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Department of Internal Medicine 3 (S.K., J.G.) and Institute of Medical Informatics and Biometry (E.K.), Carl Gustav Carus Medical School, University of Technology Dresden, Dresden D-01307; and Institute of Bioinorganic and Radiopharmaceutical Chemistry (J.P.), Research Centre Rossendorf, Dresden D-01314, Germany
Address all correspondence and requests for reprints to: Dr. Steffi Kopprasch, University of Technology Dresden, Carl Gustav Carus Medical School, Department of Internal Medicine 3, Pathological Biochemistry, Fetscherstrasse 74, D-01307 Dresden, Germany. E-mail: Steffi.Kopprasch{at}mailbox.tu-dresden.de.
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Abstract |
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Introduction |
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Decreased levels of PON1 activity have been found in patients with overt DM in several studies (6, 7, 8), and they have been causally related to diabetic microvascular and macrovascular complications. The PON1 status in an early disease state has not been evaluated yet. The aim of the present study was to determine serum PON1 activities in IGT subjects and patients with newly diagnosed DM and to relate them to levels of circulating oxLDL.
The PON1 gene polymorphisms at position 55 (methionine/leucine) and at position 192 (glutamine/arginine, Q/R) have been inconsistently associated with vascular disease. Several studies showed a positive association between the PON1 R allele and the risk of coronary heart disease. In the present study, we investigated whether the PON1 Q/R phenotype predicted the level of oxLDL.
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Subjects and Methods |
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The study comprised 603 Caucasian subjects who were attending the RIAD (Risk Factors in Impaired Glucose Tolerance for Atherosclerosis and Diabetes) study. In brief, middle-aged subjects (40–70 yr) who were at risk for development of diabetes owing to a family history of DM, obesity, and/or hyper/dyslipoproteinemia were examined. Clinically overt diabetes and medication affecting glucose tolerance were exclusion criteria. The study was conducted in accordance with the guidelines proposed in the Declaration of Helsinki. All subjects consented to participate in the study, which was approved by the local Ethics Committee.
The diagnosis of IGT and DM was confirmed by an oral glucose tolerance test (oGTT; 75 g oral glucose challenge) according to World Health Organization guidelines and criteria (9): 2 h post-challenge plasma glucose concentration between 7.8–11.1 mmol/liter for IGT, and more than 11.1 mmol/liter for DM. According to oGTT criteria, individuals were grouped into subjects with normal glucose tolerance (NGT, n = 403), subjects with IGT (n = 125), and patients with newly diagnosed DM (n = 75). Several follow-up studies documented that persons with IGT are at higher risk to develop DM than NGT subjects (10, 11). Moreover, therapies including change in lifestyle aimed to improve glucose tolerance in subjects with IGT were able to reduce the incidence of DM (12). Therefore, IGT subjects in the present study are described as a prediabetic group, independently of the fact that not all of these IGT subjects consequently will develop overt DM. Subjects included into the DM group were diagnosed by their first pathological oGTT. Notably, they had no clinical symptoms of diabetes mellitus.
Baseline clinical and biochemical characteristics of the study groups are given in Table 1
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Blood samples were centrifuged within 20 min at 4 C. Serum, citrate, and EDTA plasma was separated from cells immediately after centrifugation and stored at -80 C until analyzed.
Plasma glucose was measured by the hexokinase method (Roche Molecular Biochemicals, Mannheim, Germany) and HbA1c by HPLC on a Diamat analyzer (Bio-Rad Laboratories, Inc., Hercules, CA). Plasma triglycerides, total cholesterol, and HDL cholesterol were measured as previously described (13). LDL cholesterol was calculated using the Friedewald formula. For determination of insulin and C-peptide aliquots were shock-frozen in liquid nitrogen and stored in cryovials at -80 C. Plasma insulin was assayed by specific enzyme immunoassay (Biosource Technologies Europe, Brussels, Belgium; within-day precision less than 3.8%, between-day precision less than 6.5%; no cross-reaction with human proinsulin and insulin, respectively). C-peptide was measured by specific enzyme immunoassay (Biosource Technologies Europe; within-day precision less than 3.5%, between-day precision less than 6.0%; no cross-reaction with human proinsulin and insulin).
oxLDL
Serum levels of total oxLDL particles were directly measured by a sandwich ELISA assay (Mercodia AB, Uppsala, Sweden; within-day precision less than 3.5%, between-day precision less than 6.5%) based on monoclonal antibody mAB-4E6 used by Holvoet et al. (14).
