function in the low frequency band. No significant differences were found at p < 0.05, when groups were compared for differences by Student's t-test or the Mann-Whitney rank-sum for data failing tests of normality. Data are given as means ± SD.

Fig. 1 :

 1 Fig. 1: Baroreflex sensitivity determined by phenylephrine (BRS phe ) is directly related to carotid distensibility in controls (circles, r = 0.71, p < 0.05) but no relation exists in TGA patients (triangles).

Table 1 : Clinical characteristics, carotid artery dimensions and elastic variables of TGA-patients and control subjects BMI

 1 -body mass index; SBP -systolic brachial pressure; DBP -diastolic brachial pressure; MBP -mean brachial pressure; HR -heart rate; D -carotid end-diastolic diameter; ∆Dpulsatile distension; DC -distensibility coefficient. Groups were compared for differences by Student's t-test or the Mann-Whitney rank-sum for data failing tests of normality. Data are given as means ± SD. *significantly different from control at p < 0.05.

	Patients	Controls

Table 2 : HRV and spontaneous BRS-indices of TGA-patients and control subjects

 2

Clinical Science Immediate Publication. Published on 23 Feb 2007 as manuscript CS20060363 © 2007 The Authors Journal compilation © 2007 Biochemical Society

This work was supported by the Hungarian National Research Fund, grant OTKA -T 049690 and the Ministry of Health, grant ETT-054/2006


MD Niels P Riksen
email: n.riksen@aig.umcn.nl

Marlies Bosselaar

Stephan J L Bakker

Robert J Heine

Gerard A Rongen

Cees J Tack

Paul Smits

Acute elevation of plasma free fatty acids increases pulse wave velocity and induces peripheral vasodilation in humans in vivo

Keywords: adenosine, free fatty acids, arterial compliance, blood flow, autonomic nervous system ClinicalTrials

Plasma free fatty acid (FFA) concentrations are elevated in patients with obesity. We aimed to provide an integral haemodynamic profile of elevated plasma FFA by the simultaneous assessment of blood pressure, pulse wave velocity, forearm blood flow (FBF), and sympathetic nervous system activity during acute elevation of FFA. Secondly, we hypothesized that FFA-induced vasodilation is mediated by adenosine receptor stimulation. In a randomized crossover trial in healthy subjects, Intralipid was infused for 2 hours to elevate plasma FFA. Glycerol was administered as Control infusion. We assessed blood pressure, pulse wave velocity, FBF (venous occlusion plethysmography), and sympathetic nervous system activity by measurements of noradrenaline and adrenaline. During the last 15 minutes of Intralipid/Control infusion, the adenosine receptor antagonist caffeine (90 g/min/dl) was administered into the brachial artery of the nondominant arm. Compared with Control infusion, Intralipid increased pulse wave velocity, systolic blood pressure and pulse pressure, as well as FBF (FBF increased from 1.8 0.2 to 2.7 0.6 and from 2.3 0.2 to 2.7 0.6 ml/min/dl for Intralipid versus Control infusion; P<0.05, n=9). Although in a positive control study caffeine attenuated adenosine-induced forearm vasodilation (P<0.01, n=6), caffeine had no effect on Intralipid-induced vasodilation (P=0.5). In conclusion, elevation of plasma FFA levels increases pulse wave velocity, systolic blood pressure, and pulse pressure. Also, FBF is increased, either by baroreflex-mediated inhibition of the sympathetic nervous system, or by a direct vasodilating effect of FFA. As the adenosine receptor antagonist caffeine could not antagonize the vasodilator response, it is not mediated by adenosine receptor stimulation.

