NEFROLOGÍA. Vol. XXV. Número 2. 2005 Lipoprotein oxidation profile in end-stage renal disease patients. Role of vitamin C supplementation R. Ramos*, N. Gómez-Geriqué** and A. Martínez-Castelao* *Nephrology Department and **Experimental Research Unit (URE). Ciutat Sanitària i Universitària de Bellvitge. L'Hospitalet de Llobregat. Barcelona. SUMMARY Background: The aim of the study was to analyze the lipid and lipoprotein oxidation profile in patients with end stage renal disease who started haemodialysis and also to evaluate the possible effect of haemodialysis and vitamin C supplementation on lipoprotein oxidation one year after the initiation of the therapy. Methods: Forty-one end stage renal disease patients who started haemodialysis between january 1999 and january 2000 were enrolled in the study. The patients were randomised to receive 1,000 mg /day of vitamin C or placebo and then hemodialysis was initiated. We measured the lipid profile and the susceptibility of LDL and HDL to oxidation using cooper ions, at the moment of inclusion and oneyear after the treatment. Results: No significant differences were observed among the vitamin-C treated patients and those who received placebo. Our results show that haemodialysis by itself did not induce deletereous effects on the lipid profile, which was slightly improved. A small decrease in total cholesterol - 183 to 164 mg/dl (group A), 170 to 144 mg/dl (group B); in LDL cholesterol (100 mg/dl to 79 mg/dl (group A), 88 mg/dl to 73 mg/dl (Group B); and in phospholipids [198 to 188 mg/dl, group A (Group A), 195 mg/dl to 178 mg/dl (Group B)], was observed in all the patients one year after starting haemodialysis. When considering oxidation-derivative products, the lag phase of LDL-cholesterol and HDL-cholesterol was enlarged but without statistical significance. A tendency to increase the vitamin E generation in HDL and LDL lipoproteins was observed in vitamin-C treated patients, but the difference still remained not significant. Conclusions: Haemodialysis by itself could improve lipid profile in patients with a previous pro-oxidative state such as uraemia. Although our results have failed to demonstrate significant differences between vitamin C-treated and not treated patients, the tendency to decrease oxidation products by supplementation of vitamin C could mean a beneficial effect on oxidation parameters. In order to improve oxidative stress, the use of lipophylic more than hydrophilic vitamins could be evaluated in randomized studies with a more important number of patients. Key words: Antioxidants. Oxidative stress. Vitamin C. LDL oxidation. Haemodialysis. Correspondence: Alberto M. Castelao, MD Servicio de Nefrología Ciutat Sanitària i Universitària de Bellvitge C/ Feixa Llarga, s/n 08907 l'Hospitalet del Llobregat (Barcelona) E-mail: amcastel@terra.es 178 OXIDATIVE STRESS OF LIPOPROTEINS ESTRÉS OXIDATIVO DE LAS LIPOPROTEÍNAS Y EFECTO ANTIOXIDANTE DE LA VITAMINA C UN AÑO DESPUÉS DE INICIAR HEMODIÁLISIS RESUMEN Hemos estudiado el perfil lipídico y el estado oxidativo de las lipoproteínas en pacientes con insuficiencia renal crónica terminal (IRCT) que inician programa de hemodiálisis, evaluando el posible efecto de la hemodiálisis y de la administración de un antioxidante, como la vitamina C, al cabo de un año. Métodos: Se incluyeron 41 pacientes con IRCT que iniciaron programa de hemodiálisis crónica entre enero del 1999 y enero del 2000 y que fueron randomizados para recibir 1.000 mg /día de vitamina C o placebo antes del inicio del tratamiento sustitutivo renal (TSR). Se analizó el perfil lipídico y la susceptibilidad de la LDL y de la HDL a la oxidación, utilizando sulfato de cobre, en el momento de la inclusión y un año después de haber empezado el TSR y el antioxidante. Resultados: No se hallaron diferencias significativas en cuanto al estado oxidativo entre los pacientes tratados y los que no recibieron tratamiento con vitamina C. En nuestros pacientes, la hemodiálisis por sí, no sólo no provocó efectos deletéreos sobre el perfil lipídico sino que incluso, éste mejoró discretamente, paralelamente a la corrección parcial de la uremia, observándose descenso de colesterol