Title: Serum paraoxonase 1 activity is paradoxically maintained in nonalcoholic fatty liver disease despite low HDL cholesterol
Abstract: Nonalcoholic fatty liver disease (NAFLD) is characterized by low HDL cholesterol, but the activity of the HDL-associated antioxidative enzyme paraoxonase-1 (PON-1) remains unclear. To determine the association of PON-1 with suspected NAFLD, we measured serum enzyme activity in 7,622 participants of the Prevention of Renal and Vascular End-Stage Disease cohort. A fatty liver index (FLI) ≥60, a proxy of NAFLD, was present in 2,083 participants (27.3%) and coincided with increased prevalence of T2D, metabolic syndrome (MetS), (central) obesity, elevated triglycerides, and low HDL cholesterol (all P < 0.001). In men and women combined, serum PON-1 activity did not vary according to elevated FLI (P = 0.98), whereas in men with elevated FLI PON-1 activity was increased (P = 0.016). In multivariable linear regression analyses (adjusted for age, sex, T2D, MetS, alcohol use, and smoking), PON-1 activity was unexpectedly associated with elevated FLI (β = 0.083; P < 0.001). In a sensitivity analysis (n = 5,126) that excluded subjects with positive cardiovascular history, impaired estimated glomerular filtration rate, elevated urinary albumin excretion, and drug use, PON-1 activity was also independently associated with elevated FLI (β = 0.045; P = 0.017). These results indicate that PON-1 is paradoxically maintained and may even be increased in NAFLD despite inverse associations with metabolic disorders and low HDL cholesterol. Nonalcoholic fatty liver disease (NAFLD) is characterized by low HDL cholesterol, but the activity of the HDL-associated antioxidative enzyme paraoxonase-1 (PON-1) remains unclear. To determine the association of PON-1 with suspected NAFLD, we measured serum enzyme activity in 7,622 participants of the Prevention of Renal and Vascular End-Stage Disease cohort. A fatty liver index (FLI) ≥60, a proxy of NAFLD, was present in 2,083 participants (27.3%) and coincided with increased prevalence of T2D, metabolic syndrome (MetS), (central) obesity, elevated triglycerides, and low HDL cholesterol (all P < 0.001). In men and women combined, serum PON-1 activity did not vary according to elevated FLI (P = 0.98), whereas in men with elevated FLI PON-1 activity was increased (P = 0.016). In multivariable linear regression analyses (adjusted for age, sex, T2D, MetS, alcohol use, and smoking), PON-1 activity was unexpectedly associated with elevated FLI (β = 0.083; P < 0.001). In a sensitivity analysis (n = 5,126) that excluded subjects with positive cardiovascular history, impaired estimated glomerular filtration rate, elevated urinary albumin excretion, and drug use, PON-1 activity was also independently associated with elevated FLI (β = 0.045; P = 0.017). These results indicate that PON-1 is paradoxically maintained and may even be increased in NAFLD despite inverse associations with metabolic disorders and low HDL cholesterol. Nonalcoholic fatty liver disease (NAFLD) is emerging as the most common cause of chronic liver disease in the developed world, and its increasing prevalence parallels the obesity epidemic (1Loomba R. Sanyal A.J. The global NAFLD epidemic.Nat. Rev. Gastroenterol. Hepatol. 2013; 10: 686-690Crossref PubMed Scopus (1193) Google Scholar, 2Benedict M. Zhang X. Non-alcoholic fatty liver disease: An expanded review.World J. Hepatol. 2017; 9: 715-732Crossref PubMed Scopus (425) Google Scholar, 3Puoti C. Elmo M.G. Ceccarelli D. Ditrinco M. Liver steatosis: The new epidemic of the Third Millennium. Benign liver state or silent killer?.Eur. J. Intern. Med. 2017; 46: 1-5Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). The spectrum of NAFLD comprises simple hepatic steatosis, nonalcoholic steatohepatitis, fibrosis, and eventually cirrhosis (1Loomba R. Sanyal A.J. The global NAFLD epidemic.Nat. Rev. Gastroenterol. Hepatol. 