NDT Advance Access originally published online on September 22, 2006
Nephrology Dialysis Transplantation 2006 21(12):3443-3449; doi:10.1093/ndt/gfl443
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Low albumin levels increase endothelial NO production and decrease vascular NO sensitivity
1Laboratory of Vascular Medicine, 2Department of Nephrology and Hypertension, University Medical Centre Utrecht, Institute and Graduate School of Biomembranes, 3Faculty of Science, Section Interface Physics, Utrecht University, Utrecht, The Netherlands and 4Division of Nephrology and Hypertension, Departments Medicine, Physiology and Biophysics, University of California, Irvine, USA
Correspondence and offprint requests to: Jaap A. Joles, DVM, PhD, Department of Nephrology and Hypertension F03.223, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Email: J.A.Joles{at}med.uu.nl
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Abstract |
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Background. Hypoalbuminaemia is associated with increased risk of cardiovascular disease. It is unclear whether endothelial dysfunction is a direct result of low albumin or whether it is caused by factors like chronic inflammation or dyslipidaemia. In this study, the effect of low albumin concentrations on endothelial nitric oxide synthase (eNOS)-dependent NO production was determined in vitro and ex vivo.
Methods. eNOS activity, assessed by arginine–citrulline conversion, and NO production, determined by 4,5-diaminofluorescein diacetate, electron paramagnetic resonance and Griess colorimetry, were measured in cultured endothelial cells expressing high levels of eNOS (bEnd.3) after exposure to albumin concentrations ranging from 0.5 mmol/l (33 g/l) to 0 mmol/l. Analbuminaemic and control rat plasma NO metabolites and aortic eNOS protein mass were determined, and aortic endothelium-independent and endothelium-dependent vasodilator tone were measured ex vivo under albumin-free conditions.
Results. In vitro, eNOS activity was significantly increased in the absence of albumin (75 ± 2 vs 26 ± 6 pmol/min/mg protein; P < 0.01). Low albumin levels consistently increased NO production in endothelial cells. Plasma NO metabolites were increased (18.2 ± 1.9 vs 12.5 ± 0.8 µmol/l; P < 0.05) and endothelium-independent relaxation was markedly blunted in analbuminaemic rats, resulting in a considerably higher ED50 (80 ± 2 vs 1.1 ± 0.2 nmol/l, P < 0.01), while endothelium-dependent dilatation was slightly, but significantly, increased. Aortic eNOS protein mass was not affected. This implies that in vivo hypoalbuminaemia reduces vascular NO sensitivity.
Conclusion. We show that low albumin as such seems to enhance, rather than diminish, eNOS-mediated endothelial NO production.
Keywords: analbuminaemia; endothelial nitric oxide synthase; NO
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Introduction |
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Hypoalbuminaemia is associated with endothelial dysfunction [1,2], all-cause mortality in patients with chronic kidney disease (CKD) [3] and cardiovascular mortality in patients with end-stage renal disease [4]. However, it is unclear whether endothelial dysfunction is a direct result of the decreased levels of albumin or whether it is caused by factors such as chronic inflammation [5] and dyslipidaemia [1], present in most conditions associated with hypoalbuminaemia. Albumin may be viewed as a negative acute phase protein [6] and hypoalbuminaemia may well be a marker for chronic inflammation rather than a direct cause of endothelial dysfunction.
In the present study, we hypothesized that acute and chronic exposure to low-albumin levels as such, in the absence of other systemic factors, does not decrease endothelial nitric oxide synthase (eNOS) activity, diminish nitric oxide (NO) production, or cause endothelial dysfunction. Vasodilatation is a response involving the interaction of endothelium with vascular smooth muscle. Therefore, possible effects of hypoalbuminaemia on endothelium and vascular smooth muscle should be considered separately.
