Marcel Cg Van De Poll

Hc Cornelis

Marc Ajg Dejong

Aalt Fischer

Ger H Bast

Koek

Mcg Van De Poll

Chc Dejong

Majg Fischer

A Bast

MD G H Koek

Decreased hepatosplanchnic antioxidant uptake during hepatic ischemia-reperfusion in patients undergoing liver resection

Keywords: Liver, Ischemia Reperfusion, Oxidative Stress, Glutathione, Uric Acid, Xanthine oxidase, Antioxidants, Carotenoids

Introduction

Minimization of blood loss during liver resection is essential to limit complications. To diminish blood loss during liver resection hepatic inflow occlusion or "Pringle manoeuvre", was introduced by JH Pringle in 1908 [START_REF] Pringle | Notes on the arrest of hepatic hemorrhage due to trauma[END_REF]. However, the Pringle manoeuvre leads to hepatic ischemia-reperfusion injury. Ischemia is characterized by ATP depletion and necrotic cell death [START_REF] Malhi | Apoptosis and necrosis in the liver: A tale of two deaths?[END_REF], subsequent reperfusion aggravates cell injury. An important factor in this process is the formation of reactive oxygen species (ROS) immediately after reperfusion [START_REF] De Groot | Reoxygenation injury in rat hepatocytes: mediation by O2/H2O2 liberated by sources other than xanthine oxidase[END_REF][START_REF] Caraceni | Proteins but not nucleic acids are molecular targets for the free radical attack during reoxygenation of rat hepatocytes[END_REF]. This leads to peroxidation of membrane phospholipids [START_REF] Fukai | Lipid peroxidation during ischemia depends on ischemia time in warm ischemia and reperfusion of rat liver[END_REF] and oxidative damage to intracellular proteins [START_REF] Caraceni | Proteins but not nucleic acids are molecular targets for the free radical attack during reoxygenation of rat hepatocytes[END_REF]. Classically, the generation of ROS has been ascribed to an increased conversion of xanthine dehydrogenase into xanthine oxidase during ischemia [START_REF] Granger | Superoxide radicals in feline intestinal ischemia[END_REF], but concern about the validity of this concept has been raised [START_REF] De Groot | Reoxygenation injury in rat hepatocytes: mediation by O2/H2O2 liberated by sources other than xanthine oxidase[END_REF][START_REF] Jaeschke | Xanthine oxidase-induced oxidant stress during hepatic ischemia-reperfusion: are we coming full circle after 20 years?[END_REF]. More recently, attention has moved to the mitochondrial respiratory chain as the principal source of ROS during ischemia and reperfusion [START_REF] De Groot | Reoxygenation injury in rat hepatocytes: mediation by O2/H2O2 liberated by sources other than xanthine oxidase[END_REF]8], although the hypothesis that xanthine oxidase activity is an important source of ROS formation during hepatic ischemia-reperfusion is still debated [START_REF] Fernandez | Preconditioning protects liver and lung damage in rat liver transplantation: role of xanthine/xanthine oxidase[END_REF]. ROS oxidize glutathione (GSH) to glutathione disulfide (GSSG) [START_REF] Anderson | Glutathione: an overview of biosynthesis and modulation[END_REF] which renders cells prone to cell death [START_REF] Nagai | Reduced glutathione depletion causes necrosis and sensitization to tumor necrosis factor-alpha-induced apoptosis in cultured mouse hepatocytes[END_REF][12][START_REF] Chen | Glutathione depletion renders rat hepatocytes sensitive to nitric oxide donormediated toxicity[END_REF]. Endogenous and exogenous antioxidants offer protection against the action of ROS, consequently antioxidants may become depleted during excessive oxidative stress. Most data on hepatic ischemia reperfusion damage are derived from animal studies, which generally rely on prolonged continuous ischemia to render maximal effects. The severity of these models complicates immediate translation of these results to human clinical practice.

Altough ROS formation is generally associated with cell death as outlined above, recent data suggest that ROS are important signal molecules that activate

Materials and Methods

Patients

Ten patients (male:female 6:4) undergoing partial hepatectomy for secondary liver malignancies (colorectal cancer (n=9), insulinoma (n=1)) were studied after an overnight fast. All patients had normal liver function as assessed by preoperative laboratory parameters. They were on their standard diets in the pre-study period.

Patient characteristics are summarized in Table 1. The protocol has been reviewed and approved by the Medical Ethical Committee of the University Hospital Maastricht and all subjects provided written informed consent. The study adhered to the declaration of Helsinki.

Anesthesia and surgical procedure

All patients had radial artery and central venous catheters inserted pre-operatively to monitor arterial and central venous blood pressure as part of standard anesthetic care. Surgical procedures were performed as detailed elsewhere [START_REF] Dejong | Neoplasms of the liver[END_REF]. Liver surgery was performed using a Cavitron Ultrasonic Surgical Aspirator (Valleylab, Tyco healthcare, Boulder, CO, USA) and electrocautery or ligation of vessels as appropriate.

Intermittent Pringle manoeuvre

During

Sample collection

Using the same approach as we have published recently [START_REF] Patel | Early stress protein gene expression in a human model of ischemic preconditioning[END_REF], liver biopsies and blood samples were taken prior to and following ischemia and after reperfusion according to the schedule shown in Figure 1. Liver-tissue samples (approximately 200 mg) were excised with a scalpel from nontumorous regions of the liver, distant from the transection plane. Simultaneously blood was drawn from the arterial line and from a hepatic vein that was draining the (post-)ischemic part of the liver, allowing the measurement of AV differences across the hepatosplanchnic bed. Post-ischemia blood samples were taken immediately after hepatic blood flow was restored.

Sample processing

Blood was collected into EDTA containing tubes (Becton, Dickinson Vacutainer, Franklin Lakes, NJ,USA). All blood samples were immediately put on ice, and kept on ice during sample preparation. Plasma was obtained by centrifugation (800 x g for 10 min at 4°C) and stored at -80°C until analysis. Liver biopsies were divided in two pieces and rapidly frozen in liquid nitrogen. Prior to analyses liver biopsies were homogenized in milliQ water (150 mg tissue/ml)

Laboratory analyses

MDA assay

The assay is based on the formation of a coloured adduct of MDA-like breakdown products of lipids with 2-thiobarbituric acid (TBA) [START_REF] Lepage | Preparative steps necessary for the accurate measurement of malondialdehyde by highperformance liquid chromatography[END_REF][START_REF] Guillen-Sans | The thiobarbituric acid (TBA) reaction in foods: a review[END_REF]. One hundred µl of plasma was added to 1 ml of reagent (containing 0.012 mol/l TBA, 0.32 mol/l o-phosphoric acid, 0.68 mmol/l BHT and 0.01 % (m/v) EDTA). The mixture was incubated for 1 hour at 100° C in a water bath. After cooling, the TBA-product was extracted with 500 µl butanol. Thirty µl of the butanol layer was injected on an HPLC system (Agilent, Palo Alto, CA, USA) equipped with a fluorescence detector, set on an excitation wavelength of 532 nm and emission wavelength of 553 nm, and a Nucleosil C18 column, 150 x 3.2 mm, particle size 5 µm (Supelco Inc., Bellefonte, PA, USA).