2013; 10: 686-690Crossref PubMed Scopus (1193) Google Scholar). NAFLD is commonly seen as the liver manifestation of metabolic syndrome (MetS) (4Bugianesi E. McCullough A.J. Marchesini G. Insulin resistance: a metabolic pathway to chronic liver disease.Hepatology. 2005; 42: 987-1000Crossref PubMed Scopus (678) Google Scholar) but in itself may represent a risk marker for the development of MetS and T2D (5Ballestri S. Zona S. Targher G. Romagnoli D. Baldelli E. Nascimbeni F. Roverato A. Guaraldi G. Lonardo A. Nonalcoholic fatty liver disease is associated with an almost twofold increased risk of incident type 2 diabetes and metabolic syndrome. Evidence from a systematic review and meta-analysis.J. Gastroenterol. Hepatol. 2016; 31: 936-944Crossref PubMed Scopus (421) Google Scholar). 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Prevalence and determinants of non-alcoholic fatty liver disease in lifelines: a large Dutch population cohort.PLoS One. 2017; 12: e0171502Crossref PubMed Scopus (58) Google Scholar), which probably at least in part explains why patients with NAFLD may also be predisposed to atherosclerotic CVD (9Hamaguchi M. Kojima T. Takeda N. Nagata C. Takeda J. Sarui H. Kawahito Y. Yoshida N. Suetsugu A. Kato T. et al.Nonalcoholic fatty liver disease is a novel predictor of cardiovascular disease.World J. Gastroenterol. 2007; 13: 1579-1584Crossref PubMed Scopus (426) Google Scholar, 10Kunutsor S.K. Bakker S.J.L. Blokzijl H. Dullaart R.P.F. Associations of the fatty liver and hepatic steatosis indices with risk of cardiovascular disease: Interrelationship with age.Clin. Chim. Acta. 2017; 466: 54-60Crossref PubMed Scopus (17) Google Scholar). Paraoxonase-1 (PON-1) is a calcium-dependent esterase enzyme that can hydrolyze lipid peroxides (11Deakin S.P. James R.W. 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High plasma C-reactive protein (CRP) is related to low paraoxonase-I (PON-I) activity independently of high leptin and low adiponectin in type 2 diabetes mellitus.Clin. Endocrinol. (Oxf.). 2009; 70: 221-226Crossref PubMed Scopus (51) Google Scholar, 14Kappelle P.J.W.H. de Boer J.F. Perton F.G. Annema W. de Vries R. Dullaart R.P.F. Tietge U.J.F. Increased LCAT activity and hyperglycaemia decrease the antioxidative functionality of HDL.Eur. J. Clin. Invest. 2012; 42: 487-495Crossref PubMed Scopus (52) Google Scholar, 15Ebtehaj S. Gruppen E.G. Parvizi M. Tietge U.J.F. Dullaart R.P.F. The anti-inflammatory function of HDL is impaired in type 2 diabetes: role of hyperglycemia, paraoxonase-1 and low grade inflammation.Cardiovasc. Diabetol. 2017; 16: 132Crossref PubMed Scopus (62) Google Scholar) that likely contribute to its alleged protection against atherosclerotic vascular damage (16Mackness B. Quarck R. Verreth W. Mackness M. Holvoet P. 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Human paraoxonase-1 (PON1): gene structure and expression, promiscuous activities and multiple physiological roles.Gene. 2015; 567: 12-21Crossref PubMed Scopus (204) Google Scholar, 24Dullaart R.P.F. Otvos J.D. James R.W. Serum paraoxonase-1 activity is more closely related to HDL particle concentration and large HDL particles than to HDL cholesterol in Type 2 diabetic and non-diabetic subjects.Clin. Biochem. 2014; 47: 1022-1027Crossref PubMed Scopus (37) Google Scholar, 25Deakin S. Moren X. James R.W. Very low density lipoproteins provide a vector for secretion of paraoxonase-1 from cells.Atherosclerosis. 2005; 179: 17-25Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The transfer of circulating HDL-associated PON-1 to cell membranes may also contribute to the oxidative damage protective properties of the enzyme (26Deakin S.P. Bioletto S. Bochaton-Piallat M-L. James R.W. HDL-associated paraoxonase-1 can redistribute to cell membranes and influence sensitivity to oxidative stress.Free Radic. Biol. Med. 2011; 50: 102-109Crossref PubMed Scopus (77) Google Scholar). Several cross-sectional small-scale reports have shown that serum PON-1 activity is decreased in the context of chronic liver disease, showing a reduced ability of HDL to retard LDL oxidation and an inverse association in regulating oxidative stress, fibrosis, and cell apoptosis (21Ferré N. Marsillach J. Camps J. Mackness B. Mackness M. Riu F. Coll B. Tous M. Joven J. Paraoxonase-1 is associated with oxidative stress, fibrosis and FAS expression in chronic liver diseases.J. Hepatol. 