Phospholipid plasmalogen, a surrogate marker of oxidative stress, is associated with increased cardiovascular mortality in patients on renal replacement therapy

Peter Stenvinkel, Ulf Diczfalusy, Bengt Lindholm and Olof Heimbürger

Departments of Renal Medicine, Baxter Novum and Clinical Chemistry, Huddinge University Hospital, Karolinska Institutet, Stockholm, Sweden

Correspondence and offprint requests to: Peter Stenvinkel, MD, PhD, Department of Renal Medicine K56, Huddinge University Hospital, 141 86 Stockholm, Sweden. Email: peter.stenvinkel{at}klinvet.ki.se



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. There is a high incidence of premature cardiovascular disease (CVD) in patients with end-stage renal disease (ESRD). Free radical-induced tissue damage is thought to play a major role in the pathogenesis of atherosclerosis, and there are several reports indicating an increased oxidative stress in ESRD. However, it is not well established that increased oxidative stress predicts cardiovascular mortality in ESRD. Plasmalogens, a group of phospholipids with a vinyl ether bond in the sn-1 position, are considered to be sensitive markers of oxidative stress.

Methods. Cardiovascular and non-cardiovascular mortality was recorded (follow-up time 1860±94 days) in 105 ESRD patients (mean age 51±2 years) in whom the fasting ratio of the erythrocyte levels of plasmalogens (DMA 16/C16:0 and DMA 18/C18:0) had been determined at the start of renal replacement therapy (RRT). The prevalence of malnutrition (subjective global assessment), diabetes mellitus (DM), smoking habits and CVD was also determined.

Results. Thirty-eight patients who died of CVD (0.066±0.003) had a significantly lower DMA 16/C16:0 ratio than 15 patients who died of non-cardiovascular causes (0.078±0.005; P<0.05) and 52 patients alive at follow-up (0.075±0.003; P<0.05). A Cox proportional hazard model analysis showed that a low (<median) DMA 16/C16:0 ratio at the start of RRT was associated (relative risk 1.50; confidence interval 1.07–2.16; P<0.05) with CVD mortality independently of age, gender, DM and CVD.

Conclusion. These results suggest that increased oxidative stress may be a risk factor for cardiovascular mortality in ESRD.

Keywords: cardiovascular mortality; oxidative stress; plasmalogen



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Premature atherosclerotic cardiovascular disease (CVD) is a leading cause of morbidity and mortality in patients with end-stage renal disease (ESRD) [1]. Although traditional Framingham risk factors, such as hypertension, dyslipidaemia and diabetes mellitus (DM), may account for a large proportion of the excessive burden of CVD in this patient population, non-traditional risk factors, such as inflammation and oxidative stress, may also contribute [2]. Although oxidative stress has been proposed as a main mechanism by which the accelerated rate of CVD observed in ESRD may be explained [3,4], there are still limited data showing that various surrogate markers of oxidative stress predict cardiovascular morbidity and mortality in this patient group.

In search of valid surrogate markers of oxidative stress, we have been interested in a phospholipid subgroup found in cell membranes and plasma lipoproteins, plasmalogens (DMA 16/C16:0 and DMA 18/C18:0). Previous studies have suggested that plasmalogens may be protective by serving as antioxidants during lipoprotein oxidation [5,6]. Moreover, plasmalogens may serve as storage depot of esterified arachidonic acid [7] and as structural elements stabilizing transmembrane proteins [8]. We [9] and others [10] have previously documented decreased concentrations of phospholipid plasmalogens in ESRD patients, confirming the presence of increased oxidative stress burden in this patient group. Moreover, as we [9] found lower relative plasmalogen levels in erythrocyte membranes in malnourished patients, it could be speculated that increased oxidative stress may contribute to the excessive cardiovascular morbidity and mortality observed in malnourished ESRD patients [11].

The aim of the present study was to evaluate if lower relative levels of plasmalogens DMA 16/C16:0 and DMA 18/C18:0 at the start of renal replacement therapy (RRT) predict all-cause and CVD mortality during long-term follow-up.



   Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this post hoc analysis, we evaluated the outcome of 105 ESRD patients (61 males) in whom the fasting relative plasmalogen levels of erythrocyte membranes had been measured previously [9]. Thirty-one patients were smokers, whereas 19 were ex-smokers and 55 non-smokers (smokers and ex-smokers are grouped together). During the observation period (1860±94 days; range 21–3213 days), 53 patients died, 38 of complications related to CVD (cardiovascular n = 22, cerebrovascular n = 13, peripheral vascular disease n = 2, and aortic aneurysm n = 1) and 15 of non-cardiovascular or unknown causes (cancer n = 4, unknown n = 3, infectious complications n = 3, wasting n = 2, fatal car accident n = 1, dementia n = 1, and refusal of treatment n = 1). The effects of age, gender, CVD, DM, nutritional status, inflammation, smoking habits and oxidative stress (DMA 16/C16:0 ratio) were determined with the Cox proportional hazard model, and adjusted relative risks of both CVD and all-cause mortality were calculated. Sixty of the patients started continuous ambulatory peritoneal dialysis (CAPD) whereas 43 patients started haemodialysis (HD) and two received a kidney transplant before starting dialysis treatment. Routine bicarbonate HD was carried out three times a week (4–5 h per session) using standard cellulose acetate or polysulfone dialysis membranes. Survival was measured from the day of examination until death or censoring, which was at the end of the follow-up (October 6, 2003). Fifty-seven patients received a kidney transplant subsequent to entering the study and were followed in the same way as those that did not receive a transplant. Many patients were on antihypertensive medications, such as angiotensin-converting enzyme inhibitors (n = 44), ß-blockers (n = 52) and calcium blockers (n = 58), as well as other commonly used drugs in ESRD such as phosphate and potassium binders, diuretics and vitamins B, C and D. Whereas none of the patients was on thiazolidinediones, seven were on lipid-lowering therapy. The methods used to analyse relative phospholipid plasmalogenes of erythrocyte membranes and evaluate nutritional status by subjective global assessment (SGA) have been described previously [9]. The Ethics Committee of Karolinska Institutet approved the study protocol at Huddinge University Hospital, Stockholm, and informed consent was obtained from all patients.

Statistical methods
Values are presented as mean±SEM or medians, with P<0.05 taken to indicate significance. Comparisons between groups of patients were performed using non-parametric Kruskall–Wallis and Mann–Whitney's U-test. Survival analyses were made with Kaplan–Meier or Cox regression analyses.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The clinical characteristics of the 105 patients are shown in Table 1. As discussed previously [9], both malnutrition and DM are associated with lower levels of erythrocyte plasmalogens. However, no difference in either DMA 16/C16:0 (0.072±0.002 vs 0.074±0.003) or DMA 18/C18:0 (0.172±0.004 vs 0.176±0.006) ratios were observed comparing inflamed [C-reactive protein (CRP) >=10 mg/l] and non-inflamed ESRD patients. The 38 patients who died of CVD had a significantly lower (0.066±0.003) DMA 16/C16:0 ratio than the 15 patients that died of unknown or non-cardiovascular causes (0.078±0.005; P<0.05) and 52 survivors (0.075±0.003; P<0.05). On the other hand, the difference in the DMA C18/C18:0 ratio observed comparing 38 patients who died of cardiovascular causes (0.164±0.006) with 15 patients who died of non-cardiovascular causes (0.187±0.007) and 52 survivors (0.178±0.005) did not attain statistical significance (Kruskal–Wallis P = 0.08).


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Table 1. Clinical characteristics of patients in the present study

 
By using the Kaplan–Meier log rank test, we studied the relationship between both all-cause and cardiovascular mortality and the DMA 16/C16:0 ratio (divided into medians). Whereas a low DMA 16/C16:0 ratio was associated with higher cardiovascular mortality (Figure 1), the difference between the two DMA C16/C16:0 groups with regard to all-cause mortality did not attain statistical significance (log rank 3.4, P = 0.06). The Cox proportional hazard model was used to adjust both all-cause and cardiovascular mortality for age, gender, DM, CVD and four additional risk factors: inflammation (CRP >=10 mg/l), oxidative stress (DMA 16/C16:0 <median), malnutrition (SGA >1) and smoking (current and ex-smokers vs non-smokers) as shown in Table 2. Following adjustment for age, gender, DM and CVD, only oxidative stress (P<0.05) and malnutrition (P<0.05) were independently associated with cardiovascular mortality. The additional adjustment for mode of therapy (CAPD vs HD) did not affect the risk ratios for either oxidative stress or malnutrition.



