Markus Karlsson

Tino Kurz

Ulf T Brunk
email: ulf.brunk@liu.se

Sven E Nilsson

Christina I Frennesson

Prof Robert Hider

What does the commonly used DCF-test for oxidative stress really show?

Keywords: DCF, lysosomes, oxidative stress, ROS, transition metals AMD, age-related macular degeneration, AO, acridine orange, CP22, 1-propyl-2-methyl-3hydroxypyrid-4-one, DCF, dichlorofluorescein, DMEM, Dulbecco

Dihydrodichlorofluorescein (H 2 DCF-DA) is widely used to evaluate "cellular oxidative stress". After passing through the plasma membrane, this lipophilic and non-fluorescent compound is deesterified to a hydrophilic alcohol (H 2 DCF) that may be oxidized to fluorescent DCF by a process usually considered to involve reactive oxygen species (ROS). It is, however, not always recognized that, being a hydrophilic molecule, H 2 DCF does not pass membranes, except for the outer, fenestrated mitochondrial ones. It is also not generally realized that oxidation of H 2 DCF is dependent either on Fenton-type reactions or on unspecific enzymatic oxidation by cytochrome c, for neither superoxide, nor hydrogen peroxide, directly oxidizes H 2 DCF. Consequently, oxidation of H 2 DCF requires the presence of either cytochrome c or of both redox-active transition metals and hydrogen peroxide. Redox-active metals exist mainly within lysosomes, while cytochrome c resides bound to the outer side of the inner mitochondrial membrane. Following exposure to H 2 DCF-DA, weak mitochondrial fluorescence was found in both the oxidation-resistant ARPE-19 cells and the much more sensitive J774 cells. This fluorescence was only marginally enhanced following short exposure to hydrogen peroxide, showing it by itself being unable to oxidize H 2 DCF. Cells that were either exposed to the lysosomotropic detergent MSDH, exposed to prolonged oxidative stress, or spontaneously apoptotic showed lysosomal permeabilization and strong DCF-induced fluorescence. The results suggest that DCF-dependent fluorescence largely reflects relocation to the cytosol of lysosomal iron and/or mitochondrial cytochrome c.

INTRODUCTION

The molecular mechanisms behind age-related macular degeneration (AMD) are much debated but far from understood, even though it is the most common cause of visual impairment among the elderly in the western world [START_REF] Foran | Causes of visual impairment in two older population cross-sections: the Blue Mountains Eye Study[END_REF][START_REF] Vingerling | The prevalence of age-related maculopathy in the Rotterdam Study[END_REF][START_REF] Klein | Prevalence of age-related maculopathy. The Beaver Dam Eye Study[END_REF]. However, based on recent evidence, oxidative stress within retinal pigment epithelial (RPE) cells, with the ensuing accumulation of lysosomal lipofuscin and thereby depressed phagocytosis/autophagocytosis, is considered a major etiological factor behind AMD [START_REF] Sundelin | Lipofuscin accumulation in cultured retinal pigment epithelial cells reduces their phagocytic capacity[END_REF][START_REF] Winkler | Oxidative damage and age-related macular degeneration[END_REF][START_REF] Beatty | The role of oxidative stress in the pathogenesis of age-related macular degeneration[END_REF][START_REF] Cuervo | Autophagy and aging: the importance of maintaining "clean" cells[END_REF][START_REF] Schloendorn | Medical bioremediation: a concept moving toward reality[END_REF]. Consequently, in relation to various experimental studies on cultures of RPE cells, the extent of cellular oxidative stress is often being evaluated.

A method that is frequently relied upon for this purpose is the DCF-test [START_REF] Halliwell | Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean[END_REF][10][START_REF] Dikalov | Measurement of reactive oxygen species in cardiovascular studies[END_REF]. Dihydrodichlorofluorescein diacetate (H 2 DCF-DA) is then added to cells in culture and the intracellular oxidation of H 2 DCF to DCF documented over time. H 2 DCF-DA is a non-fluorescent lipophilic ester that easily crosses the plasma membrane and passes into the cytosol, where it is rapidly cleaved by unspecific esterases [START_REF] Bass | Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation[END_REF]. One of the reaction products is the non-fluorescent alcohol H 2 DCF. The oxidation of this molecule to the fluorochrome DCF results in green fluorescence when excited with blue light. The brightness of this fluorescence is usually considered to reflect the extent to which 'reactive oxygen species' (ROS) are present, unfortunately without any further definition of what kind of ROS may give rise to the oxidation [START_REF] Wang | Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader[END_REF]14]. Furthermore, it is often wrongly assumed that H 2 DCF is evenly distributed in the cell following the intracellular cleavage of the added diacetate ester, neglecting the inability of hydrophilic molecules to traverse membranes. Moreover, the evaluation of the DCF-test commonly involves plate readers or flow cytofluorometers, which do not allow any careful morphological analysis of individual cells that, if undertaken, might have disclosed unexpected cellular variations. Thus, the DCF-test often seems to be somewhat uncritically used.

We previously showed that oxidation of H 2 DCF to DCF is not a result of exposure to reactive oxygen species (ROS) in general, but rather indicates the specific impact of hydroxyl radicals formed during Fenton-type reactions [START_REF] Baird | Metallothionein protects against oxidative stress-induced lysosomal destabilization[END_REF]. Here we also stress that cytochrome c, as has been previously pointed out [16][START_REF] Lawrence | Evidence for the role of a peroxidase compound I-type intermediate in the oxidation of glutathione, NADH, ascorbate, and dichlorofluorescin by cytochrome c/H 2 O 2 . Implications for oxidative stress during apoptosis[END_REF][START_REF] Burkitt | Cytochrome c is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis[END_REF], operates as an unspecific peroxidase with H 2 DCF as a target. It is, therefore, plausible to suppose that lysosomal membrane permeabilization (LMP), with release to the cytosol of redox-active iron, and/or mitochondrial release of cytochrome c is required for the induction of strong cytosolic DCF-mediated fluorescence. We also point out that in the absence of LMP and apoptosis/necrosis, H 2 DCF only occurs in the cytosol and the mitochondrial intermembranous space, where it gives rise to weak cytosolic and somewhat stronger mitochondrial fluorescence that indicates oxidation. Most probably, this faint fluorescence, that regularly seems to be considered background and to be over-looked, results from the normal mitochondrial production of hydrogen peroxide that diffuses all over the cell, the presence of cytochrome c in the mitochondrial inter-membranous space as well as of minute amounts of labile iron under transport in the cytosol. Consequently, if lysosomal membrane permeabilization (LMP) and related mitochondrial damage with relocation of cytochrome c has taken place, a positive DCF-test might be interpreted as a sign of oxidative stress even if that would not necessarily be the case. Similarly, a low or undetectable level of DCF fluorescence may not necessarily indicate the absence of ROS but rather designate stable lysosomal and mitochondrial compartments.

London, (UK) and Dr. Gene N. Dubowchik (Wallingford, CT, USA), respectively. All other reagents were from Sigma (St. Louis, MO, USA).

Cells and culture conditions

ARPE-19 (human immortalized retinal pigment epithelial) cells and murine J774 macrophagelike histiocytic lymphoma cells (both obtained from ATCC, Manassas, VA, USA) were grown at 37°C in humidified air with 5% CO 2 in DMEM and F12 (1:1) supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin. Cells were sub-cultivated twice a week, seeded in 6-well plates with or without cover slips at a concentration of 5x10 5 cells/well (J774), while the much larger ARPE-19 cells were seeded at 2x10 5 cells/well with or without cover slips. Cells were subjected to experiments 12, 24 and 48 h following subcultivation.

Conditions for basic studies with H 2 DCF-DA only

Small amounts of stock solutions, containing 1 to 10 mM H 2 DCF-DA in DMSO, were added to fresh complete growth medium. The carefully blended solution was added to the culture dishes following removal of the old medium. Cells on cover slips were in that way exposed to 3-30 µM H 2 DCF-DA in medium with 0.03-3% DMSO for 30 min at otherwise standard culture conditions. The cells were then rinsed in HBSS, directly mounted in HBSS, and within 5-6 min subjected to laser scanning confocal microscopy using a Nikon Eclipse C1 laser scanning confocal microscope (Tokyo, Japan).

Following tests with varying concentrations of H 2 DCF-DA and DMSO, a stock solution of 10 mM H 2 DCF-DA in DMSO was selected for both cell types and added to medium in such a way that the final concentrations of H 2 DCF-DA and DMSO was 10 µM and 0.1%, respectively. The cells were observed and photographed using the above confocal microscope, r and the Nikon EZ-C1 V3.70 software for image acquisition. DCF fluorescence was detected using a 515/30 nm band pass filter. Since the DCF fluorescence of control cells is rather weak, the medium size pinhole and an electronic gain of 6.5 were applied. All photographs were taken at a resolution of 2 Mpixels. To allow comparison between experiments, these settings were then kept the same for all experiments involving evaluation of DCF-induced fluorescence, even when the intensity of fluorescence was much enhanced due to the conditions being applied and, consequently, a small pinhole and less amplification would have given better resolution and prevented over-exposure of strongly fluorescent cells.

Localisation of mitochondria using the mitochondria-specific dye TMRE

Mitochondria were demonstrated using the cationic and lipophilic dye tetramethylrhodamine ethyl ester (TMRE), which accumulates in the matrix of normal mitochondria. Cells were incubated with TMRE in complete culture medium (100 nM; 15 min; 37°C) and observed using the abovementioned Nikon confocal microscope. Since mitochondrial TMRE fluorescence is strong, optimal documentation conditions were used (the smallest pinhole and a low gain).

