Accepted Manuscript

Title: Vitamin E-coated dialysis membranes reduce the levels

of oxidative genetic damage in hemodialysis patients

Authors: Lara Rodrı́guez-Ribera, Zuray Corredor, Irene Silva,

Juan Manuel Diaz, José Ballarin, Ricard Marcos, Susana
Pastor, Elisabet Coll

PII: S1383-5718(16)30358-8
DOI: http://dx.doi.org/doi:10.1016/j.mrgentox.2017.01.003
Reference: MUTGEN 402802

To appear in: Mutation Research

Received date: 3-11-2016

Revised date: 18-1-2017
Accepted date: 18-1-2017

Please cite this article as: Lara Rodrı́guez-Ribera, Zuray Corredor, Irene Silva, Juan
Manuel Diaz, José Ballarin, Ricard Marcos, Susana Pastor, Elisabet Coll, Vitamin
E-coated dialysis membranes reduce the levels of oxidative genetic damage in
hemodialysis patients, Mutation Research/Genetic Toxicology and Environmental
Mutagenesis http://dx.doi.org/10.1016/j.mrgentox.2017.01.003

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Vitamin E-coated dialysis membranes reduce the
levels of oxidative genetic damage in
hemodialysis patients

1 1 2 2 2
Lara Rodríguez-Ribera , Zuray Corredor , Irene Silva , Juan Manuel Diaz , José Ballarin ,
1,3 1,3,* 2,*
Ricard Marcos , Susana Pastor , Elisabet Coll

1
Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Edifici C, Universitat
Autònoma de Barcelona, 08193 Bellaterra, Cerdanyola del Vallès, Spain.
2
Fundació Puigvert, Barcelona, Spain
3
CIBER Epidemiología y Salud Pública, ISCIII, Madrid, Spain

* Corresponding authors
Susana Pastor
E-mail address: susana.pastor@uab.cat

Elisabet Coll,
E-mail address: ecoll@terra.com
Telephone number: +34 934169700
FAX: +34 934169730

1
Highlights

 Use vitamine E-coated hemodialysis membranes decrease the oxidative damage

 Hemoglobin values increased with the use of vitamin E-coated membranes
 The use of this membranes was also effective in correcting vitamin E deficiency

2
ABSTRACT

End-stage renal disease patients present oxidative stress status that increases when

they are submitted to hemodialysis (HD). This increase in oxidative stress can affect

their genetic material, among other targets. The objective of this study was to evaluate

the effect of using polysulfone membranes coated with vitamin E, during the HD

sessions, on the levels of genetic damage of HD patients. Forty-six patients were

followed for 6 months, of whom 29 changed from conventional HD to the use of

membranes coated with vitamin E. The level of genetic damage was measured using

the micronucleus and the comet assays, both before and after the follow-up period.

Serum vitamin E concentration was also checked. The obtained results showed that

24% of our patients presented vitamin E deficiency, and this was normalized in those

patients treated with vitamin E-coated membranes. Patients with vitamin E deficiency

showed higher levels of oxidative DNA damage. After the use of vitamin E-coated

membranes we detected a significant decrease in the levels of oxidative damage.

Additionally, hemoglobin values increased significantly with the use of vitamin E-coated

membranes. In conclusion, the use of vitamin E-coated membranes supposes a

decrease on the levels of oxidative DNA damage, and improves the uremic anemia

status. Furthermore, the use of this type of membrane was also effective in correcting

vitamin E deficiency.

Keywords: Hemodialysis, Vitamin E-coated membranes, Oxidative DNA damage,

Anemia, Vitamin E deficiency

3
1. Introduction

Patients suffering from chronic kidney disease (CKD) present high incidence of

cancer [1], and increased levels of DNA damage [2, 3]. Moreover, CKD patients also

present elevated levels of oxidative stress [4] which can be associated with the adverse

outcomes observed in these patients. Oxidative stress has been postulated as one of

the causes of the high levels of DNA damage observed in CKD patients [5]. The

presence of high levels of reactive oxygen species (ROS) in these patients has been

related to the accumulation of oxidative compounds resulting from both the pathology

and the depletion of their antioxidant mechanisms. Although high levels of ROS are a

characteristic of CKD patients, these levels are particularly important in those patients

undergoing hemodialysis (HD) [6]. During this process ROS can result from the

interaction of blood with non-biological materials of the extracorporeal circuit, mainly

dialysis membranes [7].

