Proteomic and lipidomic analyses of paraoxonase defined high density lipoprotein particles: Association of paraoxonase with the anti-coagulant, protein S.

PURPOSE: Characterizing high density lipoprotein (HDL) particles and their relevance to HDL function is a major research objective. One aim is to identify functionally distinct particles. To try to limit both functional and compositional heterogeneity the present study focused on paraoxonase-1 (PON1) as a target for isolation of a minor HDL subfraction. EXPERIMENTAL DESIGN: Immunoaffinity techniques were applied to isolate PON1-containing HDL (P-HDL) and total HDL (T-HDL), which were subsequently characterized and compared. RESULTS: Analyses of the lipidomes showed significant differences between the fractions in the relative concentrations of individual lipid subspecies, notably reduced levels of unsaturated lysophosphatidylcholine (p < 0.05) in P-HDL (reflected in a significantly reduced total lysophosphatidylcholine polyunsaturated fatty acid content, p < 0.004). Significant differences were also observed for the proteomes. P-HDL was highly enriched in the anti-coagulant, vitamin K activated protein S (prot S) (p < 0.0001), and alpha2 macroglobulin (p < 0.01), compared to T-HDL. Conversely, procoagulant proteins kininogen 1 and histidine-rich glycoprotein were largely excluded from P-HDL. Immunoabsorption of PON1 from plasma significantly reduced prot S anti-coagulant activity. CONCLUSIONS AND CLINICAL RELEVANCE: The P-HDL lipidome and proteome showed significant differences from T-HDL. Enrichment in anti-coagulation proteins indicates complementary functionalities within P-HDL particles and underlines their anti-atherosclerotic potential.

HDL subfraction distribution of paraoxonase-1 and its relevance to enzyme activity and resistance to oxidative stress.

The subfraction distribution of HDL-associated peptides has implications for their functions and the impact of pathological modifications to lipoprotein metabolism on these functions. We have analyzed the subfraction distribution of paraoxonase-1 (PON1) and the consequences for enzyme activity and stability. HDL subfractions were defined by the presence (LpA-I,A-II) or absence (LpA-I) of apolipoprotein A-II (apoA-II). PON1 was present in both subfractions, although increased concentrations of HDL were associated with significantly increased PON1 in LpA-I. ApoA-II did not modify the capacity of native human HDL or reconstituted HDL to promote PON1 secretion from cells or to stabilize enzyme activity, nor did apoA-II decrease PON1 activity when added to rabbit serum normally devoid of the apolipoprotein. LpA-I,A-II particles isolated from human serum or reconstituted HDL (LpA-I,A-II) showed a significantly greater capacity than HDL(LpA-I) to stabilize secreted PON1 and purified recombinant PON1 added to such particles. PON1 associated with apoA-II-containing particles showed greater resistance to inactivation arising from oxidation. ApoA-I, apoA-II, and LpA-I,A-II, but not LpA-I, were independent determinants of serum PON1 concentration and activity in multivariate analyses. PON1 is at least equally distributed between LpA-I and LpA-II,A-II HDL particles. This dichotomous distribution has implications for PON1 activity and stability that may impact on the physiological role of the enzyme.

HDL oxidation compromises its influence on paraoxonase-1 secretion and its capacity to modulate enzyme activity.

OBJECTIVE: The purpose of this study was to analyze the consequences of HDL oxidation for paraoxonase-1 metabolism and function. METHODS AND RESULTS: HDL was oxidized with AAPH, copper ions, and hypochlorite. Secretion studies were performed using human paraoxonase-1-transfected cells lines and primary rat hepatocytes. Stability studies were performed with recombinant paraoxonase. Conditioned medium had significantly reduced paraoxonase-1 when Cu or AAPH-oxidized HDL was the acceptor complex (P<0.01); reduction was dose-dependent on the degree of oxidation. Oxidized HDL had a reduced capacity to stabilize/improve activity of secreted paraoxonase-1. Reduced secretion could not be attributed to enzyme inactivation by lipoperoxides, reduced binding affinity of HDL, or oxidation of the lipid component alone. Hypochlorite oxidation of HDL did not modify HDL-mediated paraoxonase-1 release, but activity of HDL-associated paraoxonase-1 was particularly sensitive to such treatment. CONCLUSIONS: AAPH and copper, but not hypochlorite, oxidation of HDL compromises its ability to promote release of paraoxonase-1 and stabilize enzyme activity. HDL-associated paraoxonase-1 is highly sensitive to hypochlorite. Reducing paraoxonase-1 renders HDL susceptible to oxidation, which may compromise HDL function. It provides a novel example at the HDL level of the detrimental effects of oxidative stress, and underlines the need for further evaluation of the consequences of HDL oxidation.