Overlays of DCF/TMRE pictures could not be produced because TMRE is a metachromatic fluorophore that provides both red and green fluorescence when activated by a 488 nm laser and the green one is much stronger than that of DCF in control cells. TMRE fluorescence was detected using a 590/50 nm band pass filter.

Studies aiming at varying cytosolic labile iron concentrations before exposure to H 2 DCF-DA

To evaluate the influence on DCF-induced fluorescence of different concentrations of labile iron in the cytosol, as well as in the mitochondrial inter-membranous space, cells were initially exposed to H 2 DCF-DA as above and then mounted in HBSS with 500 µM ferric ammonium citrate (FAC) to enhance labile iron. Separately, cells were incubated with H 2 DCF-DA together with 100 µM of the iron chelator 1-propyl-2-methyl-3-hydroxypyrid-4-one (CP22) to ligate labile iron and then mounted A c c e p t e d M a n u s c r i p t in HBSS in the continuous presence of CP22. CP22 (MW 203.7) is an effective, water-soluble bidentate iron chelator with a complex constant of 10 36 [START_REF] Porter | Iron mobilization from hepatocyte monolayer cultures by chelators: the importance of membrane permeability and the iron-binding constant[END_REF]. When complexed with CP22, iron is prevented from redox-cycling and unable to support Fenton-type reactions.

Studies aiming at evaluating DCF-fluorescence in relation to oxidative stress

1. Exposure to hydrogen peroxide. In order to induce oxidative stress, ARPE-19 and J774 cells were initially exposed to H 2 DCF-DA as above and then mounted in HBSS with 500 µM H 2 O 2 . To induce apoptosis of the ARPE-19 (very resistant to oxidative stress) and J774 (sensitive) cells, they were exposed to 15 mM and 100 µM H 2 O 2 in HBSS, respectively, for 30 min and then kept at standard culture conditions for about 6 h when they were incubated with H 2 DCF-DA and studied as described above. In order to further emphasize the differences in lysosomal membrane stability between cell types, both of them were exposed to 100 µM H 2 O 2 in HBSS for 30 min and then kept at standard culture conditions for about 6 h, when they were exposed to the lysosomotropic, metachromatic fluorochrome acridine orange (AO) as described before [START_REF] Antunes | Apoptosis induced by exposure to a low steady-state concentration of H 2 O 2 is a consequence of lysosomal rupture[END_REF][START_REF] Kurz | ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron[END_REF][START_REF] Kurz | Lysosomes in iron metabolism, ageing and apoptosis[END_REF][START_REF] Yu | Intralysosomal iron: a major determinant of oxidant-induced cell death[END_REF][START_REF] Zdolsek | Photooxidative damage to lysosomes of cultured macrophages by acridine orange[END_REF]. A 488 nm argon laser was used for excitation and AO fluorescence was detected using 515/30 nm (green) and 590/50 nm (red) band pass filters.

2. Exposure to a lysosomotropic detergent. In order to induce lysosomal rupture by other means than oxidative stress, ARPE-19 cells were initially exposed to H 2 DCF-DA as above. They were then mounted in HBSS with 100 µM of the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), which is known to induce lysosomal labilization [START_REF] Li | Induction of cell death by the lysosomotropic detergent MSDH[END_REF], before being studied over a 15 min period of time. In separate experiments, designed to verify the LMPeffect of MSDH, cells were initially exposed to AO as above and then mounted in HBSS with MSDH and followed over time.

Evaluation of DCF-induced fluorescence in apoptotic ARPE-19 cells

Apoptotic and post-apoptotic necrotic cells are always present in cell cultures, especially shortly after subcultivation, because a number of cells do not survive that procedure. Such apoptotic ARPE-19 cells were identified, 12 h following seeding, based on their characteristic morphology that includes chromatin condensation, nuclear fragmentation, plasma membrane blebbing, and formation of apoptotic bodies. DCF-induced fluorescence was evaluated following exposure to H 2 DCF-DA as described above.

Assessment of the capacity of cytochrome c to oxidize H 2 DCF

Using a modification [START_REF] Baird | Metallothionein protects against oxidative stress-induced lysosomal destabilization[END_REF] of a technique described by Myhre et al. [26], H 2 DCF was exposed to cytochrome c +/-the small iron chelator CP22 (at 1 and 20 µM, respectively). Briefly, a standard consisting of ferric iron (10 µM) was reduced to its ferrous form by cysteine (100 µM) in a HEPES buffer (pH 7.0; 150 mM). Hydrogen peroxide (100 µM) was added to initiate oxidative conversion of non-fluorescent H 2 DCF (5 µM) to fluorescent DCF [H 2 DCF was obtained by hydrolyzing its diacetate ester (H 2 DCF-DA)]. DMSO (10%) and CP22 (20 µM) were used to demonstrate the involvement of HO • and iron, respectively.

The capacity of cytochrome c to oxidize H 2 DCF was then compared to that of the Fe(II) standard. Fluorescence was measured in a FL600 Microplate Fluorescence Reader (Bio-Tek, Winooski, VT, USA) at ex 485 nm and em 530 nm.

Statistics

DCF-induced fluorescence intensity was measured using the NIH ImageJ software v. 1.42q. Following delineation of each cell, their mean fluorescence was calculated and analysis performed using Student's t-test.

RESULTS

Biochemical

Normal cells show a weak cytosolic and a somewhat stronger mitochondrial-type DCFfluorescence pattern, while apoptotic cells demonstrate strong diffuse fluorescence

As mentioned in the Methods section, initial experiments indicated that a final concentration of 10 µM H 2 DCF-DA in complete culture medium, obtained by using a stock solution of 10 mM H 2 DCF-DA in DMSO, gave acceptable results and allowed reasonable documentation of both normal and apoptotic ARPE-19 and J774 cells using the same confocal settings. It should be pointed out that the fluorescence obtained from normal cells was low (particularly in J774 cells). In studies using flow-or plate-reader fluorometry, it would have been considered non-specific background. The final concentration of DMSO did not influence the results as long as it was below 1%. Normal ARPE-19 and stretched-out J774 cells showed a mitochondrial DCF-induced weak fluorescence in combination with a very faint cytosolic fluorescence (Figures 1A and2A), while apoptotic ARPE-19 and J774 cells presented strong DCF-induced fluorescence that was 6-14 times higher than that of the control cells (Figures 1C, 2B and2D) whether the apoptosis was spontaneous (ARPE-19 cells; Figure 1C) or due to exposure to oxidative stress (Figure 2, B andD). It needs to be emphasized that in opposition to the ARPE-19 cells that adhere firmly to their substratum, the J774 cells tend to round up and easily detach, especially when becoming apoptotic. Therefore, the rinsing steps that are needed for the H 2 DCF-DA-exposure makes it very difficult to find more than occasional apoptotic cells in the mounted cultures that are studied in the confocal microscope (see further legends to Figure 2).

The initially weak mitochondrial-type DCF fluorescence pattern from normal cells became slightly stronger after a few seconds of exposure to blue light, which is considered an oxidative effect and probably due to formation of singlet oxygen. Due to the low intensity of fluorescence, the smallest pinhole and a low electronic gain, normally chosen in order to obtain the sharpest possible confocal microscopy pictures, could not be used. Nevertheless, it was clear that the DCF-mediated fluorescence of non-apoptotic cells originated in organelles that morphologically looked like mitochondria. Using the mitochondria-specific stain TMRE on cells not exposed to H 2 DCF-DA, we found an identical pattern of fluorescence, proving that, apart from the weak cytosolic fluorescence, the DCF-induced fluorescence of normal cells originates in mitochondria (Figure 1B).

Both cell types showed a slight, but significant, increase in DCF-mediated fluorescence intensity when mounted in 500 µM hydrogen peroxide shortly before microscopy, suggesting the presence of small amounts of labile iron in the cytosol (Figure 1D and2C).

In normal cells, DCF-induced fluorescence is a function of cytosolic Fenton-type reactions and mitochondrial enzymatic oxidation by cytochrome c

As already mentioned, the main portion of the DCF-induced fluorescence of normal cells of both types showed a distinct mitochondrial pattern, although better visualized in the thin and flat ARPE-19 cells than in the thicker and more rounded up J774 cells (Figures 1A and2A). This was confirmed by the identical DCF-and TMRE-induced fluorescences (although overlay pictures could not be produced because of the green TMRE-induced fluorescence following excitation with blue light -TMRE is a metachromatic fluorophore). Since hydrophilic molecules do not penetrate cellular membranes, it seems safe to assume that the DCF-induced fluorescence originates in the mitochondrial intermembranous space where cytochrome c is present, together with traces of low mass labile iron compounds under transport to the mitochondrial matrix.

Following exposure to FAC, the fluorescence from both compartments was substantially enhanced (Figure 3A), indicating the presence of H 2 DCF all over the cytosol as well as in the mitochondrial intermembranous space. This finding shows that oxidation of H 2 DCF requires the presence of low mass iron in redox-active form, which was confirmed by the observation that cells exposed to the small iron chelator CP22 presented a clear depression of both cytosolic and mitochondrial DCF-induced fluorescence (Figure 3B).