Moreover, HD patients follow a restrictive diet low in fruits and vegetables in order to

reduce the intake of potassium. The association of restrictive diet with malnutrition and

loss of some antioxidants during HD has been postulated as a cause of antioxidant

deficit in HD patients [8].

Although vitamin E supplementation has been shown to decrease oxidative stress in

the majority of the studies performed [8], this treatment is not routinely used in HD

patients. Another alternative is the use of dialysis membranes coated with vitamin E,

that also has shown to be effective in decreasing oxidative stress in HD patients [9,10].

With respect to the potential advantages of the use of vitamin E coated membranes to

reduce the levels of DNA damage in HD patients, only a pilot study with nine patients

has been conducted until now, and no effects were observed using the comet assay

[11].

In this context, the aim of our study was to evaluate the effect of vitamin E-coated

polysulfone (PSE) membranes in genomic and DNA oxidative damage levels,

measured before and after six months of using PSE membranes. The comet assay

4
(with and without FPG) and the micronucleus test were carried out using peripheral

blood lymphocytes. Moreover, plasma antioxidant capacity was measured only in the

group treated with PSE membranes in order to investigate the mechanism of action of

these dialyzers. To evaluate the effectiveness to correct vitamin E deficiency, serum

levels of vitamin E were also measured.

5
2. Materials and methods

2.1. Study population

The study involved a total of 46 HD patients. The inclusion criteria were as follows:

aged over 18; undergoing conventional HD for more than three months with three

weekly sessions of 3.5 to 4 hours and with a stable regimen of anticoagulation and

erythropoietin. Patients with known coagulation problems were excluded from the study

as were as those with a survival rate lower than 6 months or with expected kidney

transplantation in the next 6 months.

All participants were recruited at the hospital Puigvert Foundation (Barcelona,

Spain). Two blood samples were obtained for each patient before the HD session, at

the beginning of the study and after 6 months of the follow up period. Patients were

randomly distributed in two groups: PSE membranes and control. During that period of

time, 29 patients switched from conventional HD with polysulfone (PS) membranes to

HD with PSE membranes. The remaining patients were also followed and did not

change any parameter of the HD technique. PSE membranes (VIE-L®) were supplied

by Izasa Hospital, Barcelona, Spain. A descriptive of the general characteristics of the

patients is indicated on Table 1. All the information was obtained from clinical data, as

gender, age, time in HD, previous transplantation, hypertension, cardiovascular

pathologies, previous cancer incidence, diabetes, dyslipidemia, body mass index, and

vitamin supplementation, among others.

Standard blood analysis included the determination of calcium, phosphorus, glucose,

cholesterol, triglycerides, albumin and haemoglobin, among other habitual parameters

in HD patients. Moreover ferritin, iron, transferrin saturation, parathyroid hormone and

C-reactive protein were also analysed.

All individuals participating in the study provided written informed consent, and blood

samples were collected under protocols approved by the Ethics Committee of the

Puigvert Foundation. Blood samples were sent to the Universitat Autònoma de

6
Barcelona and immediately processed to analyse the levels of genomic damage and

the antioxidant capacity of the serum patients.

2.2. Serum concentrations of vitamin E

Venous blood (2.5mL) was collected, serum was separated immediately and

samples were frozen at -20ºC and stored until analysis. Serum levels of vitamin E were

measured by high resolution liquid chromatography. In adults, the reference values are

5-20 mcg/mL [12].

2.3. Comet assay

The levels of DNA breaks present in peripheral blood lymphocytes were measured

using the comet assay, performed following the standard protocol used in our

laboratory [3]. To determine the levels of oxidized bases present in the lymphocytes,

FPG enzyme was used. This enzyme detects oxidized bases and induces single-strand

breaks after their excision [13]. Net oxidative DNA damage was calculated by

subtracting the damage scored in the samples incubated with buffer alone from those

incubated with buffer containing FPG. The % tail DNA was used as a measure of DNA

damage [14].

