MD PhD FESC Stefan Agewall
email: stefan.agewall@karolinska.se.

Åsa Hernberg

Atorvastatin Normalizes Endothelial Function in Healthy Smokers

Keywords: Atherosclerosis, endothelial function, intima-media thickness, statins

Endothelial function predicts cardiovascular disease. The aim of this study was to examine whether endothelial function in smokers with normal cholesterol levels could be normalized by treatment with 80 mg atorvastatin in comparison with a control group. Healthy smokers (n=20) and non-smokers (n=20) were randomized to 80 mg atorvastatin or to placebo in an open, randomized cross-over study. Endothelial functional (Flow-mediated dilatation; FMD) was measured. At baseline smokers had a lower FMD compared to the non-smoking group, 2.2 % ± 0.5 and 4.5 % ± 0.9, respectively (p<0.05). In the smoker group FMD increased to 4.0 % ± 0.8 during treatment with atorvastatin (p<0.05) and returned to basal level during placebo, 2.3 % ± 0.6. In the non-smoking group FMD was unaffected by both atorvastatin and placebo. The net change of total cholesterol or LDL cholesterol was not associated with the net change of FMD when the group was considered as a whole or the smoking group was considered separately. In conclusion, an improved endothelial function was observed in a group of smokers, when they were on 80 mg atorvastatin, whereas atorvastatin had no effect on endothelial function in the non-smoking group. The improved FMD among smokers was independent of the LDL decrease during atorvastatin treatment. The results show that poor endothelial function in smokers with normal lipid levels can be improved by a statin treatment.

INTRODUCTION

Both primary and secondary prevention studies have demonstrated that statin treatment reduces cardiovascular end points [START_REF] Shepherd | Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. West of Scotland Coronary Prevention Study Group[END_REF][START_REF]Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease[END_REF][START_REF] Schwartz | Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) Study Investigators. Effects of atorvastatin on early recurrent ischemic events in acute coronary syndromes: the MIRACL study: a randomized controlled trial[END_REF]. There are several ultrasound and angiographic studies showing that a lipid-lowering treatment is associated with a reduction of the intimamedia thickness or improvement of the lumen area compared with placebo [START_REF] Hodis | Reduction in carotid arterial wall thickness using lovastatin and dietary therapy: a randomized controlled clinical trial[END_REF][START_REF] Waters | Effects of monotherapy with an HMG-CoA reductase inhibitor on the progression of coronary atherosclerosis as assessed by serial quantitative arteriography. The Canadian Coronary Atherosclerosis Intervention Trial[END_REF][START_REF] Pitt | Pravastatin limitation of atherosclerosis in the coronary arteries (PLAC I): reduction in atheroscerotic progression and clinical events. PLAC I investigation[END_REF]. However, the clinical benefit of the drugs used in the clinical studies [START_REF] Schwartz | Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering (MIRACL) Study Investigators. Effects of atorvastatin on early recurrent ischemic events in acute coronary syndromes: the MIRACL study: a randomized controlled trial[END_REF] was manifest early in the course of lipid-lowering therapy before regression of atherosclerosis could occur. Therefore, other mechanisms than cholesterol-lowering may be responsible for the positive outcome after treatment with statins. Modifications of endothelial function, inflammatory responses and plaque stability have been suggested as possible mechanisms.

Impaired endothelium-dependent relaxation has been observed in subjects with atherosclerosis [START_REF] Furchgott | The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine[END_REF]. Moreover, studies have demonstrated that the presence of cardiovascular risk factors such as cigarette smoking [START_REF] Celermajer | Cigarette smoking is associated with dose-related and potentially reversible impairment of endothelium-dependent dilatation in healthy young adults[END_REF] and hypercholesterolaemia [START_REF] Leung | Beneficial effect of cholesterol-lowering therapy on coronary endothelium-dependent relaxation in hypercholesterolaemic patients[END_REF][START_REF] O'driscoll | Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month[END_REF][START_REF] Egashira | Reduction in serum cholesterol with pravastatin improves endothelium-dependent coronary vasomotion in patients with hypercholesterolemia[END_REF] is associated with endothelial dysfunction. Furthermore, cholesterol lowering has been shown to improve the endothelial function [START_REF] Leung | Beneficial effect of cholesterol-lowering therapy on coronary endothelium-dependent relaxation in hypercholesterolaemic patients[END_REF][START_REF] O'driscoll | Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month[END_REF][START_REF] Egashira | Reduction in serum cholesterol with pravastatin improves endothelium-dependent coronary vasomotion in patients with hypercholesterolemia[END_REF]. In one study an improvement of the endothelial function after 1 month on simvastatin treatment was demonstrated in hypercholesterolemic subject [START_REF] O'driscoll | Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month[END_REF],

whereas most other studies have reported an improvement after 6 months or more [START_REF] Leung | Beneficial effect of cholesterol-lowering therapy on coronary endothelium-dependent relaxation in hypercholesterolaemic patients[END_REF][START_REF] Egashira | Reduction in serum cholesterol with pravastatin improves endothelium-dependent coronary vasomotion in patients with hypercholesterolemia[END_REF]. However, there are also studies failing to show a positive effect on endothelial function with statin treatment in certain populations [START_REF] Beishuizen | The effect of statin therapy on endothelial function in type 2 diabetes without manifest cardiovascular disease[END_REF][START_REF] Balletshofer | Intense cholesterol lowering therapy with a HMG-CoA reductase inhibitor does not improve nitric oxide dependent endothelial function in type-2-diabetes--a multicenter, randomised, double-blind, three-arm placebocontrolled clinical trial[END_REF].

The underlying pathophysiological explanation behind the improved endothelial function after treatment with statins in subjects with hypercholesterolaemia is not known. However, it has been observed that statins activate endothelial nitric oxide synthase independent of its cholesterol-lowering actions and this has been suggested to be a possible explanation for the positive outcome after treatment with statins [START_REF] Laufs | Upregulation of endothelial nitric oxide synthase by HMG CoA reductase[END_REF][START_REF] Kaesemeyer | Pravastatin sodium activates endothelial nitic oxide synthase independent of its cholesterol-lowering actions[END_REF]. It is unclear if different cardiovascular risk factors, mediate endothelial dysfunction via the same pathophysiological pathway.

Recently, it has been shown that pretreatment with vitamin C attenuates the endothelial dysfunction after acute cigarette smoking [START_REF] Stamatelopoulos | Oral administration of ascorbic acid attenuates endothelial dysfunction after short-term cigarette smoking[END_REF]. Cigarette smoking is also associated with increased monocyte-endothelial cell adhesion and in a study, L-arginine (the physiologic substrate for nitric oxide), but not antioxidants reversed that abnormality [START_REF] Adams | Cigarette smoking is associated with increased human monocyte adhesion to endothelial cells: reversibility with oral L-arginine but not with vitamin C[END_REF]. Thus, there is a possibility that an agent, such as a statin, which activates nitrate oxide formation, may improve the endothelial function in smokers, independent of a cholesterol-lowering effect.

The aim of this study was to examine whether endothelial function in smokers with normal cholesterol levels could be normalized by treatment with 80 mg atorvastatin in comparison with a non-smoking group.

METHODS

Subjects

Healthy smokers (> 10 cigarettes daily), under the age of 40 were recruited from the department and by advertisement (n=20). An aged matched non-smoking group (n=20) was recruited in the same way. Exclusion criteria: signs of cardiovascular disease, pharmacological treatment, diabetes mellitus and pregnancy. Contraception was necessary for included women. Snuff and nicotine replacement treatment were not allowed. All subjects gave informed consent after written and oral information. The ethics committee of the Karolinska Institute at Huddinge University Hospital approved the study.

Design

An open, randomized cross-over study. The subjects were, after an initial physical examination, examined with a flow-mediated dilatation examination (FMD) and randomized to active treatment with 80 mg atorvastatin daily or to no treatment. The non-smoking group underwent the same procedure. After 4 weeks each subject came back for a second examination of the endothelial function. There was a cross-over between the treatment arms and the subjects were re-examined after another 4 weeks with a last examination of the endothelial function. See flow-chart in Figure 1.

Measurements

Total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, plasma-glucose, Hb, CK,ASAT,ALAT) were controlled at baseline, 4 and 8 weeks. Resting blood pressure was measured in the right arm after 10 min supine rest. Smoking was assessed by a questionnaire.

No subject had any side effect of the drug or elevated CK, ASAT or ALAT.

Flow mediated dilatation (FMD) and intima-media thickness of the brachial artery

The ultrasound procedures for assessing endothelium-dependent FMD were performed as previously described [START_REF] Agewall | Intima-media complex of both the brachial artery and the common carotid artery are associated with left ventricular hypertrophy in patients with previous myocardial infarction[END_REF][START_REF] Agewall | Does a glass of red wine improve the endothelial function ?[END_REF]. The subjectss were examined in the morning after fasting since midnight. A high-resolution ultrasound scanner (System Five, GE Vingmed, Horten, Norway) with a 10.0-MHz linear array transducer was used. After 10-minute equilibration period at rest in the recumbent position, a single dedicated ultrasonographer performed measurements of the left brachial artery FMD. Scans of the brachial artery were taken proximal to the antecubital fossa and saved on videotape. The transducer was fixed with a stand during the FMD examinations. Baseline diameter recordings were obtained after which arterial occlusion was performed by inflating a forearm blood pressure cuff (12,5 cm wide) to 250 mmHg for 4.5 min. After cuff-release, diameter recordings were repeated during the post-occlusive increase in brachial artery blood flow. Arterial flow velocity recordings were obtained using pulsed wave Doppler at 60 ° to the vessel with the sample volume (1.5 mm) in the centre of the artery. The complete experimental sequence was performed twice at 30 min interval.