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Fig. 1. Kaplan–Meier curve showing cardiovascular mortality in relation to the plasma ratio (median) of DMA16/C16:0.

 

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Table 2. Unadjusted and adjusted hazard ratios for all-cause and cardiovascular mortality in patients starting renal replacement therapy

 


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we demonstrate that a low ratio of phospholipid plasmalogen (DMA 16/C16:0) predicts cardiovascular mortality in ESRD patients on RRT for 5 years. It is notable that following the correction for age, gender and co-morbidity (DM and CVD), the relative risk of a low erythrocyte DMA 16/C16:0 ratio was of a similar magnitude to that observed for malnutrition in predicting cardiovascular mortality. Much recent evidence suggests that ESRD is a disease state characterized by increased oxidative stress, and it has been speculated that oxidative stress may be a potentially important source of cardiovascular morbidity and mortality in this patient population [3,4]. Myeloperoxidase (MPO), a bactericidal enzyme secreted by activated phagocytes that catalyses the production of hypochlorous acid and promotes oxidative stress, has been implicated as a mediator of atherosclerosis in both non-renal [12] and renal [13] patients. However, the mechanisms by which MPO initiates or promotes atherosclerosis are not fully understood. It is therefore of interest that recent data suggest that low-density lipoprotein (LDL) plasmalogens may be attacked by MPO-derived reactive chlorinating species resulting in the production of novel lipid species that could have a role in atherosclerosis [14].

Although recent evidence from different non-renal patient groups, such as patients with burn injury [15], coronary artery disease [16] and congestive heart failure [17], demonstrates that various surrogate markers of oxidative stress parameters predict mortality, data from the renal patient population are scarce. Recently, Bayés et al. [18] demonstrated that elevated oxLDL antibodies, a surrogate marker of lipid peroxidation, were associated with all-cause mortality in 94 HD patients followed for 24 months. However, in this study, no corrections were made for the impact of DM [19] and malnutrition [9], two common clinical features of ESRD that may be associated with increased oxidative stress.

Several lines of indirect evidence suggest that oxidative stress is implicated in the atherogenic process of ESRD patients [3]. First, serum malondialdehyde (MDA), a commonly used surrogate marker of oxidative stress, has been shown to be a strong predictor of prevalent CVD in HD patients. Secondly, Drüeke et al. [20] showed a correlation between another surrogate marker of oxidative stress, advanced oxidation protein products (AOPPs), and carotid artery atherosclerosis. Finally, recent randomized studies have shown that two different anti-oxidative treatment strategies (vitamin E and acetylcysteine) reduced the number of cardiovascular events in ESRD patients [21,22].

Several shortcomings of the present study should be considered. First, oxidative stress did not have a predictive effect on all-cause mortality (Table 2). Clearly, all-cause mortality is a more solid outcome measure than CVD mortality. As we could not clarify the exact cause of death in some patients, we cannot exclude that some patients were misclassified. It is also possible that erythrocyte plasmalogens are only weak indicators of reactive oxygen species burden and underestimate the true impact of oxidative stress on mortality in ESRD. Secondly, as the number of patients was limited, the present study may not have been sufficiently powered to prove that oxidative stress is an independent outcome predictor. Finally, as this was a post hoc analysis, this may also limit the value of the study.

In summary, the present study indicates that a surrogate marker of oxidative stress, low relative phospholipid plasmalogen level of erythrocyte membranes, predicts cardiovascular mortality in ESRD patients on RRT. As plasmalogens did not predict all-cause mortality, possibly due to an insufficient sample size, larger studies are needed to establish the relationship between increased oxidative stress and outcome in this patient population.



   Acknowledgments
 
Söderbergs Foundation (PS) and the Swedish Heart Lung Foundation (UD) supported the present study.

Conflict of interest statement. B. Lindholm is employed by Baxter HealthCare Inc.



   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 30. 6.03
Accepted in revised form: 20.11.03





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