Postprandial modulation of serum paraoxonase activity and concentration in diabetic and non-diabetic subjects.

OBJECTIVES: To analyse the HDL associated anti-oxidant enzyme paraoxonase-1, during postprandial hyperlipaemia. METHODS AND RESULTS: Type 2 diabetic patients (n=72), glucose intolerant patients (n=10) and controls (n=38) consumed a high fat:high carbohydrate meal. Blood samples were collected up to 4h and analysed for lipids and paraoxonase-1. In vitro studies examined HDL function with respect to the enzyme. There were significant postprandial increases in serum triglycerides. Paraoxonase-1 activity decreased significantly throughout the postprandial phase. Concentrations of the enzyme initially decreased significantly, but returned to fasting concentrations at 4h. Specific activities were significantly lower at 4h, compared to fasting. The decrease in specific activity was linked to the dynamic phase of postprandial lipoprotein metabolism. Apo AI limited loss of paraoxonase-1. HDL isolated after being subjected to postprandial conditions in vitro had reduced capacity to associate with and stabilise PON1. CONCLUSIONS: Postprandial hyperlipaemia was associated with changes to serum paraoxonase-1, consistent with a reduced anti-oxidant potential of HDL. No differences were observed between diabetic and non-diabetic patients, suggesting that the effect was linked to postprandial hyperlipaemia. Modifications to paraoxonase-1 could contribute to increased risk of vascular disease associated with postprandial lipaemia, particularly in diabetic patients, who are already deficient in serum paraoxonase-1.

Paraoxonase-1 and serum concentrations of HDL-cholesterol and apoA-I.

Paraoxonase-1 (PON1) and HDL are tightly associated in plasma, and this is generally assumed to reflect the need for the enzyme to associate with a hydrophobic complex. The association has been examined in coronary cases and age-matched controls. Highly significant (P < 0.0001), positive associations were observed between PON1 activities and concentrations and HDL-cholesterol and apolipoprotein A-I (apoA-I) concentrations in cases and controls. Corrected slopes were significantly different in cases (cases vs. controls: arylesterase, r = 0.19 vs. 0.38, P < 0.02 for apoA-I and r = 0.15 vs. 0.34, P < 0.02 for HDL-cholesterol) such that if PON1 should influence serum HDL, it would be less effective in coronary cases. When examined as a function of the PON1 gene promoter polymorphism C-107 T, highly significant differences (P < 0.001) in HDL-cholesterol and apoA-I were observed between genotypes for controls, with high expresser alleles having the highest HDL concentrations. This relationship was lost in cases with coronary disease. The coding region polymorphisms Q192R and L55M of the PON1 gene showed no association with HDL. The promoter polymorphism was an independent determinant of HDL concentrations in multivariate analyses. These data are consistent with an impact of PON1 on plasma concentrations of HDL, with detrimental modifications to the relationship in coronary cases.

Very low density lipoproteins provide a vector for secretion of paraoxonase-1 from cells.

Paraoxonase-1 (PON1) requires a suitable acceptor complex for its secretion from producing cells. The serum lipoprotein, high-density lipoprotein (HDL) has been shown to accomplish this function, whereas low-density lipoproteins are ineffective. The present study examined the influence of the third serum lipoprotein subclass, very low density lipoproteins (VLDL), on PON1 secretion. VLDL were shown to promote secretion of PON1 from a transfected Chinese hamster ovary model and from transfected hepatocytes in a high-affinity, saturable manner. The effects of HDL and VLDL were not additive, suggesting that they may employ a common secretion pathway. VLDL was able to stabilise secreted PON1 enzyme activity, but less effectively than stabilisation by HDL. Following co-incubation of VLDL and HDL, the majority of PON1 accumulated in HDL even if HDL was added after initial association of the enzyme with VLDL. VLDL to HDL transfer of PON1 was rapid and did not require lipolysis of VLDL. Low levels of active PON1 were associated with VLDL in human serum, and VLDL-associated enzyme activity was proportional to serum triglyceride concentrations. Serum triglycerides were positively associated with whole serum PON1 mass but negatively associated with specific activity. PON1-enriched VLDL was more resistant to oxidation in vitro. The present study suggests that the triglyceride transport vector, VLDL, can modulate PON1 metabolism and activity. This is due, in part, to an influence of the lipoprotein on PON1 secretion. PON1 was associated with VLDL in human serum, where triglycerides correlated independently with variations in serum mass and activity of the enzyme. VLDL-associated PON1 exerted an anti-oxidative effect, which may be of physiological benefit.