Samples were eluted with 65 % (v/v) MilliQ, 35 % (v/v) methanol, containing 0.05% trifluoric acid. A calibration curve was constructed using malonaldehyde bis(diethylacetal) as standard.

Uric acid assay

Uric acid, the reaction product of xanthine oxidase activity was determined using an HPLC method described by Lux et al. [19] Plasma antioxidant capacity (TEAC) assay Plasma antioxidant capacity was assayed as described by Fischer et al. [START_REF] Fischer | Determination of the antioxidant capacity in blood[END_REF] Briefly, 950 µl of ABTS •+ radical solution was incubated for 1 minute at 37°C, thereafter 50 µl deproteinized plasma was added. After 5 minutes incubation the absorption at 734 nm was measured. The decrease in absorption after 5 minutes relative to blank (buffer) was related to that of trolox calibrators. The resulting value is expressed as

Hepatic GSH and GSSG content

GSH and GSSG concentrations were measured in the supernatant of homogenized liver biopsies as described by Anderson [START_REF] Anderson | Determination of glutathione and glutathione disulfide in biological samples[END_REF]. Total protein concentrations in the homogenates was measured using the bicinchoninic acid method as described by Smith et al. [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF]. Final concentrations of GSH and GSSG were expressed per mg protein.

Hepatic levels of carotenoids and -tocopherol

The analysis of the concentrations of the different carotenoids and tocopherols in the supernatant of homogenized liver biopsies was performed as described by Broekmans et al. [23] Liver biopsies were homogenized in MilliQ (150 mg tissue per mL. Final concentrations of carotenoids and alpha tocopherol were expressed per mg protein.

Statistics

Values are expressed as mean ± SEM. Overall changes in plasma and tissue levels, during the experiment were assessed by one-way ANOVA and Bonferroni posttesting, comparing values at each timepoint with baseline.

For arteriovenous gradients a fictive timepoint with an arteriovenous gradient of zero was included in the ANOVA and Bonferroni post-testing was performed for values at each timepoint versus baseline as well as versus this theoretical zero gradient. This approach is in fact a modification of the one-sample t-that which calculates whether

Results

MDA in plasma

The mean arterial plasma level of malondialdehyde (MDA), was 1.2 ± 0.1 µmol/l. This systemic level did not change significantly during the intermittent Pringle manoeuvre (Figure 2a). In contrast, the hepatosplanchnic MDA gradient tended to increase already after 15 minutes ischemia (95% CI of the mean increase vs baseline: -0.10 -0.26 µmol/l, Bonferroni's test), indicating the occurrence of oxidative stress and lipid peroxidation during ischemia. After a cumulative ischemia time of 30 minutes, hepatosplanchnic MDA release tended to remain elevated above baseline values (95% CI of the mean increase vs baseline: -0.05 -0.31 µmol/l, Bonferroni's test) during the second reperfusion period (Figure 2b).

Hepatosplanchnic uric acid release

Mean (SEM) arterial plasma level of uric acid (250 ± 16 µmol/l at baseline) increased significantly after 15 minutes of hepatic ischemia (Figure 3a). Hepatosplanchnic uric acid release, indicative of xanthine oxidase activity, was not significantly different from zero at baseline, but increased significantly following both periods of 15' hepatic ischemia. Upon reperfusion hepatosplanchnic uric acid release returned to baseline values (Figure 3b).

Plasma antioxidant capacity during intermittent Pringle manoeuvre

A gradual decline of plasma antioxidant capacity (TEAC -[uric acid]) was found during the experiment (Figure 4a). At baseline there was a negative hepatic venousarterial gradient of plasma antioxidant capacity, indicating net hepatosplanchnic uptake of antioxidants from the plasma. Net hepatosplanchnic uptake however, disappeared gradually during the intermittent Pringle manoeuvre (Figure 4b).

GSH and GSSG during intermittent hepatic ischemia and reperfusion

Hepatic tissue GSH content increased significantly during hepatic inflow occlusion and returned to baseline values during reperfusion (Figure 5a), no evidence for GSH depletion was found. Also hepatic GSSG content increased significantly after 15' ischemia but tended to keep increasing after 5' reperfusion (95% CI of the mean increase vs baseline: -0.03 -0.74 µmol/g protein, Bonferroni's test). Thereafter a rapid decline of hepatic GSSG content was found, resulting in a return to baseline values (Figure 5b). The ratio GSSG:GSH, indicative for cellular redox state showed a transient increasing tendency during the study (Figure 5c).

Carotenoids and -tocopherol

Lycopene and -carotene were the most abundant carotenoids in the liver (mean ± SEM) 61.8 ± 20.5 nmol/g protein and 16.6 ± 2.8 nmol/g protein respectively.

Expressed per gram liver hepatic carotenoids levels were found to be 0.5 ± 0.09 nmol/g liver for lutein, 7.5 ± 2.4 nmol/g liver for lycopene and 2.0 ± 0.4 nmol/g liver for -carotene, these values are in approximate agreement with previous human data [START_REF] Tanumihardjo | Retinyl ester (vitamin A ester) and carotenoid composition in human liver[END_REF][START_REF] Schmitz | Concentrations of selected carotenoids and vitamin A in human liver, kidney and lung tissue[END_REF]. Hepatic tissue levels of carotenoids and -tocopherol were not significantly affected by intermittent hepatic ischemia and reperfusion (Table 2).

Discussion

According to our knowledge, this is the first study which meticulously describes the development of oxidative stress during hepatic ischemia and reperfusion and its effect on antioxidant status in humans. During the Pringle manoeuvre the hepatoduodenal ligament is temporarily ligated, resulting in total obstruction of both portal venous and hepatic arterial inflow and in an obstruction of biliary outflow. As pointed out earlier, human studies on this subject are scarce and are limited to the description of changes in systemic plasma markers of oxidative stress. In these studies no changes in malondialdehyde and GSH levels could be detected in peripheral blood [START_REF] Vriens | The role of allopurinol in human liver ischemia/reperfusion injury: a prospective randomized clinical trial[END_REF]. By measuring transorgan gradients and by taking liver biopsies however, we could reveal a profound effect of ischemia and reperfusion on hepatic oxidative stress and antioxidant status. No differences were observed between cases where a selective or a complete hepatic inflow occlusion was used. In addition it may be expected that the intermittent hepatic inflow occlusion provided some ischemic preconditioning during the second period of ischemia [START_REF] Patel | Early stress protein gene expression in a human model of ischemic preconditioning[END_REF]. This may hamper translation of the present findings to situations where a similar cumulative ischemic period is applied in a continuous fashion. Most likely prolonged ischemia exaggerates oxidative stress upon reperfusion [START_REF] Fukai | Lipid peroxidation during ischemia depends on ischemia time in warm ischemia and reperfusion of rat liver[END_REF].

