Haemolysis in haemodialysis patients: evidence for impaired defence mechanisms against oxidative stress

Talia Weinstein1, Avry Chagnac1, Asher Korzets1, Mona Boaz2, Yaacov Ori1, Michal Herman1, Tsipora Malachi1 and Uzi Gafter1,

1 Department of Nephrology, Rabin Medical Centre–Golda Campus, Petah-Tikva and 2 Department of Nephrology, Wolfson Hospital, Sackler Medical School, Tel-Aviv University, Israel



   Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background. Uraemic patients have a decreased ability to withstand oxidative stress. It is postulated that their antioxidant capacity is reduced, yet the mechanism remains unclear. Recently 33 haemodialysis (HD) patients were exposed to chloramine contamination in the water supply. This led to haemolysis in 24 patients, while nine were unaffected. In the former group haemoglobin decreased from 11.7±1.1 to 8.5± 1.4 g/dl (P<0.0001) and returned to 11.4±0.9 g/dl (P<0.0001) following recovery. During haemolysis, haptoglobin was 38.4±10.6 vs 138.1±8.3 ng/dl (P<0.0001) following recovery.

Methods. To explore the factors affecting the severity of haemolysis we studied extracellular and intracellular anti-oxidant defence mechanisms 3 months after recovery. In 29 patients and 20 controls we determined plasma glutathione (GSH), and the erythrocyte enzymes glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rx), and superoxide dismutase (SOD). Serum malondialdehyde (MDA) was measured as a marker of oxidative stress.

Results. Plasma GSH was lower in patients as compared to controls (5.49±0.26 vs 7.4±0.5 µmol/l, P<0.005). There was an inverse correlation between GSH and the degree of haemolysis (r=-0.42, P<0.02). Patients had higher GSH-Rx (4.64±0.15 vs 3.97±0.12 U/gHb, P<0.02), lower GSH-Px (29.7±1.85 vs 35.5±1.62 U/gHb, P<0.001), and similar SOD (0.63±0.02 vs 0.51±0.02 U/mgHb) as compared to controls. There was no correlation between the enzyme levels and the degree of haemolysis. MDA was higher in patients (2.37±0.07 vs 0.97±0.1 nmol/ml, P<0.0001). There was a correlation between MDA and the years patients were on HD (r=0.43, P<0.02).

Conclusions. These data indicate that HD patients have an impaired anti-oxidant response, which may be attributed in part, to plasma GSH deficiency. Patients with the lowest plasma GSH levels are more susceptible to oxidative stress and consequent haemolysis.

Keywords: chloramines; glutathione; haemodialysis; haemolysis; malondialdehyde



   Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Oxygen-derived free radicals are highly reactive oxygen species (ROS) capable of reacting with lipids, proteins, carbohydrates, and nucleic acids. Healthy organisms combat oxygen toxicity with a variety of defence mechanisms including the intracellular erythrocyte enzymes glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Rx), and superoxide dismutase (SOD), as well as extracellular modes such as glutathione (GSH), vitamins C, E, ß-carotene, and others [1]. In pathological states, these mechanisms are attenuated, leading to sequelae such as inflammation, cancer, haemolysis, and arteriosclerosis [2,3]. ROS attack on cell membranes results in formation of lipid peroxidation products such as malondialdehyde (MDA).

There is considerable evidence that haemodialysis (HD) patients are in a continuous state of oxidative stress, perhaps provoked by bioincompatibility of the dialyser membrane, which may induce the formation of ROS [4]. HD patients may be exposed to potentially toxic substances in the dialysis water supply, and adverse reactions have been reported in patients exposed to dialysis water containing various contaminants [5,6].

We report an outbreak of haemolysis in a haemodialysis facility, traced to chloramine contamination in the dialysis water supply. Twenty-four patients developed various degrees of haemolysis, while nine patients were unaffected.

The aim of this study was to explore a possible association between the diversity in the haemolytic response and the scavenging capacity of ROS in these HD patients. The study addressed extra-and intracellular anti-oxidant defence mechanisms. We determined plasma GSH levels and the activity of the intracellular erythrocyte enzymes GSH-Px, GSH-Rx, and SOD 3 months after the haemolytic episode, when there was no evidence of haemolysis. Serum MDA levels were determined as a marker of oxidative stress.



   Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The HD facility is located in a community centre in the city of Ramat-Gan, Israel. Thirty-three HD patients were treated at the centre in 1996. Four patients who were hepatitis C positive were not included in the study. The studied group included 18 men and 11 women (Table 1Go). Their mean age was 63.8±2.2 years (range 36–79). They had been on dialysis for a mean of 6.1±0.85 years (range 2–18), and for no less than 2 years. All patients were dialysed thrice weekly (a minimum of 4 h each session) using cellulose triacetate membranes (Sureflux, Nipro, Japan). Dialysers were not reused. Twenty-one patients were treated with recombinant erythropoietin at an average dosage of 3000 U/week (range 2000–8000), and 16 with intravenous iron sucrose (Venofer, Vifor, Gallen, Switzerland) at an average dosage of 40 mg/week (range 25–75; Table 1Go).


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Table 1. Patients included in study

 
In September 1996, routine monthly blood tests revealed in 24 patients a decrease in haemoglobin (Hb) (H-2, Technicon, Miles Inc., USA). Three days after performance of the blood tests, samples of the dialysis water supply were analysed for organic and inorganic substances. The results of the initial water analysis demonstrated a chloramine level of 0.19 mg/l, while the recommended levels are less than 0.1 mg/l. The following measures were immediately undertaken: emptying of the reverse osmosis storage tank, cessation of chlorine pump activity, and addition of another carbon column. We also performed daily chloramine monitoring. The day after implementing these safety measures, chloramine levels returned to normal, yet it is not clear how many days the water was contaminated. It is possible that some patients were subjected to more contaminated dialysate than others.

Erythropoietin dosage was increased and blood transfusions were administered as required. Reticulocytes, haptoglobin (HPT reagent, Beckman Instruments Inc., Ireland) and iron status prior to, and following haemolysis were determined. Within 2 weeks, the patients returned to their pre-haemolysis Hb levels.

In order to investigate the variance in the haemolytic response to oxidative stress, we studied plasma GSH and the erythrocyte enzymes GSH-Px, GSH-Rx and SOD 3 months after the haemolytic episode, when there was no evidence of haemolysis. Serum MDA levels were also determined. Blood was drawn from the arteriovenous access immediately prior to a midweek haemodialysis treatment. Twenty healthy gender- and age-matched subjects served as controls. Informed consent was obtained from all participants.

Materials
DTNB, GSSG-reductase, N-ethylmaleimide (NEM), Sep-Pak C18, sulphosalicylic acid, NaOH, cumene hydroperoxide, ferricytochrome C, and 2-thiobarbarbituric acid (TBA) were purchased from Sigma (St Louis, MS, USA).

Total and oxidized glutathione
Twenty-five patients were studied. Blood (5 ml) was collected into a heparinized tube. Plasma glutathione levels were determined according to the methods of Adams and Griffith [7,8]. Total glutathione (oxidized and reduced) and the oxidized form (GSSG) were measured: the reduced glutathione (GSH) was calculated as the difference between the total and oxidized forms. Total glutathione concentration [(GSH)+2(GSSG)] was determined using 10 mmol/l DTNB after reduction of the GSSG by GSSG reductase. The resulting amount of GSH was determined using a standard curve (0–2.5 nmoles GSSG) and recorded at 412 nm by spectrophotometry as the change in absorbance per minute, during the reduction with GSH-reductase. To measure the oxidized glutathione (GSSG) in plasma, alkylation of SH groups was carried out with freshly prepared 10 mmol/l NEM. To prevent a NEM effect on the reaction, NEM was separated on a Sep-Pak (C18). To avoid spontaneous oxidation of plasma GSH, plasma was separated within 4 min, and acidified (1 ml plasma/50 µl of 50% sulphosalicylic acid). Prior to GSH determination, plasma was titrated with 0.2 mol/l NaOH. The plasma oxidative state was expressed as the ratio 2(GSSG)/(GSH).

Erythrocyte GSH-Px and GSH-Rx
Erythrocyte GSH-Px was measured indirectly by a coupled reaction with erythrocyte GSH-Rx as described by Palgia and Valentine [9], using cumene hydroperoxide as a substrate. The product of GSH-Px and cumene hydroperoxide act with glutathione as a substrate for GSH-Rx and oxidized NADPH to NADP. NADPH oxidation was measured spectrophotometrically by following absorbance at 340 nm. Each NADPH molecule that is oxidized by GSH-Rx corresponds to one molecule of cumene hydroperoxide reduced by GSH-Px. The activity was expressed as µmol NADPH oxidized per minute per gram haemoglobin (using as an extinction coefficient 6.2 mmol/cm). The non-enzymatic NADPH oxidized was subtracted. GSH-Rx was measured as described by Calberg and Mannervic [10]. GSH-Rx catalyses the reduction of GSSG by oxidizing NADPH to NADP. The activity was measured at 340 nm and expressed as µmol NADPH oxidized per min per gram haemoglobin.

