Current Topics in Nutraceutical Research Vol. 1, No. 3, pp. XX-XX, 2003 ISSN 1540-7535 print, Copyright © 2003 by New Century Health Publishers, LLC All rights of reproduction in any form reserved Printed in the USA by Hauser Printing

FOLIC ACID SUPPLEMENTATION INCREASES SERUM PARAOXONASE ACTIVITY: EVIDENCE FROM A RANDOMIZED DOUBLE BLIND ORAL SUPPLEMENTATION TRIAL IN MEN Voutilainen S,1 Rissanen TH,1,2 Virtanen JK,1 Porkkala-Sarataho E,1 Kaikkonen J,1,3 Seppänen K,4 Tuomainen TP,1 Lehtimäki T,5 Rontu R,5 Hämelahti P,5 Penttilä I,6 Mursu J1 and Salonen JT1,2,6 From the Research Institute of Public Health, University of Kuopio, Kuopio, Finland; 1 the Department of Public Health and General Practice, University of Kuopio, Kuopio, Finland; 2 Oy Jurilab Ltd, Kuopio, Finland; 3 Department of Clinical Chemistry, Kuopio University Hospital, Kuopio, Finland;4 Department of Clinical Chemistry, Laboratory of Atherosclerosis Genetics, Tampere University Hospital, and University of Tampere, Medical School, Tampere, Finland;5 and the Inner Savo Health Centre, Suonenjoki, Finland.6

[Received November 15, 2002; Accepted March 10, 2003] ABSTRACT: Recent studies suggest that the human serum paraoxonase, antioxidative enzyme in high-density lipoproteins that eliminates radicals in the circulation, protects against atherosclerosis and coronary heart disease. As paraoxonase in one of the key enzymes in homocysteine metabolism, our aim was to examine the effect of oral folic acid supplementation on serum paraoxonase activity in a placebo controlled double blind folic acid supplementation trial. Forty healthy voluntary men aged 19-36 years were randomized to receive either 300 mg of folic acid or placebo three times a day for 12 wk. Laboratory analyses were measured before and after supplementation. The greater the increase of erythrocyte folate concentration the larger the elevation of serum paraoxonase activity during the study period (correlation 0.36, P = 0.023). A rise in serum paraoxonase activity was also associated with a reduction in plasma total homocysteine concentration (correlation 0.25, P = 0.121). In a linear regression model the nongenetic variables with the strongest associations with the change in serum paraoxonase activity were the change in erythrocyte folate concentration (standardized coefficient 0.42, P = 0.009), and age (0.27, P = 0.086). Our results indicate that folic acid supplementation can elevate serum paraoxonase activity. This could be a novel mechanism through which folic acid can protect against coronary heart disease.

Corresponding Author:Dr. J T Salonen, Research Institute of Public Health, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland. E-mail: [email protected]. KEY WORDS: Folic acid, Genotype, Homocysteine, Paraoxonase, Polymorphism, Supplementation Corresponding Author: INTRODUCTION There is a large body of epidemiologic evidence showing

that persons with elevated high density lipoprotein (HDL) cholesterol concentrations are at reduced risk of coronary heart disease (CHD) events, suggesting that HDL protects against CHD (Salonen et al., 1991; Stampfer et al., 1991; Kriterowich, 1998). HDL has a role in the reverse transport of cholesterol and it also has been shown to possess antioxidative properties (Stein and Stein, 1999). As increased lipid peroxidation has been shown to associate with accelerated progression of atherosclerosis in man (Salonen et al., 1992), the antioxidative properties of HDL might conceivably contribute to the protection by HDL against CHD. The human serum paraoxonase/arylesterase (PON) is an antioxidative enzyme in HDL, which eliminates radicals in the circulation and protects against coronary diseases (Mackness et al., 2000a). PON has been suggested to account for an important part of the antioxidative property of HDL (Mackness et al., 2000a) and it has been shown that PON protects LDL against oxidation (Aviram et al., 1999). Its activity is modulated by two common amino acid polymorphisms at positions 192 (Gln Q > Arg R) and 55 (Met M > Leu L) in the paraoxonase gene PON1. A lowered PON activity has been reported also in patients with atherosclerotic heart disease (McElveen et al., 1986; Sanghera et al., 1998; Schmidt et al., 1998; Ayub et al., 1999; Imai et al., 2000). Low PON activity or polymorphisms in PON1 gene that are associated with paraoxonase levels in serum are also associated with CHD in some prospective studies (Salonen et al., 1999). Homocysteine (Hcy) is a sulphur-containing amino acid, which is formed from the essential amino acid methionine. Although results from prospective studies are inconsistent, elevated plasma total homocysteine (tHcy) levels have been suggested to be an independent risk factor for atherosclerosis (Boushey et al., 1995; Refsum et al., 1998; Ford et al., 2002). Defects in intracellular Hcy metabolism lead to the elevation of plasma tHcy. These metabolic defects can have a genetic background, i.e. an inherited enzyme deficiency of cystathione β-synthetase or 5,10-methylenetetrahydrofolate

