Serum paraoxonase activity, high-sensitivity C-reactive protein, and lipoprotein disturbances in end-stage renal disease patients on long-term hemodialysis
Hanaâ Lahrach, Noreddine Ghalim, Hassan Taki, Anass Kettani, Loubna Er-Rachdi, Benyounes Ramdani, Rachid Saïle*
Laboratory of Research on Lipoproteins and Atherosclerosis, Faculty of Sciences Ben M’sik, B.P. 7955, Sidi Othman, Casablanca, Morocco (Drs. Lahrach, Taki, Kettani, and Saïle); Laboratory of Biochemistry, Pasteur Institute of Morocco, Casablanca, Morocco (Drs. Lahrach and Ghalim); Nephrology and Haemodialysis Department, Ibn Rochd University Hospital, Casablanca, Morocco (Drs. Er-Rachdi and Ramdani)
BACKGROUND: Hemodialysis patients are at high risk for atherosclerotic events. Enhanced oxidant stress, dyslipidemia, and inflammation may have a major role in this risk. In this work, we assessed lipoprotein status, total homocysteine, high-sensitivity C-reactive protein (hs-CRP), and paraoxonase activity in hemodialysis patients to determine the correlations among these parameters and to compare these values with those measured in normal control subjects.
METHODS: We enrolled 109 end-stage renal disease patients on long-term hemodialysis and 100 age- and gender-matched healthy subjects. Total cholesterol, triglycerides, and high-density lipoprotein cholesterol levels were evaluated using colorimetric methods. Low-density lipoprotein (LDL) choles- terol was calculated according to the Friedewald formula. Serum levels of hs-CRP, apolipoproteins (Apo) AI, B, E, and lipoprotein(a) were measured by nephelometry. Lipoprotein particle (Lp) A-I and LpA-I:A-II were determined by immunoelectrophoresis. Total homocysteine levels were evaluated by the fluorescence polarization immunoassay method. Paraoxonase activity was determined using the paraoxon-like substrate.
RESULTS: Compared with controls, hemodialysis patients had more frequent atherogenic dyslipi- demia, hyperhomocysteinemia, and elevated hs-CRP levels. These latter findings inversely correlate with ApoA-I and LpA-I:A-II and positively with ApoB, lipoprotein(a), and ApoB/ApoA-I ratio.
Homocysteine levels correlated positively with age. Paraoxonase activity was decreased in hemodial- ysis patients, especially in elderly patients. This enzyme activity positively correlated with LpA-I:A-II, and inversely with hs-CRP, LDL-cholesterol, and ApoE levels.
CONCLUSION: The present study demonstrated an abnormal lipoprotein profile associated with increased hs-CRP and decreased paraoxonase activity in hemodialysis patients. Hence, inflammation, dyslipidemia, and increased oxidant stress linked to uremia may be contributors to increased cardio- vascular risk in this population.
© 2008 National Lipid Association. All rights reserved.
KEYWORDS:
Hemodialysis;
hs-CRP;
Lipoproteins;
Paraoxonase activity;
tHcy
* Corresponding author.
E-mail address: sailerachid@yahoo.fr
Submitted November 30, 2007. Accepted for publication December 23, 2007.
1933-2874/$ -see front matter © 2008 National Lipid Association. All rights reserved.