A microvascular endothelial cell line (bEnd.3), expressing high levels of eNOS but lacking neuronal or inducible NOS [7], was used to determine the effect of acute exposure to albumin levels, ranging from a physiological concentration of 0.5 mmol/l (33 g/l) to 0 mmol/l, on eNOS activity and NO production in vitro. Consistent with our hypothesis this experiment showed that acute exposure to low-albumin levels does not decrease eNOS activity or diminish NO production. On the contrary eNOS activity and NO production were consistently enhanced. Thus we tested our hypothesis under conditions of chronic exposure to low albumin in analbuminaemic rats [8] by measuring plasma NO metabolites and eNOS protein mass in vivo, and endothelium-independent and endothelium-dependent aortic vascular function ex vivo under albumin-free conditions.
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Cell culture
An immortalized bEnd.3 cell line was provided by Dr Alan Schwartz (University of Washington, St Louis, MO). These cells, that only contain eNOS, show substantially higher NO production than other endothelial cells such as HUVEC or HMEC [7]. Cells were cultured as described previously [7]. Prior to each experiment, cells were starved for 16 h in Dulbecco's Modified Eagles Medium (DMEM) containing only 0.1% bovine serum albumin (BSA). Culture media were obtained from Life Technologies (Burlington, ON, Canada).
Measurement of osmolality and colloid osmotic pressure (COP)
Osmolality and COP in phosphate buffered saline (PBS) containing different concentrations of BSA were measured as previously described [8]. No differences were found in osmolality, but there were substantial differences in COP (Table 1).
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Measurement of NO—4,5-diaminofluorescein diacetate assay
To measure intracellular NO production, the cell-permeable fluorescent NO indicator, 4,5-diaminofluorescein diacetate (DAF-2DA; Calbiochem), was used as described previously [7]. Briefly, bEnd.3 cells were loaded with 5 µmol/l DAF-2DA and incubated for 20 min at 37°C in the presence or absence of the NOS inhibitor N
-nitro-L-arginine methyl ester (L-NAME) (300 µmol/l; Sigma, St Louis, MO). Different concentrations of fatty acid free BSA (Sigma) were added, after which fluorescence was measured every 2 min for 20 min (excitation wavelength, 485 nm; emission wavelength, 538 nm; Fluoroskan Ascent, Labsystems, Helsinki, Finland). Note that no stimulus for NO production was given to the cell cultures. Endogenous NO production was calculated by determining slopes of each line by linear regression analysis. Reactivity of DAF-2DA towards NO was calibrated by exposing bEnd.3 cells to 10–100 µmol/l diethylenetriamine NONOate (DETA/NO; Sigma), as described previously [7]. Results were expressed, relative to 0.5 mmol/l BSA (33 g/l), as eNOS-dependent NO production (total NO production minus NO production in the presence of L-NAME). Inter-assay variability was 3.2 ± 1.3%, and intra-assay variability was 4.0 ± 1.2%.
Measurement of nitrite—griess assay
Both intra- and extracellular nitrite levels were determined using the Nitrate/Nitrite Colorimetric Assay Kit of Cayman Chemical (ITK Diagnostics, Uithoorn, The Netherlands). Cells were incubated for 20 min with different concentrations of BSA in the presence or absence of 50 ng/ml vascular endothelial growth factor (VEGF; Sigma) or 300 µmol/l L-NAME at 37°C. Absorbance was measured at 540 nm using a plate reader (Multiskan Ascent, Labsystems, Helsinki, Finland). Results were expressed, relative to control, as eNOS-dependent NO production (total NO production minus NO production in the presence of L-NAME).
Measurement of NO—electron spin resonance
NO trapping with Fe-DETC complexes was initiated by replacing the medium with 10 ml fresh DMEM containing 2.5 mmol/l diethyl dithiocarbamate (DETC) and 10 µmol/l ferrous sulfate (Fluka Buchs, Switzerland). After 10 min, the medium was replaced with a medium containing either no BSA or 0.5 mmol/l BSA in the presence or absence of 300 µmol/l L-NAME. After 20 min of NO trapping at 37°C, the flask was placed on ice for 2 min to terminate enzymatic activity. Note that no stimulus for NO production was given to the cell cultures. Cells were scraped and harvested in 10 ml incubation medium. The cell fraction with Fe-DETC complexes was separated by ultracentrifugation (1000 g for 10 min at 4°C), resuspended in 350 µl incubation medium, drawn into a syringe (id 4.8 mm) and snap frozen in liquid nitrogen until electron paramagnetic resonance (EPR) assay.