2

Folic acid supplementation and serum paraoxonase activity

reductase (MTHFR) or a nutritional background, i.e. an inadequate intake of folate or vitamin B6 or B12 that serve as cofactors or substrates to the enzymes involved in the Hcy metabolism (Nygård et al., 1999). Approximately two thirds of the cases with elevated tHcy levels have been estimated to be due to low or moderate concentrations of these vitamins, of which folate is considered the most important (Ubbink et al., 1994; Voutilainen et al., 2001). The mechanisms by which hyperhomocysteinemia may induce atherogenesis are only partially understood (Nygård et al., 1999), but promotion of low density lipoprotein (LDL) oxidation and endothelial injury have been suggested. In human plasma Hcy exists in various forms: less than 1 % is in the reduced (sulfhydryl) form, remaining part is oxidized and exists as various disulphides, such as Hcy thiolactone (Ueland, 1995). Hcy thiolactone is formed in all cell types in human and because of inadvertent reactions of thiolactone with proteins are potentially harmful; the ability to detoxify Hcy thiolactone is essential for biological integrity (Jakubowski, 2000). Dr. Jakubowski reported recently that the enzyme Hcy thiolactonase, that hydrolyzes Hcy thiolactone to Hcy, could be in fact paraoxonase (Jakubowski, 2000). If it is so, paraoxonase can hydrolyze Hcy thiolactone back to Hcy and Hcy may be converted either back to methionine (by reaction which needs folate and vitamin B12 as co-factors), or condensed with serine to form cystathionine in a reaction that is dependent on vitamin B6. In light of Dr Jakubowskis’ study, it is possible that folic acid supplementation decreases plasma tHcy (and plasma Hcy-thiolactone levels) and affects serum PON activity by this mechanism. Only few previous studies have reported modifiable, nutritional factors, which affect serum PON activity. We have earlier shown that elevated fasting plasma tHcy levels are associated with enhanced in vivo lipid peroxidation in men (Voutilainen et al., 1999). In the present study, we examined the effect of folic acid supplementation on serum PON activity in healthy young men. SUBJECTS AND METHODS Subjects The folic acid supplementation study was a double blind placebo controlled randomized trial. Healthy nonsmoking male subjects were recruited among the staff and students at the University of Kuopio. Exclusion criteria included regular intake of drugs, vitamins or antioxidants, obesity (BMI > 30 kg/m2), and severe diseases. Forty men were randomized to

receive either placebo (microcrystalline cellulose) or 300 µg folic acid (Foliren®) three times a day for 12 wk. All the subjects provided a written informed consent. The Research Ethics Committee of the University of Kuopio approved the study protocol. Measurements Venous blood samples were drawn between 8 and 10 in the