2006; 45: 51-59Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27Kilic S.S. Aydin S. Kilic N. Erman F. Aydin S. Celik I. Serum arylesterase and paraoxonase activity in patients with chronic hepatitis.World J. Gastroenterol. 2005; 11: 7351-7354Crossref PubMed Scopus (68) Google Scholar). Furthermore, it has been suggested that PON-1 measurement could improve the assessment of impaired liver function (28Camps J. Marsillach J. Joven J. Measurement of serum paraoxonase-1 activity in the evaluation of liver function.World J. Gastroenterol. 2009; 15: 1929-1933Crossref PubMed Scopus (46) Google Scholar). Surprisingly limited data are available with respect to possible alterations in serum PON-1 in the context of NAFLD despite extensive evidence that shows that serum PON-1 is decreased in MetS and T2D, particularly in relation to low HDL cholesterol (11Deakin S.P. James R.W. Genetic and environmental factors modulating serum concentrations and activities of the antioxidant enzyme paraoxonase-1.Clin. Sci. (Lond.). 2004; 107: 435-447Crossref PubMed Scopus (232) Google Scholar, 13Dullaart R.P.F. de Vries R. Sluiter W.J. Voorbij H.A.M. High plasma C-reactive protein (CRP) is related to low paraoxonase-I (PON-I) activity independently of high leptin and low adiponectin in type 2 diabetes mellitus.Clin. Endocrinol. (Oxf.). 2009; 70: 221-226Crossref PubMed Scopus (51) Google Scholar, 29Abbott C.A. Mackness M.I. Kumar S. Boulton A.J. Durrington P.N. Serum paraoxonase activity, concentration, and phenotype distribution in diabetes mellitus and its relationship to serum lipids and lipoproteins.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1812-1818Crossref PubMed Scopus (366) Google Scholar, 30Dullaart R.P.F. Kwakernaak A.J. Dallinga-Thie G.M. The positive relationship of serum paraoxonase-1 activity with apolipoprotein E is abrogated in metabolic syndrome.Atherosclerosis. 2013; 230: 6-11Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In rat and sheep models, diet-induced (e.g., a high-fat diet) hepatic steatosis may reduce serum PON-1 activity (23Wang B. Yang R-N. Zhu Y-R. Xing J-C. Lou X-W. He Y-J. Ding Q-L. Zhang M-Y. Qiu H. Involvement of xanthine oxidase and paraoxonase 1 in the process of oxidative stress in nonalcoholic fatty liver disease.Mol. Med. Rep. 2017; 15: 387-395Crossref PubMed Scopus (19) Google Scholar, 31Cao Y. Zhang J. Yang W. Xia C. Zhang H-Y. Wang Y-H. Xu C. Serum paraoxonase as an indicator for fatty liver in sheep.J. Vet. Res. 2017; 61: 97-102Crossref PubMed Scopus (5) Google Scholar). So far, only a few studies have addressed the impact of NAFLD on serum PON-1 regulation in humans. In a Turkish study, serum PON-1 activity toward paraoxon was decreased in 49 individuals with NAFLD confirmed by ultrasound compared with 25 control subjects (32Atamer A. Bilici A. Yenice N. Selek S. Ilhan N. Atamer Y. The importance of paraoxonase 1 activity, nitric oxide and lipid peroxidation in hepatosteatosis.J. Int. Med. Res. 2008; 36: 771-776Crossref PubMed Scopus (17) Google Scholar). Likewise, serum PON-1 activity toward paraoxon was impaired in 50 Egyptian subjects with NAFLD compared with 20 healthy subjects (33Samy W. Hassanian M.A. Paraoxonase-1 activity, malondialdehyde and glutathione peroxidase in non-alcoholic fatty liver disease and the effect of atorvastatin.Arab J. Gastroenterol. 2011; 12: 80-85Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In contrast, a report from Iran demonstrated that serum PON-1 activity, measured as its arylesterase activity, was elevated in 83 patients with NAFLD compared with 138 healthy subjects (34Hashemi M. Bahari A. Hashemzehi N. Moazeni-Roodi A. Shafieipour S. Bakhshipour A. Ghavami S. Serum paraoxonase and arylesterase activities in Iranian patients with nonalcoholic fatty liver disease.Pathophysiology. 