TY - JOUR
AU - Rechke, Jean‐Pierre
AB - Abstract Background. Oxidative stress has been shown in haemodialysis patients in relation with an increased production of free radicals due to membrane‐induced complement and leukocyte activation. In order to minimize membrane bioincompatibility and thereby oxidative stress, more compatible filters have been perfected. Among them, a high‐flux vitamin E‐coated membrane (CL‐EE) has been proposed recently. In vivo, little data is available on the consequences of the use of vitamin E‐coated membranes. In the present study, the effects of a 3‐month use of CL‐EE dialysis membranes compared to conventional membranes have been evaluated in 12 haemodialysis patients on the blood oxidative stress status before and after the dialysis session. Methods. We determined the lipid peroxidation status (plasma thiobarbituric acid‐reactive substances) and antioxidant defence (erythrocyte Cu,Zn‐superoxide dismutase and plasma and erythrocyte glutathione peroxidase activities, plasma vitamin E, β‐carotene, vitamin A and total antioxidant status). Also, we simultaneously determined the antioxidant content and the copper oxidizability of isolated low density‐ and high density‐lipoproteins (LDLs and HDLs). Results. The main consequence observed under these conditions was a marked enrichment of plasma with vitamin E, which was also significantly and selectively noted in HDLs (no changes in LDL vitamin E content), perhaps related to a specific storage capacity for vitamin E in HDLs of haemodialysis patients. The β‐carotene content of plasma, LDLs and HDLs was also higher after use of vitamin E‐coated membranes than after use of high‐flux biocompatible membranes. HDL copper oxidizability was reduced (as shown by an increased lag time) before dialysis after use of CL‐EE membranes compared to conventional membranes, whereas LDL oxidizability remained unchanged. Conclusion. A 3‐month use of vitamin E‐coated membranes resulted in a significant increase in plasma and HDL vitamin E content, associated with a lower oxidizability of HDLs, which could be beneficial for haemodialysis patients. antioxidant, haemodialysis, high density lipoprotein, lipid peroxidation, low density lipoprotein, vitamin E Introduction Patients with chronic renal failure, especially those receiving regular haemodialysis, have a high incidence of premature cardiovascular disease. During haemodialysis, complement and leukocyte activation by contact with artificial membranes promotes the production of free radicals [1] which are known to be involved in the pathogenesis of atherosclerosis [2]. Thus, lipid peroxidation and decreased antioxidant status have generally been found in haemodialysis patients [3–6]. Moreover, it is unclear whether low density lipoproteins (LDLs), whose oxidation would play a major role in their atherogenicity, are oxidatively modified or more susceptible to oxidation in patients on haemodialysis [7,8]. In addition, little data is provided on the oxidizability of the high‐density lipoproteins (HDLs) in these patients. In order to decrease membrane bioincompatibility and thereby minimize oxidative stress in haemodialysis patients, more compatible filters have been elaborated. With regard to lipoprotein oxidizability, only one study monitored LDL and HDL resistance to copper‐induced oxidation in patients on haemodialysis according to the type of membranes [9]. Among the most recent compatible filters, vitamin E‐coated membranes have been proposed and referred to as CL‐E membranes for low‐flux membranes and CL‐EE membranes for high‐flux membranes. Preliminary characterization of vitamin E‐coated membranes has shown a decreased activation of polymorphonuclear cells and monocytes, a lower free radical production [10,11] and a high biocompatibility [12]. Moreover, lipid peroxidation in plasma and red blood cells was decreased after a 30‐day period of use of this membrane in haemodialysis patients [13]. Nevertheless, this study did not take into account the effects of the use of this membrane on the oxidizability of plasma lipoproteins. Only one study conducted in vitro to mimic a dialysis session demonstrated a decreased oxidizability of LDLs after a 3‐hour contact of these LDLs with a vitamin E‐coated membrane (CL‐E) compared to a cellulose membrane [14]. Also, a recent in vivo two‐year‐study compared the effects of this vitamin E‐coated membrane dialyser and of an ordinary cellulose membrane dialyser on lipid metabolism and on the progress of atherosclerosis [15]. According to this study, use of the vitamin E‐coated membrane dialyser for 6 months and one or two years resulted