morning. Subjects were instructed to abstain from ingesting alcohol for three days and from smoking and eating for 12 hours before the drawing. Erythrocyte and plasma folate concentrations were measured by radioimmunoassay (Quanta phase II, Bio-Rad, Hercules, California, USA). The whole blood sample for erythrocyte folate determination was hemolyzed and stabilized with ascorbic acid immediately after blood drawing and kept frozen until measured in batches within three months. The between batch coefficients of variation (CV) of quality control serums (Lyphochek Immunoassay Plus Control 1, 2, 3, Bio Rad Laboratories, ECS Division, Anaheim, California) concentrations of 4.8, 11.6 and 25.1 nmol/L were 5.4, 5.5 and 6.4 percent, respectively (n=10). CV% was 4.2 (n=9) measured in aliquots of a stabilized whole blood hemolysate pool frozen and stored at 80 °C. Erythrocyte folate concentrations were corrected with hematocrit. Serum PON activity was measured based on its capacity to hydrolyse paraoxon to p-nitrophenol when formation was assessed. In the assay, 25 µL serum was added into 800 µL of 0.0125 M borate buffer (pH 7.5) containing 300 µM CaCl2. After preincubation for two minutes, the reaction was started by adding the substrate (100 µL of 3 mM paraoxon) and the reaction mixture was incubated at 37 °C for 30 minutes. The reaction was stopped by adding 100 µL of 0.5 M trichloroacetic acid. The absorbance of the sample was monitored at 405 nm and paraoxonase activity was calculated using p-nitrophenol as a standard. DNA was extracted from EDTA blood with a salting-out method after lysing red cells with 10 mM NaCl/10 mM EDTA. A 170-bp DNA-fragment of PON1 gene was amplified using the oligonucleotide primers and Hsp92II enzyme digestion as described (Salonen et al., 1999). The allele resulting in the formation of an 170 bp fragment (HSP92II cutting site absent) was designated as L allele and that generating both 126 bp ans 44 bp fragments as M allele. The verification of correct genotypes in gel was based on comparison of previously known genotype standards. Genotyping was done blindly and none of the other data were available in the genotyping laboratory. The C677T mutation of MTHFR genotypes were determined using PCR amplification, and subsequent restriction analysis by Hinf1 enzyme, as described previously (Wirta et al., 1998) in Research Laboratory for Atherosclerosis Genetics, University Hospital of Tampere, Tampere, Finland. Blood samples for Hcy determination were drawn in lithium heparin tubes (Venoject, Terumo, Belgium) and sodium fluoride was added to a final concentration of 4 g/L blood (Moller and Rasmussen, 1995). Plasma was separated by centrifuging immediately at +4 °C. Plasma was frozen at -70 °C. THcy was determined at the Department of Clinical Chemistry at the Kuopio University Hospital, Finland using fluorescence polarization immunoassay technology (Abbot IMx, Abbot Park, IL, USA) (Shiphandler and Moore, 1995). Serum total cholesterol and triglyceride concentrations were

Folic acid supplementation and serum paraoxonase activity 3

determined enzymatically with an automatic analyzer (Kone Specific, Konelab, Espoo, Finland). Serum LDL cholesterol was precipitated by using polyvinyl sulfate (Konelab, Finland). Serum HDL cholesterol concentration was determined after precipitation with magnesium chloride dextran sulfate. Statistical analyses The statistical analyses were performed with SPSS 10.0 for Windows. The data are expressed as mean ± SD. Correlations were calculated as Pearson’s correlation coefficients. The nonparametric Mann-Whitney test was used to compare differences in changes in erythrocyte folate concentrations, serum PON activity and plasma tHcy concentrations between folic acid and placebo groups. Linear regression analysis was used to estimate and test the associations of changes in serum PON activity and erythrocyte folate concentration, age, serum HDL cholesterol and plasma tHcy. Confidence intervals were estimated based on the assumption of asymptotic normality of the estimates. All tests of significance were two-tailed. RESULTS The main characteristics of the study subjects are shown in Table 1. There were no statistically significant differences in the baseline characteristics between the folic acid and placebo group. The 12 week folic acid supplementation increased significantly both erythrocyte and plasma folate concentrations and decreased significantly plasma tHcy levels as compared with placebo (P < 0.001 for all changes, Figure 1). In the folic acid group erythrocyte folate levels increased 103% (Mean ±

SD, min-max 437.7 ± 143.5, 195.2 - 768.2 nmol/L), plasma folate 395% (34.9 ± 25.6, 17.2-134.2 nmol/L) and plasma tHcy level decreased 24% (-2.18 ± 1.51, -4.82-0.47 µmol/L). Study subjects kept diary concerning their supplement intake during the study. Although 50% of subjects reported mistakes, these contains only few % of total amounts of pills and all study subjects are then included to the analyses. During the study period, the greater the increase of erythrocyte folate concentration, the larger the elevation of serum PON activity (correlation 0.36, P = 0.023, Figure 2). The mean serum PON activity increased by 4.0% (2.11 U/L) in the folic acid group and decreased by 3.6% (-2.87 U/L) in the placebo group (P = 0.010 for the difference between groups). The simple correlation coefficient for the association between changes in erythrocyte folate concentration and plasma tHcy concentration was -0.62 (P < 0.001), and that for changes in plasma tHcy concentration and serum PON enzyme activity -0.25 (P = 0.121). Baseline serum PON activity and changes in it in different PON1 and MTHFR genotypes are presented in the Table 2. We found not threshold values in association between changes in erythrocyte folate concentration and serum PON activity. We also standardized serum PON to HDL levels. In the folic acid group mean (± SD) change in serum PON/HDL levels was 71.81U/mmol (±179.39), and in the placebo group –1.19 U/mmol (±110.86) (P = 0.133 for the difference between groups).