doi:10.1016/j.jacl.2007.12.003
Cardiovascular diseases are the most common cause of death in chronic hemodialysis patients.1Atherosclerosis de- velopment can be mediated by several mechanisms simul- taneously. Dyslipidemia with both quantitative and, espe- cially, qualitative aspects characteristic of chronic renal failure are among the major causes of this pathology. Re- cently, inflammation and oxidative stress that appear in uremia have been associated with carotid atherosclerosis.2 C-reactive protein (CRP), an inflammatory protein, is a marker of overall and cardiovascular death in the general population and in dialysis patients.3Increased concentration of blood CRP precedes monocyte appearance in the arterial intima,4mediates low-density lipoprotein (LDL) uptake by monocytes/macrophages,5and leads to activation of expres- sion of adhesion molecules and chemokines.6,7In contrast, experimental studies suggest that homocysteine can en- hance lipoprotein oxidation, reduce glutathione peroxidase activity, and increase affinity of lipoprotein(a) [Lp(a)] for plasmin-modified fibrin.8,9
Oxidative stress and inflammation may be linked in patients with end-stage renal disease (ESRD). It has been suggested that inflammation and duration of dialysis are the most important determinants of oxidative stress in hemodialysis patients.10 Handelman and colleagues11 found an association between F2-isoprostanes and CRP levels in these patients. A significant positive correlation was also found between acute-phase proteins and markers of oxidative stress in ESRD patients.12 A tentative asso- ciation exists between increased oxidative stress and in- flammation, which are both common features of ESRD.
This may contribute to endothelial dysfunction and an increased risk of coronary heart disease.13 Serum para- oxonases (PON) are able to decrease the risk of coronary artery disease by destroying proinflammatory molecules involved in the initiation and progression of atheroscle- rotic lesions.14PONs (aryldialkylphosphatase, EC 3.1.8.1) are a series of serum esterase enzymes synthesized in the liver.
PON1 is characterized by its ability to hydrolyze the organo- phosphate substrate paraoxon, which is the toxic metabolite of the insecticide parathion. It belongs to the family of serum paraoxonases, consisting of PON1, PON2, and PON3. PON1 and PON3 are secreted from the liver into the blood circula- tion, where they are associated with high-density lipoprotein (HDL) particles.15
Rosenblat and colleagues16 have recently described the role of PON1 hydrolytic activity to be in mediating inhibi- tion of LDL oxidation and stimulation of cholesterol efflux from macrophages. The authors also reported that apoli- poprotein (Apo) A-I in HDL stimulates PON1 lactonase activity.16Furthermore, Mackness and colleagues17demon- strated that PON1 could prevent accumulation of lipoper- oxides in LDL.17
In the present study, we investigated lipoprotein status, high-sensitivity CRP (hs-CRP), total homocysteine (tHcy), and PON activity in ESRD patients on long-term hemodi- alysis treatment and compared these findings with those in healthy controls. We have also tested for possible correla-
tions between PON activity, hs-CRP, tHcy, and lipoprotein profile.
Methods
Study subjectsTwo-hundred and seventy-six ESRD patients on long- term hemodialysis treatment were initially examined for participation in this study. Exclusion criteria included dia- betes, antioxidant and/or hypolipemic medication, and clin- ical signs of infectious diseases. One-hundred and nine ESRD patients were finally enrolled in this study. Clinical data were obtained for all patients. All patients were given calcium. Sixteen percent were given various vitamin D supplements and 20% were receiving antihypertensive drugs. The main clinical characteristics are summarized in Table 1. One-hundred healthy subjects matched for age and gender served as controls. Mean age of controls was 46.83
⫾ 11.79 years. Patients gave informed consent, and the study was approved by the local ethics committee.
Laboratory tests
Blood samples were collected after 12 hours (overnight) fasting prior to initiating a scheduled hemodialysis session.
Table 1 Main clinical characteristics in end-stage renal disease patients enrolled in this study
Parameters
Hemodialysis patients
Age (y), mean⫾SD 44.92⫾14.4
Gender (male/female), n 62/47
Dialysis
Length of treatment (y), mean⫾SD
9.44⫾4.42 Duration of session 4 h/3 sessions/wk
Membrane Polysulfone
Solution Bicarbonate
Hemoglobin (g/dL), mean⫾SD
10⫾6.96 Albumin (g/dL),
mean⫾SD
40.26⫾4.16 Blood pressure (mmHg),
mean⫾SD
Systolic 122.11⫾20.78
Diastolic 75.64⫾18.65
Hypertension (%) 20
Anemia (%) 3
Obesity (%) 8
Angina pectoris (%) 9
Myocardial infraction (%) 6
Serum was stored at⫺20°C until analysis at the Laboratory of Biochemistry at Pasteur Institute of Morocco.