in a significant reduction in malondialdehyde‐rich LDLs and oxidized LDLs (evaluated by ELISA) [15]. This strongly suggested that the vitamin E‐coated membrane exhibited an antioxidant effect. Therefore, our study was aimed at evaluating the consequences of a 3‐month use of high‐flux biocompatible vitamin E‐coated membranes (CL‐EE) compared to high‐flux conventional membranes, both on the blood oxidative stress status and on the in vitro oxidizability of ultracentrifugally isolated LDLs and HDLs. The blood oxidative stress status was classically assessed both by plasma lipid peroxidation products determined as thiobarbituric acid‐reactive substances (TBARS) and by antioxidant defences. Among the latter, we determined the activity of two major enzymatic systems, namely erythrocyte Cu,Zn‐superoxide dismutase (SOD) and plasma and erythrocyte glutathione peroxidase (GSH‐Px) for which selenium is an essential element. With regard to the non‐enzymatic defence systems, we assayed vitamin E, β‐carotene and vitamin A in plasma. In order to get a global assessment of the antioxidant defences, we also measured before and after dialysis the plasma total antioxidant status, for which conflicting data has been reported [6,16] and for which no data was available after use of vitamin E‐coated membranes. Finally, we determined the oxidizability and the antioxidant content of both LDLs and HDLs isolated from the plasma of haemodialysis patients after the 3‐month use of the CL‐EE membranes, in comparison with conventional dialysis membranes. Subjects and methods Patients Twelve uraemic patients (4 females and 8 males), median age 50 (range: 35–73) years, treated with high‐flux biocompatible conventional haemodialysis membranes (6 PMMA, 2 triacetate, 1 polysulfone and 3 AN69) for a median period of 18 (range: 4–54) months were included in the study. They were dialysed three times a week, each session lasting 4 h. Inclusion criteria were the absence of diabetes, cancer, HIV infection and hepatitis. Patients' plasma cholesterol concentration was 5.2 mmol/l (3.4 to 6.1). They were not given any supplementation with vitamin E, vitamin A, β‐carotene or selenium. No patient received transfusions in the preceding two months. Patients gave their informed consent to be included in the study. A first double determination (before and after dialysis session) was made on patients treated with conventional membranes (MC). Patients were then treated for three months with a CL‐EE membrane. A double determination (before and after dialysis session) was made after these three months (ME) (Tables 1 and 2). Venous blood samples were collected in heparinized Vacutainer® tubes (Beckton‐Dickinson) protected from light. Samples were processed within three h of sampling. Plasma was obtained by centrifugation (4000 rpm for 15 min at 4°C). The vitamin E‐coated (CL‐EE) membrane (Excebrane®) was manufactured by Terumo (Japan) and was provided by Nephrotek (Rungis, France). It was characterized by the presence of a skeleton of cellulose coated with a copolymer which is composed of (i) a hydrophilic polymer and (ii) a hydrophobic part (fluorocarbon resin which constitutes a hydrophobic support for the binding of oleic alcohol). Vitamin E is hydrophobically bound to the surface of the copolymer block which also exerts an inhibitory function on platelet activation. Under these conditions, vitamin E remains fixed to the membrane. Table 1. Blood oxidative stress markers and plasma total antioxidant status in the 12 haemodialysis patients dialysed on conventional membrane (MC) and after a 3‐month use of the vitamin E‐coated dialysis membrane (ME). Results are medians (range)   MC     ME     Laboratory refrence    Before haemodialysis   After haemodialysis   Before haemodialysis   After haemodialysis   range   Plasma TBARS    1.85    2.05    1.65    1.72  0.60–1.20     (μmol/l)   (0.95–2.40)   (1.05–2.65)   (0.75–2.25)   (1.02–3.00)    Plasma TAS    1.15    0.94    1.25    0.92  1.30–1.90     (mmol/l)   (1.00–1.31)   (0.71–1.25)**   (1.11–1.71)   (0.72–1.58)****    Calculated plasma TAS    0.84    0.55    0.81    0.55  –     (mmol/l)   (0.63–1.03)   (0.44–0.85)****   (0.73–1.13)   (0.46–0.96)****    Erythrocyte Cu,Zn‐SOD  577  581  659  677  359–671     (U/g Hb)   (463–740)   (437–840)   (403–887)   (476–909)    Plasma GSH‐Px  210  237  184  239  480–650     (U/l)   (121–310)   (130–317)****   (135–329)   (152–367)***  480–650  Erythrocyte GSH‐Px   42   44   42   41  29–43     (U/g Hb)    (29–65)    (29–66)    (27–68)    (27–68)    Plasma selenium    0.80    0.95    0.80    0.95  0.90–1.30     (μmol/l)   (0.55–1.00)   (0.55–1.20)**   (0.55–1.05)   (0.60–1.05)**  0.90–1.30  Plasma vitamin E   34.4   38.5   38.0   44.2  20–37     (μmol/l)   (25.3–44.3)c   (27.2–54.6)****a   (30.1–59.1)c   (29.7–74.6)****a    Plasma vitamin A    5.73    6.51    6.38    7.12  1.5–2.6     (μmol/l)   (3.86–9.13)   (4.35–10.44)***   (3.49–9.78)   (3.51–10.7)****    Plasma β‐carotene    0.46    0.47    0.51    0.59  0.20–0.80     (μmol/l)   (0.27–0.81)   (0.29–0.76)a   (0.41–0.89)   (0.45–0.94)****a    Plasma TBARS:total lipid    0.276    0.222    0.193    0.151  –     (μmol/g)  (0.121–0.371)  (0.100–0.363)  (0.121–0.343)  (0.104–0.376)    Plasma vitamin    6.69    6.64    7.71    8.03  –     E:cholesterol   (5.88–9.16)a   (5.78–8.75)a   (6.28–9.65)a   (5.94–10.36)a       (μmol/mmol)            Plasma β‐    0.088    0.089    0.107    0.113  –     carotene:cholesterol   (0.064–0.153)c   (0.059–0.123)*b   (0.069–0.171)c   (0.063–0.148)b       (μmol/mmol)              MC     ME     Laboratory refrence    Before haemodialysis   After haemodialysis   Before haemodialysis   After haemodialysis   range   Plasma TBARS    1.85    2.05    1.65    1.72  0.60–1.20     (μmol/l)   (0.95–2.40)   (1.05–2.65)   (0.75–2.25)   (1.02–3.00)    Plasma TAS    1.15    0.94    1.25    0.92  1.30–1.90     (mmol/l)   (1.00–1.31)   (0.71–1.25)**   (1.11–1.71)   (0.72–1.58)****    Calculated plasma TAS    0.84    0.55    0.81    0.55  –     (mmol/l)   (0.63–1.03)   (0.44–0.85)****   (0.73–1.13)   (0.46–0.96)****    Erythrocyte Cu,Zn‐SOD  577  581  659  677  359–671     (U/g Hb)   (463–740)   (437–840)   (403–887)   (476–909)    Plasma GSH‐Px  210  237  184  239  480–650     (U/l)   (121–310)   (130–317)****   (135–329)   (152–367)***  480–650  Erythrocyte GSH‐Px   42   44   42   41  29–43     (U/g Hb)    (29–65)    (29–66)    (27–68)    (27–68)    Plasma selenium    0.80    0.95    0.80    0.95  0.90–1.30     (μmol/l)   (0.55–1.00)   (0.55–1.20)**   (0.55–1.05)   (0.60–1.05)**  0.90–1.30  Plasma vitamin E   34.4   38.5   38.0   44.2  20–37     (μmol/l)   (25.3–44.3)c   (27.2–54.6)****a   (30.1–59.1)c   (29.7–74.6)****a    Plasma vitamin A    5.73    6.51    6.38    7.12  1.5–2.6     (μmol/l)   (3.86–9.13)   (4.35–10.44)***   (3.49–9.78)   (3.51–10.7)****    Plasma β‐carotene    0.46    0.47    0.51    0.59  0.20–0.80     (μmol/l)   (0.27–0.81)   (0.29–0.76)a   (0.41–0.89)   (0.45–0.94)****a    Plasma TBARS:total lipid    0.276    0.222    0.193    0.151  –     (μmol/g)  (0.121–0.371)  (0.100–0.363)  (0.121–0.343)  (0.104–0.376)    Plasma vitamin    6.69    6.64    7.71    8.03  –     E:cholesterol   (5.88–9.16)a   (5.78–8.75)a   (6.28–9.65)a   (5.94–10.36)a       (μmol/mmol)            Plasma β‐    0.088    0.089    0.107    0.113  –     carotene:cholesterol   (0.064–0.153)c   (0.059–0.123)*b   (0.069–0.171)c   (0.063–0.148)b       (μmol/mmol)            Significant difference between MC and ME: a,P<0.05; b,P<0.02; c,P<0.01. Significant difference between before and after haemodialysis: *P<0.05; **P<0.02; ***P<0.01; ****P<0.005. View Large Lipid components Plasma triglyceride, phospholipid, and cholesterol concentrations were determined by enzymatic methods [17–19]. Total lipid concentration was calculated as the sum (expressed in g/l) of unesterified cholesterol, cholesteryl esters, phospholipids and triglycerides. The amount of cholesteryl esters was estimated as 1.67×esterified cholesterol, this factor representing the ratio of the average molecular weight of cholesteryl ester to unesterified cholesterol. Blood oxidative stress markers Oxidative stress markers were determined as previously described [20]. Plasma TBARS were assayed using a spectrofluorimetric method after condensation with thiobarbituric acid. Plasma vitamin E (α‐tocopherol), vitamin A and β‐carotene were determined by reverse phase HPLC. Plasma selenium was assayed by electrothermal atomic absorption spectrophotometry at 196 nm, on a Perkin Elmer 5000 spectrophotometer equipped with an HGA 400 graphite furnace. Erythrocyte SOD, erythrocyte and plasma GSH‐Px activities were determined using Randox kits (Roissy, France) with adaptation on a Hitachi 911 analyser (Boehringer, Mannheim, Germany). Briefly, the determination of SOD activity was based on the production of O2− anions by the xanthine/xanthine oxidase system. GSH‐Px catalysed the oxidation of reduced gutathione in the presence of cumene hydroperoxide. Plasma total antioxidant status (TAS) The total antioxidant status was measured in plasma by means of a commercial kit (Randox, Roissy, France), based on the method developed by Miller et al. [21] using 2,2′‐azino‐di‐(3‐ethylbenzthiazoline‐6‐sulphonic acid) (ABTS). In