LDL y de los fosfolípidos en todos los pacientes al cabo de un año de TSR. Respecto a los productos derivados de la oxidación, aunque las diferencias no resultaron significativas, los parámetros descendieron discretamente en aquellos pacientes que habían recibido el tratamiento antioxidante. Conclusiones: La corrección parcial de la uremia mediante el inicio de hemodiálisis puede mejorar el perfil lipídico de los pacientes con Insuficiencia renal crónica terminal, los cuales presentan un estado pro-oxidativo atribuible a la uremia per se. Aunque nuestros resultados no mostraron diferencias significativas entre aquéllos tratados con vitamina C frente a los que recibieron placebo, pareció existir una tendencia al cabo de un año a la disminución de los productos derivados de la oxidación en nuestros pacientes, lo que podría implicar un efecto beneficioso sobre la peroxidación lipídica. Palabras clave: Antioxidantes. Estrés oxidativo. Vitamina C. Oxidación de LDL. Hemodiálisis. INTRODUCTION Oxidative stress defines an unbalance between generation of oxygen reactive species (ROS) and antioxidant defense mechanisms. In recent years, there are cumulative evidences that show the association between uremia, oxidative stress increase and high incidence of premature atherosclerosis1. Uremia associated to dyslipemia, arterial hypertension or the cause of renal disease itself, e.g. diabetes, has been implicated as an underlying mechanism. Some studies refer a relative risk for myocardial infarction death of at least five fold in patients on renal replacement therapy as compared to the general popula- tion2. It has also been suggested that replacement therapy in uremic patients with hemodialysis or peritoneal dialysis may particularly contribute to oxidative stress and reduce antioxidants levels in these patients. Loss or deficit in antioxidant activity, for example of vitamin C, may also contribute to the oxidative stress increase in uremia. Boaz et al.3 have shown that in patients on a periodic hemodialysis program and with cardiovascular disease, supplements of 800 IU/day of vitamin E reduce total cardiovascular episodes and myocardial infraction. It has also been shown that cardiovascular episodes in hemodialysis patients may be prevented by administration of acetylcysteine4. 179 R. RAMOS y cols. However, other authors have not confirmed these findings. Schulz et al., in a study on 12 patients, have analyzed the possible accelerative effect on atherosclerosis attributed to cuprophan membranes, failing to demonstrate any reduction in some of the antioxidant defenses of lipoproteins in activated cells during hemodialysis sessions5. Interest on oxidative stress has markedly increased in recent times due to several studies that have evaluated the effect of antioxidant therapies to prevent cardiovascular diseases, with varied and controversial outcomes. The aim of our study was to analyze the lipidic profile and the lipidic peroxidation status in patients that start on renal replacement therapy by hemodialysis, and to assess whether antioxidant effect of vitamin C may be effective and contribute in this way to atherosclerosis prevention in these patients. PATIENTS AND METHODS Patients In end-stage renal insufficient patients, several lipidic oxidation parameters were studied at baseline (before initiation of periodic hemodialysis program) and one year after initiation, comparing the results after administration of vitamin C 1 g/day versus placebo. Vitamin C administration was started the first day after initiation of replacement therapy. Some nutritional (plasma albumin) and inflammatory (C reactive protein, CRP) parameters were also recorded. Forty-one consecutive patients that initiated hemodialysis in our center were included from January of 1999 to January of 2000. All signed the informed consent before being randomized to receive vitamin C 1 g/day (n = 21; group A) or placebo (n = 20; group B). Only 34 patients ended the study (11 women and 23 men). Mean age was 57 ± 17 years. The remaining 7 patients did not end up the study because of death (two patients as a result of cardiovascular complications), renal transplantation (3 patients), and