EPR spectra were recorded at 77 K on a modified X-band ESP 300 radiospectrometer (Bruker BioSpin, Karlsruhe, Germany) operating near 9.54 GHz with 20 mW power. Frozen samples were placed in a quartz liquid finger dewar at the centre of a Bruker ER4103TM cavity. Field modulation was 0.5 mT, gain 2 x 105, time constant and analog-to-digital (ADC) conversion time 82 ms. Nine scans were accumulated to reduce instrumental noise. Spin densities were calibrated with frozen reference solutions of NO-Fe2+-(MGD)2 in PBS buffer.
Measurement of eNOS activity—arginine-citrulline conversion assay
NOS activity was measured by determining the formation of L-3H-citrulline from L-3H-arginine. Cells were incubated for 20 min with either none or 0.5 mmol/l BSA and homogenized in a buffer consisting of 50 mmol/l Tris buffer, pH7.4, 320 mmol/l sucrose, 1 mmol/l ethylene-diaminetetraacetic acid, 1 mmol/l dithiothreitol (DTT), 2 mg/l aprotinin and 100 mg/l phenylmethylsulfonylfluoride. Homogenate (50 µl) was incubated in a final volume of 100 µl at 37°C for 30 min with 1 mmol/l L-citrulline, 300 µmol/l tetrahydrobiopterin, 3000 U/ml calmodulin, 0.5 mmol/l nicotinamide adenine dinucleotide phosphate (NADPH), and 1 mmol/l CaCl2, all dissolved in a buffer consisting of 50 mmol/l KH2PO4 containing 1 mmol/l DTT and 10 µmol/l L-3H-arginine. Prior to incubation, L-14C-citrulline was added to correct for procedural losses. Substituting NADPH by 100 mmol/l L-NAME corrected for non-specific activity. The incubation was stopped by placing the tubes on ice and adding 20 mmol/l ice-cold Hepes buffer, pH 5.5, followed by separation of arginine and citrulline on Dowex 50X8-200 ion exchanger resin and liquid scintillation counting of the citrulline fraction. All measurements were performed in triplicate. Results are expressed as picomole per minute per milligram protein. Inter-assay variability was 2.2 ± 0.8%, and intra-assay variability was 2.2 ± 0.3%.
Animals
Adult (3 to 4 month-old) male Sprague–Dawley rats (SDR; 200–350 g; Harlan–Olac, The Netherlands) and Nagase analbuminaemic rats (NAR; 200–350 g; Central Laboratory Animal Institute, Utrecht, The Netherlands) were housed in a temperature- and light-controlled room. Rats had free access to standard rat chow (Special Diet Services, Witham, Essex, UK) and tap water. Sentinel animals were monitored regularly for infection by nematodes and pathogenic bacteria, as well as antibodies for a large number of rodent viral pathogens, and were consistently negative throughout the course of the experiments. The Utrecht University board for studies in experimental animals approved the studies.
Organ chamber experiments
Under general barbiturate anaesthesia rats were euthanized by exsanguination. Plasma nitrite plus nitrate levels were measured with the Griess assay as aforedescribed. Thoracic aortas were dissected free and immersed in a carbogenized Krebs–Ringer buffer, pH 7.4. Indomethacin (10 µmol/l) was added to the buffer to prevent the formation of endogenous prostaglandins. Aortas were carefully cleaned of blood clots and peri-aortic tissue and cut into rings of similar weight and dimensions (2–4 mm long). Care was taken not to damage the endothelium. Rings were mounted horizontally between two stainless steel hooks in organ chambers filled with 10 ml of Krebs–Ringer buffer at 37°C plus indomethacin and gassed with 95% O2-5% CO2. One hook was anchored in the organ chamber and the other was connected to a strain gauge transducer for the measurement of isometric tension. Note that these ex vivo measurements were done under albumin-free conditions. Aortic rings were progressively stretched to an optimal basal tension of 1 g, and the contraction ability of the rings to a saturated solution of KCl was checked. Only rings that could generate a 2 g contraction force were studied. All drugs were dissolved and diluted in saline (0.9% NaCl), except KCl which was dissolved in demineralized water. Relaxation to sodium nitroprusside (SNP; 0.01 nmol/l–100 µmol/l; Sigma) or acetylcholine (ACh; 0.01–100 µmol/l; Sigma) was examined and compared between SDR and NAR. Relaxation was studied after precontraction by sufficient phenylephrine (Sigma) to generate a 2 g contraction force. ACh measurements were also performed after pre-incubation for 90 min with the arginine-analogue NG-nitro-L-arginine (L-NNA; 1 mmol/l Krebs buffer). L-NNA was also added to the organ chamber. At least four rats and three rings per rat were used in each condition. The delay between killing the rats and measurement of relaxation was standardized at 90 min.