2012; 19: 115-119Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). In the absence of large-scale studies among Caucasian subjects that aim to investigate the impact of NAFLD on serum PON-1, we initiated this study to determine the extent to which serum PON-1 activity is associated with NAFLD. To this end we carried out a cross-sectional analysis among 7,622 men and women participating in the Prevention of Renal and Vascular End-Stage Disease (PREVEND) cohort, which comprises a large and well-characterized population from the north of the Netherlands. The study was approved by the Medical Ethics Committee of the University Medical Center Groningen and was performed in accordance with Declaration of Helsinki guidelines. The study included participants of the PREVEND cohort study (35Kappelle P.J.W.H. Gansevoort R.T. Hillege J.L. Wolffenbuttel B.H.R. Dullaart R.P.F. Apolipoprotein B/A-I and total cholesterol/high-density lipoprotein cholesterol ratios both predict cardiovascular events in the general population independently of nonlipid risk factors, albuminuria and C-reactive protein.J. Intern. Med. 2011; 269: 232-242Crossref PubMed Scopus (50) Google Scholar, 36Borggreve S.E. Hillege H.L. Wolffenbuttel B.H.R. de Jong P.E. Bakker S.J.L. van der Steege G. van Tol A. Dullaart R.P.F. The effect of cholesteryl ester transfer protein -629C->A promoter polymorphism on high-density lipoprotein cholesterol is dependent on serum triglycerides.J. Clin. Endocrinol. Metab. 2005; 90: 4198-4204Crossref PubMed Scopus (56) Google Scholar). Pregnant women and diabetic subjects using insulin were not allowed to participate. All participants with a urinary albumin concentration ≥10 mg/l were invited to our clinic together with randomly selected subjects with a urinary albumin concentration <10 mg/l. The initial study population comprised 8,592 subjects who completed the total screening program. All participants gave written informed consent. For this study, we excluded subjects for which data on serum PON-1 activity and liver function tests were not available, leaving a study population of 7,622 participants. BMI was calculated as weight (kilograms) divided by height (meters) squared. Waist circumference was measured as the smallest girth between the rib cage and iliac crest. The waist-hip ratio was determined as the waist circumference divided by the largest girth between the waist and thigh. Blood pressure was measured using an automatic device. T2D was defined as a fasting glucose ≥7.0 mmol/l, a random glucose ≥11.1 mmol/l, self-report of a physician diagnosis, or the use of glucose-lowering drugs. Alcohol consumption was defined as ≥10 g/day, with one alcoholic drink being assumed to contain 10 g alcohol. Smoking was categorized as either current or never/former. Urinary albumin excretion (UAE) was measured as described in two 24-h urine collections, and the results were averaged for analysis. The estimated glomerular filtration rate (eGFR) was calculated by applying the combined creatinine cystatin C-based chronic kidney disease epidemiology collaboration equation (37Inker L.A. Schmid C.H. Tighiouart H. Eckfeldt J.H. Feldman H.I. Greene T. Kusek J.W. Manzi J. Van Lente F. Zhang Y.L. Coresh J. Levey A.S. Estimating glomerular filtration rate from serum creatinine and cystatin C.N. Engl. J. Med. 2012; 367: 20-29Crossref PubMed Scopus (2508) Google Scholar). Information on medication use was combined with information from a pharmacy-dispensing registry that has complete information on drug usage of >95% of subjects in the PREVEND study. Venous blood samples were drawn after an overnight fast after the participants had rested for 15 min. For the diagnosis of NAFLD, the algorithm of the fatty liver index (FLI) was used in subjects from the PREVEND cohort study (10Kunutsor S.K. Bakker S.J.L. Blokzijl H. Dullaart R.P.F. Associations of the fatty liver and hepatic steatosis indices with risk of cardiovascular disease: Interrelationship with age.Clin. Chim. Acta. 2017; 466: 54-60Crossref PubMed Scopus (17) Google Scholar). The FLI was calculated as [e (0.953 × loge (triglycerides + 0.139 × BMI + 0.718 × loge (GGT) + 0.053 × waist circumference – 15.745)/[1 + e (0.953 × loge (triglycerides) + 0.139 × BMI + 0.718 × loge (GGT) + 0.053 × waist circumference – 15.745)] × 100, where GGT is γ-glutamyltransferase (38Bedogni G. Bellentani S. Miglioli L. Masutti F. Passalacqua M. Castiglione A. Tiribelli C. The fatty liver index: a simple and accurate predictor of hepatic steatosis in the general population.BMC Gastroenterol. 