To the Editor:
We read with great interest the recent report by Soyman and colleagues (1) detailing relations between the high-density lipoprotein (HDL) associated enzyme paraoxonase (PON1), the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA), and flow-mediated dilatation (FMD) of the brachial artery, which is a non-invasive measure of endothelial function, in women with polycystic ovary syndrome (PCOS). Numerous other clinically relevant anthropometric, biochemical, and hormonal parameters were reported and compared with PON1 activity with the substrate paraoxon (ie, paraoxonase activity), ADMA, and FMD using Pearson correlation analyses. The finding of lower PON1 activity in the PCOS population is intriguing, but unfortunately difficult to evaluate based on the available data.
PON1 hydrolyzes numerous substrates, including certain organophosphates, esters, and lactones (2). Importantly, PON1 has a common polymorphism, either a glutamine (Q) or arginine (R), at amino acid position 192 that effects the enzyme’s hydrolytic rates with some, but not all substrates. In this study the substrate paraoxon (paraoxonase activity) was used to determine serum PON1 levels in the subjects. Paraoxon is hydrolyzed at about a six fold greater rate by the R isoenzyme compared to the Q isoenzyme. Therefore, without PON1 192 phenotype or genotype information, use of the substrate paraoxon to compare PON1 levels between individuals does not necessarily yield meaningful data.
The distribution of Q and R alleles differs in many populations. For example, the Hardy-Weinberg gene distribution in Caucasian populations is 0.7 and 0.3, respectively, whereas this is inverted in African and Asian populations. Gene frequencies in the Turkish population, which is presumably the group evaluated in the Soyman study, have been reported to be 0.63 and 0.37 for the Q and R alleles, respectively (3). Reporting only paraoxonase activities therefore overlooks the potential for PON1 Q192R phenotypes to influence findings, particularly in small patient groups.
It is currently unclear what role the Q192R polymorphism has in determining, or which enzymatic activity best reflects, PON1’s cardioprotective capacity. However, most studies suggest that PON1 protein levels are most indicative of its physiologic effectiveness in protecting against lipid peroxidation. PON1 activity with the substrate phenylacetate (arylesterase activity) is not influenced by the Q192R polymorphism. Arylesterase activity is therefore considered a more practical assessment of PON1 protein levels. Furthermore, by measuring the ratio of serum PON1 activity with two different substrates, e.g. either arylesterase/paraoxonase or diazoxonase/paraoxonase, an individual’s Q192R phenotype can readily be determined (4).
The PON1 literature contains numerous examples where paraoxonase activity was used exclusively to assess PON1 status. Addition of arylesterase activity and Q192R phenotype information in samples from the PCOS population, and those from other populations, may provide greater insight into the potential associations between PON1, ADMA, FMD, and additional measures related to lipid peroxidation and endothelial dysfunction. We invite readers interested in PON1 research to read reports from Richter (4) and Parra (5) for detailed discussions on this issue.
Scott S. Billecke, Ph.D.a
John F. Teiber, Ph.D.b
a William Beaumont Hospital
Ministrelli Women’s Heart Center
Royal Oak, Michigan
a Department of Internal Medicine
Division of Epidemiology
University of Texas Southwestern Medical Center
1. Soyman Z, Noyan V, Tulmac M, Yucel A, Sagsoz N, Bayrak T et al. Serum paraoxonase 1 activity, asymmetric dimethylarginine levels, and brachial artery flow-mediated dilatation in women with polycystic ovary syndrome. Fertil Steril 2011;95:1067-72.
2. Billecke S, Draganov D, Counsell R, Stetson P, Watson C, Hsu C et al. Human serum paraoxonase (PON1) isozymes Q and R hydrolyze lactones and cyclic carbonate esters. Drug Metab Dispos 2000;28:1335-42.
3. Karakaya A, Suzen S, Sardas S, Karakaya AE, Vural N. Analysis of the serum paraoxonase/arylesterase polymorphism in a Turkish population. Pharmacogenetics 1991;1:58-61.
4. Richter RJ, Jarvik GP, Furlong CE. Determination of paraoxonase 1 status without the use of toxic organophosphate substrates. Circ Cardiovasc Genet 2008;1:147-52.