eNOS expression in aorta
Frozen tissue was processed for determination of eNOS protein abundance. eNOS monoclonal antibody, and monoclonal eNOS, as well as peroxidase-conjugated goat anti-mouse immunoglobulinG antibody were purchased from Transduction Laboratories. Total protein was determined with a kit (Bio-Rad Laboratories, Hercules, CA).
Statistical analysis
Results are expressed as mean ± SEM. In pre-contracted rings, ACh and SNP responses are expressed as percent relaxation. Dose-response data were compared with two-way analysis of variance. If a variance ratio reached statistical significance, the studentized Newman–Keuls test was performed as post hoc test. Unpaired data were compared with a t-test. P < 0.05 was considered significant.
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Acute effects of low albumin on eNOS activity and NO production in endothelial cells
Exposure time to BSA in the cell experiments was based on time course studies. No significant differences in DAF signal were found between 20, 60 and 180 min of incubation (1.00 ± 0.04, 0.99 ± 0.11 and 1.14 ± 0.13, respectively). As longer incubations had no effect, we chose to incubate for 20 min.
Arginine–citrulline conversion was used to assess the acute in vitro effect of low-albumin on eNOS activity. The activity of eNOS was significantly increased in the absence of bovine serum albumin (BSA; 75 ± 2 vs 26 ± 6 pmol/min/mg protein; P < 0.01; Figure 1). Measurement of NO production was critical for our question. Thus to measure the effects of low albumin on NO production, three different methods were used. The NO-induced DAF signal (Figure 2A) and both basal as well as VEGF-induced nitrite levels (Figure 2B) were all inversely related to BSA concentration. Since these two techniques are indirect ways to measure NO production, EPR was used to determine NO production directly. A typical EPR spectrum (Figure 2C) showed a clear triplet hyperfine structure centred at g = 2.035 as expected for ferrous mono-nitrosyl iron-dithiocarbamate complexes (MNIC). As expected for biological samples [10], a small contribution from paramagnetic Cu2+-DETC complexes was superposed. The intense central hyperfine line of these Cu2+-DETC complexes is visible near g = 2.01 (Figure 2C). Pre-incubation for 20 min with 300 µmol/l L-NAME reduced the MNIC yield in a single 75 cm2 flask to below the detection limit of ca 10 pmol at the given spectrometer settings. NO production was significantly increased in the absence of BSA (55.3 ± 3.3 pmol per flask) as compared with 0.5 mmol/l BSA (35.4 ± 5.0 pmol per flask; P = 0.034). Note that after incubation with or without BSA cells were always washed before the in vitro measurements were performed.
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Plasma NO metabolites, vascular function and eNOS mass in aorta from NAR
To assess the chronic effect of hypoalbuminaemia we used hereditary analbuminaemic rats (NAR, n = 6) [8] and compared them with control SDR (n = 6). Plasma NO metabolites were increased in vivo (18.2 ± 1.9 vs 12.5 ± 0.8 µmol/l; P < 0.05). Endothelium-independent vasodilatation induced by SNP is a measure of the sensitivity of the vascular smooth muscle to NO. The SNP response measured under albumin-free conditions ex vivo was significantly blunted in NAR aortic rings (Figure 3). This resulted in a considerably higher ED50 in NAR vs SDR (80 ± 2 vs 1.1 ± 0.2 nmol/l, P < 0.01). Endothelium-dependent vasodilatation as induced by ACh is a measure of NO bioavailability and, indirectly, eNOS activity. Ex vivo ACh-induced relaxation was slightly, but significantly (P < 0.05), stronger in NAR than in SDR (Figure 4). Pre-incubation with L-NNA completely prevented ACh-mediated relaxation in both strains. Protein abundance of eNOS in the aorta was similar in NAR and SDR (Figure 5).