addition, we evaluated a calculated TAS (cTAS) by taking into account plasma concentrations of albumin, bilirubin and uric acid as follows: cTAS (mmol/l)=(0.63×[albumin])+(1.02×[uric acid])+(1.50×[bilirubin]), according to Miller et al. [21] (concentrations of albumin, uric acid and bilirubin should be expressed in mmol/l). Copper‐induced oxidizability of plasma LDLs and HDLs LDLs (1.019<d<1.063) and HDLs (1.063<d<1.21) were isolated from plasma by sequential ultracentrifugation [22], at 4°C in the presence of EDTA (1 g/l final concentration) with a Beckman XL‐80 ultracentrifuge and a 6513 Kontron rotor. Lipids (i.e., total and unesterified cholesterol, phospholipids and triglycerides) were assayed in lipoprotein fractions by enzymatic methods as previously described for plasma. Proteins were determined by using a pyrogallol technique (Elitech Diagnostics, Sees, France) [23]. The concentration of total lipoprotein (expressed in g/l) was calculated as the sum of the lipid and protein concentrations (expressed in g/l) in each fraction. Vitamin E and β‐carotene were assayed in LDLs and HDLs by the same procedure as described for plasma. The antioxidant content in these lipoproteins was expressed as μmol antioxidant/mol cholesterol (cholesterol being the main LDL component) but also as μmol antioxidant/g total lipid (since HDLs do not contain a major lipid component, so that total lipids must be taken into account). Lipoprotein fractions were dialysed in 10−2 mol/l phosphate buffer saline (PBS) for 18 h at 4°C in the dark. 0.5 g/l (final concentration expressed as total lipoprotein) of each lipoprotein fraction was oxidised at 37°C with 5 μmol/l CuSO4 (final concentration). This oxidation was continuously monitored by measuring the differential absorbance at 234 nm every 5 min for 200 min, according to the procedure of Esterbauer et al. [24]. The rise of this differential absorbance as a function of the oxidation time corresponds classically to the formation of conjugated dienes which are early products of lipid peroxidation. During the lag phase, it is commonly admitted that the lipophilic antioxidants protect the polyunsaturated fatty acids of lipoproteins against oxidation. The longer the lag phase, the more resistant lipoproteins are to oxidation. After consumption of these antioxidants, the lipid peroxidation process can begin the propagating chain reaction phase. A tangent to the curve was drawn during the propagation phase and extrapolated to the time axis. The time interval between the addition of copper ions (time 0) and the intersection point of the tangent on the time axis was defined as the lag phase (expressed in minutes). The propagation rate was calculated from the slope of the tangent, using a molar extinction coefficient for conjugated dienes at 234 nm equal to 29 500 mol−1.l.cm−1 [24], and was expressed as μmol of conjugated dienes formed per litre and per minute. Statistical analysis Statistical analysis was performed using the non‐parametric paired test of Wilcoxon both for the comparison of the data obtained before and after haemodialysis, and to appreciate the effects of the use of CL‐EE membranes in comparison to conventional membranes. Correlations were tested by the Spearman rank test. We considered P<0.05 to be statistically significant. All results are given as median and range unless otherwise noted. Results Table 1 shows the blood oxidative stress markers and plasma total antioxidant status in the 12 haemodialysis patients dialysed on conventional membranes (MC) and after a 3‐month use of the high‐flux biocompatible vitamin E‐coated membrane (ME). In each case, data before and after the haemodialysis session is reported. Plasma TBARS and TAS are not significantly different on conventional membranes (MC) and CL‐EE membranes (ME), before as well as after dialysis session. Nevertheless, measured and calculated TAS were always lower after than before haemodialysis (P<0.02 at MC and P<0.005 at ME for the measured TAS; P<0.005 at MC and at ME for the calculated TAS), mainly due to the presence of high urate concentrations [16] before dialysis (sample MC: 408 (267–591) μmol/l before dialysis vs 108 (63–186) μmol/l after dialysis). Cu,Zn‐SOD and GSH‐Px activities, plasma selenium and vitamin A concentrations were unchanged by the use of CL‐EE membranes. We found only after the dialysis session, higher levels of these three markers than before dialysis, at MC as well as at ME (differences statistically significant only for plasma GSH‐Px activity and for selenium and vitamin A concentrations). As expected, the selenium level was positively correlated with plasma