Paraoxonase activity and phenotyping
Serum PON1 activity was measured spectrophotometrically using three synthetic substrates: paraoxon, phenylacetate, and 4-nitrophenylacetate essentially as described by Dantoine (15). PON1 activities are expressed as µkat/liter. To determine individual phenotypes, the dual substrate method was applied (16). The ratio of the activity with paraoxon to the activity with phenylacetate was used to assign individuals to one of the three possible phenotypes QQ, QR, and RR. The phenotyping of PON1 by the dual substrate predicts the genotype of the enzyme with high accuracy (17).
Statistical analysis
All data are expressed as means ± SD or means ± SEM. Phenotype distribution and gene frequency were analyzed by 
2 test. The means of the three groups NGT, IGT, and DM were compared by one-way ANOVA. The confounding effect of HDL on PON activities was assessed by ANOVA with HDL as covariate. ANOVA was coupled with a post hoc Bonferroni correction analysis when appropriate. Spearman’s rank correlation coefficients (
) were used to express relation between PON activities and oxLDL levels. The effect of the R-carriers and study groups on oxLDL levels was evaluated by univariate variance analysis.
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Results |
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. Considering the age of the NGT group (57 ± 8 yr), these subjects were normoglycemic, normolipidemic, and only mildly obese. As compared with NGT individuals, IGT subjects and patients with newly diagnosed DM were significantly older, had a higher systolic blood pressure, and were significantly more obese. IGT and DM subjects had significant higher levels of glycosylated hemoglobin, insulin, C-peptide, and HOMA (homeostasis model assessment) indices. The HOMA index is an accepted marker for individual insulin resistance and was calculated by the formula: fasting plasma glucose (millimoles/liter) x fasting plasma insulin (picomoles/liter)/154.8. HDL cholesterol levels were lower in IGT subjects and significantly decreased in the DM group. Total cholesterol and LDL cholesterol did not differ significantly between the groups.
Table 2 shows the PON1 activities and phenotype distribution in the three groups. Because the physiological substrate of PON1 in humans is not known yet, it may be not accurate to estimate PON1 activity using exclusively paraoxon as substrate. Therefore, we determined serum PON1 activity using three different model substrates. The results show that PON1 activity was not different in NGT, IGT, and newly diagnosed DM subjects using either paraoxon, phenylacetate, or p-nitrophenylacetate as substrate.
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the individual activities toward paraoxon and phenylacetate are presented. The plot shows three groups of individuals corresponding to the three paraoxonase phenotypes QQ, QR, and RR.
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). In DM patients, the heterozygous QR phenotype dominated with 58.7%. There was no significant difference in the phenotype distribution between the groups. The allele frequency differed between the NGT and DM group being Q = 0.75, R = 0.25 in NGT and Q = 0.68 and R = 0.32 in DM. However, the differences were not statistically significant (2 test: 3.24, P = 0.198) The gene frequency data of NGT and DM subjects in the present study obtained by phenotyping of the enzyme were in excellent accordance with results of PON1 genotyping in a control and DM group of Caucasians in a study by Mackness et al. (7).
In Fig. 2 variations in PON1 activities between NGT, IGT, and DM subjects stratified by individual PON1 192 phenotype are illustrated. As assessed by Bonferroni-adjusted P values, no significant differences between NGT, IGT, and DM subjects within one phenotype were found.
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). No correlations could be found between serum PON1 activities and circulating levels of oxLDL in either study group (see Table 3). There was no tendency of carriers of the R allele in either group to have increased levels of oxLDL. As estimated by ANOVA, no specific effect of the R allele on oxLDL levels in all study groups could be found.
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Discussion |
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In the present study, PON1 activities were measured exactly as described by Dantoine et al. (15). Dantoine measured PON1 activities in 185 apparently healthy subjects. Their data are in very good accordance with our not HDL-adjusted results in NGT subjects (paraoxon: 3.67 ± 2.83 µkat/liter/Dantoine vs. 4.67 ± 3.83 µkat/liter/present study; phenylacetate: 1667 ± 550 µkat/liter vs. 1650 ± 467 µkat/liter; p-nitrophenyl-acetate: 31.7 ± 15.0 µkat/liter vs. 46.7 ± 13.3 µkat/liter). As a result of this comparison, it is unlikely that in the present study PON1 activities were already diminished in the high risk NGT group. From the assessment of PON1 activities in the present investigation, two major results were obtained: 1) serum PON1 activity is not altered in IGT and early DM when compared with NGT; and 2) serum PON1 activity variations and phenotype are not correlated with the levels of oxLDL in the circulation. From similar PON1 activities in NGT, IGT, and newly diagnosed DM, we conclude that PON1 activity loss is not an early event, but develops in later states of diabetes mellitus.