Lipid peroxidation, evidenced by MDA release, occurred already during ischemia, within 15 minutes of hepatic inflow occlusion. Moreover, we simultaneously observed a significant increase of hepatosplanchnic release of uric acid. Uric acid is generated upon oxidation of xanthine by xanthine oxidase, yielding equimolar amounts of uric acid and superoxide anion radicals [START_REF] Granger | Superoxide radicals in feline intestinal ischemia[END_REF]. These data suggest that oxidative stress and lipid peroxidation occurs within 15 minutes of hepatic ischemia in vivo in humans and that xanthine oxidase forms a significant source of ROS during ischemia. The it has been suggested that retrograde perfusion from the hepatic veins sustains some blood and oxygen supply to the liver during the Pringle manoeuvre [START_REF] Smyrniotis | Effects of hepatovenous back flow on ischemic-reperfusion injuries in liver resections with the pringle maneuver[END_REF]. In vivo studies in rats also showed progressive lipid peroxidation during continuous ischemia up to 120 minutes [START_REF] Fukai | Lipid peroxidation during ischemia depends on ischemia time in warm ischemia and reperfusion of rat liver[END_REF]. Hepatosplanchnic MDA and uric acid release ceased within 5 minutes of reperfusion following the initial 15 minutes of hepatic ischemia, suggesting that xanthine oxidase activity during reperfusion is limited. During reperfusion however, a marked increase in the oxidation of GSH to GSSG was observed as evidenced by increasing GSSG levels and an increased GSSG:GSH ratio. These data suggest ongoing or additional oxidative stress which must probably be ascribed to other ROS generating systems than xanthine oxidase, presumably the mitochondrial respiratory chain [START_REF] De Groot | Reoxygenation injury in rat hepatocytes: mediation by O2/H2O2 liberated by sources other than xanthine oxidase[END_REF]8].

After a second period of 15 minutes hepatic ischemia (again leading to significant release of uric acid and MDA), MDA was persistently released during reperfusion, reflecting a sustained lipid peroxidation within the hepatosplanchnic area after a cumulative ischemia time of 30 minutes. In contrast, uric acid release rapidly returned to baseline values during reperfusion. Thus our data consistently suggest that xanthine oxidase may particularly be involved in ROS generation during ischemia.

Hepatic GSSG content returned to baseline values during the second cycle of the intermittent Pringle manoeuvre. A possible explanation for this somewhat surprising observation is an increased activity of the enzyme glutathione reductase. An increased efflux of GSSG during oxidative stress has been demonstrated in rats [START_REF] Masuda | K(+)-driven sinusoidal efflux of glutathione disulfide under oxidative stress in the perfused rat liver[END_REF].

However, hepatocellular GSSG efflux, will probably not decrease total hepatic GSSG content during ischemia, since the absence of blood and bile flow prohibits drainage of GSSG accumulated in these extracellular fluids. Subsequently, we studied the consequences of the intermittent Pringle manoeuvre on some important antioxidants, i.e. GSH, plasma antioxidants and hepatic carotenoids and -tocopherol. An additionally unexpected finding was the increase in hepatic GSH content during ischemia. Since hepatic GSH content rapidly decreased to normal values during reperfusion this transient increase of GSH content during inflow occlusion must probably be ascribed to the accumulation of constitutively synthesized GSH during inflow occlusion, due to absence of hepatic blood flow and the consequent absent washout, which is restored upon restoration of blood and bile flow. Our data show no evidence for the occurrence of hepatic GSH depletion during the intermittent Pringle manoeuvre. Important lipophilic antioxidants are carotenoids and -tocopherol. Carotenoids are derived from vegetable intake and are stored intracellularly. In line with data on hepatic (hydrophilic) GSH levels, we found no timedependent effects of intermittent Pringle manoeuvre on hepatic levels of lipophilic carotenoids and -tocopherol. A substantial amount of the body's antioxidant system is present in plasma as amongst others glutathione, albumin, bilirubin and ascorbic acid [START_REF] Cao | Comparison of different analytical methods for[END_REF]. Upon progressive ischemia systemic plasma antioxidant capacity gradually declined (approximately 10% after 30 minutes ischemia and 10 minutes reperfusion).

At baseline there was a negative hepatosplanchnic balance of plasma antioxidant capacity, indicating hepatosplanchnic uptake of antioxidants from the plasma.

Simultaneously with decreasing plasma antioxidant capacity, this net uptake declined and ultimately disappeared throughout the study. One may speculate that this When considering the effects of ischemia and reperfusion on ROS formation and antioxidant status, it must be realized that ROS also are important signalling molecules. In line with this recent data suggest that oxidative stress may lead to a rapid induction of cytoprotective pathways during intermittent Pringle manoeuvre and ischemic preconditioning [START_REF] Chandel | Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing[END_REF]. Therefore, in future studies concerning antioxidant supplementation during liver ischemia changes in the induction and expression of cytoprotective mechanisms should be assessed as well as changes in hepatocellular integrity and liver function.

In conclusion, short term hepatic ischemia and reperfusion in man leads to xanthine oxidase activity and lipid peroxidation within 15 minutes. Prolonged cumulative ischemia-reperfusion injury leads to sustained oxidative stress, demonstrated by lipid peroxidation and to systemic antioxidant depletion. The observed gradual decline of plasma antioxidant capacity and net hepatosplanchnic antioxidant uptake during prolonged cumulative ischemia, interestingly did preserve both hydrophilic and lipophilic hepatic antioxidant levels. Decreasing plasma levels and net hepatosplanchnic uptake of plasma antioxidants may warrant antioxidant supplementation when cumulative ischemia time exceeds 30 minutes although potential deleterious effects of antioxidant supplementation on ischemic preconditioning should not be disregarded. Schematic representation of the protocol. An intermittent Pringle manoeuvre (15'-5' schedule) was applied during liver transection in 10 patients undergoing liver resection for secondary tumors in an otherwise normal liver. Before and after each event, arterial and hepatic venous blood was drawn and liver biopsies were taken (arrows). Post-ischemia blood samples were drawn seconds after restoration of hepatic blood flow, liver biopsies immediately prior to reperfusion.

  liver transection, an intermittent Pringle manoeuvre was applied by tightening a rubber tube around the entire hepatoduodenal ligament (total Pringle manoeuvre) or selectively around the left or right portal vein and hepatic artery as considered appropriate for the intended resection (selective Pringle manoeuvre, n=4). At least S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society two cycles of 15 minutes occlusion of the portal vein and hepatic artery and 5 minutes reperfusion were required to complete hepatic transection.