loss of follow-up (two patients were transferred to another hospital). Mean body mass index was 23.11 ± 2.1 kg/m2. All patients were treated with intravenous iron from the start of hemodialysis treatment with an iron dose between 62.5 mg and 125 mg per week. Hemodialysis characteristics remained constant throughout the study. Polysulfone membranes were used and patients received 3 hemodialysis sessions per week, with a mean duration of 255 minutes each. The dialysis bath flow was kept constant throughout the study period (500 180 mL/min) and blood flow (Qb) was between 270 and 300 mL/min. All patients received standard low molecular weight heparin doses during hemodialysis sessions. Twenty patients started hemodialysis through a native arterial-venous fistula (59%) and 14 did so through a provisional catheter (14%). Twentyeight patients were on antihypertensive treatment (blockers: n = 6; angiotensin converting enzyme inhibitors (ACEI): n = 5; others: n = 7). Diabetic patients, those presenting malignant tumors or those on hypolipidemic treatment with statins were excluded from the study. Methods Blood sample was obtained at the last patients' visit to the Clinic, before starting the first dialysis session and treatment with vitamin C. The follow-up period lasted for 365 days during which all patients received three hemodialysis sessions per week with hemophan or cellulose nitrate membranes. An additional blood sample was obtained at the end of the study. In each sample, a 10 mL volume of whole blood was collected in standard agar tubes and centrifuged at 3,000 g, at 4º C for 10 min. Then, chemical, hematological and hemostasia determinations were done on plasma by HITACHI 747 and Boehringer Mannheim (Germany) reactives. Apolipoproteins A and B, (a) lipoprotein and lipid peroxides (LPO) were determined. Reactive substances to thiobarbiturate acid (TBARs) were determined, defined as the amount of chromogen condensation products formed in plasma after adding thiobarbiturate acid against a curve elaborated with malondialdehyde Standard. LDL and HDL isolation High and low density lipoproteinic fractions (HDL and LDL) were obtained by ultracentrifugation preparative for lipoproteins with a rotor Beckman SW 41 at 40,000 rpm and 10º C for 24 hours. (Lipid Research Clinics-Quantilop, Immuno, Vienna). Lipoproteins composition The content on proteins, free total cholesterol, triglycerides, and phospholipids was measured both on LDL and HDL in all samples. TBARs and LPO were also determined for each lipoprotein f ra c t i o n . OXIDATIVE STRESS OF LIPOPROTEINS Table I. Lipid profile before and within one year of hemodialysis initiation Group A (Vit. C) Before HD Total cholesterol (mg/dL) Triglycerides (mg/dL) Phospholipids (mg/dL LDL) LDL cholesterol (mg/g LDL) HDL cholesterol (mg/g HDL) LPO (mg/dL) Lp (mg/dL) 183.31 ± 57.3 132.63 ± 86.5 198.31 ± 44.7 100.34 ± 50.58 50.27 ± 23.10 20.97 ± 15.71 48.47 ± 49.29 One year post-HD 164.81 ± 30.6 ns 151.35 ± 171 ns 188.39 ± 44.5 ns 79.40 ± 30.97 ns 47.18 ± 31.3 ns 85.84 ± 96.11 ns 52.97 ± 62.52 ns Group B (placebo) Before HD 170.22 ± 39.1 144.78 ± 60.8 195 ± 29.22 88.60 ± 27.03 48.7 ± 16.8 26.6 ± 30.91 38.33 ± 52 One year post-HD 144.83 ± 24.2 ns 114.78 ± 46 ns 178.11 ± 27.02 ns 73.19 ± 22.66 ns 40.05 ± 15.59 ns 47.37 ± 26.70 ns 31.46 ± 45.58 ns Lipoproteins oxidation Isolated LDL and HDL fractions were diluted with saline phosphate (PBS) buffered solution at a 50 µg of protein/mL concentration. Cupper chlohydric ion was added to obtain a concentration of 2 µM of Cu2+ ions and fractions were incubated at 37º C. Dienic complexes (DC) production was monitored continuously being recorded with a spectrophotometer at 234 nm absorbance. Besides, one spectrophotometer output was used to analyze the lag phase at the beginning of oxidation. Vitamin E content in LDL and HDL LDL and HDL samples were examined for vitamin E according to Tsen's methodology. Statistical analysis Results are expressed as mean ± standard deviation. Student-Fisher t test was used to estimate differences between both groups (A = treated with vitamin C; B = placebo) before initiation of hemodialysis replacement therapy and