and erythrocyte GSH‐Px activity, both at MC and at ME (data not shown). The major consequence of the use of the CL‐EE membranes was a significant increase of plasma vitamin E (by 10% before dialysis (P<0.01) and by 15% after dialysis (P<0.05) in the ME sample compared to MC sample. This increase was relevant as it was observed in all but one of the patients included in the study and remained significant when the plasma cholesterol level was taken into account. Thus, vitamin E : cholesterol ratio was significantly higher in ME than in MC: 15% higher before dialysis (P<0.05) and 21% higher after dialysis (P<0.05) (Figure 1a). Similarly, plasma β‐carotene level after dialysis was 26% higher in ME than in MC (P<0.05); β‐carotene : cholesterol ratio was higher in ME than in MC, before dialysis (22%, P<0.05) as well as after dialysis (27%, P<0.02) (Figure 1b). With regard to the oxidizability of the LDLs and HDLs of the haemodialysis patients, comparative results between MC and ME are reported in Table 2. The three main features of this oxidizability, namely the lag phase, the propagation rate and the maximum formation of conjugated dienes, were not modified in LDLs by the use of the CL‐EE membranes instead of the conventional membranes. In contrast, the lag phase preceding conjugated diene formation was significantly enhanced in HDLs before dialysis in ME compared to MC (P<0.05) (Figure 2), and the propagation rate of conjugated diene formation in HDLs after haemodialysis was lower in ME than in MC (P<0.05). Moreover, as previously observed in plasma, vitamin E content of HDLs was significantly enhanced (by 34%) before dialysis by use of CL‐EE membranes instead of conventional membranes. When the vitamin E : total lipid ratio in HDLs was considered, this enrichment was even more marked (170%, P<0.01) and was also observed after dialysis (48%, P<0.05). This enrichment was not observed in LDLs. With regard to β‐carotene, a marked enhancement of the β‐carotene : cholesterol ratio was shown in LDLs before (by 31%, P<0.02) and after (by 47%, P<0.02) dialysis. In HDLs, the enrichment with β‐carotene was especially very marked when the β‐carotene content was expressed as the β‐carotene : total lipid ratio (192% enrichment before dialysis, P<0.005). Fig. 1. View largeDownload slide Effect of a 3‐month use of vitamin E‐coated membranes on plasma vitamin E : cholesterol ratio (A) and β‐carotene : cholesterol ratio (B), in comparison with conventional membranes. (1) Patients treated with conventional membranes (MC), before dialysis session; (2) patients treated with conventional membranes (MC), after dialysis session; (3) patients treated for three months with vitamin E‐coated membranes (ME), before dialysis session; (4) patients treated for three months with vitamin E‐coated membranes (ME), after dialysis session. Fig. 1. View largeDownload slide Effect of a 3‐month use of vitamin E‐coated membranes on plasma vitamin E : cholesterol ratio (A) and β‐carotene : cholesterol ratio (B), in comparison with conventional membranes. (1) Patients treated with conventional membranes (MC), before dialysis session; (2) patients treated with conventional membranes (MC), after dialysis session; (3) patients treated for three months with vitamin E‐coated membranes (ME), before dialysis session; (4) patients treated for three months with vitamin E‐coated membranes (ME), after dialysis session. Fig. 2. View largeDownload slide Effect of a 3‐month use of vitamin E‐coated membranes on the lag phase of HDLs upon copper‐induced oxidation, in comparison with conventional membranes (the longer the lag phase, the more resistant HDLs are to oxidation). (1) Patients treated with conventional membranes (MC), before dialysis session; (2) patients treated with conventional membranes (MC), after dialysis session; (3) patients treated for three months with vitamin E‐coated membranes (ME), before dialysis session; (4) patients treated for three months with vitamin E‐coated membranes (ME), after dialysis session. Fig. 2. View largeDownload slide Effect of a 3‐month use of vitamin E‐coated membranes on the lag phase of HDLs upon copper‐induced oxidation, in comparison with conventional membranes (the longer the lag phase, the more resistant HDLs are to oxidation). (1) Patients treated with conventional membranes (MC), before dialysis session; (2) patients treated with conventional membranes (MC), after dialysis session; (3) patients treated for three months with vitamin E‐coated membranes (ME), before dialysis session; (4) patients treated for three months with vitamin E‐coated membranes (ME), after dialysis session. Table 