Chemicals 2 , 2 '

 22 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), uric acid and butylated hydroxytoluene (BHT) were obtained from Sigma Chemical Co., St. Louis, USA. 2,2'-S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society azino-bis(2-amidinopropane) dihydrochloride (ABAP) was obtained from Brunschwig Chemicals, Amsterdam, The Netherlands. All chemicals were of analytical purity.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society trolox equivalent antioxidant capacity (TEAC). Since TEAC is partly determined by uric acid concentration, the raw data from this assay were corrected for the uric acid content of the sample (plasma antioxidant capacity = TEAC -[uric acid]).

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society the mean of a study population significantly differs from a theoretical value [24] and is frequently used in organ balance studies [25]. This approach allows the simultaneous calculation of the difference of arteriovenous gradients at multiple timepoints both versus baseline and zero using a single statistic test while correcting for multiple comparisons. Statistics were performed using GraphPad Prism 4.0 for Windows (GraphPad Software, San Diego, CA, USA)..A p-value <0.05 was considered to indicate statistical significance. S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society occurrence of lipid peroxidation and xanthine oxidase activity during ischemia may appear paradoxal since these processes are considered to depend on the presence of molecular oxygen. However, hypoxic ROS generation has been observed previously in vitro in heart [29], lung [30] and murine macrophages [31]. Alternatively,

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society decreasing hepatosplanchnic net uptake of hydrophilic antioxidants is causally related to equally diminishing arterial antioxidant concentration.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Figure legends

Figure 2 .a.

 2 Figure 2.

Figure 3 .a.

 3 Figure 3.

Figure 4 a.

 4 Figure 4

Figure 5 .a.

 5 Figure 5.

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 1 S

 1 Figure 1 S t a g e 2 ( a ) P O S T -P R I N T

Figure 2a Figure 2b S

 2a2b Figure 2a

Figure 5a Figure 5b S

 5a5b Figure 5a

Table 1 . Patient characteristics (n=10)

 1

		Median (range)	Reference values
	Gender (M/F)	6/4*
	Age (years)	58 (52-71)
	AST (IU/L)	21 (6-35)	<35
	Bilirubin (µM)	11.8 (7.3-17.6)	2.0-17
	PT (s)	10.4 (9.8-11.9)	9.9-11.5
	Creatinine (µM)	80 (67-116)	53-110
	Urea (mM)	4.5 (2.3-6.6)	3.0-7.0

* ratio, AST=aspartate amino transferase, PT=prothrombin time S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 9 Nov 2007 as manuscript CS20070317 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070317 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

Table 2 . Hepatic carotenoids and -tocopherol content (nmol/g protein) during intermittent hepatic ischemia reperfusion

 2

		Baseline		15' ischemia (1) 5' reperfusion (1) 15' ischemia (2) 5' reperfusion (2)
		mean	SEM	mean	SEM	mean	SEM	mean	SEM	mean	SEM	1-way ANOVA
	-carotene	16.6	2.8	18.2	2.6	15.8	3.3	19.7	5.2	18.1	4.2	0.729
	lycopene	61.8	20.5	70.2	23.3	62.3	23.5	58.1	20.1	65.6	21.9	0.738
	lutein	4.2	0.6	4.7	0.7	4.3	0.6	4.5	0.6	4.5	0.6	0.488
	zeaxanthin	1.7	0.3	1.9	0.3	1.7	0.4	1.8	0.4	1.7	0.3	0.532
	cryptoxanthin	4.5	0.5	5.2	0.6	4.6	0.7	4.6	0.8	5.1	0.9	0.065
	-tocopherol	0.43	0.04	0.50	0.06	0.48	0.07	0.47	0.08	0.46	0.06	0.379

This study was supported by grants from The Netherlands Organization for Health Research and Development to MCGvdP (920-03-317 AGIKO) and CHCD (907-00-033 Clinical Fellowship).


John Lekakis

Sotirios Tsiodras

Dr Ignatios Ikonomidis

John Palios

Garyfalia Poulakou

Loukianos Rallidis

Anastasia Antoniadou

Periklis Panagopoulos

Antonios Papadopoulos

Helen Giamarellou

Dimitrios T Kremastinos

HIV POSITIVE PATIENTS TREATED WITH PROTEASE INHIBITORS HAVE VASCULAR CHANGES RESEMBLING THOSE OBSERVED IN ATHEROSCLEROTIC CARDIOVASCULAR DISEASE

Keywords: HIV, endothelial, HAART, coronary artery disease, IMT, FMD Abbreviations IMT=intimal media thickness FMD= brachial artery flow-mediated dilatation HAART=

A metabolic syndrome associated with atherosclerosis and cardiovascular disease has been described in HIV positive individuals. We investigated whether HIV individuals and CAD patients have similarities in their vascular function and structure.

In a case-control study, we compared measurements of carotid artery intima media thickness (IMT) and brachial artery flow-mediated dilatation (FMD) in HIV individuals with age and sex-matched controls with similar risk factors and patients with established CAD.

Seventy-one HIV patients age 42  13.9 yrs, 91 % male) were compared to 29 CAD patients and 25 controls. HIV patients had higher IMT than controls and similar IMT with CAD patients (0.64±0.2 vs. 0.55±0.05 vs. 0.66±0.08 mm respectively, F=4.2, p=0.01) Patients on protease inhibitors, had higher IMT (0.69±0.2 vs. 0.57±0.15 mm p=0.01), blood pressure, cholesterol and triglycerides than those without (p<0.05). In multiple regression analyses increasing blood pressure (b: 0.37, p=0.001), glucose (b: 0.26, p=0.016), cholesterol (b: 0.24, p=0.033), duration of HIV disease (b: 0.33, p=0.008) and use of protease inhibitors (b: 0.27, p=0.04) were the most important determinants of IMT respectively. FMD was associated only with triglyceride measurements.

Patients with HIV present arterial changes resembling those found in patients with atherosclerotic cardiovascular disease. These vascular changes are closely related to protease inhibitor-induced changes of metabolic parameters. Thus, intensive treatment of metabolic parameters might retard atherosclerosis in HIV patients.

INTRODUCTION

The introduction of highly active antiretroviral therapy (HAART) dramatically reduced the HIV-associated morbidity and mortality [START_REF] Palella | Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators[END_REF] prompting increasing concerns related to the chronic management of these patients. Metabolic alterations, namely dyslipidemia , insulin resistance and lipodystrophy , are common side-effects of HAART [START_REF] Tsiodras | Effects of protease inhibitors on hyperglycemia, hyperlipidemia, and lipodystrophy: a 5year cohort study[END_REF] and there is increasing concern that these changes will translate in the long-term into an increased incidence of cardiovascular atherosclerotic disease [START_REF] Leitner | Age and sex modulate metabolic and cardiovascular risk markers of patients after 1 year of highly active antiretroviral therapy (HAART)[END_REF].