Erythrocyte SOD activity
SOD activity was measured using the reaction reported by McCord and Friedrich [11], which is based on the capacity of SOD to inhibit the reduction of ferricytochrome C by the xanthine/xanthine oxidase system. A standard curve was prepared using commercially available SOD. The development of the reaction was monitored spectrophotometrically at 405 nm. One SOD unit was defined as the amount of enzyme that inhibited the rate of cytochrome C reduction by 50%. Carried out in lysate of red cells from which the haemoglobin was precipitated, the reaction was expressed as units per mg haemoglobin.

Serum MDA
Serum MDA was determined as described in our previous study [12]. Five millilitres of blood were collected and centrifuged at 1500 g for 10 min at 4°C. MDA was determined spectrophotometrically with a TBA solution. After boiling in a water bath and cooling, the absorbance at 532 nm was measured.

Statistical analysis
Results are presented as mean±SE. The data were analysed using SPSS software v. 8.0 (Chicago, Il). Pearson's correlation coefficient was used to determine association between the change in haemoglobin and numeric variables. Comparisons between patients and controls were performed using Student's t-test. Two-tailed P values <0.05 were considered significant.



   Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Hb and haptoglobin levels
In the patient group that developed haemolysis, Hb decreased from 11.7±1.1 to 8.5±1.4 g/dl (P<0.0001) (Figure 1Go). Following recovery Hb levels reached 11.4±0.9 g/dl (P<0.0001). At the peak of haemolysis, haptoglobin levels were 38.4±10.6 vs 138.1±8.3 ng/dl (P<0.0001) following recovery. The average reticulocyte count was 2.3% (range 1–4.4%).



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Fig. 1. Hb and haptoglobin levels in patients who developed haemolysis. White bars, Hb; shaded bars, haptoglobin. (*P<0.0001, comparison of Hb levels during haemolysis to levels before and after haemolysis; **P<0.0001, comparison of haptoglobin levels during and after haemolysis).

 

Plasma GSH
Plasma GSH was lower in HD patients in comparison to controls (5.49±0.26 vs 7.40±1.3 µmol/l; P<0.005) (Table 2Go). In the patient group there was an inverse correlation between plasma GSH and the degree of haemolysis as estimated by the change in Hb (r=-0.42; P<0.02; Figure 2Go).


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Table 2. Levels of GSH, GSH-Px, GSH-Rx, and MDA

 


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Fig. 2. Inverse correlation between GSH levels and the degree of haemolysis as determined by the decrease in Hb.

 

Erythrocyte GSH-Px and GSH-Rx
Erythrocyte GSH-Px was significantly lower in HD patients as compared to controls (29.7±1.85 vs 35.5±1.62 U/gHb; P<0.001); erythrocyte GSH-Rx was significantly higher (4.64±0.15 vs 3.97 ±0.12 U/gHb; P<0.02; Table 2Go). There was no correlation between the levels of either GSH-Px or GSH-Rx and the change in Hb during haemolysis.

Erythrocyte SOD activity
Erythrocyte SOD activity was 0.63±0.02 U/mgHb in HD patients and 0.51±0.02 U/mgHb in controls. This difference was not significant. There was no correlation between the level of SOD and the change in Hb during haemolysis.

MDA
In the HD group, MDA levels were higher than the controls (2.37±0.07 vs 0.97±0.10 nmol/ml; P<0.0001; Table 2Go). There was a correlation between MDA levels and the number of years patients were treated by dialysis (r=0.43; P<0.02; Figure 3Go). There was no correlation between MDA and haemolysis as reflected by the change in Hb.



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Fig. 3. Correlation between MDA levels and the number of years patients had been treated by HD.

 
None of the variables was significantly correlated with age in this sample. None of the variables differed significantly by gender. When using Pearson's correlation, partial correlations controlling for age and gender did not alter findings.

Iron status
Serum levels of iron and transferrin and iron saturation did not change following haemolysis (Table 3Go). Ferritin levels tended to rise following haemolysis (P<0.07). No correlation was observed between the change in Hb and these parameters before and after haemolysis.


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Table 3. Iron status in patients before and after haemolysis

 



   Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
This study shows that in a population of HD patients subjected to oxidative stress, the patients with the lowest levels of plasma GSH developed a more severe haemolysis. It also shows that MDA levels, which reflect exposure to oxidative stress, increased with the time patients had been treated by HD.