To demonstrate the capacity of cytochrome c to catalyze oxidation of H 2 DCF to DCF in the presence of hydrogen peroxide, we applied the in vitro DCF test as it was described before [START_REF] Baird | Metallothionein protects against oxidative stress-induced lysosomal destabilization[END_REF]. As shown in Figure 4 (A andB), cytochrome c, as well as hydroxyl radicals, readily induce such oxidation, which is in accordance with previous findings of other groups [16][START_REF] Lawrence | Evidence for the role of a peroxidase compound I-type intermediate in the oxidation of glutathione, NADH, ascorbate, and dichlorofluorescin by cytochrome c/H 2 O 2 . Implications for oxidative stress during apoptosis[END_REF][START_REF] Burkitt | Cytochrome c is a potent catalyst of dichlorofluorescin oxidation: implications for the role of reactive oxygen species in apoptosis[END_REF].

Strong cytosolic DCF-induced fluorescence is a function of LMP-dependent relocation to the cytosol of redox-active iron, as well as of release of cytochrome c following MMP

As pointed out before, short exposure to hydrogen peroxide (100 µM) is insufficient to induce LMP, as assayed by the AO-uptake method, in the very oxidative-stress-resistant ARPE-19 cells [START_REF] Kurz | ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron[END_REF]. The J774 cells are much more sensitive and apoptosis can easily be initiated by exposure for 30 min to 100 µM hydrogen peroxide (Figure 5). However, no immediate LMP occurs, not even if cells are exposed to higher concentrations, because peroxidation and ensuing fragmentation of the lysosomal membranes require some time [START_REF] Antunes | Apoptosis induced by exposure to a low steady-state concentration of H 2 O 2 is a consequence of lysosomal rupture[END_REF]. We hypothesized, based on the results following the addition of FAC to the cells (see above), that LMP might induce DCF fluorescence by allowing lysosomal transition metals (mainly Fe) to relocate to the cytosol and meet H 2 DCF and catalyze its oxidation. LMP also results in MMP with release to the cytosol of cytochrome c with ensuing enzymatic oxidation of H 2 DCF (see further below).

Following exposure of cells to the metachromatic and lysosomotropic chromophore acridine orange (AO), normal ARPE-19 and J774 cells show a large number of distinct and bright red lysosomes when activated with blue light, while the cytosol simultaneously shows moderate green fluorescence (Figures 5 A andC).

As pointed out above, many J774 cells that were exposed to 100 µM hydrogen peroxide started to show signs of early apoptosis (nuclear pycnosis and plasma membrane blebbing). They also showed a reduced number of lysosomes and enhanced cytosolic green fluorescence following exposure to AO (Figure 5D). Meanwhile the ARPE-19 cells, which are resistant to oxidative stress, remained normal when similarly treated (Figure 5B). As mentioned earlier, apoptotic and necrotic cells show a reduced number of intact lysosomes [START_REF] Kurz | ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron[END_REF][START_REF] Kurz | Lysosomes in iron metabolism, ageing and apoptosis[END_REF][START_REF] Kurz | Intralysosomal iron chelation protects against oxidative stress-induced cellular damage[END_REF].

LMP induced without oxidative stress also causes strong diffuse DCF-induced fluorescence

The above-described results, obtained by exposing the cells to hydrogen peroxide (see also Figures 1D and2C), suggested that oxidative stress per se does not necessarily give rise to more than a slight and almost insignificant increase in DCF-induced fluorescence. In order to further substantiate that hypothesis, we wanted to induce LMP by other means than oxidative stress. As shown before, lysosomotropic detergents induce LMP [START_REF] Li | Induction of cell death by the lysosomotropic detergent MSDH[END_REF][START_REF] Kagedal | Sphingosine-induced apoptosis is dependent on lysosomal proteases[END_REF]. By exposing ARPE-19 cells to MSDH at 100 µM and then comparing the resulting time-dependent increase in DCF-fluorescence intensity with the decreased number of intact lysosomes, as visualized with the AO-uptake test, it became clear that LMP and enhanced DCF-induced fluorescence were strongly linked (Figure 6, A andB). This finding confirms that a high DCF-induced fluorescence is not necessarily a consequence of oxidative stress but rather a function of relocation to the cytosol of lysosomal redox-active iron and mitochondrial cytochrome c. However, it should be kept in mind that lysosomal rupture, with release to the cytosol not only of low mass iron but also of cathepsins, certainly secondarily induces enhanced production of superoxide and hydrogen peroxide from damaged mitochondria.

DISCUSSION

The findings of this study show that significant cytosolic oxidation of H 2 DCF to DCF depends on the combined effect of Fenton-type reactions and enzymatic activity of cytochrome c. Consequently, it should not be considered a result of the presence of unspecified 'ROS'. Rather, the reaction is apparently dependent on the relocation of transition metals in redox-active form from lysosomes and/or of cytochrome c from mitochondria. Moreover, because H 2 DCF is a hydrophilic alcohol, it would not under normal conditions be expected to traverse cellular membranes, apart from the fenestrated mitochondrial outer membrane, which is known to allow molecules up to 5,000 Da to pass [START_REF] Mannella | The 'ins' and 'outs' of mitochondrial membrane channels[END_REF].

The observed mitochondrial fluorescence from normal cells, albeit low, was also observed by at least one other group of investigators [START_REF] Swift | Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress[END_REF]. It reflects the combined presence of redox-active iron, hydrogen peroxide and enzymatically active cytochrome c in the intermembranous space. Hemeproteins, such as cytochrome c are iron-containing proteins that act as pseudoperoxidases due to their content of compound I, which consists of a ferryl Fe(IV) species and a porphyrin radical state of heme [16]. Since the water soluble, low molecular weight iron chelator CP22 was found to somewhat depress both the weak mitochondrial as well as the even weaker cytosolic fluorescence of normal cells, it is likely that some low mass iron in labile form is responsible for the formation of DCF in the cytosol of normal cells and, partly, in the mitochondria. Most probably, such iron is under transport from lysosomes, where it has been liberated following degradation of autophagocytosed ferruginous macromolecules [31], for storage in ferritin or for synthesis of ironcontaining macromolecules. As is well known, many metabolic synthetic steps that involve iron take place both in the cytosol and, above all, in the mitochondrial matrix.

When cells were initially exposed to FAC, the cytosolic and mitochondrial DCF-induced fluorescence was clearly enhanced. This observation indicates that following exposure to H 2 DCF-DA, the cleaved reaction product H 2 DCF is evenly distributed in the cytosol and also enters the mitochondrial intermembranous space. The finding that the cytosolic fluorescence of cells not exposed to FAC is weak, even after a short exposure to hydrogen peroxide, suggests that the amount of labile cytosolic iron is normally minute and that iron under transport from the lysosomal compartment may well be carried in a non-redox-active form [START_REF] Kurz | Lysosomes in iron metabolism, ageing and apoptosis[END_REF].

Only when H 2 DCF interacts with significant amounts of redox-active low mass transition metals or with cytochrome c may we expect a more pronounced general cellular DCF-induced fluorescence. It is now well established that the only cellular organelle that contains such metals (mainly iron) in significant amounts, is the lysosomal compartment [START_REF] Kurz | Lysosomes in iron metabolism, ageing and apoptosis[END_REF][START_REF] Yu | Intralysosomal iron: a major determinant of oxidant-induced cell death[END_REF]31]. We have previously shown, using the calcein method for detection of 'labile iron', that when lysosomes rupture, low mass iron is relocated to the cytosol [32]. Thus it is reasonable to assume that LMP, rather than oxidative stress per se, would be the major mechanism behind any obvious DCFinduced fluorescence. Therefore, it was no surprise to find that the exposure of cells to the lysosomotropic detergent MSDH in amounts that induce LMP and ensuing apoptosis/necrosis initiated strong DCF-induced fluorescence. Under normal conditions, hydroxyl radicals are regularly produced intralysosomally because the hydrogen peroxide that is normally present in small amounts in the cell permeates lysosomes and initiates Fenton-type reactions. These radicals are, however, not detected by the DCF-method because H 2 DCF is a hydrophilic, non-permeating molecule. Following LMP and ensuing relocation of lysosomal redox-active iron to the cytosol, the very same amount of iron now gives rise to DCF-fluorescence since occurred LMP allows H 2 DCF, redox-active iron, and hydrogen peroxide to meet. Consequently, at the very same degree of 'oxidative stress' it is the stability of the lysosomal membranes that influences the outcome of the DCF-test. Importantly, when the test is positive no higher 'oxidative stress' necessarily needs to be present than when it is negative.

Importantly, oxidative stress in the form of a brief exposure to hydrogen peroxide only slightly changed the fluorescence of both ARPE-19 cells, which are highly resistant to oxidative stress, and J774 cells that are much more sensitive. The J774 cells, however, following a 30 min period of oxidative stress and a several hours long period at standard culture conditions, showed both LMP and a strong DCF-induced fluorescence. This shows that hydrogen peroxide by itself does not A c c e p t e d M a n u s c r i p t induce oxidation of H 2 DCF, unless small amounts of labile iron are present, but also that peroxidation/fragmentation of lysosomal membranes following oxidative stress is a time-dependent process [START_REF] Antunes | Apoptosis induced by exposure to a low steady-state concentration of H 2 O 2 is a consequence of lysosomal rupture[END_REF]. Since both the studied cell types degrade hydrogen peroxide to a similar degree [START_REF] Kurz | ARPE-19 retinal pigment epithelial cells are highly resistant to oxidative stress and exercise strict control over their lysosomal redox-active iron[END_REF], it is reasonable to assume that their large differences in lysosomal stability against oxidative stress reflect much different intralysosomal concentrations of redox-active transition metals.