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In this study, we demonstrated that low-albumin levels, often viewed as causal factor in the pathogenesis of endothelial dysfunction and cardiovascular disease in CKD and proteinuric conditions, does not reduce eNOS activity and NO production in the absence of other systemic factors. Albumin concentrations ranging from a physiological concentration of 0.5 mmol/l (33 g/l) to 0 mmol/l were used. Using several different experimental methods for the critical measurement of NO production, we consistently found that NO production in vitro was not decreased, but in fact increased in the absence of albumin. Our findings are in line with observations by others that the causes of hypoalbuminaemia, such as malnutrition, dyslipidaemia [1,2], insulin resistance [1] and inflammation [6], rather than the reduced albumin levels themselves, explain the relation with endothelial dysfunction in CKD and proteinuric conditions [4].
The NAR, a mutant SDR [8], has extremely low-albumin levels in the absence of chronic inflammation. Little is known on regulation of vascular function as such in NAR. In NAR the half-life of NO from an exogenous donor was reduced by 50% and plasma S-nitrosothiols were much lower than in control rats and barely increased in response to a NO-donor [11]. However, arterial pressure is at control levels [12], suggesting that a systemic increase in oxidative stress due to lack of albumin [13] may be adequately compensated despite a deficiency of nitrosylated albumin. Such compensation could involve constitutive up-regulation of vascular NO synthase activity. Indeed we found an increased plasma level of NO metabolites and blunted endothelial-independent relaxation. Furthermore, ACh-dependent relaxation was enhanced, also suggesting high levels of NO production, even though eNOS protein mass was not increased. All these observations support the notion that hypoalbuminaemia, as such, does not impede endothelial function. In fact, the stimulatory effects of acute exposure to low albumin levels on eNOS in vitro appear to persist under chronic conditions in vivo judging by plasma NO metabolites. Evidence to support this can be gathered when studying isolated vascular tissue ex vivo under albumin-free conditions.
In order to exclude osmotic or oncotic influences, osmolality and COP were measured. As expected we found no differences in osmolality, but we did find substantial differences in COP. However, additional experiments with an oncotic control (e.g. ficoll) have not been performed for two reasons. First, it was not the specific aim of the study to differentiate between hypoalbuminaemia and low COP because this is not clinically relevant. Second, the reduction in COP in adult male NAR is only about 4 mmHg because of an increase of other plasma proteins [8]. One would expect that the reduced sensitivity of NAR aorta to SNP should also imply reduced sensitivity to endogenous NO induced by ACh. Another dilatory factor, apart from NO, such as endothelial-derived hyperpolarizing factor (EDHF), might play a role in the ACh-induced relaxation. However, EDHF-mediated relaxation is defined as relaxation induced by a circulating non-NO, non-prostanoid factor, upon stimulation by a transmitter such as ACh or bradykinin. As can be seen in Figure 4, relaxation to ACh within the dose-range studied was absent in both rat strains in the presence of L-NNA (plus indomethacin). Thus within this dose range, ACh does not induce appreciable EDHF release in the rat aorta. Although, we cannot exclude that a different endothelium-dependent relaxing agent and/or higher concentrations of ACh would have stimulated EDHF release, it is doubtful whether this is relevant in vivo.