2.4. Lymphocyte culture and micronucleus (MN) assay

Blood samples from heparinised vacutainers were processed as described

previously using cytochalasin-B to arrest cytokinesis [15]. Briefly, whole blood cultures

were established and 1% of phytohemagglutinin was added to the culture. After 44 h, 6

µg/mL of cytochalasin-B were added to the culture to arrest cytokinesis, and after 72 h

of incubation, cultures were harvested by centrifugation and hypotonic treatment was

applied. Then, samples were dropped and stained with 10% Giemsa. To determine the

frequency of binucleated cells with micronuclei (BNMN), 1000 binucleated lymphocytes

per sample were blind scored, on coded slides, according to standard criteria [16]. In

addition, 500 cells with one, two or more nuclei were scored to calculate the

cytokinesis-block proliferation index (CBPI) [17].

7
2.5. Trolox equivalent antioxidant capacity (TEAC) assay

The plasma antioxidant capacity was measured using the TEAC assay as already

described [18] with minor modifications. Briefly, venous blood samples from EDTA

tubes were centrifuged at 170 g during 5 min to obtain plasma and it was stored at

-80 ºC. Ten μL of plasma or Trolox standard reacted with 6.20 μM myoglobin solution

(20 μL), 183 μM ABTS solution (125 μL) and 10 mM H2O2 (25 μL) on a microplate.

Reaction was followed at 405 nm with the plate reader Sunrise (Tecan Trading AG,

Switzerland). Lag time from kinetic curves and Trolox calibration curve was calculated

and plasma TEAC was expressed as Trolox equivalent.

2.6. Statistical analyses

Data was assessed for normality using the Kolmogorov-Smirnov test. TEAC and MN

assay data was analysed using Student t-test test for paired samples. For quantitative

data, a Student t-test was used and for qualitative data, a χ2 test was used.

Correlations were assessed using the Pearson test. Data is presented as mean ±

standard error of the mean (SEM). Statistical significance was defined as a P walue

lower than 0.05.

For the statistical analysis of the genomic damage obtained by the comet assay,

non parametric tests were used. Comparisons between the two groups were analysed

using the Wilcoxon test for continuous variables, and Mann-Whitney test for discrete

variables. Correlation coefficients were assessed by Spearman´s Rho. The

Erythropoiesis Stimulating Agents (ESA) resistance index (ERI) was determined as the

weekly weight-adjusted ESA dose (IU/week/kg) divided by the product of the patient’s

weight (Kg) and the haemoglobin level (g/dL).

8
3. Results

Selected patients were included in the HD program of the Puigvert Foundation and

underwent conventional HD for 3.5 to 4 h, on a thrice weekly dialysis schedule, using

synthetic low permeability membranes (ultrafiltration coefficient of 13 mL/h/mm Hg) of

polyethersulfone with a surface of 1.8 m2, with a bicarbonate dialysate. The average

time on HD was 23.21± 27.73 months. Twenty-nine patients included in the study

switched to HD treatment with PSE membranes for 6 months, the other 17 patients

stayed on the same treatment being used as a control group. PSE membranes had a

surface of 1.8 m2 and low permeability (ultrafiltration coefficient of 13 mL/h/mm Hg).

General characteristics of the selected patients are listed in Table 1. Main causes of

end-stage renal disease (ESRD) were glomerular disease (23.9%),

nephroangiosclerosis (21.17%), interstitial nephritis (13%) and diabetes mellitus

(10.9%).

Blood chemical data of patients before and after 6 months is shown in Table 2. Only

haemoglobin and ferritin levels increased significantly after 6 months in PSE (P=0.012

and P=0.01, respectively). Vitamin E deficiency was present in 24% of all studied

patients. After treatment with PSE membranes vitamin E levels increased slightly from

8.28 to 11.12 mcg/mL (P<0.05) (Table 2) but all patients with vitamin E deficiency

normalized this parameter (22.2% pre-treatment and 0% post-treatment, P<0.001;

Figure 1) whereas patients not treated remained almost unchanged (Table 2).