Indeed, the Data Collection on Adverse Events of Anti-HIV Drugs (DAD) Study Group [START_REF] Friis-Moller | Combination antiretroviral therapy and the risk of myocardial infarction[END_REF] reported that HAART was associated with 26% increase in the rate of myocardial infarction per year of exposure to antiretroviral drugs .

Careful cardiovascular evaluation in the course of HIV disease can identify changes early enough to intervene. In addition to the routine blood lipids and glucose measurements, it seems reasonable to estimate surrogate markers for atherosclerosis in HIV positive subjects. Endothelial function and carotid artery intima-media thickness are surrogate markers commonly used in atherosclerotic patients and have been proven to be valuable for the prediction of acute ischemic events [START_REF] Bots | Common carotid intima-media thickness and risk of stroke and myocardial infarction: the Rotterdam Study[END_REF][START_REF] Neunteufl | Late prognostic value of flow-mediated dilation in the brachial artery of patients with chest pain[END_REF][START_REF] Schachinger | Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease[END_REF] Both endothelial function indices and carotid IMT have been shown to be abnormal in HIV patients compared to normal controls [START_REF] Hsue | Progression of atherosclerosis as assessed by carotid intima-media thickness in patients with HIV infection[END_REF][START_REF] Solages | Endothelial function in HIV-infected persons[END_REF]. To date no study has been published comparing patients with HIV disease to patients with atherosclerotic heart disease.

We hypothesized that these two groups of patients might have similarities in their vascular function and structure and therefore we designed a study to investigate the carotid artery IMT and the flow-mediated dilatation of the brachial artery, a reliable estimate of endothelial function, in HIV patients, subjects with proven coronary artery disease and healthy individuals.

METHODS

Study population

We prospectively enrolled 71 HIV positive patients (mean age 42  13.9 yrs, 91 % male) attending a tertiary care center with no previous medical history of coronary artery disease. Twenty nine patients with angiographically documented coronary artery (positive control) and 25 subjects of similar age, sex and atherosclerotic risk factors to the HIV patients served as positive and control groups respectively. The HIV and control subjects had no history of cardiovascular disease, normal physical

Endothelial function

Endothelium-dependent, flow-mediated dilatation (FMD) of the right brachial artery was determined in all patients. All subjects were studied in the morning, having abstained from alcohol, caffeine and food for 8 hours before the study; all vasoactive medications were withheld for 48 hours before the study. Optimal imaging of the brachial artery was obtained and a resting scan was recorded using an Echo-Doppler ultrasound system (Vivid 7, GE Medical systems, Horton, Norway) and a 10 MHz transducer. Reactive hyperemia was then induced by inflation of a blood pressure cuff on the forearm at a pressure of 200 mmHg for 5 min and subsequent deflation; brachial artery was scanned continuously 30 sec before and 90 sec after cuff deflation.

All images were recorded on super VHS videotape for offline analysis. Artery diameter measurements were made using electronic calipers from the anterior to the posterior m-line. FMD was calculated as the percent increase in arterial diameter during hyperemia as compared to resting scan, as previously described [START_REF] Celermajer | Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis[END_REF]. Inter and intra-observer variability of these measurements in our laboratory has been previously presented [START_REF] Lekakis | Endothelial dysfunction of conduit arteries in insulin-dependent diabetes mellitus without microalbuminuria[END_REF]. At the end of the protocol, images were obtained again 4 min after sublingual nitroglycerin (0.4 mg) for measurement of nitrate-induced, endothelium-independent vasodilation.

Measurement of carotid atherosclerosis

B-mode ultrasonographic examination was performed in all patients using the same Echo-Doppler ultrasound. Scanning included left and right carotid arteries for the measurement of the intima-media thickness (IMT); all scans were performed by the same operator. The carotid artery image was focused in the far wall and 2 segments were identified on each side: the distal 1.0 cm of the common carotid proximal to the bifurcation and the carotid bulb [START_REF] Lekakis | Endothelial dysfunction of conduit arteries in insulin-dependent diabetes mellitus without microalbuminuria[END_REF].The mean value of all 4 sites was used for analysis. All scans were digitally stored (Echopac, GE Medical systems, Horton, Norway) ,and recorded on S-VHS videotape for off-line analysis.

Statistical analysis

All variables are expressed as mean±SD. Skewed variables were log-transformed to normalize their distribution. Categorical data were compared by means of the χ 2 -test.

Differences in IMT or FMD between the 3 study groups (HIV,CAD, healthy controls) were examined by full factorial ANOVA (General linear model) using as covariables BMI, smoking status, blood pressure (systolic or diastolic separately), cholesterol, triglycerides, serum glucose, creatinine clearance, use of b-blockers, diuretics, nitrates and statins. The interaction terms between the variable of the 3 study groups and the above covariables were also examined. Using ANOVA, differences in IMT or FMD between the 3 study groups were also examined in a model including the presence of metabolic syndrome as covariable. The interaction term between the variable of the 3 study subgroups and metabolic syndrome was also examined. Post hoc comparisons were performed with Bonferroni's correction. Differences in variables between patients with and without protease inhibitors, nucleoside and non-nucleoside reverse transcriptase inhibitors or between the above patient subgroups and controls were assessed by two-tailed unpaired t-test. Using simple linear regression the association of IMT or FMD (dependent variables) with age, sex, BMI, atherosclerotic risk factors (blood pressure, smoking, serum cholesterol, triglycerides and glucose levels), presence of metabolic syndrome, creatinine clearance, duration of disease, minimum CD4 lymphocyte counts and maximum viral load, current viral load and current CD4 lymphocyte counts and duration of HAART was examined. Additionally, exposure to HAART, as well as a viral load of over 100,000 c/ml for a period of more than 6 months or CD4 count <200 for 6 months were examined as dichotomous variables.

Variables with a P-value of <0.2 in univariate analysis or of major clinical significance (e.g. sex, BMI, blood pressure, glucose, cholesterol, triglycerides, smoking, pulse pressure) were entered in multiple regression models. All covariables included in the final models were tested for interactions. The following variables were

RESULTS

Both control groups had similar age, sex, atherosclerotic risk factors, creatinine clearance, incidence of metabolic syndrome and use of statins with HIV patients (table1). The CAD group had slightly higher mean age than the two other groups that did not reach statistical significance. All diabetic patients were on oral antidiabetic treatment. The CAD patients were treated to a higher percent with b-blockers than the other 2 groups (p<0.05) and 4 of them were receiving long-acting nitrates. The remaining medication was similar among HIV patients and control groups. Among HIV patients those receiving protease inhibitors (n=37) had higher mean and diastolic blood pressure and higher levels of cholesterol, triglycerides and glucose compared to patients on other antiretroviral or no treatment (table 1, for all comparisons, p<0.05).