In a linear regression model the non-genetic variables with the strongest associations with the change in serum PON activity, selected by stepwise analysis (P in 0.05, P out 0.20), were the change in Table 1. Baseline characteristics of the subjects in the folic acid supplementation study* erythrocyte folate concentration (stanAll Folic acid group Placebo group dardized coefficient 0.42, P = 0.009), (n = 40) (n = 20) (n = 20) and age (0.27, P = 0.086) (adjusted R Square for the model 0.15, P = 0.018). Serum folate (nmol/L)† 9.7 ± 3.8 8.8 ± 3.3 10.5 ± 4.1 Erythrocyte folate (nmol/L)† 437.7 ± 112.6 423.9 ± 91.7 451.4 ± 131.3 After adding the serum HDL cholesPlasma homocysteine (mmol/L)† 9.12 ± 2.65 9.13 ± 1.95 9.11 ± 3.27 terol and plasma tHcy concentration to Serum PON (U/L) 69.9 ± 46.8 60.0 ± 33.2 80.2 ± 56.2 the model, standardized coefficient was Age (y)† 25.6 ± 3.7 25.2 ± 3.8 26.1 ± 3 .7 0.36 for the change in the erythrocyte Serum total cholesterol (mmol/L)† 4.48 ± 0.76 4.23 ± 0.76 4.73 ± 0.68 Serum LDL cholesterol (mmol/L)† 2.60 ± 0.73 2.36 ± 0.76 2.85 ± 0.64 folate concentration, 0.27 for age, -0.12 Serum HDL cholesterol (mmol/L)† 1.27 ± 0.17 1.26 ± 0.16 1.28 ± 0.19 for HDL cholesterol, and -0.09 for the Serum triglycerides (mmol/L)† 1.03 ± 0.34 0.98 ± 0.34 1.08 ± 0.33 change in the plasma tHcy concentraGenotype‡ tion (adjusted R Square for the model PON1192 Q/Q 60.0 70.0 50.0 Q/R 37.5 30.0 45.0 0.12, P = 0.070). R/R 2.5 0 5.0 Two paraoxonase genotypes PON1192 PON154 L/L 40.0 45.0 35.0 (Q→R) and PON155 (L→M), and the L/M 45.0 40.0 50.0 MTHFR677

M/M C/C C/T T/T

5.0 52.1 42.5 5.4

15.0 45.0 55.0 0

15.0 30.0 60.0 10.0

* LDL, low-density lipoprotein; HDL, high-density lipoprotein; PON, paraoxonase; MTHFR, methylenetetrahydrofolate reductase. † Mean ± SD. ‡ %.

MTHFR677 (C→T) genotype were determined in our study subjects. PON1192Q allele frequency was 40% and PON155M allele frequency was 50% (Table 1). Both the PON1192 and PON155 polymorphisms were associated with the baseline serum PON activi-

4

Folic acid supplementation and serum paraoxonase activity

7.98 and 0.45 ± 2.70, P for difference 0.029). There were no statistically significant differences in changes of serum PON activity between subjects with different PON155 genotypes. Neither the baseline serum PON activity, nor the change in serum PON activity during the study differed statistically significantly between MTHFR genotypes. There were no statistically significant differences in changes in plasma tHcy concentration across either PON1 or MTHFR genotypes during the study period.

Figure 1. Changes in erythrocyte folate and plasma total homocysteine concentration in the placebo and folic acid group during the folic acid supplementation study.