Introduction

The metabolic syndrome is characterized by (central) obesity, insulin resistance, glucose intolerance, dyslipidaemia, and hypertension. Less well-known but consistently reported features of the metabolic syndrome include a hyperdynamic circulation and sympathetic activation [START_REF] Stern | Hyperdynamic circulation and the insulin resistance syndrome ("syndrome X")[END_REF][START_REF] Palatini | Parental hyperdynamic circulation predicts insulin resistance in offspring: The Tecumseh Offspring Study[END_REF][START_REF] Jiang | Hyperdynamic circulation and cardiovascular risk in children and adolescents. The Bogalusa Heart Study[END_REF][START_REF] Huggett | Sympathetic neural activation in nondiabetic metabolic syndrome and its further augmentation by hypertension[END_REF]. In patients with obesity and the metabolic syndrome, the plasma concentration of free fatty acids (FFA) is increased and evidence is accumulating that FFA play a central role in the clustering of the various characteristics of the metabolic syndrome [5;6]. Therefore, in the last decade, several studies explored the effect of experimental elevations of plasma FFA on haemodynamic parameters. Systemic administration of a triglyceride emulsion and heparin (to activate the lipoprotein lipase) is a well-validated and generally accepted model to study the effects of an acute elevation of plasma FFA [START_REF] Kearney | Acute haemodynamic effects of lipolysis-induced increase of free fatty acids in healthy men[END_REF][START_REF] Boden | Mechanisms of fatty acid-induced inhibition of glucose uptake[END_REF][START_REF] Dresner | Effects of free fatty acids on glucose transport and IRS-1-associated phosphatidylinositol 3-kinase activity[END_REF]. Most studies agree that acute experimental elevation of FFA increases skeletal muscle blood flow [7;10-14]. Most [7;12;15-17], but not all [10;14], studies have shown an increase in mean arterial blood pressure. Also the reported effects of FFA on the activity of the sympathetic nervous system are controversial: some studies have demonstrated in rats and humans that acute elevation of FFA can increase sympathetic activity [17;18], but other studies have reported the opposite [19;20].

Recently, we presented a novel hypothesis that explains the occurrence of a hyperdynamic circulation and sympathetic activation in individuals with the metabolic syndrome [START_REF] Bakker | The potential role of adenosine in the pathophysiology of the insulin resistance syndrome[END_REF]. We postulated that in these patients, elevated cytosolic concentrations of co-enzyme A derivatives of long-chain fatty acids (LCFA-CoA), the intracellular equivalent of FFA, inhibit the mitochondrial adenine nucleotide translocator. Consequently, intra-and extracellular concentrations of the endogenous nucleoside adenosine increase. Adenosine receptor stimulation induces various effects, including vasodilation in most vascular beds, renal sodium retention and activation of the sympathetic nervous system [21;22]. As such, elevated systemic concentrations of adenosine could induce the phenotype of a hyperdynamic circulation and sympathetic activation.

The aim of the present study is two-fold: to provide an integral haemodynamic profile of elevated plasma FFA and, more specifically, to study whether adenosine receptor stimulation is involved in the vasodilator response to FFA. Therefore, we performed a randomized controlled cross-over study in healthy volunteers in whom systemic administration of a triglyceride emulsion (Intralipid) was used to elevate plasma FFA, and we simultaneously measured forearm blood flow (FBF), intra-arterial blood pressure, pulse wave velocity, and estimated the activity of the sympathetic nervous system by plasma noradrenaline measurements. In addition, the adenosine receptor antagonist caffeine was administered into the brachial artery of the non-dominant arm to study whether the FFA-induced increase in FBF is due to direct adenosine receptor stimulation in the forearm vascular bed.

Methods

Subjects

The study conformed with the principles outlined in the Declaration of Helsinki and was approved by the Institutional Review Board of the Radboud University Nijmegen Medical Centre. Fifteen young healthy volunteers signed written informed consent before participation. All participants underwent a physical examination and electrocardiography to exclude hypertension and cardiovascular or pulmonary disease. As caffeine is an effective adenosine receptor antagonist [START_REF] Fredholm | Actions of caffeine in the brain with special reference to factors that contribute to its widespread use[END_REF], all volunteers were asked to abstain from caffeinecontaining beverages for at least 24 hours before the study.

Instrumentation

All experiments were performed in the morning after an overnight fast in a temperaturecontrolled room (23 C) with the subjects in supine position. After local anaesthesia (xylocaine 2%), the brachial artery of the non-dominant arm was cannulated with a 20-gauge catheter (Angiocath, Becton Dickinson, Sandy, UT, USA) for intra-arterial administration of caffeine (automatic syringe infusion pump, type STC-521, Terumo Corporation, Tokyo, Japan) and blood pressure recording (Hewlett Packard GmbH, Böblingen, Germany). Forearm blood flow (FBF) was recorded simultaneously on both arms by venous occlusion plethysmography using mercury-in-silastic strain gauges (Hokanson EC4, Hokanson, Inc., Washington, USA), as previously described [START_REF] Rongen | Preserved vasodilator response to adenosine in insulin-dependent diabetes mellitus[END_REF]. Before each recording a wrist cuff was inflated to 200 mmHg to exclude the hand circulation.