Fresh sera were used for determination of glucose, total cholesterol (TC), triglycerides (TG), and HDL cholesterol (HDL-C) using enzymatic methods. LDL cholesterol (LDL-C) was calculated according to the Friedewald for- mula.18
Determination of apolipoproteins, lipoprotein particles, and hs-CRP
ApoA-I, B, and E were determined by nephelometry (Dade Behring nephelometer BN100; Marburg GmbH, Marburg, Germany). Lipoprotein particle (Lp) A-I and LpA-I:A-II were determined by immunoelectrophoresis on agarose gels as previously described.19 Lp(a) was deter- mined by Dade Behring N Latex agglutination assay. Serum levels of hs-CRP were measured by Dade Behring Cardio- phase hs-CRP assay.
Determination of total homocysteine levels
We evaluated total L-homocysteine by the AxSYM Ho- mocysteine assay based on the Fluorescence Polarization Immunoassay method on the Abbott AxSYM system.
Measurement of PON activity
PON activity was determined using paraoxon-like sub- strate as previously described.20First, the rate of hydrolysis of paraoxon was measured by monitoring the increase in absorbance at 412 nm and 25°C. The amount of p-nitrophe- nol generated was calculated from the molar absorptivity (17,100 M⫺1cm⫺1) at pH 8.0. PON activity is expressed in units per milliliter of serum.
Statistical analysis
Data were summarized as mean⫾ SD. Variables were compared by Student’st-test. Analysis of variance post-hoc test was performed for comparison among three groups.
Correlation coefficients between all parameters studied were calculated by Pearson’s correlation analysis with SPSS 13.0 (SPSS, Inc, Chicago, IL).Pvalues⬍ 0.05 were con- sidered statistically significant.
Table 2 Parameters studied in long-term hemodialysis patients compared with control subjects
Parameters Hemodialysis patients Control subjects Pvalue
Total cholesterol (g/L) 1.60⫾0.30 1.56⫾0.18 NS
Triglycerides (g/L) 1.57⫾0.37 0.80⫾0.34 ⬍0.001
HDL cholesterol (g/L) 0.42⫾0.09 0.54⫾0.13 ⬍0.01
LDL cholesterol (g/L) 0.86⫾0.27 0.76⫾0.17 0.023
Apolipoprotein AI (g/L) 1.18⫾0.23 1.27⫾0.28 0.006
Apolipoprotein B (g/L) 0.84⫾0.25 0.67⫾0.21 ⬍0.001
Apolipoprotein E (mg/L) 0.05⫾0.02 0.07⫾0.02 ⬍0.001
LpA-I (g/L) 0.39⫾0.16 0.46⫾0.11 ⬍0.001
LpA-I:A-II (g/L) 0.79⫾0.24 0.81⫾0.11 ⬍0.001
Lp(a) (g/L) 0.20⫾0.13 0.17⫾0.08 ⬍0.001
hs-CRP (mg/L) 5.98⫾7.61 2.06⫾0.97 0.003
Paraoxonase activity (U/ml) 116.97⫾84.59 138.31⫾82.50 0.030
Total homocysteine (mol/L) 44.32⫾15.46 13.88⫾4.49 0.008
HDL, high-density lipoprotein; hs-CRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein; LpAI:A-II, lipoprotein particle AI:A-II; Lp(a), lipoprotein(a); NS, not significant.