Images were digitally acquired from the videotape and measured in random order by a single observer blinded to the conditions under which the ultrasonic images were obtained.

Measurement of the brachial artery diameter was defined as a distance from the leading edge of the near wall intima-lumen echo to the leading edge of the far wall lumen-intima echo along a line perpendicular to the artery's long axis. A computer system [START_REF] Wendelhag | A new automated computerized analysing system simplifies reading and reduces the variability in ultrasound measurements of intima-media thickness[END_REF] with automated tracing of echo interfaces and measurements of distances between the wall echoes within a 5 mm long section of the brachial artery was used. Brachial artery diameter was calculated in diastolic frames taken coincidentally with the R wave on the electrocardiogram twice at rest and then 45, 60 and 75 seconds after cuff deflation. A mean of the diameters after 45, 60 and 75 seconds was calculated. Diameter changes were expressed as the percentage change relative to the mean baseline value. The mean of two FMD examinations was used.

The intima-media thickness was defined as the distance between the leading edge of the lumen-intima echo and the leading edge of the media-adventitia echo.

Statistical analysis

Descriptive measures are reported as means and standard deviations of the mean.

Experimental measures are reported as means and SE of the mean. Changes within a study group were compared with a paired t test, whereas comparisons between the study groups were made using an unpaired t test. Pearson's correlation coefficients were calculated. In a clinical trial using this technique a mean improvement in flow mediated dilatation of at least 2 % would usually be required to detect a treatment benefit. In a cross-over trial design, with 80 % power and 95 % confidence interval, a less than 1.5 % change in flow-mediated dilatation will be detected with 20 subjects [START_REF] Sorensen | Non-invasive measurement of human endothelium dependent arterial responses: accuracy and reproducibility[END_REF]. With the technique in our hands the coefficient of variation is 2 % and a significant enhancement of 1.7 % induced by a treatment was detected in a crossover design in 12 subjects [START_REF] Agewall | Does a glass of red wine improve the endothelial function ?[END_REF].

RESULTS

Baseline characteristics of all individuals enrolled are shown in Table 1. The only observed difference between the study groups was a higher HDL cholesterol in non-smoking subjects compared to the smokers (p<0.05). The ultrasound data at baseline is given in Table 2. The lumen and IMT of the brachial artery did not differ between the study groups, whereas smokers had a lower FMD compared to the non-smoking group, 2.2 % ± 0.5 and 4.5 % ± 0.8, respectively (p<0.05).

In Figure 2 the effect of 80 mg atorvastatin on total and LDL cholesterol is presented. Total cholesterol decreased from 4.2 ± 0.4 mmol/l to 2.6 ± 0.6 mmol/l in the smoking group during treatment with 80 mg atorvastatin (p<0.05), whereas the corresponding levels in the nonsmoking group was 4.4 ± 0.7 mmol/l and 2.7 mmol ± 0.6 mmol/l p<0.05). LDL cholesterol decreased from 2.4 ± 0.5 mmol/l to 1.0 ± 0.5 mmol/l in the smoking group during treatment with 80 mg atorvastatin (p<0.05). In the non-smoking group LDL cholesterol was 2.4 ± 0.6 mmol/l at baseline and 1.0 mmol ± 0.4 mmol/l during atorvastatin treatment (p<0.05).

None of the measured lipid levels during the placebo phase differed from the baseline values.

The triglyceride level decreased from 1.0 ± 0.4 mmol/l at baseline to 0.6 ± 0.2 mmol/l during atorvastatin treatment in the non-smoking group (p<0.05) (Figure 3).

During the open-labelled placebo all lipid levels return to the basal levels in both study groups.

The FMD measurements are presented in Table 1 and in Figure 4. In the smoker group FMD was 2.2 % ± 0.5 at baseline. FMD increased to 4.0 % ± 0.8 during treatment with atorvastatin (p<0.05) and returned to basal level during placebo, 2.3 % ± 0.6. In the non-smoking group FMD was unaffected by both atorvastatin and placebo. Thus, FMD increased 1.8 % in the smoker group during atorvastatin treatment whereas

The net change of total cholesterol or LDL cholesterol was not correlated with the net change of FMD when the group was considered as a whole or the smoking group was considered separately.

DISCUSSION

In this study we confirm that cigarette smokers have a worse flow-mediated dilatation compared to an aged matched non-smoking group. In the smoking group, endothelial function was improved to the same level as in a non-smoking group, when they were on 80 mg atorvastatin, whereas atorvastatin had no effect on endothelial function in the nonsmoking group. The improved FMD among smokers was independent of the LDL decrease during atorvastatin treatment. Despite a clearly low FMD, the intima media-thickness of the brachial artery was not enlarged in the smoker group. It is more common to use intima-media thickness of the common carotid artery as a surrogate measure for atherosclerosis, however we have Copyright 2006 The Biochemical Society previously reported that the brachial artery intima media thickness is significantly associated with the artery intima media thickness in the common carotid artery [START_REF] Agewall | Intima-media complex of both the brachial artery and the common carotid artery are associated with left ventricular hypertrophy in patients with previous myocardial infarction[END_REF]. In a recent autopsy study Sorensen et al [22] the grade of lesion severity in the brachial and the left anterior descending coronary artery was significantly correlated and so was the severity in the brachial and carotid arteries. Thus, the finding in that autopsy study supports that also intima media thickness of the brachial artery may serve as a surrogate variable when studying atherosclerosis. However it is important to know that it is not possible to differentiate between intimal and medial thickening with ultrasound technique. Wiesmann et al [START_REF] Sorensen | Atherosclerosis in the human brachial artery[END_REF] found, using high-resolution MRI, a poor central vascular distensibility in both the common carotid arteries and at multiple sites in the aorta in a group of smokers compared to nonsmokers. The discordance between this study and the study by Wiesmann is probably explained by different examination methods and examination of different vessel beds.

The underlying mechanism why cigarette smoking deteriorates endothelial function is not completely known. FMD gives us an indirect measure of NO bioavailability in the brachial artery. Since also snuff and nicotine spray [START_REF] Wiesmann | Global impairment of brachial, carotid, and aortic vascular function in young smokers: direct quantification by highresolution magnetic resonance imaging[END_REF][START_REF] Rohani | Oral snuff impairs endothelial function in healthy middleaged snuff users[END_REF] decrease FMD, nicotine has been suggested to be responsible for this action. However, because there are numerous known and unknown components of cigarette smoke whose metabolic fate in the human body is unknown, an appropriate in vitro model of cigarette smoking exposure remains to be established. Previous reports suggest statins activate endothelial nitric oxide synthase [START_REF] Laufs | Upregulation of endothelial nitric oxide synthase by HMG CoA reductase[END_REF][START_REF] Kaesemeyer | Pravastatin sodium activates endothelial nitic oxide synthase independent of its cholesterol-lowering actions[END_REF] and our observation of an improved FMD in the smoker group only, might be explained by a deactivated NO synthase among smokers, which returns back to a normal activation level during statin treatment, whereas the NO synthase acitivity among non-smokers already is activated and therefore the statin treatment has no effect. The In the present study we found no relationship between improvement of FMD in the smoking group and the improved lipid profile. This observation supports the hypothesis that statins activate endothelial nitric oxide synthase independent of the cholesterol-lowering actions.

Other studies have suggested damage of NO by free radicals [START_REF] Neunteufl | Contribution of nicotine to acute endothelial dysfunction in long-term smokers[END_REF] as the potential mechanisms through which smoking impairs endothelial function. This theory was supported by an observation by Papamichael et al [START_REF] Murohara | Cigarette smoke extract contracts isolated porcine coronary arteries by superoxide anion-mediated degradation of EDRF[END_REF], who found that simultaneous consumption of red wine when smoking, partly decreased the harmful acute effect of smoking through its effect on FMD. Furthermore, treatment with vitamin C attenuates the endothelial dysfunction after acute cigarette smoking [START_REF] Stamatelopoulos | Oral administration of ascorbic acid attenuates endothelial dysfunction after short-term cigarette smoking[END_REF].

We found a lower HDL cholesterol in the smoking group, which is a known phenomena since before [START_REF] Papamichael | Red wine's antioxidants counteract acute endothelial dysfunction caused by cigarette smoking in healthy nonsmokers[END_REF]. The mechanisms responsible are not clearly elucidated, and the role of dietary differences between smokers and non-smokers is unknown. As expected we observed dramatic decrease of total and HDL cholesterol in both study groups but the improved FMD level in the smoking group was independent of these lipid level changes.

In this study FMD was used as a surrogate variable and one can question the relevance of this observation. However, we know from numerous studies that FMD is associated with cardiovascular risk factors including cigarette smoking [START_REF] Celermajer | Cigarette smoking is associated with dose-related and potentially reversible impairment of endothelium-dependent dilatation in healthy young adults[END_REF][START_REF] Leung | Beneficial effect of cholesterol-lowering therapy on coronary endothelium-dependent relaxation in hypercholesterolaemic patients[END_REF][START_REF] O'driscoll | Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month[END_REF][START_REF] Egashira | Reduction in serum cholesterol with pravastatin improves endothelium-dependent coronary vasomotion in patients with hypercholesterolemia[END_REF] and we know that FMD predicts a poor outcome in different patient groups [START_REF] Craig | Cigarette smoking and serum lipid and lipoprotein concentrations: An analysis of published data[END_REF][START_REF] Gokce | Risk stratification for postoperative cardiovascular events via noninvasive assessment of endothelial function: a prospective study[END_REF]. Our results are supported in a previous study [START_REF] Neunteufl | Late prognostic value of flow-mediated dilatation in the brachial artery of patients with chest pain[END_REF]. The results may suggest the cardiovascular risk in smokers with normal lipid levels can be reduced by a statin treatment. However, to confirm that information a large study with hard end-points is needed. Copyright 2006 The Biochemical Society In summary, we observed an improved endothelial function in a group of smokers, when they were on 80 mg atorvastatin, whereas atorvastatin had no effect on endothelial function in the non-smoking group. The improved FMD among smokers was independent of the LDL decrease during atorvastatin treatment. Copyright 2006 The Biochemical Society

Figure 1

 1 Figure 1: Flow-chart

Clinical Science Immediate Publication. Published on 12 Apr 2006 as manuscript CS20060033

Clinical Science Immediate Publication. Published on 12 Apr 2006 as manuscript CS20060033

Table 1 .