In the main study two catheters were inserted into large antecubital veins of both arms.

Intralipid or glycerol was administered intravenously into the dominant arm (vide infra).

Venous blood was drawn from the non-dominant arm for the determination of FFA, glycerol, triglycerides, and glucose. Arterial and venous adrenaline and noradrenaline levels were assessed in blood simultaneously drawn from the brachial artery and antecubital vein from the non-dominant arm. Plasma insulin concentration was determined in arterial blood.

Validation study

Six healthy volunteers participated in the validation study. Thirty minutes after cannulation of the brachial artery of the non-dominant arm, FBF was measured for 5 minutes during saline infusion. Then, adenosine was administered into the brachial artery (5 g/min per dl of forearm tissue) for 10 minutes and FBF was measured. After 20 minutes wash-out, adenosine infusion was repeated, 5 minutes later followed by co-infusion of caffeine (90 g/min/dl).

Main study

For this study 9 subjects were studied twice according to a randomized crossover design. All subjects were asked to avoid excessive exercise for 24 hours before the tests and to have a similar dinner the evening before both experiments. The second experiment was conducted one month after the first. In one experiment (Intralipid experiment), subjects received an intravenous dose of a triglyceride emulsion (Intralipid 20%, 90 ml/h) with co-administration of heparin (bolus 500 IU, followed by continuous infusion of 0.2 IU/kg/min intravenously) to activate lipoprotein lipase [START_REF] Kearney | Acute haemodynamic effects of lipolysis-induced increase of free fatty acids in healthy men[END_REF]. In the other experiment (Control experiment), glycerol (50% solution; 3 gram/h) and heparin were administered to control for the elevated glycerol and heparin concentration during the Intralipid experiment. This design has previously been described by Krebs et al [START_REF] Krebs | Free fatty acids inhibit the glucosestimulated increase of intramuscular glucose-6-phosphate concentration in humans[END_REF].

In both experiments, FBF was measured for 5 minutes during saline infusion, thirty minutes after cannulation. Thereafter, Intralipid or Control infusion was started. In both experiments, FBF was recorded for 5 minutes and blood was drawn for the determination of FFA, triglycerides and glycerol 60 and 120 minutes after initiation of infusion. Concentrations of insulin, adrenaline, and noradrenaline were determined on t=0 and t=120 minutes. Finally, caffeine was administered for 15 minutes into the brachial artery (90 g/min/dl) during continuous Intralipid or Control infusion and with uninterrupted measurement of FBF.

During the 10 minutes immediately before the start of administration of Intralipid/Control and during the last 10 minutes of infusion, heart rate was recorded by surface electrocardiography, and blood pressure was recorded in the brachial artery. For each heartbeat, we calculated the time difference between the electrocardiographic R wave and arrival of the pressure wave at the brachial artery and averaged this value over these 10 minute periods (MIDAC testorganiser, Radboud University Nijmegen Medical Centre). The pressure wave signal was first filtered with a lowpass filter (first order, 10 Hz), after which the local maxima (systole) and minima (diastole) were determined. Subsequently, for each pressure wave, the best line was fit with linear regression through the rising edge between 10% and 90% of the maximumminimum amplitude. The intersection between this line and a horizontal line (placed at the level of the diastolic value) was taken as the arrival of the bloodpressure wave. As the travel distance is the same for each subject during the two experiments, this time difference can be considered as an measure for pulse wave velocity.

Analytical procedures

Blood samples for the determination of glycerol, FFA and triglycerides were collected in precooled EDTA-containing vacutainers and centrifuged at 1000 g for 10 min at 4°C. Aliquots of plasma were immediately stored at -80°C. FFA (NEFA-C; Wako Chemicals, Neuss, Germany), free glycerol (148270; Roche Diagnostics, Indianapolis, IN), and triglyceride (GPO-Trinder 337B; Sigma Diagnostics, St Louis, MO) concentrations were analyzed with a COBAS semiautomatic analyzer (Roche). Due to technical problems, baseline plasma glucose levels were only determined in our laboratory in the first five experiments.