The levels of genetic damage, before and after the switch to PSE HD, are described

in Table 3. No differences were observed in BNMN and CBPI values between sampling

times. Regarding the comet assay, no significant differences were observed in the

basal damage between the two sampling periods. Nevertheless, when the comet assay

was complemented with FPG enzyme to determine the levels of oxidized DNA bases, a

significant reduction was observed after 6 months using PSE (P = 0.012). Among the

group treated with PSE membranes only 17 patients decreased their oxidative damage

and the other 12 did not change or presented a slight increase (Figure 2). The group

9
that decreased oxidative damage had the highest basal oxidative damage. Serum

levels of vitamin E correlated negatively with oxidative DNA damage, demonstrating

that patients with low levels of vitamin E presented the highest levels of DNA oxidative

damage (rho=-0.402, P=0.038). TEAC assay was only measured in the PSE group and

we didn’t found changes in the antioxidant capacity when PSE membranes were used

(Table 3). The decrease in oxidative DNA damage do not correlate with the antioxidant

status of the patients’ plasma, evaluated by the TEAC assay (rho=0.220, P=0.252).

In addition, a negative correlation between haemoglobin levels and oxidative DNA

damage (rho=-0.373, P=0.046) was observed.

10
4. Discussion

CKD patients have been considered as a group exhibiting high levels of oxidative

stress, which takes place when the equilibrium between the generation of pro-oxidants

or ROS and the endogenous antioxidant capacity is altered. Since kidney organ is rich

in mitochondria this would means that it is highly vulnerable to damage caused by

oxidative stress. In fact, it is documented that ESRD patients have higher rates of pro-

oxidant activity and lower rates of anti-oxidant activity [19].

Genomic damage is also a characteristic of CKD patients [2,3]. Although the internal

accumulation of uremic toxins has been proposed to cause part of the DNA damage

observed in these patients, oxidative stress is also a key factor involved in such

damage. The presence of elevated levels of genomic damage can be linked to the high

incidence of cancer presented in CKD patients. Nevertheless, in these patients is not

clear if genomic damage is the cause or rather a consequence of their increased

incidence of cancer [1].

Although oxidative stress is related to CKD status itself, different authors have

postulated that when ESRD patients underwent HD, the extra-corporal flux of blood

through the dialysis membrane act as supplementary source of oxidative stress [20].

Taking into account the adverse effects related to oxidative stress, many alternatives

have been proposed including the use of antioxidant supplements in pre-dialysis

patients [21,22], or the use of dialysis membranes coated with vitamin E for those

patients subjected to HD [23,24]. The use of vitamin E-coated membranes acts as a

powerful radical scavenger, and its use showed to be effective reducing damage

caused by oxidative stress to lipids, proteins and nucleic acids during long-term HD

treatment [25]. The use of vitamin-E coated membranes is better than oral vitamin E

supplementation because ensures a better treatment adherence in patients who are

taking many drugs. Moreover, in the gastrointestinal system the absorption rate of

vitamin E varies interindividually between 20-80% and intestinal absorption is an

important factor that limits vitamin E bioavailability [26]. Vitamin E-coated membrane

11
avoids this step and ensures a direct contact between blood and this vitamin improving

bioavailability.

From our study we have determined that the use of PSE coated membranes

significantly reduces the levels of oxidized DNA bases, supporting an antigenotoxic

effect. Moreover, a significant reduction in the levels of 8-hydroxy-deoxyguanosine

have been described in leukocyte of patients moving to the use of vitamin E bonded

regenerated cellulose membranes [27,28]. There are a few studies evaluating the

effects of vitamin E supplementation on oxidative stress by measuring DNA damage

using the comet assay in hemodialysis patients. All of them have demonstrated that

vitamin E supplementation is effective in the reduction of DNA damage levels [29,30].

However, there is a lack of studies evaluating the clinical translation of oxidative DNA

damage reduction after vitamin E supplementation on cardiovascular outcome

reduction or mortality reduction.