 1 Baseline demographics

	Parameters	Smokers (n=20)	Non-smokers (n=20)
	Age (year)	30 ± 6	30 ± 5
	Blood pressure (mm Hg)	119 / 71 ± 15 / 9	116 / 72 ± 12 / 7
	Total cholesterol (mmol/L)	4.2 ± 0.4	4.4 ± 0.6
	LDL cholesterol (mmol/L)	2.4 ± 0.5	2.4 ± 0.6
	HDL cholesterol (mmol/L)	1.3 ± 0.3	1.6 ± 0.4 *
	Triglycerides (mmol/L)	1.1 ± 0.6	1.0 ± 0.5
	Glucose (mmol/lL	4.9 ± 0.3	4.7 ± 0.3
	Values are means ±SD.
	* p<0.05

Clinical Science Immediate Publication. Published on 12 Apr 2006 as manuscript CS20060033 Copyright 2006 The Biochemical SocietyTable 2 .

 2 Baseline ultrasound data of the subjects

	Brachial examination	Smokers (n=20)	Non-smokers (n=20)
	Intima-media thickness, (mm)	0.24 ±0.004	0.25 ±0.007
	Lumen diameter (mm)	3.6 ±0.09	3.5 ±0.13
	Values are means ±SEM.
	* p<0.05

Clinical Science Immediate Publication. Published on 12 Apr 2006 as manuscript CS20060033

Acknowledgements: This study was supported by an unrestricted grant from Pfizer. Clinical Science Immediate Publication. Published on 12 Apr 2006 as manuscript CS20060033 Copyright 2006 The Biochemical Society


MD. Stephan F E Praet
email: s.praet@mmc.nl

Ralph J F Manders

Ruth C R Meex

A G Lieverse

D A Coen

Harm Stehouwer

Hans A Kuipers

Luc J C Keizer

Van Loon

MD PhD H A Keizer

PhD L J C Van Loon

Glycemic instability is an underestimated problem in Type II diabetes

Keywords: CGMS, hyperglycemia, CONGA, type 2 diabetes, HbA 1 c

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Over the last 15 years, improvements in microdialysis and biosensor technology have enabled clinicians to reliably monitor plasma and/or interstitial glucose concentrations in an ambulatory and continuous way [START_REF] Maran | Continuous subcutaneous glucose monitoring in diabetic patients: A multicenter analysis[END_REF][START_REF] Jungheim | Subcutaneous continuous glucose monitoring: Feasibility of a new microdialysis-based glucose sensor system[END_REF]. These, so-called, continuous subcutaneous glucose-monitoring systems (CGMS) have proven quite useful to optimize individual exogenous insulin administration in diabetes patients [START_REF] Schaepelynck-Belicar | Improved metabolic control in diabetic adolescents using the continuous glucose monitoring system (cgms)[END_REF], since they provide information on ambulatory postprandial [START_REF] Hay | Unrecognized hypo-and hyperglycemia in well-controlled patients with type 2 diabetes mellitus: The results of continuous glucose monitoring[END_REF] and/or nocturnal glucose excursions [START_REF] Bolinder | Self-monitoring of blood glucose in type i diabetic patients: Comparison with continuous microdialysis measurements of glucose in subcutaneous adipose tissue during ordinary life conditions[END_REF]. Moreover, both in children and adults with type 1 diabetes it has been shown that average 24 h blood glucose concentrations strongly correlate with HbA 1 c concentrations [START_REF] Salardi | The glucose area under the profiles obtained with continuous glucose monitoring system relationships with hba(lc) in pediatric type 1 diabetic patients[END_REF][START_REF] Sharp | Continuous glucose monitoring and haemoglobin a(1c)[END_REF]. However, the inter-and intra-individual day-to-day variation in glycemic load, meal composition [START_REF] Wolever | The glycemic index[END_REF] and daily physical activity [START_REF] Burstein | Effect of an acute bout of exercise on glucose disposal in human obesity[END_REF] can complicate therapeutic decision-making based on these 24 h blood glucose profiles [10,[START_REF] Kerssen | Day-to-day glucose variability during pregnancy in women with type 1 diabetes mellitus: Glucose profiles measured with the continuous glucose monitoring system[END_REF]. Therefore, in order to compare CGMS results between normoglycemic and diabetic subjects, standardization of both diet [START_REF] Wolever | The glycemic index[END_REF] and physical activity [START_REF] Burstein | Effect of an acute bout of exercise on glucose disposal in human obesity[END_REF] is essential. Epidemiological studies and preliminary intervention studies have shown that postprandial hyperglycemia is a direct and independent risk factor for the development of cardiovascular disease (CVD) [12].

Importantly, the postprandial rapid increase in blood glucose concentrations or 'hyperglycemic spikes' seem to be even more relevant to the onset of cardiovascular complications than merely elevated fasting plasma glucose [13]. Therefore, therapeutic targets should be aimed at reducing postprandial blood glucose excursions. Although scientific studies on the prevalence of hyperglycemic spikes in type 2 diabetes are still scarce [13], recommendations on proper glycemic control have recently been redefined [START_REF] De Backer | European guidelines on cardiovascular disease prevention in clinical practice. Third joint task force of european and other societies on cardiovascular disease prevention in clinical practice[END_REF]15] To define abnormal postprandial blood glucose excursions and relate this to the pathogenesis of diabetic vascular complications, it is important to have more detailed information on normal postprandial blood glucose profiles in a non-insulin resistant population under exactly the same dietary ambulatory conditions. Therefore, in the present study, we investigated 24 h blood glucose profiles in type 2 diabetes patients on oral blood glucose lowering medication and healthy, normoglycemic controls under strictly standardized, but free-living conditions. As such, this study provides a frame of reference for future studies on the role of real-life postprandial hyperglycemia in the pathogenesis of diabetic complications.

SUBJECTS AND METHODS

Subjects

A total of 11 long-term diagnosed male type 2 diabetes patients and 11 healthy, age and BMI matched, normoglycemic control subjects were selected to participate in this study. Subjects' characteristics are presented in Table 1. Exclusion criteria were impaired renal or liver function, severe obesity (BMI>35 kg/m 2 ), cardiac disease, hypertension, diabetic complications, and exogenous insulin therapy. All type 2 diabetes patients were treated with oral plasma glucose lowering medication (metformin only (n=3), or in combination with sulfonylureas (n=8)). All medication was continued during the trials. All subjects were informed about the nature and the risks of the experimental procedures before their written informed consent was obtained. The study was approved by the local Medical Ethical Committee.

Screening

Before inclusion, all subjects first performed an oral glucose tolerance test (OGTT). Blood glucose lowering medication was withheld prior to the screening. After an overnight fast, subjects reported at the laboratory at 8.00 a.m. A catheter (Baxter BV, Utrecht, the Netherlands) was inserted into an antecubital vein and a resting blood sample was drawn after which a bolus of 75 g glucose (dissolved in 250 ml water) was ingested (t= 0 min). After the bolus was consumed, blood was sampled every 30 min until t=120 min. Plasma glucose concentrations were measured to determine glucose intolerance and/or type 2 diabetes according to the World Health Organization criteria of 1999 [START_REF]Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: Diagnosis and classification of diabetes mellitus[END_REF]. In addition, plasma glucose and insulin concentrations were used to assess insulin sensitivity (IS) using the oral glucose insulin sensitivity (OGIS)-index for a 2 h OGTT as described by Mari et al [17] and whole-body insulin resistance using the homeostasis model assessment insulin resistance index (HOMA-IR) [START_REF] Matthews | Homeostasis model assessment: Insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man[END_REF].

Blood sample analysis

Blood (10 ml) was collected in EDTA containing tubes and centrifuged at 1,000 g and 4°C for 10 min.

Aliquots of plasma were immediately frozen in liquid nitrogen and stored at -80°C until analyses. Glucose concentrations (Uni Kit III, Roche, Basel) were analyzed with the COBAS FARA semi-automatic analyzer (Roche). Plasma insulin was determined in duplicate and averaged by radioimmunoassay (HI-14K, Linco research Inc, St. Charles, USA). To determine HbA 1 c content a 3 ml blood sample was collected in EDTA containing tubes and analyzed by high-performance liquid chromatography (Bio-Rad Diamat, Munich, Germany).