For the determination of plasma insulin, adrenaline, and noradrenaline, blood was collected in pre-cooled lithium-heparin-containing vacutainers. Plasma concentrations of noradrenaline and adrenaline were determined by HPLC with fluorimetric detection [START_REF] Willemsen | Highly sensitive and specific HPLC with fluorometric detection for determination of plasma epinephrine and norepinephrine applied to kinetic studies in humans[END_REF]. Insulin was measured with an insulin-specific double antibody radioimmunoassay (interassay coefficient of variation, 6.2%). Plasma caffeine concentrations were determined by reversed-phase HPLC with UV-detection set at 273 nm. Forearm spillover of noradrenaline was estimated as previously described by Rongen et al [START_REF] Rongen | Comparison of two indices for forearm noradrenaline release in humans[END_REF]. To correct for changes in FBF, we also calculated appearance rate of noradrenaline [27;28]. For these calculations, forearm fractional extraction of adrenaline was used as a substitute for noradrenaline extraction.

Statistical analysis

All data are expressed as mean SE. Baseline parameters of subjects in the validation study and main study were compared with an unpaired Student's t-test and baseline values in both experiments of the main study were compared with a paired Student's t-test. Plasma concentrations of free fatty acids, glycerol, triglycerides and insulin as well as haemodynamic parameters in both groups were analyzed by repeated measures ANOVA. Forearm vascular resistance was calculated by the ratio of mean arterial pressure and FBF. SPSS for Windows (release 12.0.1) was used for statistical analyses.

Results

Baseline characteristics

There were no significant differences between subjects participating in the validation study and the main study. Average age was 21.2 0.6 years. Baseline plasma caffeine concentration was 0.29 0.17 mg/l in the validation study and 0.21 0.10 versus 0.15 0.07 mg/l in the Intralipid and Control study, respectively.

Validation study

Infusion of adenosine into the brachial artery of the non-dominant arm for 5 minutes increased FBF from 1.9 0.4 to 4.0 0.7 ml/min per dl of forearm tissue during the first period of adenosine administration and to 4.1 0.8 ml/min/dl during the second period (figure 1a). In the first period, adenosine infusion was continued resulting in a further increase in FBF up to 6.3 1.0 ml/min/dl. In the second period, caffeine was co-administered after 5 minutes, resulting in a decrease of FBF to 2.9 0.5 ml/min/dl. There was a significant difference between the change in FBF during the first 5 minutes of caffeine administration and the change in FBF observed during continuous infusion of adenosine alone (figure 1b; P<0.01).

There were no significant differences between both infusion periods regarding blood pressure, heart rate and FBF in the control arm (data not shown). These results show that caffeine in the dose administered is effective in reducing adenosine-induced vasodilation.

Main study

Haemodynamic values and plasma concentrations of FFA, glycerol, triglycerides, glucose and insulin at baseline, and after 1 and 2 hours of Intralipid/Control infusion are summarized in table 1. Plasma concentrations of FFA, triglycerides and glycerol increased significantly more during administration of Intralipid than during Control infusion. During Intralipid infusion a small yet significant increase in plasma insulin concentration was observed (table 1).

Both Intralipid and Control infusion resulted in a significant increase in systolic blood pressure, pulse pressure and heart rate (P<0.01 for all three parameters), whereas diastolic blood pressure and mean arterial pressure were not affected. However, increases in systolic blood pressure and pulse pressure were significantly more pronounced during Intralipid infusion (table 1).

Baseline FBF was significantly higher before Control infusion than before Intralipid infusion. Therefore, we calculated the change in FBF from baseline to express the effects of each intervention. Administration of Intralipid resulted in a significant increase in FBF compared with Control infusion (Figure 2; P<0.05). Also, FVR (MAP/FBF) was significantly decreased during Intralipid infusion compared with Control infusion (from 52. The time difference between the electrocardiographic R wave and arrival of the pressure wave at the brachial artery decreased during Intralipid administration compared with Control infusion (table 1), indicating that Intralipid administration significantly increased pulve wave velocity. We used the baseline measurements of the time difference in both groups (before any intervention) to calculate the coefficient of variation for this technique, which was 6.5% (average of standard deviation devided by mean for each subject). For biological variables, a coefficient of variation of <10% is considered good, and <20% is acceptable [START_REF] Scott | Reproducibility of systolic and diastolic time intervals in normal humans: an important issue in clinical cardiovascular pharmacology[END_REF].

Administration of Intralipid had neither an effect on arterial noradrenaline concentration nor on calculated forearm noradrenaline spillover compared with Control infusion. If anything, forearm appearance rate of noradrenaline tended to increase more during Control infusion (table 2).