Several DM-associated explanations of changes of PON1 activity are possible. First, conformational changes of the enzyme as a result of glycoxidation and/or lipoxidation processes (20) could alter its activity. As indication, in vitro glycation of purified paraoxonase protein by incubation in 25 mmol/liter glucose for 1 wk can cause a 40% reduction in enzymatic activity (21).
Second, changes in physicochemical properties of the HDL particle or in HDL metabolism could influence PON1 activity (22). Recently, Deakin et al. (23) found that size reduction and accumulation of unesterified cholesterol in the HDL particle may impair the capacity of HDL to facilitate PON1 release from cells and to stabilize the enzyme. In IGT and DM, composition of HDL is substantially altered (24, 25). This is followed by conformational changes in HDL particles that dramatically favors their catabolism (24, 25). There is evidence that alterations in HDL composition change binding properties of apolipoprotein A-I to the lipoprotein particle (25). It can be speculated that conformationally altered HDL also influences both binding of PON1 as well as PON1 metabolism.
Third, hormone and glucose homeostasis could affect PON1 activities. In a nondiabetic Japanese population, a positive association between HOMA index, an surrogate marker of insulin resistance, and paraoxonase activity has been found (26). Furthermore, short-term decreases of PON1 activity occur during acute hyperglycemia. In a previous study, we could show that during an oGTT PON1 activity significantly declines by 10–5% in both NGT and DM subjects (27).
Several studies reported a positive association between the PON1 gene polymorphism at codon 192 and the risk of coronary heart disease. A meta-analysis of 18 case-control studies investigating subjects with different diseases revealed a statistically significant overall association between the PON1-192R allele and the presence of coronary heart disease (28). This association was confirmed in several studies with DM patients (29, 30, 31, 32). In contrast, Cao et al. (33) did not found an association between carotid intima-media thickness as a surrogate parameter of macroangiopathy and PON1-192 gene polymorphism. In the present study, DM patients had a higher R allele frequency as compared with IGT and NGT subjects. However, the difference was not statistically significant.
The functional characteristics of the R allozyme that could be responsible for more frequent vascular disease are far from being fully understood. Regarding this, the main hypothesis is based on in vitro investigations showing that the R allele is less effective in protecting LDL against oxidative modification than the Q allele (34, 35). These investigations were performed by oxidizing LDL with copper. However, copper ions are very unlikely physiological candidates for LDL oxidation in vivo (36). Moreover, Aviram et al. (35) showed in his experiments that the effects of PON Q and PON R in preventing copper-induced LDL oxidation strongly depended on the experimental setup. If, for example, the enzymes were added simultaneously to LDL and the oxidant, PON Q had a greater protective effect than PON R. The opposite effect was observed when the allozymes were added 1.5 h after the initiation of LDL oxidation. In this case, PON R was more protective than PON Q. The authors concluded from their study that PON R and PON Q might contribute in different ways, even synergistically, to reduce LDL oxidation.
In the present investigation, IGT and DM subjects had significantly higher levels of oxLDL than NGT subjects. However, the levels of oxLDL did not correlate with serum PON1 activities. Moreover, there was no tendency of R carriers to have increased levels of circulating oxLDL. In this respect, several points could be discussed. First, PON1 is not the only antioxidant in HDL and, moreover, in the blood. Other components may have a greater impact in preventing LDL oxidation or destroying specific oxidized lipoprotein constituents in the circulation. Second, the onset of vascular complications in early states of diabetes mellitus may not necessarily be caused by a decrease in circulating PON1 activity. Third, a decrease in PON1 activity in overt diabetes mellitus may not be causally related to late diabetic complications. It develops as a result of disease-associated metabolic and hormonal changes in the circulation without effect on the integrity of lipoproteins in vivo.
In conclusion, our study does not support a causal relationship between circulating PON1 activities and oxidative modification of circulating LDL in IGT and newly diag-nosed DM.
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Acknowledgments |
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Footnotes |
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Abbreviations: DM, Diabetes mellitus type 2; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; IGT, impaired glucose tolerance; LDL, low-density lipoprotein; NGT, normal glucose tolerance; oxLDL, oxidized LDL; oGTT, oral glucose tolerance test; PON1, paraoxonase 1; Q/R, glutamine/arginine.
Received October 7, 2002.
Accepted January 3, 2003.
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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, RR phenotype; 
, QR phenotype; 
, QQ phenotype.