ty being highest in those with PON1192 R/R and PON155 L/L genotypes and lowest in those with PON1192 Q/Q and PON155 M/M genotypes. Increase in serum PON activity was significantly higher in subjects with the 192 Q/R genotype compared

with subjects with the

192 Q/Q

genotype (mean ± SD 6.00 ±

DISCUSSION We report here a novel finding indicating that dietary folic acid enhances serum PON activity. This is the first study to report that oral folic acid supplementation and elevated erythrocyte folate concentrations are associated with increased serum PON activity. Previous data concerning dietary factors affecting human serum PON activity are limited (Durrington et al., 2002). Recently Jarvik and colleagues report in their population study that serum activity of PON1 was positively correlated with the dietary and medicinal intake of vitamins C and E and with statin treatment and inversely with smoking. Sutherland and co-workers (Sutherland et al., 1999) compared, in a randomized crossover study, the effect of a meal rich in oxidized lipids in the form of fat that had been used for deep-frying in a fast food restaurant with the effect of a control meal rich in corresponding unused fat, on postprandial serum PON activity in 12 healthy men. In their study a meal rich in oxidized lipids decreased significantly postprandial serum PON activity, as compared with the control meal. Van den Gaag and co-workers studied an association between alcohol consumption and serum PON activity in a diet-controlled, randomized, cross-over study in 11 healthy men (Van den Gaag et al., 1999). They found that serum PON activity was higher after three weeks intake of alcoholic beverages as compared with mineral water and concluded that increased serum PON activity may be one of the biological mechanisms underlying the reduced CHD risk in moderate alcohol consumers. There is also some evidence in an animal model that dietary fat modulates serum PON activity in rats (Kudchodkar et al., 2000) and in rabbits (Mackness et al., 2000b). The present study has some limitations. The effect of folic acid on PON activity observed in our study was relatively small compared with overall individual variation in PON activity. Although not statistically significant, we also found difference in mean serum PON activity between placebo and folic acid group in our study baseline. This was mainly due to one relatively high serum PON activity measurement in one study subject of placebo group, both in study baseline and in the end of the study (219 and 204 U/L, respectively). We believe that this mean difference between groups in baseline measurement in serum PON activity did not explain noticed results as median activity of serum PON in study groups were almost the same (43.4 and 40.8). Because of long study period (12 weeks), we couldn’t use crossover study design that is

Folic acid supplementation and serum paraoxonase activity 5

Table 1. Baseline serum PON activity (U/L) and changes in PON activity* in different PON1 and MTHFR genotypes†

All subjects Folic acid group Placebo group Genotype PON1192 Q/Q Q/R R/R PON155 L/L L/M M/M

n

PON activity

Change

40 20 20

69.9 (42.0, 55.0 - 84,9) 80.2 (43.4, 53,9 – 106.6) 59.6 (40.8, 44.1 – 75.1)

0.38 (-0.65, -2.37 – 1.62) 2.12 (1.88, -0.93 – 4.62) -2.87 (-1.30, -5.77 – 0.04)

24 15 1

37.4 (36.7, 32.2 – 42.6) 112.0 (116.4, 99.1 - 124.8) 219.5

-0.40 (-0.45, -1.49 – 0.70) 0.84 (-0.80, -3.92 – 5.60) -18.1

16 18 6

94.2 (100.6, 66.8 – 121.56) 62.6 (41.6, 43.8 – 81.35) 27.3 (26.9, 22.6 – 31.9)

-0.05 (-0.08, -4.63 – 4.53) -1.01 (-1.45, -3.39 – 1.37) 0.65 (0.15, -0.86 – 2.16)

genetic risk factors for ischemic heart disease. Subjects with the PON1192 Q/Q genotype have lower PON activity than those with the 192 Q/R and 192 R/R genotypes. It has been demonstrated that the Q type isozyme is more efficient in protecting against LDL oxidation than the R type, but controversially the R type hydrolyzes thiolactones more readily than the Q isoform (Billecke et al., 2000). In our study serum PON activating effects of folic acid supplementation was greatest in the subjects with PON1192 Q/R genotype (as compared