In the literature, we find many examples of the DCF-test being used to demonstrate 'oxidative stress' when in fact it most probably reflects LMP. A typical example is [START_REF] Li | Metallothionein protects islets from hypoxia and extends islet graft survival by scavenging most kinds of reactive oxygen species[END_REF], where it is shown (Figure 1 of the paper) that transgenic islet cells overexpressing metallothioneins are significantly "less sensitive to oxidative stress" than control cells. Importantly, in this paper the authors found that DCF-induced fluorescence engendered in normal cells following the addition of H 2 O 2 was nearly absent in cells overexpressing metallothionein, despite the fact that the same concentration of peroxide was present. We previously showed that overexpression of metallothioneins or heat shock protein 70 (HSP70) gives rise to increased autophagy of these proteins that, in turn, creates a temporary reduction of intralysosomal redox-active iron that lasts as long as the influx of metallothioneins persists. That makes lysosomes less prone to undergo LMP following exposure to various forms of oxidative stress [START_REF] Baird | Metallothionein protects against oxidative stress-induced lysosomal destabilization[END_REF][START_REF] Kurz | Autophagy of HSP70 and chelation of lysosomal iron in a non-redox-active form[END_REF].

Another example is [35] where it is shown that radiation of cultured human prostatic cancer cells in combination with exposure to hydrogen peroxide strongly enhances formation of ROS, as evaluated with the H 2 DCF-DA method. Pretreatment with ammonium chloride abolished the formation of DCF fluorescence, although cells were then exposed to the same concentration of hydrogen peroxide. Moreover, it was demonstrated that radiation plus hydrogen peroxide ruptures most lysosomes, while they were preserved in cells pre-treated with ammonium chloride. The results strongly suggest that the appearance of cytosolic DCF-induced fluorescence requires lysosomal rupture with relocation of redox-active iron that in turn mediates oxidation of H 2 DCF to DCF. Such oxidation may very well take place without any enhanced oxidative stress at all, but rather rely on normal cellular concentrations of hydrogen peroxide for the necessary formation of hydroxyl radicals.

A clear understanding of the factors that determine DCF-induced fluorescence is required in order to appreciate what DCF-induced fluorescence really means. Presently, and based on the results of this study, we suggest that it should be considered to reflect LMP and MMP with ensuing relocation to the cytosol of redox-active iron and cytochrome c rather than to be the result of some incompletely defined 'ROS or oxidative stress'.

In summary, strong DCF-fluorescence seems to require the simultaneous presence in the cytosol of either H 2 DCF, hydrogen peroxide and Fe(II) or of H 2 DCF and cytochrome c. Relocation to the cytosol of Fe(II) or cytochrome c, in turn, requires LMP or detachment of cytochrome c from the outside of the inner mitochondrial membrane. In apoptosis LMP is often an upstream event that results in MMP with release of cytochrome c [START_REF] Kurz | Lysosomes in iron metabolism, ageing and apoptosis[END_REF]31]. Consequently, induction of DCFfluorescence may indicate LMP and/or MMP, stemming from whatever cause and not just from oxidative stress.

LEGENDS FOR FIGURES

Figure 1, A -E As for Figure 1, cells were exposed to H 2 DCF-DA (10 µM; 30 min). (A). Normal J774 cells. (B). J774 cells 6 h after a 30 min long exposure to initially 100 µM hydrogen peroxide. Due to the unavoidable loss of the rounded-up apoptotic cells during the rinsing processes, few such cells remain. They are also in another plane and barely attached. Therefore, (B) is an overlay from two slightly different focal planes (the apoptotic cell is arrowed). (C) shows J774 cells mounted in HBSS with 100 µM hydrogen peroxide (compare with 1D). Note that exposure to hydrogen peroxide only causes a minor increase of fluorescence intensity. (D). To induce oxidative stressdependent apoptosis in ARPE-19 cells they had to be exposed for 30 min to 15 mM hydrogen peroxide. Following an additional 6 h period at standard culture conditions, they showed a number of apoptotic cells with strong, diffuse DCF-induced fluorescence. Mean cellular fluorescence values are displayed in Panel (E). Conditions for confocal microscopy were as for Figure 1A. Cells were prepared as described for Figure 1A, except that they were mounted in HBSS with 500 µM ferric ammonium citrate (A) or pre-incubated for 30 min (together with H 2 DCF-DA) with 100 µM of the potent, water-soluble iron-chelator CP22 and then mounted in HBSS in the continuous presence of CP22 (B). Cells were studied and documented under the confocal microscope within 5-6 min. Compared to control ARPE-19 cells (Figure 1A), exposure to low mass iron enhanced the fluorescence, while exposure to the iron-chelator depressed it. Mean cellular fluorescence values are displayed in Panel (C). Conditions for confocal microscopy were as for Figure 1A. The two cell-types were exposed to initially 100 µM hydrogen peroxide for 30 min (during the incubation a major part of the hydrogen peroxide was degraded by the cells), followed by another 6 h at standard culture conditions, and then exposed to AO as described in the Methods section. Control ARPE-19 and J774 cells (not exposed to hydrogen peroxide) are shown in (A) and (C), respectively. The ARPE-19 cells remained unaffected with intact lysosomes (B), while the J774 cells largely rounded up and often detached (compare with Figure 2B). Remaining J774 cells often showed decreased numbers of intact lysosomes in parallel with increased cytosolic and nuclear green fluorescence (due to the relocation from lysosomes to the cytosol of the metachromatic fluorophore AO), although yet no apoptotic morphology (arrow-heads) (D). A few still not detached apoptotic cells showed nuclear fragmentation (long arrow), while occasional cells had phagocytosed apoptotic cells or fragments (short arrow). The confocal laser scanning microscopy settings were the small pinhole and gains 5.0 (red detector) and 5.6 (green detector). (A). Cells were prepared as described for Figure 1A, except that they were mounted in HBSS with 100 µM of the lysosomotropic detergent MSDH. Note increasing green DCF-induced fluorescence over a 7 min period [0 (Start), 3, 5 and 7 min]. (B). Cells were pre-loaded with AO (as for Figure 5) and then mounted in HBSS with MSDH. When followed over time the number of intact lysosomes with distinct red fluorescence diminished, while the cytosolic green-yellowish fluorescence from relocated AO increased. Panel (C) shows mean cellular fluorescence values of cells in (A). The results suggest that relocation of redox-active iron and cytochrome c from lysosomes and mitochondria, respectively, gives rise to strong DCF-induced fluorescence. The confocal laser scanning microscopy settings were for (A) the medium sized pinhole and a gain of 6.5 and for (B) the small pinhole and gains 5.0 (red detector) and 5.6 (green detector).

Biochemical

  Journal Immediate Publication. Published on 23 Mar 2010 as manuscript BJ20100208 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20100208

  Journal Immediate Publication. Published on 23 Mar 2010 as manuscript BJ20100208 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20100208 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 23 Mar 2010 as manuscript BJ20100208 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20100208

  Figure 1, A -E. Exposure to H 2 DCF-DA induces weak mitochondrial and hardly noticeable cytosolic DCF-induced fluorescence in control ARPE-19 cells that only slightly changes following exposure to hydrogen peroxide, while spontaneously apoptotic cells show strong cytosolic fluorescence. (A) ARPE-19 cells were exposed to H 2 DCF-DA (10 µM; 30 min) in complete medium 48 h after seeding, rapidly rinsed and mounted in HBSS for confocal laser scanning microscopy that was initiated within 5-6 minutes. Note the mitochondrial-type green fluorescence pattern, which was confirmed (B) by the appearance of an identical red fluorescence following exposure to the mitochondria-specific fluorochrome TMRE. Figure (C) depicts a few spontaneously apoptotic cells known to contain ruptured lysosomes (membrane blebbing typical for apoptosis are arrowed) from a culture early after subcultivation when apoptotic cells are common, while (D) shows cells a few min after being mounted in 500 µM hydrogen peroxide in HBSS, but otherwise treated as in (A). Note the small difference in fluorescence intensities between (A) and (D). In Panel (E), mean fluorescence intensity values are given for (A), (C) and (D). Note the almost 9-fold increase in DCF-fluorescence in apoptotic cells as compared to normal ones, whereas the increase in fluorescence of cells exposed to hydrogen peroxide is marginal.The medium sized pinhole and a gain of 6.5 was used for (A, C and D), which caused considerable over-exposure of the apoptotic cells in (C), while for (B) the small pinhole was applied in combination with gain 5.0. Due to the disturbing green fluorescence from the metachromatic fluorophore TMRE, it was not possible to construct overlays.