During continuous systemic administration of Intralipid, infusion of caffeine into the brachial artery of the non-dominant arm did not have any effect on FBF (figure 3; P=0.5 compared with the control arm). Also during continuous Control infusion, caffeine did not affect FBF (data not shown).

Discussion

The main finding of this study is that acute elevation of FFA induces a haemodynamic profile consisting of increased pulse wave velocity, elevated systolic blood pressure and pulse pressure and peripheral vasodilation. The observed FFA-induced forearm vasodilation is not mediated by adenosine receptor stimulation.

Previous studies have shown that a hyperdynamic circulation is associated with several characteristics of the metabolic syndrome, although these findings have not received much attention. In adults a hyperdynamic circulation is associated with increased body mass index, triglyceride levels, glucose and insulin levels [START_REF] Stern | Hyperdynamic circulation and the insulin resistance syndrome ("syndrome X")[END_REF]. Moreover, insulin resistance and obesity are associated with increased arterial stiffness [30;31], which is an important cardiovascular risk marker [START_REF] Franklin | Is pulse pressure useful in predicting risk for coronary heart Disease? The Framingham heart study[END_REF].

We hypothesized that this hyperdynamic circulation is caused by increased concentrations of plasma FFA [START_REF] Bakker | The potential role of adenosine in the pathophysiology of the insulin resistance syndrome[END_REF]. In obese subjects the release of FFA into the circulation is increased compared with lean subjects [START_REF] Heptulla | Temporal patterns of circulating leptin levels in lean and obese adolescents: relationships to insulin, growth hormone, and free fatty acids rhythmicity[END_REF]. Indeed, we demonstrated that acute experimental elevation of plasma FFA increased systolic blood pressure and pulse pressure, but we did not observe an increased activity of the sympathetic nervous system. More specifically, we hypothesized that FFA would induce a hyperdynamic circulation and sympathetic activation by increased adenosine receptor stimulation [START_REF] Bakker | The potential role of adenosine in the pathophysiology of the insulin resistance syndrome[END_REF]. In animal studies, LCFA-CoA have been shown to result in a concentration-dependent inhibition of the mitochondrial adenine nucleotide translocator in a physiological concentration range [START_REF] Chua | Reversible inhibition of adenine nucleotide translocation by long chain acyl-CoA esters in bovine heart mitochondria and inverted submitochondrial particles. Comparison with atractylate and bongkrekic acid[END_REF]. The adenine nucleotide translocator transports ADP from the cytosol into the mitochondrial matrix in exchange for ATP. As such, inhibition of this translocator results in increased cytosolic ADP concentrations. Consequently, according to the cytosolic adenylate kinase equilibrium, ATP and AMP concentrations increase and AMP is converted into adenosine. Indeed, it was shown in vitro that palmitoyl-CoA inhibits the adenine nucleotide translocator and increases extramitochondrial AMP concentrations [START_REF] Ciapaite | Modular kinetic analysis of the adenine nucleotide translocator-mediated effects of palmitoyl-CoA on the oxidative phosphorylation in isolated rat liver mitochondria[END_REF]. An increased intracellular adenosine concentration diminishes the transmembranous adenosine concentration gradient, thus limiting uptake of extracellular adenosine [START_REF] Deussen | Quantification of extracellular and intracellular adenosine production: understanding the transmembranous concentration gradient[END_REF]. By stimulation of specific membrane-bound adenosine receptors, adenosine induces various cardiovascular and neurohumoral effects [START_REF] Rongen | Cardiovascular pharmacology of purines[END_REF], such as vasodilation, and activation of the sympathetic nervous system. In addition, some studies suggest that increased systemic adenosine concentrations result in renal sodium and water retention [START_REF] Balakrishnan | Effects of intravenous adenosine on renal function in healthy human subjects[END_REF]. Together, It has been demonstrated previously that acute elevation of FFA is associated with increased forearm and leg blood flow [7;10-14]. In the present study we confirmed these findings. Subsequently, we administered the adenosine receptor antagonist caffeine into the brachial artery of the non-dominant arm in order to explore the role of local adenosine receptor stimulation in this FFA-induced vasodilation. Administration of caffeine did not affect FBF in this setting, whereas in the same dose it effectively blocked adenosine-induced vasodilation.

As such, our findings do not support a role for adenosine in the vasodilator action of FFA.