2. Oxidizability features and antioxidant content of LDLs and HDLs isolated from the plasma of the 12 haemodialysis patients dialysed on conventional membrane (MC) and after a 3‐month use of the vitamin E‐coated dialysis membrane (ME). Results are medians (range)   MC     ME       Before haemodialysis   After haemodialysis   Before haemodialysis   After haemodialysis   LDLs             Lag phase (min)  49.2  51.0  50.1  51.9     (31.2–71.0)   (41.4–68.9)   (22.9–66.7)   (31.4–59.4)  Propagation rate (μmol   1.46   1.62   1.30   1.46     CD.l−1.min−1)   (0.35–2.54)   (0.27–2.42)   (0.91–1.66)   (1.03–2.12)*  Maximum of CD  49.5  54.3  50.2  58.8     formation (μmol/l)   (12.4–68.8)   (11.1–67.8)   (43.7–61.7)   (46.4–81.4)*  Vitamin E (μmol/l LDL)  23.9  24.3  23.3  24.9     (15.2–46.6)   (16.4–45.3)   (11.7–34.6)   (10.2–36.8)  β‐carotene (μmol/l LDL)   0.575   0.590   0.665   0.735     (0.350–1.070)   (0.250–1.030)   (0.280–1.260)   (0.400–1.530)  Vitamin E:cholesterol   3.89   3.62   3.52   4.10     (μmol/mmol)   (2.87–5.57)   (2.71–5.78)   (2.98–5.44)   (3.04–4.92)  β‐carotene:cholesterol   0.090   0.086   0.118   0.126     (μmol/mmol)  (0.035–0.200)b  (0.052–0.178)c  (0.077–0.173)b  (0.087–0.167)c  Vitamin E:total lipids   2.740   3.040   2.460   3.130     (μmol/g)  (1.645–4.736)  (1.337–3.674)  (1.549–4.869)  (1.651–4.279)  β‐carotene: total lipids   0.069   0.069   0.086   0.102     (μmol/g)  (0.024–0.141)  (0.019–0.150)  (0.040–0.179)  (0.038–0.172)  HDLs            Lag phase (min)  17.9  19.2  22.1  18.8     (14.7–22.5)a   (15.3–32.5)   (15.2–33.9)a   (14.4–43.2)  Propagation rate (μmol   0.78   0.77   0.77   0.72     CD.l−1.min−1)   (0.58–1.08)   (0.52–1.28)a   (0.49–1.03)   (0.34–0.89)a  Maximum of CD  36.9  38.0  40.3  39.3     formation (μmol/l)   (29.7–45.4)a   (32.7–45.4)   (35.8–50.8)a   (34.3–55.9)  Vitamin E (μmol/l HDL)   8.8  10.4  11.8  11.2     (4.8–14.9)d   (6.3–24.2)   (9.4–29.6)d   (5.9–21.1)  β‐carotene (μmol/l HDL)   0.075   0.090   0.175   0.140    (0.050–0.280)a  (0.050–0.590)  (0.090–0.370)a  (0.050–0.500)  Vitamin E:cholesterol   5.83   5.94   6.49   7.04     (μmol/mmol)   (3.59–9.14)   (4.75–8.87)   (4.44–9.31)   (5.07–8.80)  β‐carotene:cholesterol   0.054   0.049   0.077   0.082     (μmol/mmol)  (0.034–0.179)a  (0.033–0.287)  (0.060–0.138)a  (0.050–0.143)  Vitamin E:total lipids   0.940   2.000   2.540   2.960     (μmol/g)  (0.218–3.096)c  (0.806–3.687)**a  (1.159–7.532)c  (1.714–6.583)a  β‐carotene:total lipids   0.012   0.020   0.035   0.037     (μmol/g)  (0.003–0.017)d  (0.004–0.069)***  (0.017–0.077)d  (0.017–0.122)    MC     ME       Before haemodialysis   After haemodialysis   Before haemodialysis   After haemodialysis   LDLs             Lag phase (min)  49.2  51.0  50.1  51.9     (31.2–71.0)   (41.4–68.9)   (22.9–66.7)   (31.4–59.4)  Propagation rate (μmol   1.46   1.62   1.30   1.46     CD.l−1.min−1)   (0.35–2.54)   (0.27–2.42)   (0.91–1.66)   (1.03–2.12)*  Maximum of CD  49.5  54.3  50.2  58.8     formation (μmol/l)   (12.4–68.8)   (11.1–67.8)   (43.7–61.7)   (46.4–81.4)*  Vitamin E (μmol/l LDL)  23.9  24.3  23.3  24.9     (15.2–46.6)   (16.4–45.3)   (11.7–34.6)   (10.2–36.8)  β‐carotene (μmol/l LDL)   0.575   0.590   0.665   0.735     (0.350–1.070)   (0.250–1.030)   (0.280–1.260)   (0.400–1.530)  Vitamin E:cholesterol   3.89   3.62   3.52   4.10     (μmol/mmol)   (2.87–5.57)   (2.71–5.78)   (2.98–5.44)   (3.04–4.92)  β‐carotene:cholesterol   0.090   0.086   0.118   0.126     (μmol/mmol)  (0.035–0.200)b  (0.052–0.178)c  (0.077–0.173)b  (0.087–0.167)c  Vitamin E:total lipids   2.740   3.040   2.460   3.130     (μmol/g)  (1.645–4.736)  (1.337–3.674)  (1.549–4.869)  (1.651–4.279)  β‐carotene: total lipids   0.069   0.069   0.086   0.102     (μmol/g)  (0.024–0.141)  (0.019–0.150)  (0.040–0.179)  (0.038–0.172)  HDLs            Lag phase (min)  17.9  19.2  22.1  18.8     (14.7–22.5)a   (15.3–32.5)   (15.2–33.9)a   (14.4–43.2)  Propagation rate (μmol   0.78   0.77   0.77   0.72     CD.l−1.min−1)   (0.58–1.08)   (0.52–1.28)a   (0.49–1.03)   (0.34–0.89)a  Maximum of CD  36.9  38.0  40.3  39.3     formation (μmol/l)   (29.7–45.4)a   (32.7–45.4)   (35.8–50.8)a   (34.3–55.9)  Vitamin E (μmol/l HDL)   8.8  10.4  11.8  11.2     (4.8–14.9)d   (6.3–24.2)   (9.4–29.6)d   (5.9–21.1)  β‐carotene (μmol/l HDL)   0.075   0.090   0.175   0.140    (0.050–0.280)a  (0.050–0.590)  (0.090–0.370)a  (0.050–0.500)  Vitamin E:cholesterol   5.83   5.94   6.49   7.04     (μmol/mmol)   (3.59–9.14)   (4.75–8.87)   (4.44–9.31)   (5.07–8.80)  β‐carotene:cholesterol   0.054   0.049   0.077   0.082     (μmol/mmol)  (0.034–0.179)a  (0.033–0.287)  (0.060–0.138)a  (0.050–0.143)  Vitamin E:total lipids   0.940   2.000   2.540   2.960     (μmol/g)  (0.218–3.096)c  (0.806–3.687)**a  (1.159–7.532)c  (1.714–6.583)a  β‐carotene:total lipids   0.012   0.020   0.035   0.037     (μmol/g)  (0.003–0.017)d  (0.004–0.069)***  (0.017–0.077)d  (0.017–0.122)  CD=conjugated dienes. Significant difference between MC and ME: a,P<0.05; b,P<0.02; c,P<0.01; d,P<0.005. Significant difference between before and after haemodialysis: *P<0.05; **P<0.01; ***P<0.005. View Large Discussion This study was aimed at evaluating the consequences of a 3‐month use of vitamin E‐coated dialysis membranes instead of conventional membranes, on the blood oxidative stress status and on the in vitro oxidizability of isolated plasma LDLs and HDLs, in 12 haemodialysis patients. The main consequence observed under these conditions was a marked enrichment of plasma with vitamin E, which was also significantly and selectively noted in HDLs (no changes in LDL vitamin E content). The β‐carotene content of plasma, LDLs and HDLs was also higher after use of CL‐EE membranes than after use of conventional membranes. HDL copper oxidizability was reduced, as shown by the longer lag time observed before the dialysis session, after use of CL‐EE membranes compared to conventional membranes, whereas LDL oxidizability remained unchanged. Confirmation of an oxidative stress in haemodialysis patients on conventional membranes The results we obtained with conventional membranes (MC) confirmed the presence of an oxidative stress status in haemodialysis patients. Indeed, as previously reported by several authors [1,5,6], all the haemodialysis patients exhibited a high plasma TBARS concentration, which was in favour of an oxidative stress. In accordance with others [6], we did not observe a significant variation of this concentration during the dialysis session. The oxidative stress was confirmed by the low total antioxidant capacity (TAS) values compared to those of healthy subjects (reference values). With regard to the antioxidant enzymes, plasma GSH‐Px activity was low, as previously reported by others [5,25], both before and after dialysis. GSH‐Px activity is highly dependent on selenium, as also shown in our study by positive correlations observed between selenium and GSH‐Px activity; similarly, plasma selenium concentration was also low in all the 12 haemodialysis patients, as generally reported [5,25]. It can be noted that erythrocyte GSH‐Px activity was not decreased in the haemodialysis patients, as previously observed by Ceballos‐Picot et al. [25]. Cu,Zn‐SOD activity was in the reference range and unchanged after dialysis on conventional membrane, although a decreased SOD activity has already been described in haemodialysis patients [5,25] and could lead to an increased oxidative stress in relation with an impaired detoxification of the superoxide anion. The non‐enzymatic antioxidant defences vitamin E and β‐carotene were not decreased in the haemodialysis patients, in accordance with published data [26,27]. Vitamin A was high, as reported by others [27], probably due to a decreased glomerular filtration of its carrier protein, the retinol binding protein (RBP). Effect of the use of CL‐EE membranes The major observation was the substantial enrichment of plasma with vitamin E and β‐carotene after use of CL‐EE membranes in comparison with conventional membranes. The plasma enrichment with vitamin E observed in our study could not be explained by a release of this antioxidant from the vitamin E‐coated dialysis membrane. Indeed, the binding between vitamin E and oleic alcohol is very tight and in vitro studies did not show any transfer of vitamin E from the dialysis membrane [14]. Nevertheless, it could be hypothesized that the vitamin E‐coated membranes, by scavenging oxygen free radicals in situ, decrease the local oxidative stress and thereby contribute to a sparing effect towards circulating vitamin E. Galli et al. [12], who also observed in vivo an increased plasma vitamin E after use of the CL‐E filter, suggested that this was related to an increased glutathione‐dependent protection in both plasma and red blood cells. It is noteworthy that Mune et al. [11] recently did not report in vivo any significant difference in plasma vitamin E concentration between two groups of haemodialysed patients, one being dialysed with CL‐E membranes and the other with classical membranes, with a follow‐up of six months, one year and two years. Nevertheless, the authors themselves noted that the lack of significant differences in plasma vitamin E concentrations was unclear. Maybe a temporary increase of plasma