one year after. Accepted significance was < 0.05. Descriptive analysis was done with SPSS statistical software. RESULTS Hemodialysis characteristics were kept stable throughout the study with regards to hemodialysis doses, blood flow (Qb), iron treatment, and heparin dose used. There were no observed differences in final results between dialyzed patients with arterialvenous fistula and those with catheter. Only 13% of included patients maintained residual renal function within one year of replacement therapy initiation. There were no significant differences when comparing the lipid profile of patients treated with vitamin C (group A) with regards to non-treated ones (group B), at basal laboratory work-up or one year after hemodialysis initiation. Lipid profile for both groups is summarized in Table I. With regards to (a) lipoprotein, there were no differences between both groups, observing a non-significant increase in the vitamin C treated group and a non-significant decrease in the placebo group within one year in all patients. There were no significant differences in initial lag phase absorbances of LDL or HDL, or in the dienic conjugates either (Table II). TBARs (oxidation derived products) were not significantly elevated within one year in both groups, although that increase was slightly lower in vitamin C treated patients (Table III). Vitamin E levels in LDL were significantly increased in vitamin C treated patients. Vitamin E levels in HDL were significantly increased in vitamin C treated patients and decreased in the placebo group within one year of RRT (Table IV). There were no significant differences with regards to CRP and albumin between both groups, and there was no correlation between oxidative stress markers and the inflammatory status (CRP) at the end of the study (Table V). Although in the present study oxalate levels were not assessed, no patient treated with vitamin C presented clinical signs of lithiasis or other complications attributable to vitamin C throughout the study. 181 R. RAMOS y cols. Table II. Lag phase and dienic compounds Group A (vit. C) Before HD Lag phase/LDL (min) Lag phase/HDL (min) Tmax/LDL (min) Tmax/HDL (min) Initial DC/LDL Initial DC/HDL Propagation rate/LDL Propagation rate/HDL 42' 27' 86' 63' 49 64 1.94 1.88 One year post-HD 46' 17' 93' 59' 48 52 1.75 1.35 Before HD 37' 20' 63' 68' 46 46 2.20 1.41 Group B (placebo) One year post-HD 41 18' 59' 66' 49 51 1.87 1.46 p ns ns ns ns ns ns ns 0.042 Tmax = maximum rate of oxidation. DC = dienic complexes. DISCUSSION Recently Himmelfarb et al.6 have suggested that oxidative stress could be the linkage between cardiovascular complications and uremia. Oxidative stress is considered as the «total amount of oxidation products» against the «total amount of antioxidants». As is well described by Wratten7, this definition may lead to errors in estimating the physiologic importance of oxidative stress. Contrary to what is suggested by Toborek8, who states that there exist an intensification in lipidic lipoperoxidation in those patients with chronic renal failure that receive hemodialysis, and that that replacement therapy may accelerate atherosclerosis due to an increase in peroxidation, we have found no significant differences among our patients that started on hemodialysis within one year of treatment, although there was a trend towards a decrease in total cholesterol, LDL cholesterol and phospholipids levels in both groups, which is in agreement with the results by Heimann9 and Sakurai10. Himmelfarb et al.6 state that the existing pro-atherogenic state in uremia may improve with dialytic therapy. In our case, there were no differences between oxidation derive parameters before and after initiation of hemodialysis, independently of presence or absence of vitamin C treatment (Table I). Although some parameters of the lipid profile did vary, such as total cholesterol (in both treated and nontreated groups), phospholipids, and cholesterol content in LDL, cholesterol in HDL also decreased. None of these changes reached statistical significance. This allows us to strengthen Himmelfarb's hypothesis. Some studies have demonstrated that oxidative modification