It has been shown that patients with chronic renal failure (CRF) have an impaired anti-oxidant response and therefore are at an increased risk for sequelae such as inflammation, fibrosis, cancer, haemolysis, and arteriosclerosis [3,4]. HD treatment imposes additional oxidative stress due to the bioincompatibility of dialyser membranes and activation of macrophages, as well as the presence of potentially toxic substances in the dialysis water supply [4,6]. Protection from such oxidant injury involves numerous enzymatic and non-enzymatic pathways. One of the most important non-enzymatic protective mechanisms involves the glutathione pathway. In the steady state, 99.8% of the glutathione is reduced (GSH) and only 0.2% is oxidized (GSSG). GSH is constantly being oxidized in the selenium-dependent glutathione peroxidase reaction in which GSH eliminates potentially damaging peroxides. In the absence of an efficient GSH-generating system, GSH cannot be maintained in the reduced state when subjected to oxidative stress. Accumulating peroxides and GSSG may produce the cellular damage, which ultimately leads to shortening of the erythrocyte life span.

Chloramine contamination in the dialysis water supply may lead to massive haemolysis due to oxidation of the erythrocyte membrane [5,13]. In our centre, chloramine exposure induced a decrease in Hb levels in 24 HD patients, while nine were unaffected. We hypothesized that the difference in the severity of haemolysis was due, at least in part, to variations in their anti-oxidant defence mechanisms.

This study shows that HD patients have a significantly lower level of plasma GSH in comparison to healthy controls. GSH levels were inversely correlated with the severity of haemolysis. Plasma GSH is an important line of defence against acute ROS exposure, prior to involvement of the intracellular GSH enzymatic system. Overproduction of ROS by chloramines results in the oxidation of reduced GSH to oxidized GSH. Therefore the subgroup of patients with the lowest GSH levels were probably at a higher risk for haemolysis. The disturbance in the glutathione system has been well documented in HD patients. HD and peritoneal dialysis patients were shown to have significantly lower whole blood and erythrocyte glutathione levels than normal subjects [14]. In another study, HD patients were found to have plasma GSH similar to controls [15]. In a further evaluation of the intracellular enzymatic system responsible for detoxifying ROS, we have found elevated levels of erythrocyte GSH-Rx, decreased values of GSH-Px, and unchanged SOD in blood samples obtained prior to one HD session. However, none of these erythrocyte enzyme levels was correlated with the degree of haemolysis. Previous studies of the individual antioxidant enzymes in uraemic patients have yielded varying results; we cannot reconcile these discrepancies. GSH-Rx has been reported elevated and normal [16]. GSH-Px has been reported low in HD patients [17,18]. GSH-Px is a selenium-dependent enzyme, and its activity is related to the blood selenium level, which is lower than normal in dialysis patients. SOD has been reported to be low [18], or similar to controls [17]. SOD is zinc and copper dependent, and therefore decreased serum ion levels in HD patients could contribute to its inactivity. It thus appears that the deficiency in the GSH detoxifying system is already detectable in uraemic patients not yet on maintenance HD, suggesting that uraemia itself could alter the antioxidant system. This is worsened by the additive effect of the HD procedure, which triggers renewed production of ROS. An additional insult to the system is the presence of free iron in the circulation, which aggravates the toxicity of ROS [19]. However, in this study we did not find a correlation between iron levels and haemolysis.

Oxidative processes favour the occurrence of atherosclerosis, probably via interference with lipoproteins and the production of reactive lipid peroxidation products such as MDA [3]. Similar to previous studies, we have found increased MDA levels in HD patients, indicating that they are in a state of continuous oxidative stress [3,4,12]. Moreover, we found a correlation between MDA levels and the number of years the patient had been on HD, suggesting that prolonged HD treatment is one of the risk factors involved in the development of atherosclerosis.

Recently it has been shown that serum antioxidant activity is significantly decreased in HD patients, and that it is improved by HD treatment [20]. We suggest that plasma GSH deficiency contributes to the reduced serum anti-oxidant response in HD patients. Moreover, upon exposure to oxidative stress, the patients with the lowest serum GSH levels are at the highest risk of developing haemolysis.



   Acknowledgments
 
The authors thank Dr Shifra Sela for determining the plasma GSH levels.



   Notes
 
Correspondence and offprint requests to: U. Gafter MD PhD, Head, Department of Nephrology, Rabin Medical Centre–Campus Golda, 7 Keren Kayemet Street, Petah-Tikva 49372, Israel. Back



   References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Received for publication: 17. 6.99
Revision received 18. 1.00.