Table 3 Correlation coefficients between Log(hs-CRP) levels and lipoprotein profile in long-term hemodialysis patients
Parameters r Pvalue
Age (y) ⫺0.010 0.920
Total cholesterol (g/L) 0.057 0.553
Triglycerides (g/L) 0.046 0.630
HDL-C (g/L) ⫺0.040 0.676
LDL-C (g/L) 0.067 0.488
TC/HDL-C ratio 0.117 0.222
LDL-C/HDL-C ratio ⫺0.047 0.625
ApoB/ApoA-I ratio 0.354 ⬍0.001*
ApoA-I (g/L) ⫺0.234 0.014
ApoB (g/L) 0.192 0.044
ApoE (mg/L) 0.164 0.088
Lp(a) (g/L) 0.192 0.044
LpA-I (g/L) 0.020 0.832
LpA-I:A-II (g/L) ⫺0.232 0.015
Apo, apolipoprotein; HDL-C, high-density lipoprotein cholesterol;
hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipopro- tein cholesterol; LpAI:A-II, lipoprotein particle AI:A-II; Lp(a), lipopro- tein(a); TC, total cholesterol.
*Significant correlation with Spearman’s correlation coefficient.
Results
Lipoprotein and lipid profile in long-term hemodialysis patients
Analyses of lipid parameters were carried out on fresh sera. Frozen specimens were used for other measurements in the same period. As shown in Table 2, all parameters analyzed varied significantly in hemodialysis patients from those in control subjects, except for TC. In parallel with a significant increase in TG and LDL-C levels, HDL-C con- tent was decreased in hemodialysis patients. ApoA-I, E, LpA-I, and LpA-I:A-II were significantly decreased, whereas ApoB and Lp(a) levels were significantly increased compared to specimens from control subjects.
Hs-CRP levels in long-term hemodialysis patients
We observed higher serum levels of hs-CRP in ESRD patients than in healthy controls (Table 2). As shown in Table 3, a significant inverse correlation between serum levels of Log(hs-CRP) and those of ApoA-I and LpA-I:A-II were observed in these patients. Furthermore Log(hs-CRP) correlated positively with serum levels of ApoB, Lp(a), and ApoB/ApoA-I ratio.
We categorized our patients into three groups with low hs-CRP (1–3 mg/L), moderate hs-CRP (1 ⱕ hs-CRP ⱕ3 mg/L), and high hs-CRP (⬎3 mg/L),21thought to be associ- ated with low, moderate, and high risk of cardiovascular dis- ease in the general population. In this population of ESRD patients, we observed that 17.27% had hs-CRP ⬍1 mg/L (group 1), 32.73% of patients had 1⬍CRP⬍3 mg/L (group 2), and 50% of patients had hs-CRP⬎3 mg/L (group 3).
In the third group, those with assumed high cardiovascular risk, we noticed an increase in TC, TG, and LDL-C levels and
a decrease in HDL-C levels, although not statistically signifi- cant from the other groupings. However, a significant increase of the TC/HDL-C ratio and decreased serum levels of ApoA-I and LpA-I:A-II were observed compared to the first group.
The ApoB/ApoA-I ratio was increased significantly in both the second and third groups compared to those with hs-CRP⬍1 mg/L. Also, serum levels of ApoB and Lp(a) were significantly increased in the two highest CRP groups compared to the lowest group (Table 4).
Figure 1 Correlations of total homocysteine (tHcy) levels with age in patients on long-term hemodialysis.
Table 4 Lipids and lipoproteins in end-stage renal disease patients on long-term hemodialysis
Parameters (g/L) hs-CRP⬍1mg/L 1ⱕhs-CRPⱕ3 mg/L hs-CRP⬎3 mg/L
TC 1.56⫾0.32 1.56⫾0.31 1.63⫾0.29
Triglycerides 1.47⫾0.29 1.58⫾0.37 1.60⫾0.39
HDL-C 0.44⫾0.09 0.42⫾0.08 0.41⫾0.10
LDL-C 0.83⫾0.28 0.83⫾0.25 0.89⫾0.27
TC/HDL-C ratio 3.66⫾0.64 3.83⫾0.91 4.08⫾1.02*
LDL-C/HDL-C ratio 1.94⫾0.53 2.03⫾0.72 2.26⫾0.84
ApoA-I 1.22⫾0.26 1.23⫾0.25 1.13⫾0.19*‡
ApoB 0.72⫾0.19 0.83⫾0.23† 0.89⫾0.26**
ApoE (mg/L) 0.05⫾0.03 0.05⫾0.02 0.05⫾0.02
ApoB/ApoA-I ratio 0.60⫾0.12 0.69⫾0.23† 0.80⫾0.23***
LpA-I 0.39⫾0.13 0.39⫾0.21 0.39⫾0.13
LpA-I:A-II 0.84⫾0.23 0.84⫾0.29 0.74⫾0.21*‡
Lp(a) 0.17⫾0.12 0.18⫾0.11 0.23⫾0.14*‡
Apo, apolipoprotein; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; LDL-C, low-density lipoprotein cholesterol; LpA-I:A-II, lipoprotein particle A-I:A-II; Lp(a), lipoprotein(a); TC, total cholesterol.