  Figure 1, A -E. Exposure to H 2 DCF-DA induces weak mitochondrial and hardly noticeable cytosolic DCF-induced fluorescence in control ARPE-19 cells that only slightly changes following exposure to hydrogen peroxide, while spontaneously apoptotic cells show strong cytosolic fluorescence. (A) ARPE-19 cells were exposed to H 2 DCF-DA (10 µM; 30 min) in complete medium 48 h after seeding, rapidly rinsed and mounted in HBSS for confocal laser scanning microscopy that was initiated within 5-6 minutes. Note the mitochondrial-type green fluorescence pattern, which was confirmed (B) by the appearance of an identical red fluorescence following exposure to the mitochondria-specific fluorochrome TMRE. Figure (C) depicts a few spontaneously apoptotic cells known to contain ruptured lysosomes (membrane blebbing typical for apoptosis are arrowed) from a culture early after subcultivation when apoptotic cells are common, while (D) shows cells a few min after being mounted in 500 µM hydrogen peroxide in HBSS, but otherwise treated as in (A). Note the small difference in fluorescence intensities between (A) and (D). In Panel (E), mean fluorescence intensity values are given for (A), (C) and (D). Note the almost 9-fold increase in DCF-fluorescence in apoptotic cells as compared to normal ones, whereas the increase in fluorescence of cells exposed to hydrogen peroxide is marginal.The medium sized pinhole and a gain of 6.5 was used for (A, C and D), which caused considerable over-exposure of the apoptotic cells in (C), while for (B) the small pinhole was applied in combination with gain 5.0. Due to the disturbing green fluorescence from the metachromatic fluorophore TMRE, it was not possible to construct overlays.

Figure 2 ,

 2 Figure 2, A -E. Normal J774 cells show DCF-induced fluorescence similar to that of the ARPE-19 cells, while oxidative-stress-induced apoptotic cells display strong cytosolic fluorescence.As for Figure1, cells were exposed to H 2 DCF-DA (10 µM; 30 min). (A). Normal J774 cells. (B). J774 cells 6 h after a 30 min long exposure to initially 100 µM hydrogen peroxide. Due to the unavoidable loss of the rounded-up apoptotic cells during the rinsing processes, few such cells remain. They are also in another plane and barely attached. Therefore, (B) is an overlay from two slightly different focal planes (the apoptotic cell is arrowed). (C) shows J774 cells mounted in HBSS with 100 µM hydrogen peroxide (compare with 1D). Note that exposure to hydrogen peroxide only causes a minor increase of fluorescence intensity. (D). To induce oxidative stressdependent apoptosis in ARPE-19 cells they had to be exposed for 30 min to 15 mM hydrogen peroxide. Following an additional 6 h period at standard culture conditions, they showed a number of apoptotic cells with strong, diffuse DCF-induced fluorescence. Mean cellular fluorescence values are displayed in Panel (E). Conditions for confocal microscopy were as for Figure1A.

Figure 3 ,

 3 Figure3, A -C. Exposure to low mass iron somewhat enhances the cytosolic as well as the mitochondrial DCF-induced fluorescence of ARPE-19 cells, while exposure to an ironchelator has the opposite effect. Cells were prepared as described for Figure1A, except that they were mounted in HBSS with 500 µM ferric ammonium citrate (A) or pre-incubated for 30 min (together with H 2 DCF-DA) with 100 µM of the potent, water-soluble iron-chelator CP22 and then mounted in HBSS in the continuous presence of CP22 (B). Cells were studied and documented under the confocal microscope within 5-6 min. Compared to control ARPE-19 cells (Figure1A), exposure to low mass iron enhanced the fluorescence, while exposure to the iron-chelator depressed it. Mean cellular fluorescence values are displayed in Panel (C). Conditions for confocal microscopy were as for Figure1A.

Figure 4 ,

 4 Figure 4, A and B. Cytochrome c oxidizes H 2 DCF in the presence of hydrogen peroxide. (A). Cytochrome c (upper line), acts as a potent unspecific peroxidase that is not significantly inhibited by CP22-mediated iron-chelation (results not shown). H 2 O 2 does not by itself significantly oxidize H 2 DCF (results not shown). The hydroxyl radical-dependent H 2 DCF oxidation (Fenton reaction -middle line), is preventable by CP22-mediated iron-chelation (lower line). (B). Normalization to the cytochrome c values of (10 min) DCF fluorescence. Mean +/-STD (n=3). If error bars are not visible (as in the middle line for A), they are smaller than the symbol.Importantly, the magnitudes of the enzymatic cytochrome c reaction and the Fenton-reaction cannot be directly compared because the reducing agent cysteine, needed to reduce Fe(III) to Fe(II) for the Fenton reaction, is also an effective acceptor of hydroxyl radicals.

Figure 5 ,

 5 Figure 5, A -D. ARPE-19 and J774 cell lysosomes are differently sensitive to oxidative stress.The two cell-types were exposed to initially 100 µM hydrogen peroxide for 30 min (during the incubation a major part of the hydrogen peroxide was degraded by the cells), followed by another 6 h at standard culture conditions, and then exposed to AO as described in the Methods section. Control ARPE-19 and J774 cells (not exposed to hydrogen peroxide) are shown in (A) and (C), respectively. The ARPE-19 cells remained unaffected with intact lysosomes (B), while the J774 cells largely rounded up and often detached (compare with Figure2B). Remaining J774 cells often showed decreased numbers of intact lysosomes in parallel with increased cytosolic and nuclear green fluorescence (due to the relocation from lysosomes to the cytosol of the metachromatic fluorophore AO), although yet no apoptotic morphology (arrow-heads) (D). A few still not detached apoptotic cells showed nuclear fragmentation (long arrow), while occasional cells had phagocytosed apoptotic cells or fragments (short arrow). The confocal laser scanning microscopy settings were the small pinhole and gains 5.0 (red detector) and 5.6 (green detector).

Figure 6 ,

 6 Figure 6, A -C. Lysosomal rupture induced without oxidative stress greatly enhances cytosolic DCF-mediated fluorescence in ARPE-19 cells.(A). Cells were prepared as described for Figure1A, except that they were mounted in HBSS with 100 µM of the lysosomotropic detergent MSDH. Note increasing green DCF-induced fluorescence over a 7 min period [0 (Start), 3, 5 and 7 min]. (B). Cells were pre-loaded with AO (as for Figure5) and then mounted in HBSS with MSDH. When followed over time the number of intact lysosomes with distinct red fluorescence diminished, while the cytosolic green-yellowish fluorescence from relocated AO increased. Panel (C) shows mean cellular fluorescence values of cells in (A). The results suggest that relocation of redox-active iron and cytochrome c from lysosomes and mitochondria, respectively, gives rise to strong DCF-induced fluorescence. The confocal laser scanning microscopy settings were for (A) the medium sized pinhole and a gain of 6.5 and for (B) the small pinhole and gains 5.0 (red detector) and 5.6 (green detector).

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

ACKNOWLEDGEMENTS

We thank Mr. Stephen Hampson for linguistic editing. The financial support by Crown Princess Margareta's Foundation for the Visually Handicapped and the Linköping University Hospital Research Fund (ALF) is gratefully acknowledged.


Dr Simonetta Genovesi
email: simonetta.genovesi@unimib.it.

Prata Pizzala

Massimo Pozzi

Laura Ratti

Maria Milanese

Federico Pieruzzi

Antonio Vincenti

Andrea Stella

Giuseppe Mancia

Marco Stramba-Badiale

Daniela M Prata Pizzala

relevance of hepatic venous pressure gradient and serum calcium

Keywords: Cirrhosis, QT interval, portal hypertension, calcium, heart rate variability cirrhosis ECG, electrocardiogram, HRV, heart rate variability, HVPG, hepatic venous pressure gradient

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.

QT interval prolongation and decreased heart rate variability in cirrhotic patients:

INTRODUCTION

A wide spectrum of cardiovascular changes characterizes liver cirrhosis, ranging from the subtle subclinical alterations of preascitic stages to the hyperkinetic syndrome observed when decompensation develops [START_REF] Kowalski | The cardiac output at rest in Laennec's cirrhosis[END_REF][START_REF] Schrier | Peripheral arterial vasodilation hypothesis: a proposal for the initiation of renal sodium and water retention in cirrhosis[END_REF]. A prolongation of QT interval has been shown in patients with cirrhosis [START_REF] Bernardi | Q-T interval prolongation in cirrhosis: prevalence, relationship with severity, and etiology of the disease and possible pathogenetic factors[END_REF], and represents the most common electrocardiographic finding in this setting. Accordingly, altered ventricular repolarization is considered as part of the definition of the so-called "cirrhotic cardiomyopathy" [START_REF] Ma | Cirrhotic cardiomyopathy: getting to the heart of the matter[END_REF][START_REF] Henriksen | Dyssynchronous electrical and mechanical systole in patients with cirrhosis[END_REF][START_REF] Ytting | Prolonged Q-T(c) interval in mild portal hypertensive cirrhosis[END_REF][START_REF] Zambruni | Cardiac electrophysiological abnormalities in patients with cirrhosis[END_REF]. A prolonged QT interval is associated with a higher risk of sudden death and cardiac mortality in patients with inherited and acquired forms of long-QT syndrome, after myocardial infarction and even in healthy individuals [START_REF] Algra | QTc prolongation measured by standard 12-lead electrocardiography is an independent risk factor for sudden death due to cardiac arrest[END_REF][START_REF] Schouten | QT interval prolongation predicts cardiovascular mortality in an apparently healthy population[END_REF][START_REF] Schwartz | Prolongation of the QT interval and the sudden infant death syndrome[END_REF]. A relationship between prolonged QT interval and overall mortality in subjects with liver failure has been suggested [START_REF] Bernardi | Q-T interval prolongation in cirrhosis: prevalence, relationship with severity, and etiology of the disease and possible pathogenetic factors[END_REF], although clear evidence showing a significant increase in the incidence of sudden cardiac death in this population is still lacking. Episodes of "Torsade de pointes" in patients with liver disease have been reported, but in most cases they occurred concomitantly with the administration of drugs known to induce QT interval prolongation [START_REF] Faigel | Torsade de pointes complicating the treatment of bleeding esophageal varices: association with neuroleptics, vasopressin, and electrolyte imbalance[END_REF][START_REF] Kamisako | Torsades de pointes associated with terfenadine in a case of liver cirrhosis and hepatocellular carcinoma[END_REF][13][START_REF] Di Micoli | Terlipressin infusion induces ischemia of breast skin in a cirrothic patient with hepatorenal syndrome[END_REF]. It has been suggested that the prolongation of the QT interval in cirrhotic patients is associated with a greater severity of liver disease. However, the populations of patients studied are heterogeneous in terms of aetiology, concomitant therapies and gender [START_REF] Bernardi | Q-T interval prolongation in cirrhosis: prevalence, relationship with severity, and etiology of the disease and possible pathogenetic factors[END_REF][START_REF] Li | Clinical investigation of Q-T prolongation in hepatic cirrhosis[END_REF][START_REF] Trevisani | QT interval in patients with non-cirrhotic portal hypertension and in cirrhotic patients treated with transjugular intrahepatic porto-systemic shunt[END_REF], factors that may have affected the results. Moreover, several mechanisms may be responsible of the alterations in ventricular repolarization duration in cirrhosis, such as electrolytes' imbalance or changes in sympathetic activity: these factors should be taken into account when QT interval is analyzed in patients with advanced liver disease. The aim of the present study is to assess the potential determinants of QT interval prolongation in patients with chronic liver disease. For this purpose the duration of ventricular repolarization has been evaluated in a population of patients with significant liver disease, carefully characterized in terms of severity, aetiology and plasma electrolytes' concentration. In order to overcome the limitations of standard electrocardiogram (ECG) tracing, 24-hour Holter recordings have been analyzed, and the long term variation of QT interval has been evaluated. Furthermore, the relationship between QT interval and cardiac cycle length was analyzed as well as the heart rate variability (HRV) in the time domain. To carefully assess the influence of the severity of liver disease on QT interval, patients were classified according to the Child-Pugh score [START_REF] Pugh | Transection of the oesophagus for bleeding oesophageal varices[END_REF] and the hepatic venous pressure gradient (HVPG) was also measured.

METHODS

Study population.

Forty-eight male cirrhotic patients (median age 57, range 38-77yrs) were studied. In all patients without ascites a liver biopsy was performed for histological staging (Ishak score). Patients were classified according to Child-Pugh classification:

- alcohol consumption in the previous 6 months; treatment with beta-blockers, antivirals and any drug that may prolong-QT interval. Patients with evident cardiac disease such as valvular disease, depressed systolic function and myocardial dilatation were also excluded. The study has been performed in accordance with the Declaration of Helsinki (2000) of the World Medical Association and it has been approved by the Ethics Committee of our Institution. All patients signed an informed consent.

Portal pressure measurements HVPG measurements have been performed in all patients. A 7F venous introducer (Cordis Corporation, Miami, U.S.A.) was inserted in the right jugular vein under local anaesthesia and a 6F catheter (Meditech, Boston Scientific Corporation, Waterton, U.S.A.) was positioned under fluoroscopic guidance in a hepatic vein. The catheter was then substituted with a 6F balloon catheter which was used for measurements. HVPG was obtained by means of a pressure recorder (Sirecust 1260, Siemens Medical Electronics, Danvers, U.S.A.) in the occluded position and then after deflation of the balloon, after having checked that the tip of the catheter was freely floating in the middle of the hepatic vein. HVPG was calculated as the difference between the occluded and free hepatic venous pressure (mmHg). Three measurements were averaged.

Electrocardiographic Holter recordings and analysis.

A 24-hour ECG Holter monitoring was recorded in each subject. Holter recording was performed within 24-48 hours of HVPG measurement. All recordings were obtained using a portable batteryoperated 3-channel Holter recorder. The digitized three-channel ECG signals were processed by the Synescope Holter analysis software (ELA Medical, Mountrouge, France), which sampled the 24hour recording into 2880 templates obtained by 30-second time intervals. To improve the signal-tonoise ratio, one median complex was computed every 6 seconds from the consecutive sinus beats: then the five median beats within each 30-second template were averaged in order to obtain single representative PQRST complexes for each of the 2880 templates. For each template, an algorithm automatically measured the QT and the RR interval (ms). Measurements from the channel of lead V5 were used for the analysis. Each QT value was plotted against the cycle length, and the program automatically computed the linear regression (QT/RR) for the entire 24-hours or for pre-specified periods, and automatically provided the slope, the intersect and the correlation coefficient of the linear regression line. The program also provided for each hour the mean of QT intervals corrected for HR according to the Bazett's formula (QTc). Mean QTc, the coefficient of variation of mean QTc (CV QTc = SD QTc /mean QTc *100) and QT/RR slopes were calculated: the analysis was performed for the whole 24-hours and the periods of wakefulness/sleep. HRV has been analyzed by time domain parameters. Mean and standard deviation of RR intervals (SD RR ) were analyzed for the whole 24-hours and the periods of wakefulness/sleep. Also the coefficients of variation of RR intervals (CV RR =SD RR /mean RR *100) have been calculated for the same periods. The SD of the averages of RR intervals in all 5 minute periods (SDANN) and the % of RR intervals differing by more than 50 ms from the adjacent RR interval (pNN50) have been calculated. All measurements have been performed by an investigator who was blinded to the patient condition.

Cardiac ultrasound examination.

The echocardiograms were obtained in the standard precordial positions using digital echocardiography equipment (Aloka ProSound SSD Alpha 10, Tokyo, Japan) with 1-5MHz transducers. We followed the recommendations for standard measurements from M-mode echocardiograms [START_REF] Lang | Recommendations for chamber quantification: a report from the American Society of Echocardiography's Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology[END_REF]. Instantaneous measurements were made from three cardiac cycles and the average values of the following parameters were obtained for each subject: septal wall thickness at end-diastole (SWT), left ventricular posterior wall thickness at end-diastole (PWT) and left ventricular internal diameter at the end of diastole (LVDd). Left ventricular mass (LVM) was A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. calculated according to the formula modified by Deveraux [START_REF] Devereux | Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings[END_REF] using the American Society of Echocardiography convention and indexed for body surface area (LVMI). The presence of left ventricular hypertrophy was defined as LVMI grater than 125g/m 2 [START_REF] Lang | Recommendations for chamber quantification: a report from the American Society of Echocardiography's Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology[END_REF]. Left ventricular enddiastolic volume (LVEDV) and end-systolic volume (LVESV) were calculated by two-dimensional measurements for volume calculations using biplane method of disks (modified Simpson's rule) in apical 4-chamber and apical 2-chamber views and indexed for body surface area. Left ventricular ejection fraction (LVEF) was calculated as LVEF= (LVEDV-LVESV)/LVEDV. Echocardiographic measurements were performed by an investigator who was blinded to the patient condition.

Blood samples.

Plasma concentration of potassium, total calcium (Ca) and ionized calcium (Ca ++ ) were measured in all patients. Plasma aldosterone was measured after 3 hours of recumbent position. Potassium was measured by indirect potenziometry ISE (OMNI-S gas analyser Roche Diagnostics GmbH, Mannheim, Germany), Ca by photometry (O-cresolphtalein Complex, Modular P Roche-Hitachi Diagnostics, Tokyo, Japan) and Ca ++ by direct potenziometry ISE (OMNI-S gas analyser Roche Diagnostics GmbH, Mannheim, Germany). Plasma aldosterone concentration was measured by radioimmunoassay (RIA Kit DiaSorin, Vercelli, Italy).

Statistical analysis.

Results are expressed as mean±standard deviation. Comparisons between groups were performed by analysis of variance (ANOVA) followed by Fisher's post-hoc test. Differences in frequency of QT interval prolongation (QTc>440msec) between groups were analyzed by chi square test. Correlations between QTc, HRV measures, electrolytes' concentration, HVPG, and Child-Pugh score and age were evaluated by univariate and multivariate regression analysis. The following parameters were included in the multivariate analysis model: SD RR , Ca ++ and HVPG. The Statview Statistical package (Abacus Concepts, version 4.5) was used for statistical analysis. P values<0.05 were considered significant.

RESULTS

Characteristics of the patients.

Clinical, portal hemodynamic and biochemical data of the patients are shown in Table1. Child scores were significantly different among the three groups (p<0.001). HVPG values were significantly lower in Child A as compared to both Child B and Child C patients (p=0.001). None of the patients had clinical history of heart failure.

QT interval during Holter monitoring.

None of the patients showed complex ventricular arrhythmias during Holter recording and the incidence of ectopic beats did not differ between the three groups. Mean 24-hour QTc was progressively prolonged from Child A to Child C group. QTc was 425+24 msec in Child A, 452+30 msec in Child B, and 465+24 msec Child C (p=0.001, ANOVA. Child A vs Child B: p=0.002, Child A vs Child C: p=0.001). These differences in QTc were present both during the day and at night. Specifically, QTc was greater than 440 in 5/26 of Child A patients (19%), in 10/15 (67%) of Child B and in 6/7 (86%) of Child C (p<0.001). However, when we analyzed our data according to the aetiology, irrespective of disease severity, we found that 15/18 (83%) patients with alcohol-related disease had a QTc>440 msec, whereas QTc was prolonged in only 6/30 (20%) patients with post-viral cirrhosis (p=0.001). Interestingly, the patient with the Heart rate and heart rate variability. Mean 24-hour RR intervals were shorter in Child C patients as compared with the other two groups.