Study protocol

All experimental trials described in this study are part of a greater project investigating the effects of nutritional interventions to improve glycemic control in type 2 diabetes patients. On the first day subjects reported to the laboratory in the afternoon and were instructed about their diet, and on the use of the food intake and physical activity diaries. Next, subjects received instructions in the use of the capillary blood sampling method (Glucocard Memory PC, A. Menarini Diagnostics, Firenze, Italy) used for the calibration of the continuous glucose monitoring system. All subjects were instructed to measure capillary blood glucose concentrations before every meal. After the subjects were fully instructed, a microdialysis fiber (Medica, Medolla, Italy) with an internal diameter of 0.17 mm and a cut-off weight of 18 kD was inserted in the peri-umbilical region, without anesthesia, using an 18-gauge Teflon catheter as a guide, as described previously [19]. For the measurements the micro-fiber was then connected to a portable CGMS (GlucoDay ® S, A. Menarini Diagnostics, Firenze, Italy), which consists of a peristaltic pump that pumps Dulbecco's solution at 10 µL/min through the microdialysis fiber. A more detailed description of the device has been published earlier [START_REF] Maran | Continuous subcutaneous glucose monitoring in diabetic patients: A multicenter analysis[END_REF]. Briefly, the subcutaneous interstitial fluid is taken up by the microdialysis fiber and is transported to the measuring cell. The glucose sensor, consisting of immobilized glucose oxidase, measures the glucose concentration every sec and stores an average value every 3 min for Clinical Science Immediate Publication. Published on 13 Apr 2006 as manuscript CS20060041 a total of 48 h. The entire device weighs about 250 g and is worn in a pouch under the subjects' clothes.

After the CGMS was checked for proper function, subjects were provided with their diet (pre-weighed and packaged meals, drinks and snacks) and were allowed to return home and resume all their normal activities. CGMS data of the second test day (from 7.00 am to 7.00 am) were used for data analysis. The first period was used to familiarize subjects with the equipment and, therefore, not used in the data analyses.

Diet and physical activity

All subjects maintained their normal physical activity patterns throughout the entire experimental period.

Subjects refrained from heavy physical labor and exercise training for at least 3 d prior to and on the day of the trial. Subjects were asked to keep a comprehensive record of time spent performing al activities (to the nearest 10 min) including sleeping, eating sitting, standing, watching television, occupational activity and household tasks, as well as information on the duration and relative intensity (e.g. light, moderate) of all structured activities. The rate of energy expenditure for each activity was then determined using the Compendium of Physical Activities [START_REF] Ainsworth | Compendium of physical activities: An update of activity codes and met intensities[END_REF]. Daily energy expenditure did not differ between groups and averaged 13.6±0.7 and 13.3±0.6 MJ day -1 in the type 2 diabetes patients and normoglycemic controls, respectively. All meals, snacks and beverages were provided in pre-weight packages and ingested at predetermined time points to ensure fully standardized dietary modulation. On the evening prior to the 24h analyses period, all subjects received the same standardized meal (43.8 kJ kg -1 BW; consisting of 60 Energy % (En%) carbohydrate, 28 En% fat and 12 En% protein). The following day the subjects were instructed to ingest their designated meals, drinks and snacks at set time-points. Throughout this 24 h test period subjects received a standardized diet (3 meals and 3 snacks per day) representing an energy intake of 121 kJ kg -1 BW per day consisting of 64 En% carbohydrate, 25 En% fat and 11 En% protein. Before and after consuming a meal (i.e. breakfast, lunch and dinner) subjects were asked to obtain a capillary blood glucose sample (Glucocard Memory PC). The following day the subjects reported back to our laboratory to obtain a non-fasting venous blood glucose measurement and to remove the CGMS. The Clinical Science Immediate Publication. Published on 13 Apr 2006 as manuscript CS20060041 acquired data were then downloaded from the device to a personal computer with GlucoDay ® software (V3.0.5). Values reported by the CGMS were converted into glucose values using the capillary glucose measurements as calibration values.

Statistics and data analyses

Data are expressed as means±SEM. Glucose responses were calculated as mean glucose area under the curve (AUC) up to 6 h after each meal. Since the CGMS device provides an average glucose value every 3 min, AUC is expressed as mmol/L * 3 min. To quantify and compare the glucose excursions in the control and diabetes population, AUC and the amount of time during which glucose concentrations were above 10.0 mmol/L or below 3.9 mmol/L were calculated. On the first and second study day, fasting glucose was determined from the calibrated CGMS curves 10 min before breakfast and averaged. The non-fasting venous blood glucose measurement was used to calculate the coefficient of variation (CV) of the CGMS data. Relationships between CGMS parameters and standard measures of IS were calculated using linear regression models.

To assess intra-day glycemic variability, continuous overall net glycemic action (CONGA), a novel method recently described by McDonnell et al, was used [START_REF] Mcdonnell | A novel approach to continuous glucose analysis utilizing glycemic variation[END_REF]. CONGAn has been defined as the standard deviation of the differences in glucose concentration using varying time differences of n hours. We used CONGA1, CONGA2 and CONGA4, indicating intra-day glycemic variability based on 1 h, 2 h and 4 h time differences, respectively. In normal non-diabetic subjects CONGA values vary between 0.4 and 1.2, while values above 1.5 indicate glycemic lability [START_REF] Mcdonnell | A novel approach to continuous glucose analysis utilizing glycemic variation[END_REF].

Before pooling data from all 22 subjects, homogeneity of regression was tested using ANCOVA in order to exclude significant interaction. Time dependent variables were tested using repeatedmeasures ANOVA with a Tukey-Kramer post-hoc test when applicable. For non-time dependent variables, a Student's t-test for unpaired observations was applied. Significance was set at the 9

RESULTS

Baseline and postprandial blood glucose responses are provided in Table 2. Total 24 h blood glucose responses in both diabetes patients and healthy controls are illustrated in Fig. 1. Basal and mean glucose concentrations were significantly greater in the type 2 diabetes patients vs the normoglycemic controls (ttest, p<0.05). In the type 2 diabetes patients, the prevalence of hyperglycemia (>10.0 mmol/L) was 55±7% of the 24 h period. In contrast, in the normoglycemic controls, hyperglycemia was evident in 1.6±1 %. As such, hyperglycemia was present for 13.3±1.7 h and 0.38±0.2 h, respectively. The postprandial AUCs above 10.0 mmol/L following breakfast, lunch and dinner contribute, respectively 46±7%, 29±3%, 11±3% to the total amount of hyperglycemia in our type 2 diabetes patients present during the 24 h monitoring period. This breakfast-related hyperglycemia was significantly greater (ANOVA, p<0.01) compared to the amount of hyperglycemia during the evening or during the night.

Both in type 2 diabetes patients and healthy controls the average CONGA1 values following breakfast were significantly raised compared to the 6 h following lunch and dinner (Table 2, ANOVA, p<0.01).

CONGA1 values were lowest during the night (ANOVA, p<0.01) and did not differ between groups from 01.00 -06.00 h (ANOVA, p>0.05, Table 2).

In this study the coefficient of variation (CV) between interstitial CGMS glucose values and venous blood glucose was on average 8.0±1.3%

Correlations between CGMS parameters and our standard measures for glycemic control are presented in Table 3. In the diabetes patients, HbA 1 c-levels correlated well with the average 24 h blood glucose concentrations (R=0.81, P<0.01), the time during which blood glucose levels were >10 mmol/L (R= 0.70, P<0.05), and postprandial AUC following lunch (R=0.80, P<0.01) and dinner (R=0.87, P<0.01). In a subgroup of diabetes patients with apparent acceptable glycemic control (HbA 1 c ≤ 7.0, n=6), hyperglycemia was present for 46±8% of the day (11.0±1.9 h).

Both in the diabetes and control group, mean 24 h and nocturnal blood glucose concentrations correlated strongly with fasting plasma glucose levels (R between 0.61-0.86, P<0.05). In both groups, no significant correlations were reported between the 24 h CONGA indices and HbA 1 c content, however, in the diabetes patients a significant correlation was found between postprandial CONGA1 values and AUC in the 6 h following a meal (R=0.47, p<0.01). When pooling the data from both groups, the 24 h CONGAn values correlated significantly with blood HbA 1 c content (R=0.53-0.66, P<0.01), mean 24 h glucose concentrations (R=0.73-0.77, P<0.001) and to a lesser extent with mean fasting plasma glucose concentrations (R=0.50-0.52, P<0.05). Also, a significant correlation was found between postprandial CONGA1 values and AUC 6 h following a meal (R=0.60, p<0.001)

DISCUSSION

The present study shows that under normal, standardized dietary conditions, type 2 diabetes patients using oral blood glucose lowering medication experience a substantial amount of hyperglycemia for more than 13 h within a 24 h period. This disturbance in blood glucose homeostasis is predominantly present following breakfast. After comparing 24 h blood glucose profiles between healthy, normoglycemic controls and type 2 diabetes patients under usual medical care by a general practitioner, it seems clear that standard treatment schemes with oral blood glucose lowering drugs appear to have insufficient therapeutic strength to normalize postprandial hyperglycemia. Given the clinical relevance of the hyperglycemic spikes [13], CGMS provides an excellent tool to evaluate the level of glycemic stability in type 2 diabetes patients.