As a result, patients of protease inhibitors had a higher incidence of metabolic syndrome than patients on other antiretroviral or no treatment (p<0.05). Furthermore, patients receiving protease inhibitors had higher IMT (p=0.01) and similar FMD values (p=0.07) compared to patients on other antiretroviral or no treatment. Patients receiving protease inhibitors had higher maximal viral load and incidence viral load>10 5 c/ml for more than 6 months as well as reduced minimum CD4 count compared to patients on other antiretroviral or no treatment (p<0.05). However, the current CD4 count and viral load was similar between the 2 subgroups of HIV patients (table 1, p=ns).

Comparisons between HIV patients CAD patients and healthy controls

By ANOVA, HIV patients had higher IMT than controls and similar IMT with CAD patients (0.64±0.2 vs. 0.55±0.05 vs. 0.66±0.08 mm respectively, F=4.2, p=0.01, table 1). However, this difference was driven by those patients treated with PI (0.69±0.2 vs. 0.57±0.15 mm, p=0.01, table 1) as patients not receiving PI had similar IMT levels to those in controls (p=0.47). HIV patients had lower FMD than controls but higher

Determinants of vascular indices among HIV patients

In univariate regression analyses age, systolic, diastolic and mean blood pressure, serum glucose, cholesterol and triglyceride levels, creatinine clearance, duration of HIV infection, use of PI and viral load>10 5 c/ml for more than 6 months were associated with IMT (p < 0.05 for all associations, table 2). The presence of metabolic syndrome had a bordeline association with IMT (β=0.24, p=0.049) and no association with FMD (β=0.043, p=0.68). Maximal viral load, current viral load, minimum CD4 count, CD4 count <200 for >6 months were not associated with IMT or FMD (p>0.2 for all associations).

In multivariate regression analysis, weighted for the effect of age, the most important determinants of increased IMT were serum glucose, cholesterol and blood pressure (mean, systolic or diastolic) levels, HIV duration, viral load>10 5 copies/ml for more than 6 months and use of PIs after adjustment for sex, BMI, pulse pressure, smoking status, triglycerides, duration of HAART (table 2). In a similar model including the presence of metabolic syndrome instead of other associated risk factors (glucose, cholesterol, triglycerides, blood pressure parameters), the most important determinants of increased IMT were metabolic syndrome (β=0.29, p=0.01), use of PIs (β=0.24, p=0.046) and viral load>10 5 copies/ml for 6 months (β=0.23, p=0.049). In univariate and multivariate regression analysis (weighted for the effect of age) triglyceride levels were the most important determinants of decreased FMD (table 2, p=0.002) after adjustment for sex, BMI, blood pressure (mean, systolic or diastolic separately) pulse pressure, smoking status, use of HAART (any regimen, PI, NRTI or NNRTI separately), duration of HAART or HIV disease (entered separately), viral load>10 5 c/ml for more than 6 months. In a similar multivariable model, including metabolic syndrome instead of other associated risk factors (glucose, cholesterol, triglycerides, blood pressure parameters), the association between metabolic syndrome and FMD remained not significant (β=0.032, p=0.8). This study confirms previous data evaluating IMT changes in HIV patients; however none of the studies had examined patients with diagnosed CAD as controls.

Although such comparisons are affected by significant confounding the may provide significant clinical insight. In a previously reported study HIV positive subjects had higher IMT values than age matched controls, that also progressed rapidly after one year of follow-up [START_REF] Hsue | Progression of atherosclerosis as assessed by carotid intima-media thickness in patients with HIV infection[END_REF]. In concordance with our findings this study found classical cardiovascular risk factors to be associated with increased IMT [START_REF] Hsue | Progression of atherosclerosis as assessed by carotid intima-media thickness in patients with HIV infection[END_REF] ; however they also reported an association with low CD4 count-an index of immunodeficiency in HIV patients, that could not be verified in our patient population in univariate analysis. In addition higher IMT values were observed in patients with viral load>10 5 copies/ml for more than 6 months further suggesting a relation between severity of the disease and vascular abnormalities.

Another recently performed study compared HIV patients with healthy controls as well as diabetic patients [START_REF] Van Wijk | Functional and structural markers of atherosclerosis in human immunodeficiency virusinfected patients[END_REF]; IMT was similar in HIV and diabetic patients but in addition to metabolic parameters, factors relating to HIV infection and antiretroviral medications use were also related to IMT [START_REF] Van Wijk | Functional and structural markers of atherosclerosis in human immunodeficiency virusinfected patients[END_REF]. The role of duration of disease and exposure to HAART needs further elucidation. We found a significant positive association between increasing HIV duration and higher IMT weighted for the ageing effect. Similarly a recent large case-control study emphasized the independent role of HIV duration as well as exposure to HAART in the development of higher IMT values [START_REF] Lorenz | Both long-term HIV infection and highly active antiretroviral therapy are independent risk factors for early carotid atherosclerosis[END_REF]. In fact the study concludes that the "vascular age of HIV individuals is 4-5 years higher than age and sex-matched controls [START_REF] Lorenz | Both long-term HIV infection and highly active antiretroviral therapy are independent risk factors for early carotid atherosclerosis[END_REF]. Thus, chronic inflammation during the course of the disease may cause the release of several cytokines and growth factors leading functional and structural vascular changes in HIV patients. In support of this mechanism, we have observed an independent association between a viral load>10 5 copies/ml for more than 6 months, a marker of the intensity of the inflammatory process, and IMT values in our HIV patients. In the present study, we have also found that exposure to protease inhibitor treatment was related to increased IMT values independently of the examined metabolic parameters. Furthermore, in our study patients receiving protease inhibitors had higher mean diastolic blood pressure and higher levels of cholesterol, triglycerides and glucose and thus, a higher incidence of the metabolic syndrome compared to patients on other antiretroviral therapy or no treatment in agreement with previous studies [START_REF] Tsiodras | Effects of protease inhibitors on hyperglycemia, hyperlipidemia, and lipodystrophy: a 5year cohort study[END_REF]. This finding suggests that protease inhibitor treatment may contribute to the impairment of metabolic parameters in HIV patients leading to increasing IMT values. The significant adverse effects of antiretroviral treatment on cardiovascular indices have been reported in very young HIV patients without traditional risk factors; indeed in a study of 83 children under treatment, higher IMT values were noted in HIV positive compared to healthy children [START_REF] Charakida | Early structural and functional changes of the vasculature in HIV-infected children: impact of disease and antiretroviral therapy[END_REF]. These observations further highlight that the importance of antiretroviral therapy cannot be ignored.