of LDL may explain the accelerated atherogenesis in hemodialysis patients, with the absence of greater increases in native LDL. Therefore, a reduction in the lag phase could be expected in these patients11. In the present study, we observed a very moderate and non-significant shortening of the LDL lag phase, as well as a moderate and non-significant lengthening of the HDL lag phase 12 months after initiation of hemodialysis treatment. In addition, this was similar in both groups. From these data, we might infer that hemodialysis itself has not carried along an increase in the oxidative status of our patients. Differences in C reactive protein before and after hemodialysis initiation (independently of presence or absence of treatment with vitamin C) do not show either that this is the cause of the conti- Table III. TBARs levels before starting on HD and within one year of HD Group A (vit. C) Before HD TBARs LDL (mg/g LDL) TBARs HDL (mg/g HDL) 0.25 0.21 One year post-HD 0.38 0.31 Before HD 0.28 0.20 Group B (placebo) One year post-HD 0.46 0.27 p ns ns TBARs = thiobarbiturate acid reactive substances. 182 OXIDATIVE STRESS OF LIPOPROTEINS Table IV. Vitamin E levels Group A (vit. C) Before HD Vitamin E (µmol)/g LDL Vitamin E (µmol)/g HL 1.82 1.10 One year post-HD 2.96 1.60 Before HD 0.98 1.59 Group B (placebo) One year post-HD 1.05 0.59 p ns ns nuous inflammatory and pro-oxidative state in hemodialyzed patients. With regards to total thiobarbiturate acid reactive substances (TBARs), that represent one of the oxidative stress markers, some authors have found that this lipoperoxidation product is elevated in hemodialysis patients and that, in turn, there is an LPO increase1215 . In our study, we have found a non-significant TBARs increase together with an LPO increase 12 months after hemodialysis initiation. However, TBARs levels were less increased within one year of hemodialysis initiation in vitamin C treated patients, but did rise in the placebo group. This finding constitutes an argument in favor of vitamin C therapy in this type of patients. Although LPOs differences before and after replacement therapy were greater than those in TBARs, the later were balanced if we exclude one outlier (an extreme value from patient #27). On the other hand, during hemodialysis session, the concentration of water-soluble antioxidants such as vitamin C is very low16, being very difficult in this situation to achieve effective antioxidants levels. Although some authors have detected a decrease of TBARs in plasma using dietary vitamin C supplements, the results are so controversial that we believe that vitamin C usefulness should be carefully assessed17. On the other hand, it is well known that vitamin E is the major lipid phase antioxidant and that, in order to restrain the pro-oxidative state, high vitamin E content levels in LDL would have to be achieved. Our results show that vitamin E content in lipopro- teins was higher within one year of replacement therapy initiation only in vitamin C treated patients. This fact could be explained as a consequence of the vitamin C regeneration properties by increasing vitamin E; a process that would take place during water exchange in hemodialysis. Although vitamin E is located in membranes and vitamin C in aqueous phases, the later is able to regenerate in the form of vitamin E, allowing disrupting the oxidative stress chain and maintaining the vitamin E scavenger effect during lipoperoxidation while ascorbic acid from vitamin C is present18-20. In conclusion, we believe that hemodialysis, by itself, does not deteriorate, but may partially contribute to improve, the lipid profile of uremic patients with end-stage chronic renal failure that, in addition, have a pro-oxidative state in the advanced stage of renal disease. Although our results do not demonstrate significant differences with regards to oxidative stress, the trend towards a decrease in oxidation derived products --such TBARs-- in vitamin C treated patients and the increase of vitamin E levels inside LDL may represent a tendency towards improving the parameters of lipidic peroxidation. 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