The concept that oral blood glucose lowering therapy provides inadequate protection against hyperglycemia is not new [START_REF] Roy | Real world effectiveness of rosiglitazone added to maximal (tolerated) doses of metformin and a sulfonylurea agent: A systematic evaluation of triple oral therapy in a minority population[END_REF][START_REF] Davies | The reality of glycaemic control in insulin treated diabetes: Defining the clinical challenges[END_REF][24]. Epidemiological studies and preliminary intervention studies have shown that postprandial hyperglycemia is a direct and independent risk factor for the development of cardiovascular disease [12]. However, the postprandial rapid increase in blood glucose concentrations seems to be more relevant to the onset of cardiovascular complications than merely elevated fasting plasma glucose concentrations [13]. Therefore, more detailed information on 24 h blood glucose profiles in a diabetic state is essential to increase our understanding of the relationship between hyperglycemia, glucotoxicity and cardiovascular morbidity. In an attempt to assess postprandial glycemic instability in type 2 diabetes, we applied CGMS in diabetes patients and compared this with blood glucose profiles of normoglycemic subjects under strict nutritional and exercise standardization, but otherwise free-living conditions. In most of our normoglycemic subjects, hyperglycemia or glycemic instability was not detectable. In contrast, despite healthy dietary conditions and continued use of oral blood glucose lowering medication according to standard primary care [25] and international guidelines [15] , the type 2 diabetes patients were hyperglycemic during more than 13 h per day, while using exactly the same diet as the normoglycemic controls. In accordance with earlier observations by Monnier et al [START_REF] Monnier | Morning hyperglycemic excursions: A constant failure in the metabolic control of non-insulin-using patients with type 2 diabetes[END_REF], this study shows that postprandial hyperglycemia was most prominent following breakfast, and less evident during the night. As we provided a healthy, balanced diet (43.8 kJ/kg BW; consisting of 60 En%), carbohydrate, 28 En% fat and 12 En% protein), it could be speculated that the total amount of hyperglycemia may even be worse under normal, unrestricted dietary conditions. The observed levels of hyperglycemia during the day (13±2 h / 24 h) are unacceptable and likely cause the excess formation of advanced glycation endproducts [START_REF] Aronson | How hyperglycemia promotes atherosclerosis: Molecular mechanisms[END_REF], causing the macro-and microvascular damage [28].

In line with earlier studies [START_REF] Shorr | Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (ukpds 33). Uk prospective diabetes study (ukpds) group[END_REF][30][START_REF] Shichiri | Long-term results of the kumamoto study on optimal diabetes control in type 2 diabetic patients[END_REF], our findings emphasize the need for different types of interventional strategies in type 2 diabetes patients. It should be noted that there is a weak, non-significant, correlation between fasting blood glucose and the percentage of hyperglycemia (R 2 =0.25, p>0.05, Table 3). The latter indicates that FPG is unlikely to be of sufficient sensitivity to successfully evaluate new treatment strategies that focus on reducing postprandial hyperglycemia. For more long-term evaluation purposes, changes in blood HbA 1 c concentrations have generally been assessed, since blood HbA 1 c content correlates relatively well with both mean 24 h [32, 33] and postprandial glucose levels [START_REF] Salardi | The glucose area under the profiles obtained with continuous glucose monitoring system relationships with hba(lc) in pediatric type 1 diabetic patients[END_REF][START_REF] Sharp | Continuous glucose monitoring and haemoglobin a(1c)[END_REF]. In accordance, in the present study we observed strong correlations between HbA 1 c and mean 24 h glucose and postprandial glucose levels following lunch and dinner (Table 3). It should be mentioned here that even under clinically acceptable HbA 1 c levels (i.e. HbA 1 c ≤ 7.0 in 6 out of 11 diabetes patients) hyperglycemia can still be unacceptably large at 11±2 hours of blood glucose excursion >10 mmol/L per 24 h. Therefore, these results extend on earlier findings [24,34,[START_REF] Bonora | Plasma glucose levels throughout the day and hba(1c) interrelationships in type 2 diabetes: Implications for treatment and monitoring of metabolic control[END_REF], and strongly suggest that the ability of HbA 1 c to monitor postprandial hyperglycemia is debatable. Moreover, the measurement of prospective changes in blood HbA 1 c content only has sufficient sensitivity to detect changes in glucose homeostasis during middle to long-term interventions [START_REF] Miyazaki | Improved glycemic control and enhanced insulin sensitivity in type 2 diabetic subjects treated with pioglitazone[END_REF]. Therefore, the present study underlines the notion that CGMS is a promising tool when evaluating short-term (<3 months) changes in blood glucose homeostasis following pharmacological, dietary and/or exercise interventions [START_REF] Maran | Continuous subcutaneous glucose monitoring in diabetic patients: A multicenter analysis[END_REF].

Another benefit of the CGMS approach, that has potential clinical application as well, is the possibility to calculate the level of glycemic instability in insulin resistant states. This so-called Continuous Overall Net Glycemic Action (CONGAn) is probably a more appropriate measure to assess short-term changes in glucose homeostasis throughout the day [START_REF] Mcdonnell | A novel approach to continuous glucose analysis utilizing glycemic variation[END_REF]. This CGMS measure reflects the standard deviation of the differences in glucose concentration using varying time windows [START_REF] Mcdonnell | A novel approach to continuous glucose analysis utilizing glycemic variation[END_REF]. Therefore, we determined CONGAn values in both our diabetes patients and normoglycemic controls (Table 2). The proposed sensitivity of CGMS to detect subtle variations in glycemic control was confirmed in our normoglycemic control group. Interestingly, 2 of our control subjects appeared to have rather high postprandial CONGA1 values that almost approached values observed in the type 2 diabetes patients (i.e. average postprandial CONGA1 >2.1). These 2 subjects also showed the highest insulin values during the oral glucose tolerance test, and were the only 'normoglycemic' persons who showed some hyperglycemia throughout the day (data not shown). Altogether, our results suggest that more advanced CGMS analyses techniques provide promising measures to assess glycemic instability in diabetes patients [START_REF] Mcdonnell | A novel approach to continuous glucose analysis utilizing glycemic variation[END_REF]. Research is warranted to investigate the diagnostic value of CGMS in other diabetes related populations, like patients in a prediabetic and/or insulin resistant state.

In conclusion, detailed analyses of 24 h blood glucose profiles show that standard measures for glycemic stability grossly underestimate the amount of hyperglycemia during real-life conditions in type 2 diabetes patients. Given the macro-and microvascular damage caused by postprandial hyperglycemia, CGMS provides an excellent tool to more directly evaluate additional therapeutic strategies to reduce the amount of glycemic instability and risk of cardiovascular complications in type 2 diabetes patients. Hyperglycemic episodes, total time during which [glucose] levels are above 10.0 mmol/L 1 ; Hypoglycemic episodes, total time during which [glucose] levels are below 3.9 mmol/L 1 ; FPG, fasting glucose was determined from the calibrated CGMS curves 10 min before breakfast on the the first and second day; Mean Noct glucose, average glucose concentration between 24:00 and 07:00 h; CONGA1,2,4: continuous overall net glycemic action describing intra-day glycemic variability between respectively 1, 2 and 4 h time periods over 24 h ; AUC PP, area under the curve 6 h postprandial (mmol/L* 3 min ); CONGA1 glycemic variability between 1 h time periods. CONGA1 PP breakfast: 07:00 -12:00; PP lunch:13:00-18:00 ; PP dinner:19:00 -24:00; Nocturnal fasting: from 01:00 till 6:00 h under fasting conditions

Figure legends: Figure 1

 1 Figure legends:

Clinical Science Immediate Publication. Published on 13 Apr 2006 as manuscript CS20060041

Clinical Science Immediate Publication. Published on 13 Apr 2006 as manuscript CS20060041

Clinical Science Immediate Publication. Published on 13 Apr 2006 as manuscript CS20060041

Clinical Science Immediate Publication. Published on 13 Apr 2006 as manuscript CS20060041

Clinical Science Immediate Publication. Published on 13 Apr 2006 as manuscript CS20060041

Table 1

 1 Subjects' characteristics

	Controls	Diabetes

* BMI, body mass index; HbA 1 c, glycated hemoglobin; Fasting glucose was determined after 48 hour abstinence of blood glucose lowering medication and 15 min before the oral glucose tolerance test (OGGT); HOMA-IR, homeostasis model assessment insulin resistance index as desribed by Matthews et al ; OGIS, oral glucose insulin sensitivity-index for a 2 h OGTT as described by Mari et al

[START_REF] De Backer | European guidelines on cardiovascular disease prevention in clinical practice. Third joint task force of european and other societies on cardiovascular disease prevention in clinical practice[END_REF]

. *: significantly different between groups; P<0.01, †: P<0.001.

Table 2

 2 CGMS measurements

	Control subjects (n=11)	Diabetes patients (n=11)

Clinical Science Immediate Publication. Published on 13 Apr 2006 as manuscript CS20060041

ACKNOWLEDGEMENTS

The authors would like to thank A. Menarini Diagnostics BENELUX and Hanneke van Milligen for the technical support. We gratefully acknowledge our volunteers for participating in the experimental trials.


Use of the prone position in the acute respiratory distress syndrome: how should we assess benefit?

Use of the prone position in the acute respiratory distress syndrome -how should we assess benefit? Abstract

Prone positioning of patients with acute respiratory failure was first suggested over 30 years ago. In the present issue of Clinical Science, Reutershan and co-workers have studied the changes in end-expiratory lung volume in 12 patients with ARDS (acute respiratory distress syndrome) over an 8 h period following manual turning from the supine to prone position. From the data presented, the authors suggest that baseline end-expiratory lung volume could be used to identify responders, and serial measurements would permit appropriate 'dosing' of the therapy. Although this is an interesting study that provides data that have rarely been collected when assessing the response to prone positioning, there are a number of limitations that need to be considered. However, despite the limitations, the study does stimulate a number of important questions related not only to the use of the prone position, but also to the management of patients with ARDS in general.