5. Parra S, Marsillach J, Aragones G, Rull A, Beltran-Debon R, Alonso-Villaverde C et al. Methodological constraints in interpreting serum paraoxonase-1 activity measurements: an example from a study in HIV-infected patients. Lipids Health Dis 2010;9:32.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.04.016
The Authors Respond:
We would like to thank Drs. Billecke and Teiber as experts in this area for the valuable contribution to the pitfalls in the assessment of PON1 status. It is currently unclear how PON1’s cardioprotective capacity should be determined. PON1’s enzymatic activity for the hydrolysis of certain substrates (paraoxon, phenylacetate, diazoxon, etc.) and polymorphism of the PON1 gene are widely studied, yielding no clear-cut conclusions. Serum PON1 activity is determined by the enzyme genotype and several polymorphisms of the PON1 gene have been described, not only with PON1 192 but also with 55,–108,–909 and many other variants.
On the other hand, several investigators have pointed out the inadequacy of examining PON1 genotype alone as a risk factor for disease. “PON1 status” was introduced as new terminology which includes both plasma PON1 activity levels and PON1 192 genotype. Studies that have examined PON1 status have found that plasma PON1 activity level is indeed a risk factor for vascular disease, whereas there was no association observed with PON1 genotypes.
Billecke and Teiber have stated that PON1 activity with the substrate phenylacetate was not influenced by the Q192R polymorphism and arylesterase activity was therefore considered a more practical assessment of PON1 protein levels. In a large (n=1527) cross-sectional evaluation performed on PON1 genotypes, catalytic activity and bioavailability, the major determinant of paraoxon hydrolytic activity was the Q192R genotype (1). Interestingly, phenylacetate hydrolytic activity was also affected by the C-107T and L55M genotypes, indicating that arylesterase activity was also influenced by the genetic variability.
When we look at the issue from the clinical perspective, PON1 activities toward paraoxon were found to be significantly lower in patients with coronary artery disease in many studies. Mackness et al. (2) studied PON1 activity, concentration and gene distribution in 417 subjects with angiographically proven coronary heart disease and 282 controls. It has been found that PON1 activity which was determined with paraoxon was significantly lower in patients with coronary heart disease. There were no differences in the PON1-55 and -192 polymorphisms between the groups. Thus, the findings indicated that PON1 activities toward paraoxon were lower in coronary heart disease, regardless of the PON1 genotype.
In another study including 277 Turkish patients with coronary heart disease and 92 controls, genotype distributions and allele frequencies were significantly different for PON1 L55M polymorphism but not with Q192R polymorphism between the groups (3). More importantly, serum PON1 activity determined with hydrolysis of paraoxon was significantly lower in patients than the controls.
In a case-control sample including 106 carotid artery disease cases, PON1 hydrolysis rates for both paraoxon and diazoxon were significantly lower in cases than in controls and were significant predictors of carotid artery disease by use of logistic regression (4).
In conclusion, we strongly agree that there are cetain pitfalls in the assessment of PON1 status in clinical populations, but PON1 activity determined with paraoxon as the substrate was found to be lower in patients with coronary artery disease in many studies. It is by no means perfect, but it is valuable in the assessment of endothelial dysfunction.
Volkan Noyan, M.D.
Kirikkale University School of Medicine
Department of Obstetrics and Gynecology
1. Roest M, van Himbergen TM, Barendrecht AB, Peeters PHM, Van der Schouw YT, Voorbij HAM. Genetic and environmental determinants of the PON-1 phenotype. European J Clin Invest 2007;37:187-96.
2. Mackness B, Davies GK, Turkie W, Lee E, Roberts DH, Hill E, et al. Paraoxonase status in coronary heart disease. Are activity and concentration more important than genotype? Arterioscler Thromb Vasc Biol. 2001;21:1451-7.
3. Kaman D, Ilhan N, Metin K, Akbulut M, Ustundag B. A preliminary study of human paraoxonase and PON 1 L/M 55–PON 1 Q/R 192 polymorphisms in Turkish patients with coronary artery disease. Cell Biochem Funct. 2009;27:88–92.
4. Jarvik GP, Rozek LS, Brophy VH, Hatsukami TS, Richter RJ, Schellenberg GD, et al. Paraoxonase (PON1) phenotype is a better predictor of vascular disease than is PON1192 or PON155 genotype. Arterioscler Thromb Vasc Biol. 2000;20:2441-7.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.04.015