Interestingly, we were unable to find changes in the levels of genomic damage

(aneuploidies or breaks) by using the MN assay. Maybe, it should be interesting the

analysis of nucleoplasmic bridges or nuclear buds, which would give us more

information about the DNA repair. One explanation for these inconsistent data could be

the different sensitivity to DNA repair mechanisms of the comet assay and the MN

frequency test. MN frequency provides information about accumulated genomic

damage over the lifespan of the lymphocyte which can be 3-4 years and our follow-up

period was too short to induce a significant improvement in this marker [31]. On the

other hand, genomic damage measured by comet assay and DNA oxidative damage

can be repaired quickly by the cells, but DNA oxidative damage could be a more

sensitive marker for measure the effect of an antioxidant, as described previously [32].

An interesting finding of this study was that PSE membranes were more effective (in

terms of diminution of oxidative damage) in patients with higher basal oxidative

damage (as observed in Figure 2). As we described in a previous study [33] oxidative

damage was a prognosis biomarker of survival in HD patients. In order to improve the

12
prognosis of those individuals with high levels of oxidative damage, the use of PSE

membranes could be recommended. This is a cheap maneuver, well tolerated and

easy to use. Ensuring adequate intake of vitamins in CKD patients is important to avoid

subtle effects of low intake on oxidative stress and its consequences. People under

hemodialysis treatment may not meet their daily vitamin requirements for several

reasons such as restrictions on diet, interference with absorption due to medications or

altered metabolic pathways due to uremia [34].

Vitamin E is one of the main defenses against ROS and lipid peroxidation. Vitamin E is

present in some vegetables (spinach, broccoli), some fruits (kiwi, mango) and nuts

(almonds, hazelnut), that due to the diet restriction of the hemodialysis patients are

practically missing in its diet. Twenty-four percent of our studied patients showed a

vitamin E deficiency that can be corrected with the use of vitamin E-coated

membranes. It should be remarked that the supplement of vitamin E-coated

membranes does not increase significantly the levels of those patients that showed

normal levels of vitamin E. Avoiding intoxication by this way of supplement. By

contrast, this membrane is effective in correct the vitamin E deficiency. The discovery

of an association between low levels of vitamin E and high levels of oxidative DNA

damage permits us to detect the individuals that can improve their oxidative stress

status only by using this easy way of supplementation.

In HD patients, oxidative stress damages mainly erythrocytes, causing uremic

anemia [35]. Our results showing increased levels of hemoglobin and ferritin, without

changes in the dose of ESA and intravenous iron, would support amelioration on the

anemia status of our patients, related to the antioxidant effect observed in the comet

assay; nevertheless ERI did not decrease significantly. In contrast, in a meta-analysis

study [24] ERI decreased while hemoglobin level or weekly EPO dose did not show a

significant improvement; and another study concluded that these membranes may

have utility in patients with heightened ESA resistance [36].

13
 The limitations of this study are the low number of patients studied and the short

time of the use of vitamin E membranes. Studies in a large population of HD patients

are needed in order to know how much time has to be supplemented to keep regular

levels of vitamin E and ameliorate their oxidative stress. In addition, will be important to

know if the use of vitamin E membranes could be translated in a fall of cardiovascular

events and, therefore, in a decrease of global mortality of this population.

5. Conclusions

As a summary, we can indicate that moving HD patients from conventional dialysis

to PSE membranes suppose a decrease of the DNA oxidative damage and improve

the uremic anemia status. The use of PSE membranes could also correct vitamin E

deficiency.

14
Conflict of interest

There are no conflicts of interest, and the results presented in this paper have not been

published previously in whole or part, except in abstract format.

Acknowledgments

First of all we thank all the volunteers that have participated in this study. L.

Rodriguez-Ribera and Z. Corredor were supported by postgraduate fellowships from

the Universidad Autónoma de Barcelona and COLCIENCIAS, respectively. This

investigation has been supported in part by the Generalitat de Catalunya (CIRIT,

2014SGR-202) and by a grant from Fondo de Investigación Sanitaria

(FIS,PI12/02559). The research group belongs to a consolidated Research Group

(AGAUR 2009/SGR-1116) and to REDINREN (Spanish Renal Network for Research

16/06 RETICS, Instituto de Investigación Carlos III).