HRV measured in the time domain showed significant differences between groups. 24-hour SD RR , SDANN and pNN50 progressively decreased with the increase of liver diseases severity. Of note, the difference in HRV in the three groups of patients was not affected by different heart rates, as also the CV RR progressively decreased (Table2).

Echocardiographic findings.

The echocardiographic variables in the population studied did not significantly differ between groups (Table3). Specifically, all patients showed normal left ventricular systolic function. Although patients belonging to Child B and C group had a slightly greater values of LEDVI, none of the patients had left ventricular dilatation. LVMI was not different in the three groups although left ventricular hypertrophy was present in 6/25 (24%) Child A patients, in 3/14 (21%) Child B patients and in 1/6 (16%) Child C.

Electrolytes' concentration.

Calcium and Ca ++ serum concentrations differed in the three groups (Table1). In details, Ca serum levels were significantly higher in Child A group as compared to the other two groups (p=0.001), whereas the difference in Ca ++ serum levels was significant between Child C and Child A patients (p=0.05). However, Ca serum levels were below the lower normal limits (2.15mmol/L) in only 7 patients (4 Child B and 3 Child C), while Ca ++ serum levels were in the normal range in all patients.

Regression analysis.

At univariate analysis mean 24-hour QTc duration was significantly and directly correlated with HVPG values (r=0.43, p=0.003, Figure3A) and Child score (r=0.51, p<0.001). SD RR was significantly and inversely correlated with HVPG values (r=0.59, p<0.001, Figure3B). A correlation between SD RR and QTc was also found (r=0.44, p=0.002, Figure3B). Twenty-four-hour mean QTc was inversely correlated with Ca (r=0.58, p<0.001) and with Ca ++ (r=0.51, p<0.001, Figure3A) serum levels. No correlation was found between QTc, SD RR values, HVPG and the echocardiographic parameters. Also age did not correlate with electrocardiographic or portal hemodynamic parameters. At multiple regression analysis only HVPG and Ca ++ were independently correlated to QTc (p=0.002 and p=0.05, respectively).

DISCUSSION

In our study, strict inclusion criteria have been adopted. In fact, patients who had characteristics which may potentially affect ventricular repolarization duration, i.e. co-morbidities, drug able to prolong QT interval, major complications of disease, have been excluded. The present study has shown that QT interval measured from ECG Holter monitoring is prolonged in a significant portion of patients with cirrhosis. The duration of ventricular repolarization was normal in the majority of patients with less severe liver disease, while it was markedly altered in patients with more severe cirrhosis. As most of the patients with a QT interval prolongation were affected by alcohol-related cirrhosis, independently of the severity of the disease, it is possible that aetiology might have played a role. Also the relation between QT interval and cardiac cycle length in the 24 hours, expressed as the slope of the QT/RR regression line, was altered in patients with alcoholic cirrhosis, suggesting that these patients have a further QT interval prolongation when heart rate decreases. A significant correlation between QTc duration and HVPG, a reliable marker of the degree of portal hypertension, has been found. This suggests that portal hypertension may represent a major pathophysiological mechanism involved in the alterations of ventricular repolarization. The presence of severe portal hypertension was also associated with decreased heart rate variabilty, likely reflecting a higher sympathetic tone. Interestingly, QTc was inversely correlated with plasma ionized calcium concentration, although all patients showed values within the normal limits.

Mechanisms of QT interval prolongation.

QT interval was more prolonged in patients with greater Child-Pugh score, corresponding to clinically significant liver disease, in agreement with previous studies [START_REF] Henriksen | Dyssynchronous electrical and mechanical systole in patients with cirrhosis[END_REF][START_REF] Zambruni | Cardiac electrophysiological abnormalities in patients with cirrhosis[END_REF][START_REF] Bal | Prolongation of QTc interval: relationship with etiology and severity of liver disease, mortality and liver transplantation[END_REF]. The majority of patients with higher Child-Pugh score were affected by alcohol-related cirrhosis and most of them showed a prolongation of the QT interval duration independently of the severity of liver disease. Data on the role of cirrhosis aetiology in the genesis of ventricular repolarization alterations seem controversial. In the study by Bernardi [3] the prevalence of QT interval prolongation did not differ between patients affected by alcohol-related cirrhosis and those with the post-viral disease. However, only 7% of patients were affected by alcoholic cirrhosis and 12% had cirrhosis of mixed aetiology. In a study by Bal [START_REF] Bal | Prolongation of QTc interval: relationship with etiology and severity of liver disease, mortality and liver transplantation[END_REF] a prolonged QTc was seen more commonly in patients with alcoholic cirrhosis (60%) as compared to non-alcoholic cirrhosis (35%) and alcoholic cirrhosis was one of the independent predictors of QT interval prolongation. The present study suggests that the alcoholic aetiology may indeed play a role in the prolongation of QT interval in cirrhotic patients, although the relative small size of the population does not allow the exact definition of its contribution. Of note, although patients belonging to Child B and C group had a slightly greater values of left end-diastolic volumes, none of our patients had left ventricular dilation and the prevalence of left ventricular hypertrophy was not affected by aetiology. The absence of marked alterations in left ventricular function or size in our population may be partly explained by our strict exclusion criteria. In fact, patients with overt structural heart disease and those with atrial fibrillation had been excluded. Thus, it seems unlikely that the abnormalities in ventricular repolarization might have been caused by structural cardiac alterations related to long-term alcohol exposure.

In the present study, we have also measured the HVPG to better characterize the population [START_REF] Vorobioff | Hepatic venous pressure in practice: how, when, and why[END_REF]. The observation in our study that HVPG is significantly correlated with QT interval duration indicates that ventricular repolarization is prolonged particularly when clinically significant portal hypertension develops. Portal hypertension may represent a major pathophysiological mechanism involved in the alterations of ventricular repolarization. It has been shown that QT interval may be prolonged also in patients with non-cirrhotic portal hypertension and preserved liver function [START_REF] Trevisani | QT interval in patients with non-cirrhotic portal hypertension and in cirrhotic patients treated with transjugular intrahepatic porto-systemic shunt[END_REF] and it has been hypothized that in portal hypertension with portosystemic shunting a dumping into the systemic circulation of splanchnic-derived substances, such endotoxins and cytokines, or autocoids, substances that are produced locally in the heart, may contribute to the alterations in ventricular repolarization [START_REF] Zambruni | Cardiac electrophysiological abnormalities in patients with cirrhosis[END_REF]. We have found a significant negative correlation between QT interval duration in the 24-hours and both total and ionized plasma calcium values, although ionized plasma levels were within the normal limits in all patients. This finding is in agreement with our previous results obtained in a population of patients with end stage renal disease undergoing haemodialysis [START_REF] Genovesi | Electrolyte concentration during haemodialysis and QT interval prolongation in uraemic patients[END_REF]. In that study changes in plasma calcium concentration induced by the haemodialysis session were inversely correlated with changes in QT interval. It has been demonstrated that a decrease in plasma calcium concentration is associated with an increase in ventricular action potential duration and QT interval prolongation [START_REF] Ter Keurs | Calcium and arrhythmogenesis[END_REF]. In the present study we have shown that in patients with cirrhosis even small differences in calcium plasma levels are associated with significant differences in QT interval duration. As patients with advanced cirrhosis show an alteration of calcium homeostasis [START_REF] Mawer | Metabolism of vitamin D in patients with primary biliary cirrhosis and alcoholic liver disease[END_REF][START_REF] Collier | Guidelines on the management of osteoporosis associated with chronic liver disease[END_REF] a close monitoring of both total and ionized plasma calcium concentration should be performed in this clinical setting.

QT interval and its relationship with cardiac cycle length in liver cirrhosis.

The duration of ventricular repolarization is traditionally assessed by measuring the QT interval from a short surface electrocardiogram. Moreover, as the QT interval is affected by the cardiac cycle length, a correction for heart rate is necessary. Although several formulas for heart rate correction have been proposed, the most commonly used is the Bazett's formula, the standard for clinical use. In the context of studies performed in cirrhotic patients a novel formula has been proposed [START_REF] Zambruni | QT interval correction in patients with cirrhosis[END_REF] and utilized. However, even if a particular formula may be more accurate in a defined population of patients and can be utilized in clinical research, its widespread use is not warranted, as a comparison with other populations of patients with different clinical conditions becomes unfeasible.

To overcome the limitations inherent to the measurement from a short ECG tracing, at variance with previous studies, except one [27], QT interval was analyzed from a 24-hour Holter recording, by using a robust dedicated algorithm which automatically measures QT interval and RR interval [START_REF] Stramba-Badiale | Gender and the relationship between ventricular repolarization and cardiac cycle length during 24-h Holter recordings[END_REF]. As Bazett's formula may not be accurate at high and low heart rate, the relationship between the absolute value of QT interval and the cycle length expressed as the slope of linear regression line was also considered. Interestingly, patients with alcohol-related cirrhosis had a steeper slope than patients with post-viral cirrhosis, irrespective of disease severity, suggesting that also these alterations in ventricular repolarization may be partly explained by the aetiology of cirrhosis. We do not have a clear explanation of this finding that may have clinical relevance as the further prolongation of QT interval at long cycle lengths may favour the occurrence of bradycardia-related "Torsades des pointes" [START_REF] Roden | Incidence and clinical features of the quinidine-associated long QT syndrome: implications for patient care[END_REF].

