Serum paraoxonase activity is decreased in uremic patients

ELSEVIER

Clinica Chimica Acta 247 (1996) 71-80

Serum paraoxonase activity is decreased in uremic patients Renzo Schiavon *a, Emanuela De Fanti a, Davide Giavarina a, Stefano Biasioli b, Gaetano Cavalcanti b, Giancesare Guidi ~ aLaboratorio di Analisi Chimico Cliniche e Microbiologia, Ospedale di Legnago, via Gianella, 1, 37045 Legnago, Italy bServizio di Nefrologia ed Emodialisi, Ospedale di Legnago, Verona, Italy CLaboratorio di Analisi Chimico Cliniche e Microbiologia, Centro Ospedaliero Clinicizzato, Valeggio s.M., Verona, Italy

Received 10 July 1995; revision received 20 October 1995; accepted 4 November 1995

Abstract

Paraoxonase is a high-density lipoprotein (HDL)-associated enzyme capable of hydrolysing lipid peroxides. We measured the activity of serum paraoxonase together with serum concentrations of a variety of lipid constituents - - total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL), cholesterol, triglycerides, apolipoproteins A-I and B - in 60 bemodialyzed (HD) patients. We found that the paraoxonase activity was significantly reduced in HD patients compared with 64 healthy controls (mean median and interquartile values: 93, 63, 87 IU/I in HD patients and 151,120 and 135 IU/I in controls). In patients, the trimodal frequency of distribution of paraoxonase activity showed a shift toward lower levels. The effect of NaCI on enzyme activation was more pronounced in the patient group, as compared with controls, suggesting a higher frequency of the B allozyme (more responsive to NaCI) in this population. We suggest that altered HDL subfraction, present in HD patients, may be the main cause of the widespread depression of paraoxonase. Furthermore, the higher frequency of allozyme B among HD patients might increase the risk of coronary artery disease. In conclusion, paraoxonase activity may be an adjunctive index of altered lipoprotein metabolism with important repercussions on atherosclerosis. Keywords: Paraoxonase; Uremic patients; Haemodialysis; Lipoproteins; High density lipo-

proteins

* Corresponding author, Tel: +39-442-632262; Fax: +39-442-632705. 0009-8981/96/$15.00 © 1996 Elsevier Science B.V. All fights reserved S S D I 0009-8981 (95)06221-X

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1. Introduction

In patients with chronic renal failure (CRF) a decrease of HDL-C values in association with hypertriglyceridemia is the most common lipid abnormality encountered. Several enzymes are present on HDL which may be important for lipoprotein metabolism and their activity may be altered in disease conditions [1]. Paraoxonase is one of these enzymes which seems to play a role in reverse cholesterol transport [2] and in reducing LDL oxidation [3] and, consequently, atheroma generation through the uptake of oxidized LDL by macrophages. As C R F is associated with the lipoperoxidative process [4], the determination of the serum paraoxonase activity may be more informative than the simple determination of the HDL-C. Several studies have documented a decreased paraoxonase activity in some conditions associated with the development of premature atherosclerosis such as familial hypercholesterolemia, insulin-dependent diabetes mellitus [5], coronary artery disease [6]; similar conditions may also appear in their offspring [7]. On the basis of relative enzyme activities, two isotypes, A (low activity) and B (high activity), have been defined. The isotype difference has been linked to the presence of a single aminoacid polymorphism (Gln-Arg192) of the paraoxonase protein [8]. Recently, in a case control study, the B allele and AB+BB genotypes were associated with an increased risk of coronary heart disease in non-insulin dependent diabetic patients [9]. The present study aimed to investigate the activity of serum paraoxonase in uremic patients under long-term dialysis treatment (HD patients), a condition characterized by accelerated atherogenesis and derangement of lipoprotein metabolism [10]. 2. Materials and methods

2.1. Subjects HD patients: 60 uremic patients under long-term dialysis treatment, 29 males and 31 females, age (mean 4-S.D. and range, in parentheses) 56.0 4- 18.6 (17-93) and 64.9 4- 13.1 (29-82) years, respectively. Control group: 64 healthy blood donors, 38 males and 26 females, age 36.6 4- 10.6 (20-67) and 35.8 ± 12.6 (22-66) years, respectively. For paraoxonase only, the control group included 102 healthy individuals aged 41.2 ± 15.2 years (mean 4- S.D.): two different age groups were selected (30 4- 6, n = 56, and 55.1 4- 11.4 years, n = 46, respectively).

2.2. Blood Blood was withdrawn from the antecubital vein in the fasting state; all the

R. Schiavon et al. /Clinica Chimica Acta 247 (1996) 71-80

73

tests were performed on freshly separated serum. HD patients were evaluated before the first dialysis session of the week, i.e. after the longest interdialysis interval (3 days). Informed consent was obtained from both patients and blood donors. 2.3. Total and H D L cholesterol, triglycerides

Total cholesterol and triglyceride concentrations were assayed by Kodak Ektathem slides on a Kodak Ektachem 700XR Analyzer (Eastman Kodak, Rochester, NY, USA), employing enzymatic colorimetric methods. HDL cholesterol was measured by the enzymatic method on a Technicon RA-1000 autoanalyzer (Bayer, Cavenago Brianza, Milan, Italy) after precipitation of lower density lipoproteins with polyethylene glycol 6000. LDL-C was calculated according to Friedewald et al. [11]. Since triglyceride concentration never exceeded 4.5 mmol/1 (400 mg/dl), the latter formula could always be applied. Furthermore any correction for the cholesterol content of Lp(a) was omitted, as negligible interference resulted from the method of precipitation chosen [ 12]. 2.4. Apolipoprotein A-I and B assay

Apolipoprotein A-I (apo A-I) and apolipoprotein B (apo B) concentrations were measured by immunonephelometric methods on a Nephelometric Analyzer using specific antisera (Behring, Scoppito-L'Aquila, Italy) following the analysis protocol provided by the manufacturer. 2.5. Paraoxonase assay

Paraoxonase activity was assayed spectrophotometrically by the method described by Mackness [5] with minimal modifications. Briefly, the assay mixture consisted of 500 #1 of 2.22 mmol/1 paraoxon substrate solution in 0. I mol/l Tris-HCl buffer, pH 8.0, containing 2 mmol/1 CaCI2 and 50 #1 of fresh serum specimen. The absorbance was monitored photometrically at 405 nm and at 37°C on a spectrophotometer, Beckman DU-64 (Beckman Instruments, Fullerton CA, USA). A lower substrate concentration (2 mmol/l) was chosen to reduce the absorbance of the reagent blank, after determining a value of 1.2 mmol/l for the Kin. The specificity of the method was verified by comparing the enzyme activity of the serum with that of the supernatant after precipitation with PEG 6000, which is known to contain high-density lipoproteins (linear regression, serum vs. PEG: y = 1.09x- 16.8, r = 0.99 n = 12).

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R. Schiavon et al. / Clinica Chimica Acta 247 (1996) 71-80

2.6. Mixing experiments These experiments aimed to search for the presence of inhibitor(s) in the serum of HD patients. One pool of serum from four uremic patients showing low paraoxonase activity was mixed in various proportions (12.5, 25, 50, 75 and 100%) with one pool from eight healthy individuals with intermediate activity.

2. 7. Effect of dialysis on the paraoxonase activity In seven HD patients paraoxonase activity was measured immediately before and after the dialysis session.

2.8. Effect of sodium chloride on the paraoxonase activity The stimulation of the paraoxonase activity by salt was evaluated in 28 HD patients and in 26 healthy subjects by adding final concentrations of 0.5, 1 and 2 mol/1 NaC1 to the reaction mixture. The slope of the linear regression of activities vs. salt concentrations was determined.

2.9. Statistics The Wilcoxon test for non-parametric data was applied for evaluating paraoxonase and triglycerides, while Students' t-test was used for the other parameters. 3. Results

Table 1 reports data of all parameters from uremic patients and healthy individuals. In the HD patient group serum paraoxonase activity was significantly decreased (P < 0.001), while triglyceride levels were increased and those of HDL-C decreased without statistical significance. In Fig. 1 the histograms of the distribution of serum paraoxonase activity for both uremic patients (Fig. 1A) and healthy individuals (Fig. 1B) are shown. As expected, a trimodal pattern of distribution was found in both the groups, but a uniform shift towards lower levels, more evident in the percentile distribution (Fig. I C), was found in HD patients. The same effect was seen when data were expressed as the ratios of serum paraoxonase activity with HDL-C concentration, although the difference was slightly less marked (Fig. 1D). In healthy individuals the enzyme activity did not change statistically with age (Table 2) and paraoxonase was significantly decreased in HD patients compared with all age groups. By diluting the uremic serum

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with normal serum we aimed to reduce hypothetical inhibitors present in uremic serum; this procedure did not change the reaction rate beyond what was to be expected on a proportional basis (Fig. 2), thus excluding the presence of non-competitive inhibitors. The hemodialysis treatment induced a slight increase in paraoxonase activity (Table 3), which paralleled that of the other parameters, probably as a consequence of water loss: in fact, the

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R. Schiavon et al./ Clinica Chimica Acta 247 (1996) 71-80

Table 2 Paraoxonase activity in groups of healthy individuals of different ages Group I

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amount of the increase was very similar for all the considered parameters (range 19-29%), both lipids and proteins (total protein included), without evidence of statistical difference between them. Finally, the paraoxonase activity was stimulated by NaCI both in uremic patients and controls. The stimulation was positively correlated with the activity without salt and was higher in HD patients compared with healthy controls (Fig. 3).

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R. Schiavon et al. / Clinica Chimica Acta 247 (1996) 71-80

Table 3 Data collected from 20 uremic patients before and soon after a dialysis session Parameter

Unit

Before

After

A/B ratio

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IU/I retool/1 mmol/1 mmol/l go gO gO

72.5 (11.9) 0.53 (0.32) 0.51 (0.21) 1.12 (0.05) 1.37 (0.05) 1.09 (0.09) 69.1 (2.20)

85.9 (13.2) 5.51 (0.38) 1.88 (0.25) 1.35 (0.07) 1.68 (0.11) 1.3"1 (0.11) 79.7 (4.00)

1.25 (0.09) 1.23 (0.05) 1.29 (0.09) 1.21 (0.05) 1.21 (0.05) 1.20 (0.04) 1.19 (0.06)

The entity of variation is expressed by the ratio of values after and before dialysis (A/B ratio). Mean values and standard errors (in parentheses) are reported. Dialysis induced a statistically significant increase in all the parameters (P < 0.01), while A/B ratio did not differ statistically (Wilcoxon rank sum test).

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Fig. 3. The stimulation of the paraoxonase activity by NaCI. For each individual the slope (regression line of activities vs. 0.5, 1 and 2 mol/l NaCI) is plotted against the activity without NaCI addition. A positive correlation was found and the stimulation was higher in HD patients compared with healthy controls. O, HD patients: linear regression: y = 2.25x - 30.23; r = 0.96. II, Healthy subjects: linear regression: y = 1.29x - 21.78; r = 0.97.

R. $chiavon et aL / Clinica Chimica Acta 247 (1996) 71-80

79

4. Discussion In this study we observed that the activity of paraoxonase is decreased in HD patients and this is the only abnormality found among a series of lipid risk factors investigated (Table 1). In fact, triglycerides and HDL-C showed only a trend towards increased or decreased levels, respectively. Serum paxaoxonase activity was not correlated with either HDL-C or apo A-I levels (data not shown) either in HD patients or in controls. Age was not responsible for the reduced enzyme activity in HD patients (Table 2). As expected, the paraoxonase activity showed a trimodal frequency of distribution. Among the possible causes for the widespread depression of the paraoxonase in HD patients (Fig. 1), altered HDL subfraction distribution should be considered. HDL-C/apo A-I ratio tended to be reduced in HD patients, though not to a degree that was statistically significant (Table 1). This is in accordance with the pattern of lower HDL2 and normal HDL3 subfraction levels already described [10]. It has been demonstrated that with conversion of HDL3 to HDL2, the specific activity of paraoxonase increases [13]. Thus the reduction of HDL2 levels may account for the reduced paraoxonase activity in HD patients. However, other causes might be a reduced gene expression for the enzyme or the presence of an inhibitor (not confirmed by in vitro mixing experiments) (Fig. 2). The decreased paraoxonase activity in uremic patients may have some implications in the atherosclerotic process, as already demonstrated for patients with familial hypercholesterolemia, insulin dependent diabetes mellitus, coronary artery disease [5-6]. In fact, a decreased paraoxonase activity might lead to a reduction of the mechanism of reverse transport of cholesterol and of the defence against LDL oxidation [2-3]. Although paraoxonase activity was depressed in HD patients the effect of NaC1 on enzyme activation was more pronounced in the patient group as compared with controls (Fig. 3). This may be in accordance with a higher frequency of the B allozyme in the patient group as the latter shows a greater degree of stimulation of its activity by NaCI than does the A allozyme [14]. As the B allozyme was associated with an increased risk of coronary heart disease in type 2 diabetes [9] the depression of B allozyme activity may make it an even more critical factor in atherosclerosis [15]. Although the effect of NaC1 on the enzyme activity is only an indirect approach to define the allozyme and the sample of HD patients was rather small, one may suppose that individuals with the B allozyme, which does not protect well against lipid oxidation, might also be more prone to develop an accelerated kidney disease [16]. Although prospective studies are needed before we can draw any conclusions, we suggest considering the assay of paraoxonase activity as an adjunctive index of altered lipoprotein metabolism. We think that the activity of this enzyme might also be utilized in monitoring the progression of the disease.

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