15
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19
Table 1
Description of the studied groups

PSE membrane Control group

N=29 N=17
Sex (men/women) (%) 44.8 / 55.2 52.9 / 47.1
Age (years) (mean ± SE) 68.59 ± 2.46 60.70 ± 3.56
Time in HD (months) (mean ± SE) 20.69 ± 4.97 27.52 ± 7.19
RT previous (% yes/no) 13.8 / 86.2 35.3 / 64.7
Hypertension (% yes/no) 89.7 / 10.3 94.1 / 5.9
CV pathology (% yes/no) 69 / 31 70.6 / 29.4

Previous cancer (% yes/no) 20.7 / 79.3 35.3 / 64.7

Diabetes mellitus (% yes/no) 27.6 / 72.4 47.1 / 52.9
Dyslipidemia (% yes/no) 62.1 / 37.9 76.5 / 23.5
SE: Standard Error, HD: hemodialysis, RT: renal transplant, CV: cardiovascular,
PSE: polysulfone coated with vitamin E. Not statistical differences were
2
observed (P > 0.05, χ test or Student t-test).

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Table 2
Comparison of the blood chemistry data of patients before and after the follow up period

PSE group (n=29) Control group (n=17)

Before After 6 months Before After 6 months
Albumin (g/L) 40.41 ± 0.80 40.65 ± 0.71 38.74± 0.93 39.6 ± 0.99
2 2
Ca x P (mmol /L ) 3.44 ± 0.19 3.59 ± 0.19 3.53 ± 0.29 3.52 ± 0.16
PTH (7-53 ng/L) 219.54 ±26.29 255.56 ± 39.74 193.04 ± 36.5 200.92 ± 40.19
Haemoglobin (g/L) 114.82 ± 2.10 121.72 ± 2.55* 121.47 ± 3.36 120.11 ±2.84
Ferritin (µg/L) 196.01 ± 30.67 309.57±47.52** 201.44 ± 49.19 292.26 ± 64.64
Transferrin saturation (%) 25.10 ± 2.28 26.52 ± 1.87 28 ± 3.8 26.75 ± 2.4
ERI (IU/week/kg/g/dL) 13.89 ± 1.85 11.57 ± 1.46 18.31 ± 4 16.39 ± 3.2
C-reactive protein (mg/L) 9.65 ± 1.94 10.57 ± 2.31 6.9 ± 2.57 11.24 ± 3.91
Cholesterol (mmol/L) 3.81 ± 0.1 3.75 ± 0.15 4.19 ± 0.23 3.76 ± 0.28*
Triglycerides (mmol/L) 1.36 ± 0.14 1.30 ± 0.11 1.60 ± 0.20 1.61 ± 0.16
Kt/V 1.60 ± 0.07 1.67 ± 0.06 1.49 ± 0.06 1.60 ± 0.06
Vitamin E levels (mcg/mL) 8.28 ± 3.8 11.12 ± 3.2** 5.87±3.1 6.2±2.9
Vitamin E deficiency
22.2/ 77.8 0/100** 29.4/70.6 37.5/62.5
(% yes/no)
Ca x P, calcium phosphorus product; PTH, Parathyroid hormone; ERI, ESA resistance index,
Mean ± S.E. Paired samples t- test: *P ≤ 0.05; ** P ≤ 0.01

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Table 3
Levels of DNA damage and antioxidant capacity before and after the follow up period

PSE group (n=29) Control group (n=17)

Basal After 6 months Basal After 6 months

BNMN (‰) 8.69 ± 1.38 10.66 ± 1.66 7.56 ± 1.05 6.62 ± 1.11
CBPI 1.59 ± 0.03 1.55 ± 0.02 1.39 ± 0.03 1.39 ± 0.04
Comet basal damage
8.65 ± 0.735 9.49 ± 0.473 14.29 ± 0.55 14.79 ± 1.61
(% tail DNA)
Comet oxidative damage
35.83 ± 2.54 27.32 ± 1.75* 9.31 ± 0.88 11.04 ± 1.61
(% tail DNA)
TEAC (mMol/L) 0.17 ± 0.02 0.15 ± 0.02 ND ND

Mean ± S.E. ND: no data available. Paired samples t-test: *P ≤ 0.05

22
Figure 1. Evaluation of vitamin E serum levels in the 29 patients who switched to PSE
membranes.

Figure 2. Evaluation of oxidative DNA damage in the 29 patients after 6 months of treatment
with PSE membranes.

23