with R/R), who also had higher serum baseline serum PON activity than the 21 75.1 (42.7, 51.3 – 98.9) -2.05 (-0.80, -5.00 – 0.91) subjects who were PON1192 Q/Q 17 61.4 (39.5, 41.4 – 81.4) 2.22 (0.10, -0.53 – 4.97) homozygotes. Thus, it appears that the 2 87.8 -4.85 homozygosity not only lowers 192 Q/Q * Mean, (median, 95% CI); folic acid group 300mg folic acid three times/d. PON activity, but also slightly attenu† PON; paraoxonase, MTHFR, methylenetetrahydrofolate reductase. ates the PON-enhancing effect of folic acid. Whether this effect modification most reliable setting to the studies with shorter study period. concerns also other PON-indicating nutrients, remains to be As most of our subjects were students of the University of studied. As we had also study subjects with increased erythroKuopio, six months plus washout periods would be too long cyte folate concentration but not increased serum PON levels time to guarantee good compliance and minimum number of (Figure 2), it is possible that there are also other genes and/or interruptions. Amount of supplemented folic acid in our lifestyle factors that attenuated the PON-enhancing effects of study was also quite high (0.9 mg), and it is difficult to get folic acid. that amount from folate-rich or fortified foods. Folate is an important substrate in the remethylation of Hcy back to methionine and a low serum or erythrocyte folate concentration is associated with elevated plasma tHcy concentration. According to a meta-analysis (Homocysteine Lowering Trialists’ Collaboration 1998), after standardization to pretreatment plasma concentrations of tHcy of 12 µmol/L and serum folate of 12 nmol/L (approximate average concentrations for Western populations), supplemented folic acid reduced blood tHcy concentrations by 25 %, with similar effects in the range of 0.5-5 mg folic acid daily. We have earlier shown that a high fasting plasma tHcy is associated with an enhanced lipid peroxidation in vivo in men (Voutilainen et al., 1999). In this previous study, lipid peroxidation was assessed by quantifying plasma F2-isoprostane levels. HDL is MTHFR677 C/C C/T T/T

known to protect LDL against the oxidation, and paraoxonase is one of the main components of HDL responsible for this (Salonen et al. 1992; Mackness et al. 1991; Mackness et al., 1993; Hayek et al., 1997). Oxidized LDL is also known to inactivate serum PON activity (McElveen et al., 1986; Navab et al., 1997). Therefore, in theory, it is possible that folic acid reduces circulating tHcy concentrations and LDL oxidation and in this way spares the PON activity of HDL. An alternative interpretation would be that folic acid induces PON production directly. There are two polymorphisms in the coding region of the paraoxonase gene, which have been identified as potential

Figure 2. Correlation between changes in erythrocyte folate concentration and serum paraoxonase activity during the folic acid supplementation study (r=0.36, P=0.023, n=4).

6

Folic acid supplementation and serum paraoxonase activity

The association between a low dietary intake of folic acid and low circulating folate concentration and increased CVD has been reported in a number of studies (Boushey et al., 1995; Refsum et al., 1998; Nygård et al., 1999; Voutilainen et al. 2000; Voutilainen et al., 2001). None of these studies have reported an association between serum PON activity or PON genotypes and CVD in their study population. It is widely accepted that dietary folate or folic acid can decrease CVD risk by reducing circulating levels of Hcy. Although both retrospective and prospective epidemiologic studies have suggested that even moderate hyperhomocysteinemia is associated with an increased risk for premature vascular disease, the risk-increasing mechanisms of Hcy have been poorly understood. Our present study provides the novel explanation that dietary folic acid or folates either preserves PON activity of HDL or through an unknown mechanism elevates activity of this endogenous antioxidative enzyme. In summary, our findings indicate that folic acid supplementation and blood folate concentration can affect serum PON activity in humans. Together with the previous findings, our results provide additional support to the theory that folate has a role in the prevention of CHD, and suggest a novel mechanism for this protection. It is conceivable that dietary folic acid supplements and the fortification of foods with folic acid could be used to elevate serum PON activity, and thus to enhance the endogenous antioxidative capacity of humans. ACKNOWLEDGEMENTS We thank the volunteer study subjects for their participation, our staff for helping with data collection and Oy Verman Ab (www.verman.fi) for delivering supplements and for partial funding of this study. Supported by Oy Verman Ab, Juho Vainio Foundation (SV), Finnish Cultural Foundation (SV) and Academy of Finland (SV, JK, JTS). CONFLICT OF INTEREST DISCLOSURE; None declared REFERENCES Ayub, A., Mackness, M.I., Arrol, S., Mackness, B., Patel, J., Durrington, P.N. (1999) Serum paraoxonase after myocardial infarction. Arteriosclerosis Thrombosis and Vascular Biology 19, 331-335. Aviram, M., Rosenblat, M., Billecke, S., Erogul, J., Sorenson, R., Bisgaier, C.L., Newton, R.S., La Du, B. (1999) Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants. Free Radical in Biology and Medicine 7-8, 892-904. Billecke, S., Draganov, D., Counsell, R., Stetson, P., Watson, C., Hsu, C., La Du, B.N. (2000) Human serum paraoxonase (PON1) isoenzymes Q and R hydrolyse lactones and cyclic carbonate esters. Drug Metabolism and Disposition 28, 13351342.

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