The finding that IMT values in HIV patients were comparable to those observed in CAD patients is of major concern. Measurement of IMT is useful in studying atherosclerosis; it is associated with the extent of coronary athrerosclerosis [START_REF] Lekakis | Atherosclerotic changes of extracoronary arteries are associated with the extent of coronary atherosclerosis[END_REF] as well the occurrence of cardiovascular events [START_REF] Bots | Common carotid intima-media thickness and risk of stroke and myocardial infarction: the Rotterdam Study[END_REF][START_REF] Chambless | Association of coronary heart disease incidence with carotid arterial wall thickness and major risk factors: the Atherosclerosis Risk in Communities (ARIC) Study, 1987-1993[END_REF][START_REF] O'leary | Carotid-artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults. Cardiovascular Health Study Collaborative Research Group[END_REF]. Recently the value of using IMT as a predictor for cardiovascular events has been extended to younger patients i.e less than 50 years of age [START_REF] Lorenz | Carotid intima-media thickening indicates a higher vascular risk across a wide age range: prospective data from the Carotid Atherosclerosis Progression Study (CAPS)[END_REF]. Also IMT is a popular surrogate marker for the evaluation of various interventions for the retardation or even the regress of the atherosclerotic process [START_REF] Stamatelopoulos | Tamoxifen improves endothelial function and reduces carotid intima-media thickness in postmenopausal women[END_REF]. It is reasonable to hypothesize that all applications of IMT in CAD patients can also be used to HIV patients for risk stratification and evaluation of antiatherosclerotic therapies, but this hypothesis has to be proved. The independent association of IMT with blood pressure measurements, serum glucose and cholesterol values or with the presence of metabolic syndrome in our study highlights the significance of these parameters in the genesis of atherosclerosis in HIV individuals. Further it underscores the necessity for intense treatment of these risk factors and metabolic syndrome, especially in patients with higher values of IMT.

Endothelial dysfunction appears to be an early step to the atherosclerotic process and may be present even in children with cardiovascular risk factors [START_REF] Celermajer | Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis[END_REF]. Flow-mediated dilatation of the brachial artery is a reliable estimate of endothelial function, which is commonly used in clinical studies since it is noninvasive, correlates with the endothelial function in the coronary arteries and can predict cardiovascular events in high risk populations [START_REF] Anderson | Close relation of endothelial function in the human coronary and peripheral circulations[END_REF][START_REF] Corretti | Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force[END_REF][START_REF] Gokce | Predictive value of noninvasively determined endothelial dysfunction for long-term cardiovascular events in patients with peripheral vascular disease[END_REF][START_REF] Karatzis | Long-term prognostic role of flow-mediated dilatation of the brachial artery after acute coronary syndromes without ST elevation[END_REF]. Endothelial dysfunction assessed by flow-mediated dilatation was modest in our HIV patients, worse than in normal individuals and better compared to CAD patients. Endothelial dysfunction has been previously reported in HIV patients [START_REF] Solages | Endothelial function in HIV-infected persons[END_REF][START_REF] Van Wijk | Functional and structural markers of atherosclerosis in human immunodeficiency virusinfected patients[END_REF] and may be caused by HIV infection itself as well as could be the result of the antiretroviral agents used to treat HIV. Infection with HIV is associated with a systemic inflammatory environment that could affect the vascular endothelium [START_REF] Lewis | Atherosclerosis in AIDS: potential pathogenetic roles of antiretroviral therapy and HIV[END_REF][START_REF] Tsiodras | The HAART induced metabolic syndrome[END_REF]. Moreover infections with opportunistic pathogens in patients with advanced HIV disease may exacerbate endothelial damage [27]. From the available antiretroviral medications, specific protease inhibitors have been associated with metabolic changes that may lead to endothelial dysfunction [START_REF] Tsiodras | Effects of protease inhibitors on hyperglycemia, hyperlipidemia, and lipodystrophy: a 5year cohort study[END_REF][START_REF] Tsiodras | The HAART induced metabolic syndrome[END_REF], while a direct cytotoxic effect on endothelial cells has also been described [START_REF] Conklin | HIV protease inhibitor ritonavir decreases endothelium-dependent vasorelaxation and increases superoxide in porcine arteries[END_REF][START_REF] Zhong | HIV protease inhibitor ritonavir induces cytotoxicity of human endothelial cells[END_REF]. Nevertheless, an association with either protease inhibitors or viral load and FMD was not found in our study. Only triglyceride levels were an independent study predictor of decreased FMD in our HIV population suggesting that an intense treatment of elevated triglyceride levels might improve endothelial function in these patients. It is well known that hypertriglyceridemia impairs endothelial function while vasodilator function improves after lowering plasma triglycerides even without changes in confounding lipoproteins or insulin resistance [START_REF] Capell | Short-term triglyceride lowering with fenofibrate improves vasodilator function in subjects with hypertriglyceridemia[END_REF]. Triglycerides may cause endothelial dysfunction through several mechanisms including enhancement of oxidative stress, reduction of high-density lipoprotein cholesterol, influence on low-density lipoprotein (LDL) size distribution, induction of cell adhesion molecule expression, direct effects of TG-rich lipoproteins (TRLs) on the vessel wall, and increase of fatty acids [START_REF] Capell | Short-term triglyceride lowering with fenofibrate improves vasodilator function in subjects with hypertriglyceridemia[END_REF].

The triglycerides were the only independent determinant of FMD in our patients likely because of the extensive use of protease-inhibitors in our study cohort (52%). We have previously shown, that the use of these drugs [START_REF] Tsiodras | Effects of protease inhibitors on hyperglycemia, hyperlipidemia, and lipodystrophy: a 5year cohort study[END_REF] is related with 2 -fold higher incidence rate for hypertriglyceridemia than for hypercholesterolemia. Thus, the use of protease-inhibitors has a stronger association with elevated triglyceride levels than with other lipoprotein levels through mechanisms which are not fully defined and include a direct enhancement of the formation of VLDL lipoproteins [START_REF] Purnell | Effect of ritonavir on lipids and post-heparin lipase activities in normal subjects[END_REF] or reduction of lipoprotein lipase activity (LPL) [START_REF] Ranganathan | The HIV protease inhibitor saquinavir impairs lipid metabolism and glucose transport in cultured adipocytes[END_REF]. LPL is an enzyme located at the capillary endothelial surface , which hydrolyzes triglycerides to make fatty acids available to cells [START_REF] Capell | Short-term triglyceride lowering with fenofibrate improves vasodilator function in subjects with hypertriglyceridemia[END_REF][START_REF] Eckel | Lipoprotein lipase: a multifunctional enzyme relevant to common metabolic diseases[END_REF]. Thus, reduction of its activity by protease inhibitors may contribute to elevation of triglyceride levels and consequently, to endothelial dysfunction as observed in the present study.