Unfortunately, we did not measure endogenous plasma adenosine concentrations during administration of Intralipid. It is complicated to measure adenosine, because of the extremely short half life of less than 1 second [START_REF] Moser | Turnover of adenosine in plasma of human and dog blood[END_REF]. It is possible to estimate adenosine concentration by microdialysis [START_REF] Riksen | Enhanced cellular adenosine uptake limits adenosine receptor stimulation in patients with hyperhomocysteinemia[END_REF], but this technique lacks sensitivity to detect small differences in adenosine concentration.

In the present study, experimental elevation of FFA was not associated with activation of the sympathetic nervous system. If anything, a reduction of forearm appearance rate of noradrenaline was observed. Previous studies on this topic in healthy subjects yielded inconsistent results [17;20]. Also in rat models, administration of lipid emulsions has been demonstrated to either increase [START_REF] Grekin | Pressor effects of portal venous oleate infusion. A proposed mechanism for obesity hypertension[END_REF] or decrease [START_REF] Magnan | Lipid infusion lowers sympathetic nervous activity and leads to increased beta-cell responsiveness to glucose[END_REF] sympathetic activity. Because of these inconsistent results, it remains to be determined whether FFA are responsible for the reported association between the metabolic syndrome and sympathetic activation.

Interestingly, we also demonstrated that administration of Intralipid decreased the time difference between the electrocardiographic R wave and arrival of the pressure wave at the brachial artery. As the travel distance is the same for each subject during both experiments, this observation implies that elevation of FFA increases pulse wave velocity. This is in accordance with the results of two previous studies in which compliance after administration of a triglyceride emulsion was estimated by pulse wave analysis [16;39]. A plausible explanation for the haemodynamic effects of FFA in our study is that FFA increased pulse wave velocity by decreasing arterial compliance, which increased systolic blood pressure and pulse pressure by impairing windkessel function and by summation of the propagated and reflected pressure waves. Subsequently, baroreflex-mediated inhibition of the sympathetic nervous system resulted in peripheral vasodilation. A concomitant reduction in heart rate would have been expected in this situation, but it has been demonstrated in rats [START_REF] Shaltout | Mechanism of fatty acids induced suppression of cardiovascular reflexes in rats[END_REF] and humans [START_REF] Gadegbeku | Raising lipids acutely reduces baroreflex sensitivity[END_REF] that acute experimental elevation of plasma FFA reduces baroreflex sensitivity, which is considered mainly to reflect vagal activity. An alternative explanation would be that FFA also have a direct vasodilating effect on resistance arteries, not mediated by adenosine receptor stimulation, in addition to the reduction in arterial compliance. Finally, a direct effect of Intralipid administration on cardiac stroke volume could have contributed to the observed increase in pulse pressure.

A number of potential limitations of our study should be mentioned. First, the study was not blinded. Because of the different colour of the Intralipid emulsion and glycerol solution blinding of this study would have been extremely complicated. However, the test sequence was randomized, and the haemodynamic and laboratory analyses were performed blinded.

Second, plasma insulin levels were slightly but significantly increased by administration of Intralipid compared with Control. This is in accordance with the well-known effect of FFA on insulin-mediated glucose uptake [START_REF] Krebs | Free fatty acids inhibit the glucosestimulated increase of intramuscular glucose-6-phosphate concentration in humans[END_REF]. It is unlikely that this increased insulin concentration contributed to the increase in FBF because in previous studies with comparable study protocols, insulin either slightly increased [START_REF] Vigili De Kreutzenberg | Plasma free fatty acids and endothelium-dependent vasodilation: effect of chain-length and cyclooxygenase inhibition[END_REF] or did not increase at all [START_REF] Gokce | Acute hypertriglyceridemia is associated with peripheral vasodilation and increased basal flow in healthy young adults[END_REF], whereas blood flow increased in all studies. Moreover, insulin-mediated vasodilation was observed in previous studies only after much higher elevations of plasma insulin of approximately tentimes baseline levels [START_REF] Tack | Activation of the sodium-potassium pump contributes to insulin-induced vasodilation in humans[END_REF]. Third, baseline FBF was significantly higher before Control infusion than before Intralipid infusion. However, it is unlikely that this affected the vasomotor response to Intralipid/Control infusion, as there was no correlation between baseline FBF and the change in FBF in response to Intralipid or Control infusion (data not shown). Fourth, in both groups, parameters of sympathetic activity tended to increase in time (table 2). Also, systolic blood pressure and heart rate significantly increased in time in both groups. This could be stress-related, caused by discomfort of the experimental procedure. As the study had a randomized cross-over design, it is unlikely that this would have influenced our results. Finally, it has to be realized that we studied acute and not chronic elevation of plasma FFA and that the plasma FFA concentration after Intralipid infusion was higher than the FFA concentration in patients with the metabolic syndrome or diabetes mellitus [START_REF] Paolisso | Lowering fatty acids potentiates acute insulin response in first degree relatives of people with type II diabetes[END_REF]. Thus, future studies are warranted to confirm that the haemodynamic effects as observed in the present study also apply to chronic and less extreme elevations in plasma FFA concentration.