Prone positioning of patients with acute respiratory failure was first suggested over 30 years ago [START_REF] Bryan | Comments of a devil's advocate[END_REF] . Although studies have repeatedly shown improvement in arterial oxygenation in about two-thirds of patients with the acute respiratory distress syndrome (ARDS) [START_REF] Bernard | The American-European Consensus Conference on ARDS Definitions, Mechanisms, Relevant Outcomes and Clinical Trial Coordination[END_REF] , no clear outcome benefit has been demonstrated largely because nearly all the studies have been small, directed at discovering the mechanism of the benefit and at attempting to predict which patients would benefit. Central to these studies has been the assumption that improvement in oxygenation would equate with outcome benefit. The only two large randomised controlled studies of prone positioning in acute lung injury (ALI) from Italy [START_REF] Gattinoni | Effect of prone positioning on the survival of patients with acute respiratory failure[END_REF] and France [START_REF] Guerin | Effects of systematic prone positioning in hypoxaemic acute respiratory failure: A randomized controlled trial[END_REF] showed the anticipated improvement in oxygenation but there was no survival benefit. Both of these studies have been criticised on the grounds of their design, the heterogeneity of the patients enrolled, protocol violations, for only providing prone position for brief periods (≤ 8hours per day) and for failing to adjust the ventilator settings in response to the improvement in gas exchange (not 'using the gain'). A Spanish study (Mancebo et al) achieved 20 hours per day in PP, did 'use the gain' to modify the ventilator settings (reduce FIO2 and PEEP) and demonstrated a 25% relative reduction in ICU mortality and a 19% reduction in hospital mortality but, due to recruitment difficulties, it was terminated prematurely and also failed to achieve conventional significance.

In the study by Reutershan and colleagues 5 changes in end-expiratory lung volume (EELV) in 12 patients with ARDS were measured over an eight hour period following manual turning from the supine to prone position. To their credit the authors give a clear description of how the patients were supported when in the prone position.

Since change in thoraco-abdominal compliance is a major factor in the change in distribution of ventilation that results from proning, the way in which the thorax and abdomen are supported can be of great significance. EELV was measured by manually ventilating the patient on a rebreathing bag containing 1% sulphur hexafluoride in oxygen and values were derived for relative FRC (EELV/predicted FRC). Even in this small study the baseline data reveal a group of patients that is in regard to the aetiology of lung injury (pulmonary vs extrapulmonary), the time from onset of ARDS and the level of positive end-expiratory pressure (PEEP) used.

There was also a surprising variation in the relative FRC with the majority showing the expected significant volume loss but with some patients at or above predicted values. Eight patients (66%) were defined as 'responders' on the basis of ≥ 30% increase in oxygenation index (PaO2/FIO2) but the time course of this improvement and the associated increase in EELV was variable with three patients achieving maximum recruitment within a few hours while in the other five patients recruitment continued through the entire proning period. Of the baseline parameters measured it was only EELV that predicted the gas exchange response to proning but the time course of the changes was patient specific and not predictable. The authors suggest that baseline EELV could be used to identify responders and serial measurements would permit appropriate 'dosing' of the therapy. This is an interesting study that provides data that have rarely been collected when assessing the response to prone positioning but some issues need to be highlighted. The technique described to measure EELV is neither practical in routine clinical practice nor arguably desirable in view of the risk of de-recruitment associated with removing patients with severe lung injury from the ventilator, notwithstanding the authors' practice of clamping the endotracheal tube at end expiration in an attempt to maintain PEEP. The patients were also paralysed for the procedure. The fact that duplicate measurements showed no difference between the first and second measurement does not necessarily exclude the possibility that the procedure influenced the measurement, only that the effect, however large or small, is consistent. Most of the previous studies that have explored the baseline characteristics that may be predictive of benefit from proning have used a lesser ( ≈ 20%) improvement in oxygenation to define a 'responder'. As an observational study it is understandable that the clinically determined ventilator settings were not changed during the study period but the tidal volumes used were consistently above the 6ml/kg demonstrated to have survival benefit in the ARDSnet study [START_REF] Nih | Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network[END_REF] published in 2000 and the higher levels of PEEP used in some of the patients would not be supported by a more recent study from the same group [START_REF] Brower | Higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome[END_REF] . Furthermore, improvements in gas exchange achieved in the responders were not used to permit more lung friendly ventilation by reducing the inspired oxygen concentration, tidal volume and PEEP. Although no mortality benefit would be expected in such a small study, it is interesting to note that survival was higher (75%) in the 'non responders' & than in the 'responders' (63%). Despite its limitations this paper stimulates a number of important questions related not only to the use of the prone position but also to our management of patients with ARDS in general:

(i)
How should we determine the 'optimum' ventilator settings particularly with regard to PEEP? Should we be guided by gas exchange benefit alone or, as the evidence increasingly suggests, beware of producing high airway pressures and overinflating normal lung? Would measurement of EELV be an appropriate way of assessing the need for and the impact of recruitment manoeuvres?

(ii) Which measurement should be used to define a 'responder' to a therapy such as proning and which intermediate measurements are the best surrogates for outcome benefit? If we become obsessed with an inappropriate intermediary endpoint the assessment of new therapies and the design of subsequent outcome studies will be seriously compromised. Could our fixation on improvement in indices of oxygenation be flawed? A previous therapy for ARDS, inhaled nitric oxide, produced dramatic improvements in gas exchange in the short term but proved to have no long term benefit 8 and may even have been harmful. The concept of ventilator induced lung injury (VILI) has now focussed our management not only on avoiding mechanical trauma due to high alveolar pressures and lung overdistension but also on avoiding ventilatory strategies that may cause increased cytokine release which may produce distant organ damage and contribute to multiple organ failure [START_REF] Ranieri | Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: A randomized controlled trial[END_REF][START_REF] Parsons | Lower tidal volume ventilation and plasma cytokine markers of inflammation in patients with acute lung injury[END_REF] . Surely any gain in gas exchange obtained with proning should be used to reduce both the inspired oxygen concentration and PEEP to the level at which maximum recruitment of collapsed lung is achieved but without causing damaging overdistension of more normal lung (iii) Should we rely on large randomised controlled studies to decide how to manage patients with ARDS and thereby adopt a 'one size fits all' policy ignoring the heterogeneity of this patient population or attempt to tailor management to the individual patient using measurements such as EELV?

The recently introduced technique of electrical impedance tomography promises the opportunity of the bedside measurement not only of lung volumes but also the distribution of ventilation and perfusion within the lung in the ventilated patient and may prove to be a more feasible way of collecting the data that this paper suggests is important in assessing and titrating the use of prone positioning and other therapies that will hopefully improve the outcome from ARDS.


Wolfgang Lieb

MD Juliane Bolbrinker

MD Angela Döring

MD; Hans-Werner Hense

MD Jeanette Erdmann

PhD Heribert Schunkert

MD, FAHA Reinhold Kreutz
email: reinhold.kreutz@charite.de

No association of the CYP3A5*1 allele with blood pressure and left ventricular mass and geometry: the KORA/MONICA Augsburg echocardiographic substudy

Keywords: CYP3A, blood pressure, heart, hypertension, pharmacogenetics -1 -

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Recently, a polymorphism in an isoenzyme of the cytochrome P-450 3A (CYP3A) enzyme, i.e. CYP3A5, has attracted increasing attention in cardiovascular research. This results from the fact that CYP3A5 is not only involved in drug metabolism but is also expressed in the human kidney where it is the major CYP3A enzyme [START_REF] Givens | CYP3A5 genotype predicts renal CYP3A activity and blood pressure in healthy adults[END_REF][START_REF] Haehner | Bimodal distribution of renal cytochrome P450 3A activity in humans[END_REF][START_REF] Wojnowski | Genetics of the variable expression of CYP3A in humans[END_REF]. The renal expression of CYP3A5 in epithelial cells of the proximal tubule and collecting duct [4;5] represented the basis for its implication in blood pressure and volume control and thus the pathogenesis of arterial hypertension. One potential mechanism by which CYP3A5 could mediate blood pressure effects results from the fact that CYP3A enzymes metabolize endogenous corticosteroids and mineralocorticoids in humans and animals to their 6ß-hydroxylated metabolites [3;6-8]. In the kidney, this could lead to a decreased stimulation of the mineralocorticoid receptor and thus lower sodium reabsorption via the epithelial sodium channel [6;9]. Interestingly, the expression of CYP3A5 has a strong genetic basis and is linked to a single nucleotide polymorphism (A6986G) in intron 3 of CYP3A5 that disturbs correct splicing of transcripts and leads to low expression and enzyme activity. Thus, low expression of CYP3A5 is found in homozygous carriers of the CYP3A5*3 allele while homozygous and heterozygous carriers of the CYP3A5*1 allele exhibit high expression of CYP3A5 [1;3;10;11]. Therefore, the observed higher renal expression of CYP3A5 in carriers of CYP3A5*1 could affect blood pressure regulation in these individuals.

Recent studies revealed, however, controversial results as to the role of CYP3A5*1 for blood pressure regulation and its association with hypertension [1;12-15]. The aim of the current study therefore was to further test the role of CYP3A5*1 in a large population-based sample of Caucasian origin including both women and men in whichafter exclusion of individuals on antihypertensive medications -one third (33.3%) of the individuals were hypertensive (blood pressure ≥140/90 mm Hg). The functional effects of CYP3A5 in the cardiovascular system could be mediated by its effects on renal sodium reabsorption [6;9;16]. Sodium intake and salt-sensitive blood pressure regulation are significantly linked to cardiac structure, LVH [17;18] and left ventricular dysfunction [START_REF] Rothermund | Endothelin-A receptor blockade prevents left ventricular hypertrophy and dysfunction in salt-sensitive experimental hypertension[END_REF]. Furthermore, LVH represents one of the most frequent and clinically important hypertensive organ damages [START_REF] Koren | Comparison of left ventricular mass and geometry in black and white patients with essential hypertension[END_REF][START_REF] Kannel | Risk stratification in hypertension: new insights from the Framingham Study[END_REF][START_REF] Lorell | Left ventricular hypertrophy: pathogenesis, detection, and prognosis[END_REF] and salt-sensitive hypertension contributes significantly to its development [19;23]. Therefore, the effect of CYP3A5*1 on echocardiographically determined parameters of left ventricular mass and geometry was also evaluated in the present study.