The mechanism of the adaptation of vascular NO synthase activity to low-albumin levels in vivo is unclear. In the present study eNOS localization, essential for proper eNOS activity [14], was not studied. To date, no studies have been published concerning the effect of low albumin on eNOS localization. Another option is altered calcium partitioning. Since albumin binds Ca2+, its absence can increase the available pool of Ca2+ for uptake by endothelial cells. We accounted for this problem in the in vitro experiments by providing excess Ca2+ in the experimental buffers. To our knowledge, no studies have been published concerning the cytosolic calcium concentration in NAR, although it has been reported that serum ionized calcium is slightly decreased in NAR [15]. Hence, it is conceivable that in NAR, the absence of albumin results in higher cytosolic calcium concentration, leading to an increased cellular concentration of Ca2+-calmodulin complexes which are essential for eNOS translocation and activity. By the same token, the greater cytosolic calcium concentration in vascular smooth muscle cells in NAR can raise vascular tone and confer some degree of NO resistance. This may, in turn, contribute to maintenance of normal blood pressure [12], despite profound hypoalbuminaemia in NAR in vivo.
SNP-mediated relaxation was blunted in the NAR aorta suggesting reduced NO sensitivity of guanylate cyclase [17], possibly by increased basal NO production, or altered distribution of intracellular calcium towards the sarco(endo)plasmic reticulum [18]. The sensitivity of target tissues for increased NO is determined by three factors. First, the expression and function of NO-sensitive guanylate cyclase may be depressed [19]. Second, activity of cyclic guanosine monophosphate (cGMP)-dependent kinases, which mediate the effects of cGMP, may be reduced. Finally, presence and activity of phosphodiesterases responsible for cGMP breakdown may be enhanced [20]. Reducing ambient NO level, by de-endothelialization or by pharmaceutical means, sensitizes the vasodilator response to NO itself, and conversely NO desensitizes the effector pathway, also designated as ‘nitrate tolerance’ [19]. Previously, we reported normal arterial pressure, renal blood flow and glomerular filtration rate in NAR [12]. The absence of hypotension and renal hyperperfusion, despite up-regulation of NO production in vascular tissue of NAR, can be due to decreased NO export by albumin, leading to a local increase in NO which hypothetically can either lower NO production by eNOS [21] or desensitize the effector pathway, hence creating endogenous resistance to local enhanced NO-production [17]. However, when stimulated by acetylcholine the magnitude of relaxation was enhanced rather than depressed, suggesting that the sustained response to exogenous NO from a source such as nitroprusside (this study) or NOC7 [11] is impeded. This combination is suggestive of enhanced cGMP breakdown, rather than reduced NO production, guanylate cyclase expression or down-stream signalling. Nitrosylated albumin derivatives have been advanced as pharmacologically active NO transporters [21]. The present study suggests that, although possibly useful in an acute setting, such compounds may induce adaptive down-regulation of endogenous NO effector sensitivity.
In the clinical setting of hypoalbuminaemia it is not easy to isolate the effect of low albumin from other factors that have well-established effects on endothelial function: chronic renal disease, inflammation, dyslipidaemia and the sympathetic nervous system. By choosing an in vitro model to examine the effects of hypoalbuminaemia on NO production, we purposely excluded such factors. This allowed us to determine the direct effect of albumin on eNOS activity and NO production. Similar considerations motivated us to study vascular rings isolated from male analbuminaemic rats. Although this rat is obviously not the ideal model for hypoalbuminaemia, it is devoid of inflammation and renal disease, and in the males dyslipidaemia is mild [23]. To our knowledge, this is the best available ‘clean’ model of hypoalbuminaemia.
In conclusion, in vitro low albumin enhances eNOS activity and NO production from the endothelium rather than the reverse. In agreement, in vivo plasma NO metabolites were increased, and ex vivo, the relaxation of vascular smooth muscle in response to the NO donor SNP was blunted. Both observations suggest that adaptation to chronic hypoalbuminaemia raised the local NO levels in vascular tissues. The enhanced ACh response and normal eNOS protein expression disproves that isolated hypoalbuminaemia causes endothelial dysfunction. Thus, as postulated previously [4], hypoalbuminaemia is largely a surrogate marker as opposed to inflammation and dyslipidaemia, which may well be the real culprits in the pathogenesis, of endothelial dysfunction associated with hypoalbuminaemia.
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The Dutch Heart Foundation supported this study (99.041). B.B. is a fellow of the Royal Dutch Academy of Arts and Sciences.
Conflict of interest statement. None declared.
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Accepted in revised form: 26. 6.06
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