In conclusion, acute elevation of plasma FFA levels in healthy young subjects increased pulse wave velocity, systolic blood pressure, and pulse pressure, decreased forearm vascular resistance, but did not significantly increase the activity of the sympathetic nervous system.

The reduced vascular resistance could be due to baroreflex-mediated inhibition of sympathetic nerve activity. Alternatively, FFA could have direct vasodilator effects, which, however, are not mediated by local adenosine receptor stimulation.

  3 5.1 to 40.3 5.3 and from 39.2 3.6 to 43.2 7.1 for Intralipid and Control, respectively, P<0.05).

10 Clinical

 10 Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society these findings suggest a role for adenosine in causing the haemodynamic changes observed in the metabolic syndrome and in response to administration of FFA.

Figure 1 FBF

 1 Figure 1 FBF in the experimental arm in the pilot-study during administration of adenosine (filled circles) and adenosine/caffeine (open circles) in the brachial artery (a, n=6, mean SE).

Figure

  Figure 1b depicts the change in FBF after the addition of caffeine (open circles) or continued administration of adenosine only (filled circles). P<0.01 for difference between both interventions, ANOVA for repeated measures.

Figure 2 FBF

 2 Figure 2 FBF in the experimental arm, expressed as change from baseline, during Intralipid (filled circles) and Control infusion (open circles). P<0.05 for difference between both interventions, ANOVA for repeated measures (n=9, mean SE).

Figure 3 FBF

 3 Figure 3 FBF in the experimental (filled circles) and control (open circles) arm during continuous administration of caffeine into the brachial artery for 15 minutes during continuous systemic Intralipid administration. P=0.5 for comparison between both arms, ANOVA for repeated measures (n=9, mean SE).

Table 1 : 20 Clinical

 120 Haemodynamic and laboratory values in the main study (mean SE; I = Intralipid; 05 for difference between I and C at baseline. # P=0.05 and † P<0.05 for difference between I and C (repeated measures ANOVA). Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

12 Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

13 Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 2 :

 2 Plasma noradrenaline kinetics (mean SE)

	Parameter	Intralipid	Intralipid	Control	Control
		T = 0	T = 120	T = 0	T = 120
	Arterial noradrenaline	0.63 0.06	0.71 0.11	0.57 0.07	0.70 0.10
	(nmol/l)
	Arterial adrenaline	0.21 0.03	0.17 0.02	0.17 0.02	0.25 0.04
	(nmol/l)
	Spillover (pmol/min/dl	0.66 0.18	0.85 0.21	0.84 0.16	1.06 0.12
	forearm tissue)
	Appearance rate	2.10 0.59	2.82 0.82	2.30 0.70	3.30 0.59*
	(pmol/min/dl forearm
	tissue)

Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 19 Feb 2007 as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Clinical Science Immediate Publication. Published on 19 Feb

as manuscript CS20060365 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

The technical support by Petra van den Broek (Pharmacology-Toxicology, Radboud University Nijmegen Medical Centre), and Dr. Luc J van Loon (Human Biology and Movement Sciences, Nutrition Research Institute Maastricht, Maastricht University) is gratefully acknowledged. We thank Stef Mientki (Radboud University Nijmegen Medical Centre) for writing the algorithm to calculate the arrival of the pressure wave at the brachial artery. The study was supported by a grant of the Dutch Diabetes Foundation (grant 1999.007). NP Riksen is a recipient of an AGIKO-fellowship of the Netherlands Organization for Scientific Research (ZonMw). CJ Tack is a recipient of a clinical fellowship of the Dutch Diabetes Foundation. The contribution of GA Rongen has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences (KNAW).