Methods

Study Population

The subjects of this study participated in the echocardiographic substudy (total n = 1674) of the third MONICA (MONItoring of trends and determinants in CArdiovascular disease) Augsburg survey [START_REF] Schunkert | Lack of association between a polymorphism of the aldosterone synthase gene and left ventricular structure[END_REF], which is now continued in the framework of KORA (Cooperative Health Research in the Augsburg Area). Individuals were all Caucasians and originated from a gender-and age-stratified random sample of all German residents of the Augsburg area. The third survey represents individuals 25 to 74 years of age, and about 300 subjects for each 10-year increment. The population was studied by physical examination, blood testing, and a standardized interview including medical history, physical activity, medication and personal habits. Resting blood pressure was taken according to MONICA guidelines using the random zero sphygmomanometer after subjects had been resting in a sitting position. Individuals on antihypertensive medications were excluded and the mean of the second and third recording was calculated and employed to define hypertension as a blood pressure ≥ 140/90 mm Hg. Body weight in kilogram and height in meters were determined with subjects wearing light clothing. Written informed consent was obtained from all subjects, and a local ethical committee approved the study protocol.

Echocardiographic Measurements

Two-dimensional guided M-mode echocardiograms were obtained by 2 expert sonographers using the Sonos 1500 (Hewlett Packard Inc.). M-mode tracings were recorded on strip-chart paper at 50 mm/s. To reduce interobserver variability, all Mmode tracings were analyzed by a single cardiologist who was blinded for clinical and biochemical data. Measurements for M-Mode guided calculation of the left ventricular mass were taken just below the tip of the mitral valve. Only high-quality tracings that demonstrated optimal visualisations of endocardial and epicardial surfaces throughout the cardial cycle were used. This resulted in exclusion of 16% of potential subjects. Left ventricular end-diastolic diameter (LVEDD), septal wall (SWT) and posterior wall (PWT) thickness were measured according to the recommendations of the American Society of Echocardiography (ASE) [START_REF] Devereux | Standardization of M-mode echocardiographic left ventricular anatomic measurements[END_REF]. Left ventricular mass (LVM) was calculated using the formula LVM (g) = 0.8x1.04[(LVEDD+SWT+PWT)³-LVEDD³] + 0.6 as described by Devereux et al. [START_REF] Devereux | Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings[END_REF]. LVM was indexed to body surface area (BSA), LVMI=LVM/BSA. Left ventricular hypertrophy was defined as LVMI > 134 g/m² in men and LVMI > 110 g/m² in women [START_REF] Schunkert | Associations between circulating components of the renin-angiotensin-aldosterone system and left ventricular mass[END_REF].

Biochemical Analyses

Blood was drawn for biochemical analyses from non-fasting subjects. Creatinine was assessed quantitatively with an enzymatic colorimetric test (Hitachi 717, Boehringer Mannheim, Germany). Glomerular filtration rate was estimated (eGFR) using the modified MDRD (Modification of Diet in Renal Disease) equation: eGFR(ml/min/1.73m²) =186 x (Scr) -1.154 x (Age) -0.203 x (0.742 if female) [START_REF] Levey | National Kidney Foundation practice guidelines for chronic kidney disease: evaluation, classification, and stratification[END_REF].

CYP3A5 genotype determination

Genotyping was performed by real-time PCR as reported [START_REF] Kreutz | The effect of variable CYP3A5 expression on cyclosporine dosing, blood pressure and long-term graft survival in renal transplant patients[END_REF]. The ABI PRISM ® 7000 SDS instrument in conjunction with the ABI TaqMan Universal Master Mix (Applied Biosystems, Darmstadt, Germany) was used to perform the assays [START_REF] Ranade | High-throughput genotyping with single nucleotide polymorphisms[END_REF]. Appropriate primers and fluorogenic probes were designed with the Primer Express ® software. Fluorogenic probes were synthesized by TIB Molbiol (Berlin, Germany) and primers were obtained from Proligo (Paris, France). Primers were selected according to the sequence available at GenBank accession number NG_000004 [START_REF] Smith | Molecular genetics of the human cytochrome P450 monooxygenase superfamily[END_REF]. The following probes and primers were used for genetic determination of the CYP3A*1 and CYP3A5*3 alleles (nt 260167G>A): the fluorogenic probes were 5'VIC- All p-values should be considered descriptive.

Results

In total, 670 women and 689 men were successfully genotyped. 2.

Association of CYP3A5*1 with blood pressure measurements and hypertension

In the overall analysis the presence of the CYP3A5*1 allele exhibited no effect on systolic and diastolic blood pressure and pulse pressure in both men and women (Table 3). Overall, 361 (33.3%) individuals were diagnosed with hypertension (Table 3). In normotensive females a somewhat higher allele frequency for CYP3A5*1 was observed compared to the other groups.

However, no association between the presence of CYP3A5*1 and hypertension was detected in separate analysis in men and women (Table 3).

Association of CYP3A5*1 with left ventricular mass and geometry

Left ventricular mass measurements could be determined echocardiographically in 942 individuals (464 men and 478 women). No association of the CYP3A5*1 allele with LVMI, SWT, PWT and LVEDD could be found in multivariate analyses in males and females and in the combined analysis of both genders (Table 1

Association of CYP3A5*1 with eGFR

We also analyzed the association of CYP3A5*1 with renal function represented by estimated GFR (eGFR) values, which demonstrated no association in the entire popuation and in women (Table 1). In the entire population, the observed adjusted difference assuming a dominant model for the rare CYP3A5*1 allele is 1.06 (95% CI -3.02 to +5.13) In men, a weak evidence for association was found (p=0.045). The genotype distribution was not different between individuals with mildly impaired renal function (eGFR < 90 ml/min/1.73m²) and individuals with normal renal function (eGFR ≥ 90 ml/min/1.73m²), neither in the combined analysis of both genders nor in separate analysis according to gender (data not shown).

Discussion

The research on the role of the CYP3A5 polymorphism in blood pressure regulation has been stimulated by the initial study in a small group of African-Americans in which higher blood pressures were reported in individuals carrying the CYP3A5*1 allele [START_REF] Givens | CYP3A5 genotype predicts renal CYP3A activity and blood pressure in healthy adults[END_REF].

Subsequently, additional studies analyzed the role of CYP3A5 on blood pressure in different populations producing controversial results [9;12-14;29]. In this regard it is important to consider that the frequency of carriers of CYP3A5*1, i.e. high expressors of CYP3A5, shows extreme variations across human populations [START_REF] Wojnowski | Genetics of the variable expression of CYP3A in humans[END_REF] and is significantly correlated with the distance from the equator with the highest frequency of approximately 70% observed in Africans and the lowest with approximately 10% found in European Caucasians [START_REF] Thompson | CYP3A variation and the evolution of salt-sensitivity variants[END_REF]. Consequently, the relevance of CYP3A5*1 may vary population, in which we also determined lower systolic blood pressures and pulse pressures but not diastolic blood pressures in carriers of the CYP3A5*1 allele [START_REF] Kreutz | The role of the cytochrome P450 3A5 enzyme for blood pressure regulation in the general Caucasian population[END_REF]. Taken together the latter two studies are in agreement with the postulated mechanism that renal CYP3A5 expression confers a blood pressure lowering effect by decreasing renal sodium reabsorption [START_REF] Kreutz | The role of the cytochrome P450 3A5 enzyme for blood pressure regulation in the general Caucasian population[END_REF]. Nevertheless, these findings are at variance with the data obtained in the current report, in which we found no association between CYP3A5*1

and any blood pressure phenotype in both men and women. This is of interest since the subgroup analysis in the PREVEND study population according to gender revealed that CYP3A5*1 exhibited a significant effect on systolic blood pressure and pulse pressure only in females but not in males [START_REF] Kreutz | The role of the cytochrome P450 3A5 enzyme for blood pressure regulation in the general Caucasian population[END_REF].

Importantly, in the current analysis of the KORA/MONICA echocardiographic substudy population we could not find an association of the CYP3A5*1allele with left ventricular mass measurements or LVH in multivariate models. The finding on left ventricular echocardiographic parameters is in line with the observation reported by Fromm et al. who also performed echocardiographic analyses of left ventricular mass in their young male individuals and detected no effect of CYP3A5*1 on cardiac parameters although they detected significant lower systolic blood pressures in carriers of CYP3A5*1 [START_REF] Fromm | CYP3A5 genotype is associated with elevated blood pressure[END_REF].