The lack of the assessment of oxidative stress and inflammatory markers such as Creactive protein may be considered a limitation of our study because oxidative stress and low-grade inflammation may contribute in the development of vascular changes.

However, in the present study we have demonstrated an independent association between IMT and a viral load>10 5 copies/ml for more than 6 months which is a marker of the intensity of the inflammatory process in HIV patients.

In conclusion, patients with HIV present functional and structural arterial changes resembling those found in patients with atherosclerotic cardiovascular disease.

Characteristics of the HIV infection itself e.g. viral load treatment, duration of the disease, treatment with protease inhibitors as well as parameters of the metabolic syndrome e.g. serum glucose, cholesterol and triglyceride values and blood pressure measurements appear to influence the pathogenesis of these vascular changes.

Therefore intensive treatment of such changes might be helpful in preventing or retarding the atherosclerotic process in HIV patients.

  examination and normal resting and exercise electrocardiogram. The presence of metabolic syndrome was assessed among the study groups according to the criteria proposed by the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) [10]. The creatinine clearance was calculated as marker of renal function using the following formula: Creatinine Clearance = [[140age(yr)]*weight(kg)]/[72*serum Cr(mg/dL)] (multiplied by 0.85 for women). The study was approved by the hospital's scientific committee and all patients gave informed written consent. The research has been carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association.

  entered in the model separately to avoid colinearity. A) systolic, diastolic or mean blood pressure, B) use of HAART (any regimen) PI, NRTI or NNRTI, c) minimum CD4 or current CD4 lymphocyte counts, maximum viral load, current viral load or viral load>10 5 c/ml for more than 6 months, d) disease duration or HAART duration, S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Feb 2008 as manuscript CS20070353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070353 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society e) presence of metabolic syndrome or associated risk factors (glucose, cholesterol, triglycerides, blood pressure parameters). Analysis was weighted for age. The associations between vascular indices and patients' clinical characteristics are presented by means of standardized regression coefficient (β). P<0.05 was considered to be the level of statistical significance. All statistical tests were two tailed. SPSS 11.5 software (SPSS Inc.Chicago, IL) was used for statistical analysis.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Feb 2008 as manuscript CS20070353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070353 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  FMD than CAD patients [5.4±2.8 vs. 7.1±3.7 vs. 2.8±2.6 % respectively, F=11.7, p<0.01]. NMD values were similar among the study groups. Differences remained significant after adjustment for the use of statins, other medication or presence of metabolic syndrome. Additionally, the interaction terms between the variable of the 3 study groups and use of medication or presence of metabolic syndrome were not significant (data not shown).

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Feb 2008 as manuscript CS20070353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070353 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society DISCUSSION Our case-control study compared functional and structural arterial changes between HIV patients, age-and sex -matched coronary artery disease patients and controls with similar atherosclerotic risk factors. Patients with HIV had similar IMT values to those observed in patients with coronary artery disease, and higher than those found in individuals with similar atherosclerotic risk factors. Components of the HIV disease such as treatment with protease inhibitors and duration of disease as well as components of the metabolic syndrome such as higher blood pressure, cholesterol and glucose levels determined independently increasing IMT values. Treatment with protease inhibitors was related to impaired metabolic parameters and a higher incidence of the metabolic syndrome compared to other antiretroviral treatment and thus may have contributed to increasing IMT values. Regarding FMD, HIV patients showed values in-between those of matched patients with coronary artery disease and healthy controls.

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Feb 2008 as manuscript CS20070353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070353 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Feb 2008 as manuscript CS20070353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070353 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Feb 2008 as manuscript CS20070353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070353 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Feb 2008 as manuscript CS20070353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070353 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

S t a g e 2

 2 ( a ) P O S T -P R I N T Clinical Science Immediate Publication. Published on 5 Feb 2008 as manuscript CS20070353 THIS IS NOT THE FINAL VERSION -see doi:10.1042/CS20070353 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Table 1 . Clinical characteristics of the study groups

 1

		HIV	CAD	CONTROLS	P *	PI	No PI	P #
		(n=71)	(n=29)	(n=25)		(n=37)	(n=34)
	Age (years)	42±13.9	46±5.7	40±5.6	0.1	44±12	40±13	0.1
	Males	65 (91%)	24 (83%)	23 (92%)	0.2	32 (86%)	34 (97%)	0.24
	Cholesterol	200±60	220±70	223±40	0.14	224±60*	175±48	<0.001
	(mg/dl)
	Triglycerides	200±154	137±70	182±100	0.11	256±166*	139±116	<0.001
	(mg/dl)
	Glucose (mg/dl)	94±44	90±45	88±39	0.9	104±59*	83±112	<0.05
	Creatinine	0.7±0.04	0.8±0.03	0.7±0.03	0.9	0.8±0.04	0.68±0.04	0.9
	(mg/dl)
	Creatinine	142±25	138±27	150±15	0.6	140±27	144±24	0.9
	clearance(ml/min)
	Body mass index (kg/m 2 )	24±3	28±4	25±3	0.9	24±3	24.5±3	0.8
	Smoking	52 (73%)	24 (82%)	18 (72%)	0.7	26 (71%)	26 (76%)	0.7
	Hypertension	14 (20%)	11 (37%)	6 (24%)	0.5	11 (32%)	3 (9%)	<0.05
	Diabetes	5 (7%)	4 (13%)	2 (8%)	0.6	4(10%)	1(3%)	0.19
	Hyperlipidemia	15 (21%)	6 (21%)	5 (20%)	0.9	11 (30.6%)	4 (11.8%)	<0.05
	Metabolic	13 (18%)	6 (21%)	5 (20%)	0.8	10 (27%)	3 (9%)	0.04
	syndrome
	Current CD4	443.6±246.2	N/A	N/A		420.2±263.9	470.1±226.2	0.4
	(cells/ml)
	Minimum CD4	232.2±152.4	N/A	N/A		160.4±107.2	310.2±157.1	<0.001

.05 IMT intima media thickness, FMD flow mediated dilatation-brachial index, NMD nitrate mediated dilatation-brachial index, HAART, highly active antriretoviral theraphy (any regimen). Viral load: patients with viral load >10 5 for more than 6 months duration. PI, protease inhibitors ,NNRTI, non-nucleoside reverse transcriptase inhibitors NRTI, nucleoside reverse transcriptase inhibitors , SBP, Systolic , DBP, diastolic or MBP mean blood pressure, N/A =not applicable *P value for ANOVA between 3 groups, # P value for comparison between PI vs. non-PI, †P=ns for HIV vs. normal controls