Clinical relevance of electrocardiographic abnormalities.

Prolongation of QT interval is associated with an increased risk of sudden death and cardiovascular mortality in several clinical conditions [START_REF] Algra | QTc prolongation measured by standard 12-lead electrocardiography is an independent risk factor for sudden death due to cardiac arrest[END_REF][START_REF] Schouten | QT interval prolongation predicts cardiovascular mortality in an apparently healthy population[END_REF][START_REF] Schwartz | Prolongation of the QT interval and the sudden infant death syndrome[END_REF]. Sudden death is considered uncommon in cirrhosis. However, a study performed in subjects with alcoholic cirrhosis showed a higher incidence of sudden death in those with QT interval prolongation [START_REF] Day | QT prolongation and sudden cardiac death in patients with alcoholic liver disease[END_REF]. In fact QT interval prolongation was independently associated with the risk of mortality. "Torsades de pointe" have been sporadically described in cirrhotic patients, although in concomitance with the use of drugs which may induce QT interval prolongation [START_REF] Faigel | Torsade de pointes complicating the treatment of bleeding esophageal varices: association with neuroleptics, vasopressin, and electrolyte imbalance[END_REF][START_REF] Kamisako | Torsades de pointes associated with terfenadine in a case of liver cirrhosis and hepatocellular carcinoma[END_REF][13][START_REF] Di Micoli | Terlipressin infusion induces ischemia of breast skin in a cirrothic patient with hepatorenal syndrome[END_REF]. An increased sympathetic activity to the heart has been shown to favour life-threatening arrhythmias. In our study we have found that heart rate variability, which reflects the balance between the parasympathetic and the sympathetic tone to the heart, progressively decreased with the increase of portal pressure, independently of the basal heart rate. Patients with advanced cirrhosis have increased levels of plasma catecholamines [START_REF] Henriksen | Circulating noradrenaline and central haemodynamics in patients with cirrhosis[END_REF][START_REF] Nicholls | Elevated plasma norepinephrine concentrations in decompensated cirrhosis. Association with increased secretion rates, normal clearance rates, and suppressibility by central blood volume expansion[END_REF] and of muscle sympathetic nervous traffic, a direct index of autonomic nervous system activity [START_REF] Floras | Increased sympathetic outflow in cirrhosis and ascites: direct evidence from intraneural recordings[END_REF][START_REF] Pozzi | Early effects of total paracentesis and albumin infusion on muscle sympathetic nerve activity in cirrhotic patients with tense ascites[END_REF]. Cirrhotic patients with oesophageal varices are currently treated with beta-blockers to decrease portal pressure and the bleeding risk. These agents also reduce the sympathetic activity to the heart and are the treatment of choice of the long-QT syndrome, significantly improving survival in these patients as well as in those with myocardial infarction or heart failure [START_REF] Packer | The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. U.S. Carvedilol Heart Failure Study Group[END_REF][START_REF] Fauchier | Comparison of the beneficial effect of beta-blockers on mortality in patients with ischaemic or non-ischaemic systolic heart failure: a meta-analysis of randomised controlled trials[END_REF][START_REF] Gottlieb | Effect of beta-blockade on mortality among high-risk and low-risk patients after myocardial infarction[END_REF][START_REF] Dargie | Effect of carvedilol on outcome after myocardial infarction in patients with left-ventricular dysfunction: the CAPRICORN randomised trial[END_REF]. We cannot exclude that these beneficial effect of beta-blockers may have played a role in the reduction of risk of cardiovascular mortality in patients with cirrhosis. Interestingly, in cirrhotic patients with a more prolonged QT interval the administration of beta-blockers reduces sympathetic activity to the heart and decreases the duration and the spatial dispersion of QT interval [START_REF] Zambruni | Effect of chronic beta-blockade on QT interval in patients with liver cirrhosis[END_REF][START_REF] Henriksen | Acute non-selective betaadrenergic blockade reduces prolonged frequency-adjusted Q-T interval (QTc) in patients with cirrhosis[END_REF], as already demonstrated in other clinical conditions [START_REF] Stramba-Badiale | Effects of beta-adrenergic blockade on dispersion of ventricular repolarization in newborn infants with prolonged QT interval[END_REF].

In conclusion, cirrhotic patients with a more severe disease, especially of alcoholic aetiology, who show greater venous pressure gradient and lower calcium plasma levels, have an altered ventricular repolarization and a reduced vagal activity to the heart that may predispose to life-threatening arrhythmias. Further studies are necessary to prospectively assess the time-course of changes of heart rate variability and QT interval duration in cirrhotic patients. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figures and Tables

  Child A: 26 patients, 22 with a viral aetiology (18 HCV-RNA+ and 4 HBV-DNA+) and 4 with alcohol-related cirrhosis. -Child B: 15 patients, 7 with a viral aetiology (6 HCV-RNA+ and 1 HBV-DNA+) and 8 with alcohol-related cirrhosis. -Child C: 7 patients, 1 with a viral aetiology (HCV-RNA+) and 6 with alcohol-related cirrhosis. None of the patients had a mixed aetiology. Exclusion criteria were any systemic, endocrine, lung and neoplastic disease; diabetes; arterial hypertension; recent haemorrhage (<3months); anaemia (Hb<11g/dL), serum creatinine>1,5mg/dL; Clinical Science Immediate Publication. Published on 10 Dec 2008 as manuscript CS20080325 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080325A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 10 Dec 2008 as manuscript CS20080325 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080325

  Clinical Science Immediate Publication. Published on 10 Dec 2008 as manuscript CS20080325 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080325 A c c e p t e d M a n u s c r i p t longest QT interval (502 msec), although affected by alcohol-related disease, belongs to Child B group (Figure1).No differences in QTc were found between patients with HCV or HBV-related aetiology. Twenty-four-hour CV QTc did not differ between Child groups, suggesting that QT interval variability was not affected by the liver disease stage. The slope of the linear regression line, which expresses the relation between QT interval and cardiac cycle length in the 24-hours, was slightly but not significantly flatter in Child A group as compared with the other two groups (Child A: 0.16+0.04, Child B: 0.19+0.10, Child C: 0.18+0.07). Patients with alcohol-related cirrhosis had a steeper slope than patients with post-viral cirrhosis, irrespective of disease severity (0.21±0.06 vs 0.16±0.04, p=0.02),suggesting that these subjects have a further QT interval prolongation when heart rate decreases (Figure2).

Clinical

  Science Immediate Publication. Published on 10 Dec 2008 as manuscript CS20080325 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080325A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Clinical

  Science Immediate Publication. Published on 10 Dec 2008 as manuscript CS20080325 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080325

Figure 1 .

 1 Figure 1. QTc mean and single QTc values according to Child-Pugh classification. White points: patients with viral aetiology; black points: patients with alcohol-related disease. *: p<0.05 vs Child B; †: p<0.001 vs Child C.

Figure 2 .

 2 Figure 2. Example of QT/RR linear regression slopes recorded from a patient with alcoholrelated cirrhosis (A) and from a patient with viral aetiology (B).QTc: QT intervals corrected for heart rate; r: correlation coefficient.

Figure 3 .

 3 Figure 3. A) Scatter plot of QTc versus HVPG and Ca ++ values; B) Scatter plot of SD RR versus HVPG and QTc values.QTc: QT intervals corrected for heart rate; HVPG: hepatic venous pressure gradient; SD RR : standard deviation of RR intervals.

Table 1 . Demographic, clinical, portal hemodynamic and biochemical characteristics of 48 patients according to Child-Pugh classification.

 1

	HVPG: hepatic venous pressure gradient.
		Child A (n=26)	Child B (n=15)	Child C (n=7)	p (ANOVA)
	RR (msec)	850 ± 105 †	856 ± 160 † 740 ± 79	0.05
	CV RR	13.5 ± 3.4 * † 11.1 ± 3.3	9.1 ± 2.2	0.002
	pNN50 (%)	5.9 ± 7.1 †	4.5 ± 6.2	1.2 ± 0.6	0.05
	SDANN (msec) 109 ± 30 * †	88 ± 40	67 ± 17	0.008
	SD RR (msec)	115 ± 30 †	98 ± 40 †	67 ± 17	0.004

Table 2 . Heart rate variability parameters (time domain).

 2 Clinical Science Immediate Publication. Published on 10 Dec 2008 as manuscript CS20080325 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20080325 A c c e p t e d M a n u s c r i p t

	RR: RR interval; SD RR : standard deviation of RR intervals; SDANN: SD of the averages of RR intervals in
	all 5 minute periods; pNN50: percentage of RR intervals differing by more than 50 ms from the adjacent RR;
	CV RR : coefficient of variation of RR intervals.
	*: p<0.05 vs Child B; †: p<0.05 vs Child C.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 3 .

 3 Echocardiographic parameters.

SWT, septal wall thickness at end diastole; PWT, left ventricular posterior wall thickness at end diastole; LVDd, left ventricular internal dimension at end diastole; LVMI, left ventricular mass indexed for body surface area; LVEDVI, left ventricular end-diastolic volume indexed for body surface area; LVEF, left ventricular ejection fraction.

© 2008 The Authors Journal compilation © 2008 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited