Serum paraoxonase-3 concentration in HIV-infected patients. Evidence for a protective role against oxidation.

We investigated the influence of the HIV infection on serum paraoxonase-3 (PON3) concentration and assessed the relationships with lipoprotein-associated abnormalities, immunological response, and accelerated atherosclerosis. We studied 207 HIV-infected patients and 385 healthy volunteers. Serum PON3 was determined by in-house ELISA, and PON3 distribution in lipoproteins was investigated by fast-performance liquid chromatography (FPLC). Polymorphisms of the PON3 promoter were analyzed by the Iplex Gold MassArray(TM) method. PON3 concentrations were increased (about three times) in HIV-infected patients with respect to controls (P < 0.001) and were inversely correlated with oxidized LDL levels (P = 0.038). Long-term use of nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy was associated with a decrease of PON3 concentrations. In a multivariate linear regression analysis, these relationships were still strong when the main confounding covariates were considered. PON3 was mainly found in HDL in HIV-infected patients, but a substantial amount of the protein was detected in LDL particles. This study reports for the first time an important increase in serum PON3 concentrations in HIV-infected patients that is associated with their oxidative status and their treatment with NNRTI. Long-term, prospective studies are needed to confirm the possible influence of this enzyme on the course of this disease and its possible utility as an analytical biomarker.

of renal insuffi ciency, liver damage, neoplasia, or neurological disorders.
A fasting venous blood sample was obtained from all the participants. CD4+ T cells and CD8+ T cells were analyzed immediately, and serum, plasma, and leukocytes were stored at Ϫ 80°C in our biological sample bank until the other measurements were performed. We employed independent aliquots that were never thawed before this investigation, although participants in this study partially coincided with those reported in previous investigations ( 5,6 ). All the participants provided fully informed consent to participation in the study on the understanding that anonymity of all data is guaranteed. The study was approved by the Institutional Review Board of the Hospital Universitari de Sant Joan de Reus.

Biochemical and serological measurements
Serum PON3 concentrations were determined by in-house ELISA using rabbit polyclonal antibodies generated against a synthetic peptide with a sequence specifi c to mature PON3. Details of this method have been previously reported ( 18,22 ). Plasma viral load was measured with the COBAS ® TaqMan ® HIV-1 assay (Roche, Basel, Switzerland), and CD4+ T-cell and CD8+ T-cell counts were measured by fl ow cytometry (Coulter Epics XL-MLC, Beckman Coulter, Fullerton, CA). Antibodies against HCV, serum ␤ -2-microglobulin [a marker of lymphocyte destruction and progression of HIV-infection ( 23 )], and serum cholesterol, triglycerides, HDL-cholesterol, and apolipoprotein (apo)A-I were measured in an automated analyzer (UniCel TM DxI 800, Beckman Coulter, Fullerton, CA). Oxidized LDL levels were measured by ELISA (Mercodia, Uppsala, Sweden).

FPLC lipoprotein fractionation
PON3 distribution in lipoproteins was assessed by FPLC (Bio-Rad BioLogic DuoFlow 10 system, Bio-Rad Laboratories, Hercules, CA). Sera from three HIV-infected patients and three noninfected participants were pooled separately. To maximize the possible differences between groups, sera from the HIV-infected patients were chosen to have a PON3 concentration > 20 mg/l. Two-hundred microliters from each pool were injected into a Superose 6/300 GL column (GE Healthcare Europe, Glattbrugg, Switzerland), and 500 l fractions were collected. Cholesterol, triglycerides, and PON3 in each fraction were measured as described.

PON3 promoter genotyping
Genomic DNA was obtained from leukocytes (Puregene DNA Isolation reagent set, Gentra Systems, Minneapolis, MN). Selected single nucleotide polymorphisms (SNP) of the PON3 promoter were analyzed by the Iplex Gold MassArray TM method (Sequenom, San Diego, CA) at the Spanish National Genotyping Center (Centro Nacional de Genotipado, Universitat Pompeu Fabra, Barcelona, Spain).

Statistical analysis
The normality of distributions was determined with the Kolmogorov-Smirnov test. Differences between two groups were assessed with the Student t -test (parametric) or the Mann-Whitney U test (nonparametric). Differences between multiple groups were analyzed by the Kruskal-Wallis test. Pearson or Spearman correlation coeffi cients were used to evaluate the degree of association between variables. Each SNP was tested for Hardy-Weinberg equilibrium using Haploview 4.0 software ( 24 ). Estimates of linkage disequilibrium between SNPs were calculated using Fisher's test. Diagnostic accuracy for the measurement of serum PON3 concentration was calculated with ROC analysis ( 25 ). A multiple lipoprotein (LDL) oxidation and cellular oxidative stress ( 11 ). In addition, data obtained from a variety of mouse models of atherosclerosis have consistently shown that human PON1, 2, or 3 expression inhibits or reverses the development of atherosclerosis via mechanisms involving the reduction of oxidative stress, the promotion of cholesterol effl ux from macrophages, and the normalization of vascular endothelium function (12)(13)(14)(15). Moreover, recent studies showed that PON2 expression is increased in cultured hematopoietic cells and mouse thymocytes after HIV-1 infection ( 16 ). The PON family also plays a role in innate immunity and can prevent bacterial infection ( 17 ).
Although knowledge on PON1 and PON2 structure and function is rapidly expanding, data about the PON3 protein remain elusive. Its gene was identifi ed in 1996 when Primo-Parmo et al. ( 7 ) detected a large number of cDNA sequences in the Genome Data Base with signifi cant similarity to, but not identical with, human PON1. The percentage identity among human PON1, PON2, and PON3 genes is high (about 70%), and the genes are believed to derive from a common precursor ( 11 ). Clinical research on PON3 has been hampered by the lack of methods for measurement, but we recently described a high-throughput, reliable enzyme-linked immunosorbent assay (ELISA) to analyze PON3 concentration in human serum ( 18 ). The main objective of the present study was to investigate whether serum PON3 concentration may provide new information to improve our understanding of metabolic complications associated with HIV infection.

Study participants
From among the HIV-infected patients attending our clinic, 207 (139 men, 68 women; mean age, 38 years; range, 22-66 years) accepted an invitation to participate in the present study. Of these patients, 122 were coinfected by the hepatitis C virus (HCV). All patients were undergoing antiretroviral therapy with protease inhibitor (PI)-based or nonnucleoside reverse transcriptase inhibitor (NNRTI)-based schemes. The antiretroviral adjuvant drugs were zidovudine, stavudine, didanosine, or lamivudine. The exclusion criteria were age under 18 years, or renal function impairment defi ned as creatinine levels higher than 106 mol/l, or having an AIDS-related opportunistic disease at the time of the study. Twenty-fi ve patients had subcutaneous lipoatrophy, defi ned as the presence of hollow cheeks, prominent superfi cial veins in the limbs, or fl attening of the buttocks ( 19 ). Carotid and femoral ultrasound measurements were performed in 178 patients and the intima-media thickness (IMT) was measured as an estimate of the presence of subclinical atherosclerosis, as previously described ( 20 ). Patients were considered to have subclinical atherosclerosis when IMT was у 0.8 mm or when an atheromatous plaque was seen in the analyzed areas of the arteries. The main clinical characteristics of these patients are summarized in supplementary Table I. The control group consisted of 385 healthy volunteers (153 men, 232 women; mean age, 47 years; range, 19-75 years) who participated in an ongoing epidemiological study conducted in our geographic area, the details of which have been previously reported ( 21 ). All the volunteers had been invited to attend a clinical examination and to provide a fasting blood sample. There was no clinical or analytical evidence there were no signifi cant associations among serum PON3 concentrations, cholesterol, and triglycerides (supplementary Table II).

FPLC lipoprotein fractionation
In noninfected participants, PON3 immunoreactivity was observed almost exclusively in HDL fractions. However, in the HIV-infected pool, a substantial amount of this protein eluted with the smallest HDL and with LDL particles ( Fig. 2 ).

Infl uence of genotype on serum PON3 concentrations
The frequency distributions of the selected PON3 promoter gene polymorphisms are shown in Table 2 . There were no signifi cant differences between control subjects and HIV-infected patients. These polymorphisms moderately infl uenced serum PON3 concentrations in the control subjects but not in the patient group.
All PON3 promoter polymorphisms were strongly linked in a single haplotype, and we did not observe any significant differences between patients and controls (supplementary Fig. I).

Relationships among serum PON3 concentrations and the immunological and virological outcomes
Coinfection with HCV was associated with a signifi cantly higher PON3 concentration [5.8 (2.6-11.1) versus 4.5 (2.4-11.6) mg/l, respectively; P = 0.024]. There were not any signifi cant associations between serum PON3 concentrations and CD4+ T-cell and CD8+ T-cell counts, the CD4+/ CD8+ ratio, or the plasma HIV-1 viral load (supplementary Table III). There was a signifi cant direct linear relationship ( r = 0.397; P < 0.001) between serum PON3 and ␤ -2microglobulin concentrations ( Fig. 3A ). There were no signifi cant differences in serum PON3 concentrations between HIV-infected patients with or without lipoatrophy

Infl uence of treatments on serum PON3 concentrations
We observed a signifi cant inverse relationship between serum PON3 concentrations and the duration of the antiretroviral therapy in patients under the NNRTI-based scheme ( r = Ϫ 0.250; P = 0.035; Fig. 3B ) but not in patients receiving a PI-based scheme ( r = Ϫ 0.059; P = 0.408). linear regression model was fi tted to evaluate the factors that were independently associated with PON3 concentrations in HIV-infected patients. Results are shown as means and SD (parametric) or as medians and 95% confi dence interval (CI; nonparametric). The SPSS 18.0 package was used for all statistical calculations.

Relationships among serum PON3 concentrations and lipoprotein abnormalities
Serum PON3 concentrations were signifi cantly increased in HIV-infected patients with respect to the control group [5.5 (1.2-10.8) versus 1.8 (1.0-2.5) mg/l, respectively; P < 0.001; Fig. 1A ]. The results of the ROC analysis for serum PON3 concentration measurement are shown in Fig. 1B . The area under the curve (AUC) was 0.94 (95% CI: 0.92-0.97; P < 0.001), which highlights the remarkable differences in serum PON3 concentrations between patients and controls. We observed a signifi cant inverse relationship ( r = Ϫ 0.147; P = 0.038) between serum PON3 concentration and oxidized LDL levels ( Fig. 1C ) in HIV-infected patients but not in the control group ( r = 0.024; P = 0.786). HIV-infected patients were characterized by raised serum triglyceride concentration and decreased cholesterol values in HDL and LDL ( Table 1 );  Results are presented as means and SD in parentheses (parametric) or as medians and 95% CI in parenthesis (nonparametric). tions with oxidized LDL and with the use of NNRTI as the backbone of the antiretroviral therapy ( Table 3 ) DISCUSSION Viral replication and some clinical manifestations of HIV infection involve a misbalance in reduction-oxidation (redox) status and free radical production ( 26 ). Moreover, oxidative stress may be induced by antiretroviral

Multivariate analysis for PON3 concentration in HIV-infected patients
A multiple linear regression analysis showed signifi cant and independent relationships of serum PON3 concentra- to a higher cellular expression is, at present, unknown. However, a recent study reported a similar increase (fi ve times) in Pon3 mRNA expression in late gestation, a physiological state with high oxidative stress ( 33 ). Contrary to what we previously observed for PON1 in HIV-1 infection ( 5 ), serum PON3 concentrations were not signifi cantly related with markers of lymphocyte recovery. We did not observe any association with CD4+ T-cell counts, and the correlation observed with ␤ -2 microglobulin in the bivariate analysis disappeared in the multiple regression analysis. An interesting observation was the association of HCV coinfection with higher PON3 concentrations. These results confi rm a recent report from our group describing increased serum PON3 concentrations in patients with chronic liver disease, mainly secondary to HCV infection ( 34 ).
In the present study, treatment of HIV-infected patients with NNRTI, but not with PI, was associated with a significant decrease of serum PON3 concentrations. The use of some nucleoside reverse transcriptase inhibitors (NRTI) and PIs is associated with an atherogenic lipoprotein profi le ( 35 ), but NNRTIs, such as efavirenz, promote antiatherogenic changes in HDL particles and function, including normalization of size and lipid composition, enhancement of reverse cholesterol transport, and improvement of antioxidant capacity ( 36 ). A previous study from our group showed that NNRTI treatment was associated with a relative normalization of serum apoA-I and apoA-II, as well as HDL-cholesterol concentrations, in HIV-infected patients ( 37 ), which is consistent with the reported beneficial effects of this compound on HDL composition and function. However, an alternative explanation is that the infl uence of NNRTI fades with time. Similar effects have been described for some antiretroviral drugs. Shlay et al. ( 38 ) reported that NRTI are associated with positive changes in subcutaneous tissue distribution during the early periods and negative changes in the late periods of treatment.
We did not fi nd any signifi cant differences in genotype or haplotype of PON3 promoter gene polymorphisms between patients and controls, suggesting that genotype does not infl uence the course of the disease. In our previous report, we observed a moderate infl uence of some polymorphisms on serum PON3 concentration in the general treatments ( 27 ). PON3 is an enzyme with lactonase activity ( 28 ), the physiological function of which is not completely understood, but evidence suggests that it has an antioxidant role by hydrolyzing oxidized lipid peroxides, similar to PON1 and PON2. Purifi ed human and rabbit PON3 and recombinant PON3 have been shown to decrease macrophage oxidative stress and inhibit the in vitro oxidation of LDL (29)(30)(31)(32). The present study revealed a remarkable increase (about three times) in serum PON3 concentration in HIV-infected patients that may be clinically relevant. ROC analysis showed an AUC very close to 1.0, demonstrating a high sensitivity and specifi city of serum PON3 measurement in distinguishing between HIVinfected and noninfected subjects. Interestingly, oxidized LDL levels were not signifi cantly increased, but a significant inverse relationship was observed between their serum levels and those of PON3. These data support the concept that PON3 plays a protective role against oxidative stress and increased lipid peroxidation in HIV infection. Whether this increase in circulating PON3 is related  population ( 18 ), but this is not the case in HIV-infected patients. Possibly, the upregulation of PON3 expression secondary to the infection masks the small effect of these polymorphisms. Unlike PON1 ( 6 ), PON3 seems not to be associated with the presence of subclinical atherosclerosis in HIV-infected patients. Although lipid peroxidation and atherosclerosis are known to be strongly linked phenomena ( 39 ), the pathophysiology of atherosclerosis is complex, and our results suggest that the protective effects of these enzymes differ under certain situations. Perhaps PON1 is more effi cient in protecting against the alterations leading to atherosclerosis and PON3 is in some way involved in protection against infection. This is, to the best of our knowledge, the fi rst in vivo evidence suggesting such a hypothesis, and it warrants further investigation. Another interesting point is the wider lipoprotein distribution of PON3 in HIV-infected patients. PON3 is observed in substantial amounts in the smallest HDL and in LDL particles. The accepted concept to-date is that PON1 and PON3 are exclusively transported in the bloodstream by HDL ( 12 ) and are only associated with other lipoproteins in exceptional circumstances ( 40 ). Perhaps the excess PON3 produced in HIV infection cannot be properly packed in the HDL particles, as the conformation of HDLassociated apoA-I leaves little free surface area for other proteins to bind ( 41 ). This may have resulted in some PON3 redistributing to LDL. The physiological implications of this observation require further investigation.
In conclusion, this study reports for the fi rst time an important increase in serum PON3 concentrations in HIVinfected patients that is associated with their oxidative status and that could be partially attenuated by treatment with some antiretroviral agents. Long-term, prospective studies are needed to further confi rm the possible infl uence of this enzyme on the course of this disease and its possible utility as an analytical biomarker.