vitamin E concentration could occur. Indeed, Galli et al. [12] reported that a one‐month treatment with the CL‐E filter increased plasma vitamin E by 84.3% with respect to treatment with a cuprammonium rayon membrane (CL‐S filter), whereas a three‐month treatment resulted only in a 68.9% increase. We did not observe such a substantial plasma enrichment with vitamin E after a three‐month use of CL‐EE membranes as that described by Galli et al. [12], although the basal vitamin E concentrations were comparable in both studies. Kinetic studies would thus be of great interest to manage plasma vitamin E concentration as a function of the haemodialysis duration with CL‐E membranes. This plasma enrichment with vitamin E is potentially very useful in haemodialysis patients. Indeed, vitamin E supplementation results in a sparing effect on erythropoietin dosage requirement, an increase in the activities of the erythrocyte glutathione‐dependent antioxidative enzymes (namely, glutathione‐S‐transferase, glutathione reductase and glutathione peroxidase) and a decreased lipid peroxidation in plasma and erythrocytes [28]. Apart from the antioxidant properties of vitamin E, new functions of this antioxidant have been shown in polymorphonuclear leukocytes, especially a decrease of superoxide production by activated phagocytes [29]. With regard to β‐carotene, no data has previously reported a plasma enrichment with this antioxidant after use of CL‐EE membranes. This enrichment could be explained by the secondary protective effect of vitamin E itself towards β‐carotene, as already shown upon in vitro LDL oxidation. Under our conditions, the enrichment of plasma with vitamin E was exclusively found in the isolated HDLs, whereas the vitamin E content of LDLs remained unchanged. HDLs from haemodialysis patients could thus act as a storage site for vitamin E, perhaps due to a deficient function of this lipoprotein function to transfer this antioxidant to membranes [4]. This could support the specific enrichment of HDLs with vitamin E that we observed after use of CL‐EE membranes. With regard to β‐carotene, which is almost totally carried by LDLs, the enrichment was in contrast essentially observed in LDLs whereas it was less significant in HDLs. The LDLs of the 12 patients studied did not display marked changes in their copper‐induced oxidizability after a 3‐month use of CL‐EE membranes. Only the lag phase related to the HDLs isolated from plasma before dialysis was enhanced by use of CL‐EE membranes. This is in favour of a lower oxidizability of these HDLs in the ME sample than in the MC sample, which could be related to their enrichment with vitamin E. Indeed, enrichment of HDLs with vitamin E enhances their resistance to oxidation [30]. Nevertheless, it can be noted that the maximum of conjugated diene formation in HDLs was higher with ME than with MC. This could be related to a greater content of HDLs in polyunsaturated fatty acids (PUFA) with ME than with MC. Indeed, Galli et al. [12] observed that plasma exposed in vitro to CL‐E membranes, even for a short time, showed higher levels of PUFA than that exposed to conventional (cuprammonium rayon) filters. Altogether, our data shows that a 3‐month use of CL‐EE membranes instead of conventional membranes resulted in a significant increase in plasma and HDL vitamin E content, which could be beneficial for haemodialysis patients, all the more so since new antiatherogenic functions have been attributed to HDLs with regard to the oxidative hypothesis of atherosclerosis [30] (Figure 3). Indeed, the reinforcement of the antioxidant capacity of HDLs via the use of CL‐EE membranes would be of great importance, in order both to increase their protective role towards LDL oxidation and to obtain HDLs which are themselves more resistant to oxidation. Fig. 3. View largeDownload slide Hypotheses on the antiatherogenic role of HDLs. (1) inhibition of LDL oxidation; (2) stimulation of the cholesterol efflux from macrophage‐derived foam cells; (3) inhibition of the secretion of adhesion molecules by endothelial cells. Fig. 3. View largeDownload slide Hypotheses on the antiatherogenic role of HDLs. 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TI - Blood oxidative stress and lipoprotein oxidizability in haemodialysis patients: effect of the use of a vitamin E‐coated dialysis membrane
JO - Nephrology Dialysis Transplantation
DO - 10.1093/ndt/15.12.2020
DA - 2000-12-01
UR - https://www.deepdyve.com/lp/oxford-university-press/blood-oxidative-stress-and-lipoprotein-oxidizability-in-haemodialysis-rZbSTP92tA
SP - 2020
EP - 2028
VL - 15
IS - 12
DP - DeepDyve
ER -