In addition to blood pressure effects Givens et al. reported in their small study in young healthy Afro-Americans an association between CYP3A5*1 and GFR measurements, i.e. creatinine clearance [START_REF] Givens | CYP3A5 genotype predicts renal CYP3A activity and blood pressure in healthy adults[END_REF]; this observation was only made in females but not in males by comparing 10 female CYP3A5*1 carriers with 5 female non-carriers. This finding should be viewed with caution, since the numbers in the latter study were clearly very small and a confounding factor due to differences in body-mass-index in females could not be excluded in this study because values for creatinine clearance were not adjusted to body surface area [START_REF] Givens | CYP3A5 genotype predicts renal CYP3A activity and blood pressure in healthy adults[END_REF]. In the more recent study by Bochud et al. CYP3A5*1 exhibited a significant effect on GFR measured by inulin clearance (P=0.045), however, the significance disappeared after the adjustment for body surface (P=0.093), while no significant association with creatinine clearance was observed [START_REF] Bochud | Association of CYP3A5 genotypes with blood pressure and renal function in African families[END_REF]. In contrast, Ho et al.

found in their study no effect of CYP3A5*1 on creatinine clearance and urinary albumin excretion in both Afro-Americans and Caucasian individuals. Similarly, Fromm et al.

found also no effect of CYP3A5*1 on GFR measurements in their carefully studied cohort of young Caucasian individuals [START_REF] Fromm | CYP3A5 genotype is associated with elevated blood pressure[END_REF]. In this regard a limitation of our study results from fact that renal sodium handling was not studied and direct GFR measurements were not available. Nevertheless, determination of eGFR values by the MDRD equation [START_REF] Levey | National Kidney Foundation practice guidelines for chronic kidney disease: evaluation, classification, and stratification[END_REF] revealed overall no significant effect of CYP3A5*1, while only a subgroup analysis according to gender indicated higher GFR levels in male but not female carriers of CYP3A5*1. Taken together, as evident from the data reported so far consistent effect of CYP3A5*1 on GFR is far from being established.

Although Copyright 2006 The Biochemical Society elevated blood pressures [START_REF] Fromm | CYP3A5 genotype is associated with elevated blood pressure[END_REF]. In this study the authors could demonstrate that 96 individuals with the CYP3A5*3/*3 genotype demonstrated on average 5 mmHg higher systolic blood pressure values compared to the 9 carriers of the CYP3A5*1 allele, while serum aldosterone levels were significantly lower in CYP3A5*3/*3 suggesting a compensatory suppression of aldosterone in response to higher systolic blood pressures in these individuals [START_REF] Fromm | CYP3A5 genotype is associated with elevated blood pressure[END_REF]. Consequently, it appears of interest to further pursue this hypothesis and to perform both experimental and clinical studies on the role of CYP3A5 in blood pressure control.

Table 1

 1 6 individuals were excluded because of missing covariates. Furthermore, 269 individuals with antihypertensive medication (131 men, 138 women) were excluded, leaving 530 women and 554 men for the final analyses. The allele frequencies of the CYP3A5*1 and CYP3A5*3 allele were 5.8% and 94.2%, respectively. displays baseline characteristics, blood pressure levels, left ventricular mass measurements as well as eGFR measurements according to CYP3A5 genotype in the overall study population and stratified by gender. Carriers of the CYP3A5*1 allele were slightly younger than non-carriers, while no differences in BMI and the prevalence of diabetes were found in men and women between carriers and non-carriers of the CYP3A5*1 allele. The baseline characteristics of the study population, stratified by hypertension status and gender are shown in Table

	The CYP3A5*1/*1, CYP3A5*1/*3, and CYP3A5*3/*3 genotypes were found in 0.5%,
	10.6%, and 88.9% of the population. These frequencies do not deviate from those
	predicted by Hardy-Weinberg equilibrium and were similar to those reported in
	Caucasians [9].

Clinical Science Immediate Publication. Published on 6 Jul 2006 as manuscript CS20060075

  the number of 1084 individuals analyzed herein in the KORA/MONICA study population is still considerable high and exceeds by far the figures reported in Caucasians by others[1;13;14] it is still significantly lower compared to 6777 individuals analyzed in the PREVEND study[START_REF] Kreutz | The role of the cytochrome P450 3A5 enzyme for blood pressure regulation in the general Caucasian population[END_REF]. Therefore, the negative result in the current study may be in part related to a lack of statistical power. The observed adjusted

	difference between CYP3A5*1/*1 + CYP3A5*3/*1 individuals compared to
	CYP3A5*3/*3 individuals is -0.40 (95% CI -3.45 to +2.65) mm Hg for SBP and 0.31
	(95% CI -1.67 to +2.30) mm Hg for DBP. Thus, with a confidence of 95%, the true
	difference between the groups lies within this interval and a mean difference smaller
	than -3.45 or greater than 2.65 mm Hg for SBP and smaller than -1.67 or greater than
	2.30 mm Hg for DBP can be excluded. For LVMI, differences smaller than -3.81 or
	greater than 2.34 g/m² between carriers and non-carriers of the CYP3A5*1 allele can be
	excluded with 95% confidence.
	Another important issue relates to the higher frequency of individuals with hypertension
	(33.3%; defined as BP ≥ 140/90 mm Hg) analyzed in the current KORA/MONICA
	study compared to PREVEND in which hypertension was less prevalent (22%). In this
	regard it appears possible that the functional effect of CYP3A5*1 depends on the blood
	pressure level and stage of hypertension and might be more relevant -in agreement with
	our recent analysis in PREVEND [9] and the study by Fromm et al. [14] -at lower
	blood pressure values. The biological effect at higher blood pressure levels could be
	blunted or eliminated due to other genetic factors that come into play and/or

compensatory mechanisms. The potential relevance of adaptive mechanisms has been highlighted in the recent study in young male individuals with normal or mildly -11 -

Table 3 .

 3 Genotype distribution of CYP3A5 genotypes, CYP3A5*1 allele frequency in men and women with and without arterial hypertension and OR (95% CI) for hypertension.

		Men		Women
		Normotensive	Hypertensive	Normotensive	Hypertensive
	All individuals	335	219	388	142
	Genotype
	CYP3A5*3/*3, n (%)	304 (90.7)	197 (90.0)	335 (86.3)	128 (90.1)
	CYP3A5*3/*1, n (%)	31 (9.3)	21 (9.6)	50 (12.9)	13 (9.2)
	CYP3A5*1/*1, n (%)	0	1 (0.4)	3 (0.8)	1 (0.7)
	CYP3A5*1 allele frequency	0.046	0.053	0.072	0.053
	OR (95% CI), p-value	1.26 (0.69-2.32), p=0.46	0.85 (0.43-1.67), p=0.63
	Combined men and women
	OR (95% CI), p-value		1.02 (0.66-1.60), p=0.92

OR (95% CI) for hypertension, adjusted for age, BMI, and gender were computed for the analysis comparing CYP3A5*3/*3 (reference) individuals with CYP3A5*3/*1 + CYP3A5*1/*1 individuals. Two-tailed descriptive pvalues from WALD-χ ² -test are given.

Clinical Science Immediate Publication. Published on 6 Jul 2006 as manuscript CS20060075 Copyright 2006 The Biochemical Society

Clinical Science Immediate Publication. Published on 6 Jul 2006 as manuscript CS20060075

Acknowledgements: This study was supported by a grant from the Bundesministerium für Bildung und Forschung (Dr. Kreutz), Nationales Genomforschungsnetz (Grant KGCV1, 01GS0416 to Dr. Kreutz; 01GS0418 to Drs Schunkert and Erdmann).

Clinical Science Immediate Publication. Published on 6 Jul 2006 as manuscript CS20060075

Copyright 2006 The Biochemical Society between different ethnic populations [START_REF] Wojnowski | Genetics of the variable expression of CYP3A in humans[END_REF]. Due to the relative low frequency in Caucasians it appears particularly important to study large cohorts in this ethnic group.

In the study by Ho et al. no association between CYP3A5*1 and hypertension was found in white patients from North America, while the proportion of salt-sensitive subjects was higher in white hypertensive CYP3A5*1 carriers compared to non-carriers [START_REF] Ho | Association between the CYP3A5 genotype and blood pressure[END_REF]. In a population of elderly individuals (age ≥ 75 years) from Finland it was shown, that the proportion of individuals carrying the CYP3A5*1 allele was significantly higher in hypertensives compared to normotensive elderly subjects suggesting a role of CYP3A5*1 for hypertension development in elderly Caucasian subjects [START_REF] Kivisto | CYP3A5 Genotype is Associated with Diagnosis of Hypertension in Elderly Patients: Data from the DEBATE Study[END_REF]. More recently, Bochud et al. analyzed the effect of CYP3A5 polymorphisms on ambulatory blood pressure and GFR in a cohort of 375 individuals recruited from family with at least two hypertensive siblings [START_REF] Bochud | Association of CYP3A5 genotypes with blood pressure and renal function in African families[END_REF]. The authors obtained an allele frequency of 43.7% for CYP3A5*1 compared to 5.8% in the Caucasian cohort of the current report.

Although crude blood pressure levels were similar between carriers and non-carriers of the CYP3A5*1 allele in the study by Bochud et al., the authors detected a significant positive interaction between CYP3A5*1 and age for systolic and diastolic ambulatory blood pressure during daytime but not at night. They reported that CYP3A5*1 carriers demonstrated an age-related significant blood pressure increase. Interestingly, Bochud et al. also investigated the effect of CYP3A5*1 on renal sodium handling by fractional excretion of lithium and found that non-carriers of CYP3A5*1 decreased their proximal tubular sodium reabsorption with age, while CYP3A5*1 carriers did not. Further analysis between urinary sodium excretion -reflecting dietary salt intake -and ambulatory blood pressure indicated, that CYP3A5*1 carriers tended to increase their night-time blood pressure with increasing dietary salt-intake more than non-carriers.

The authors suggested [START_REF] Bochud | Association of CYP3A5 genotypes with blood pressure and renal function in African families[END_REF] that carriers of CYP3A5*1 appear to be more salt-sensitive than non-carriers and supported a role for CYP3A5*1 in salt-sensitive hypertension, which might be particularly important with increasing age [16;32].

In contrast, CYP3A5*1 was found to be associated with lower systolic blood pressure, but not diastolic blood pressure, in a homogenous group of 115 young white, male students with normal or mildly elevated blood pressures [START_REF] Fromm | CYP3A5 genotype is associated with elevated blood pressure[END_REF]