*P⬍0.05, **P⬍0.01, ***P⬍0.001 significant differences between patients with hs-CRPP⬍1 mg/L and patients who had hsCRP⬎3 mg/L.
†P⬍0.05 significant differences between patients with hs-CRP⬍1 mg/L and patients who had 1ⱕCRPⱕ3 mg/L.
‡P⬍0.05 significant differences between patients with 1ⱕCRPⱕ3 mg/L and patients with hs-CRP⬎3mg/L.
Hyperhomocysteinemia in ESRD patients
We found that the mean tHcy concentration was sig- nificantly increased in ESRD patients compared to con- trol subjects (Table 2). tHcy correlated positively with age (Fig. 1). We found no associations between tHcy levels and gender, PON activity, hs-CRP, or albumin levels.
When separated into two groups (moderate elevation of tHcy [15–30 mol/L] and intermediate elevation [30 – 100 mol/L]), the hemodialysis patients showed no sig- nificant differences in all parameters studied.
PON1 activity in long-term hemodialysis patients
In this study, hemodialysis patients showed a signifi- cant decrease in PON activity compared with control subjects (Table 2). Our analysis reveals a positive corre- lation between PON activity and serum levels of LpA-I:
A-II (Fig. 2A), and a negative correlation between this activity, LDL-C, ApoE, and hs-CRP levels (Fig. 2B through D).
To show whether PON activity varies with age, we separated the patients into two groups (40 years and younger) and (older than 40 years), which revealed a sig- Figure 2 Correlations between lipoprotein parameters, high-sensitivity C-reactive protein (hs-CRP) and paraoxonase (PON) activity.
apoE, apolipoprotein E; LDL, low-density lipoprotein; Lp, lipoprotein particle.
nificant decrease of PON activity in older patients compared with younger adult patients (Fig. 3).
Discussion
In the present study, we analyzed lipoprotein profile, inflammation, PON activity, and their impact on cardio- vascular risk. Patients on long-term hemodialysis treat- ment showed a disturbed lipid profile characterized, as reported previously, by hypertriglyceridemia, increased serum LDL-C levels, and decreased HDL-C.22,23 In ac- cordance with other studies, we did not observe signifi- cant differences in TC levels.24,25 Hypertriglyceridemia is known to be related to a decrease of lipolytic enzymes activity, such as lipoprotein lipase, and may be a conse- quence of loss of carnitine in dialysis fluid. Recently, Rashid and colleagues26demonstrated that the processing of TG-rich HDL by hepatic lipase can be considered as one of the different mechanisms that might explain the reduction in HDL-C levels in hypertriglyceridemia. Pro- duction of smaller HDL particles by this enzyme in- creases ApoA-I clearance from TG-rich HDL.26
We observed decreased serum levels of ApoA-I, ApoE, LpA-I, LpA-I:A-II; these results were supported by several studies.27,28 As reported previously,29 we also observed increased serum levels of ApoB and Lp(a). These findings may be associated with high risk of cardiovascular disease development in ESRD.
In this study, ESRD patients showed evidence of in- flammation characterized by increased serum levels of hs-CRP and tHcy. Using previous convention,2132.73%
of the patients were at moderate cardiovascular risk and 50% were at high cardiovascular risk related to hs-CRP
levels. Elevation of hs-CRP was associated with an atherogenic lipoprotein profile revealing a high cardio- vascular risk in long-term hemodialysis patients. As re- ported by Zimmermann and colleagues,30we found neg- ative and positive correlations between hs-CRP levels, ApoA-I, and both ApoB, and Lp(a) levels, respectively.
A significant increase in mortality has been noted in ESRD patients presenting with systemic inflammation (as evidenced by increased serum levels of CRP) compared with patients without systemic inflammation.31 Hence, serum CRP concentration is a strong predictor of overall and cardiovascular mortality in this disease.32
Furthermore, as demonstrated in several studies, ESRD patients present with decreased serum PON activity com- pared with healthy subjects,33–36 an effect that could be secondarily related to decreased serum levels of HDL-C, ApoA-I, LpA-I, and LpA-I:A-II in this population. We also found that PON activity correlates inversely with hs-CRP levels. PON activity seems to be remarkably lower in pa- tients with elevated hs-CRP (⬎10 mg/L). These results suggest that the HDL-C, ApoA-I, LpA-I, and LpA-I:A-II levels decrease during inflammation, leading to the ob- served reduction of PON activity.
Recently, it has been shown that PON activity, HDL-C, and ApoA-I levels were found to be significantly lower in long-term hemodialysis patients than in controls. The de- crease of PON/arylesterase activities could be related to reduced HDL-C and ApoA-I levels, as well as to increased urea and creatinine concentrations35 and reduction of HDL3, whereas involvement of a genetic PON1 polymor- phism appears to be excluded.37
Decreased PON/HDL and PON/ApoA-I ratios may also lead to reduction of antioxidant capacity of HDL.38 PON activity correlates positively with HDL-C and ApoA-I.39 Recently, Gugliucci and colleagues33 demonstrated that PON activity increases after hemodialysis related to the effectiveness of dialysis to clear creatinine and urea, and with clearance of advanced glycation end products adducts of low molecular weight. Such alteration in PON activity can be restored after renal transplantation.40
As reported in other studies, we showed hyperhomo- cysteinemia in ESRD patients.41– 43 Total homocysteine levels did not correlate with hs-CRP, PON activity, or albumin levels, but we found a correlation with age. The association between tHcy and risk for atherothrombotic disease is not a consistent finding in ESRD patients.
Several studies found an association between higher tHcy and cardiovascular events,44,45 whereas others showed that patients with higher tHcy levels had better survival rates.46,47 Furthermore, patients with lower tHcy levels had a significantly worse survival as a result of malnu- trition and hypoalbuminemia.9 Recently, Jamison and colleagues48found that treatment with high doses of folic acid and B vitamins, known for decreasing homocysteine levels, did not improve survival or reduce incidence of vascular disease in ESRD patients. Neither high nor low tHcy levels were associated with atherosclerotic indices 0
50 100 150 200 250
Age≤40 years Age>40 years
P ar ao xo n ase act iv ity (U /m l)
*
*p<0.05
Figure 3 Serum paraoxonase activity in the elderly pateints on long-term hemodialysis.
and cardiovascular events.49 Recently, Sjöberg and col- leagues50 reported that the reduced form of Hcy may be a more relevant marker of cardiovascular disease risk than tHcy level.
In conclusion, this comparative study of 109 patients with ESRD on long-term hemodialysis treatment and 100 normal volunteers found an abnormal lipoprotein profile associated with an activated acute-phase response as evi- denced by increased hs-CRP and total homocysteine serum levels in renal disease patients. Decreased PON activity was also documented and may be an additional factor contrib- uting to premature atherosclerosis. Inflammation, dyslipide- mia, and oxidant stress, probably linked to uremia, may be factors associated with increased cardiovascular risk in this population.
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