2006; 6: 33Crossref PubMed Scopus (1430) Google Scholar). The optimal cutoff value for the FLI has been documented to be 60 with an accuracy of 84%, sensitivity of 61%, and specificity of 86% for detecting NAFLD as determined by ultrasonography (38Bedogni G. Bellentani S. Miglioli L. Masutti F. Passalacqua M. Castiglione A. Tiribelli C. The fatty liver index: a simple and accurate predictor of hepatic steatosis in the general population.BMC Gastroenterol. 2006; 6: 33Crossref PubMed Scopus (1430) Google Scholar). The FLI is currently considered as one of the best-validated steatosis scores (39Marchesini G. Day C.P. Dufour J.F. Canbay A. Nobili V. Ratziu V. Tilg H. Roden M. Gastaldelli A. Yki-Järvinen H. et al.EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease.J. Hepatol. 2016; 64: 1388-1402Abstract Full Text Full Text PDF PubMed Scopus (2518) Google Scholar) and was validated in a Caucasian population from Italy that conforms to our Western European population. Alternatively, we used the hepatic steatosis index (HSI) (40Lee J-H. Kim D. Kim H.J. Lee C-H. Yang J.I. Kim W. Kim Y.J. Yoon J-H. Cho S-H. Sung M-W. et al.Hepatic steatosis index: a simple screening tool reflecting nonalcoholic fatty liver disease.Dig. Liver Dis. 2010; 42: 503-508Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar). The HSI (evaluated in an Asian population in the Republic of Korea) was estimated as follows: HSI = 8 × alanine aminotransferase (ALT)-aspartate aminotransferase (AST) ratio + BMI (+2, if diabetes; +2, if female). The cutoff value of the HSI for detecting NAFLD is 36 (40Lee J-H. Kim D. Kim H.J. Lee C-H. Yang J.I. Kim W. Kim Y.J. Yoon J-H. Cho S-H. Sung M-W. et al.Hepatic steatosis index: a simple screening tool reflecting nonalcoholic fatty liver disease.Dig. Liver Dis. 2010; 42: 503-508Abstract Full Text Full Text PDF PubMed Scopus (644) Google Scholar). MetS was defined according to the revised National Cholesterol Education Program Adult Treatment Panel III criteria (41Grundy S.M. Cleeman J.I. Daniels S.R. Donato K.A. Eckel R.H. Franklin B.A. Gordon D.J. Krauss R.M. Savage P.J. Smith S.C. et al.Diagnosis and management of the metabolic syndrome: an American Heart Association/National Heart, Lung, and Blood Institute Scientific Statement.Circulation. 2005; 112: 2735-2752Crossref PubMed Scopus (9052) Google Scholar). Three or more of the following criteria were required to categorize subjects with MetS: waist circumference >102 cm for men and >88 cm for women; plasma triglycerides ≥1.7 mmol/l; HDL cholesterol <1.0 mmol/l for men and <1.3 mmol/l for women; hypertension (blood pressure ≥130/85 mm Hg or the use of antihypertensive medication); and hyperglycemia (fasting glucose ≥5.6 mmol/l or the use of glucose-lowering drugs). Heparinized plasma and serum samples were obtained by centrifugation at 1,400 g for 15 min at 4°C. Plasma and serum samples were stored at −80°C until analysis. Glucose was measured directly after blood collection. Plasma total cholesterol, triglycerides, and HDL cholesterol were measured as described previously (35Kappelle P.J.W.H. Gansevoort R.T. Hillege J.L. Wolffenbuttel B.H.R. Dullaart R.P.F. Apolipoprotein B/A-I and total cholesterol/high-density lipoprotein cholesterol ratios both predict cardiovascular events in the general population independently of nonlipid risk factors, albuminuria and C-reactive protein.J. Intern. Med. 2011; 269: 232-242Crossref PubMed Scopus (50) Google Scholar, 36Borggreve S.E. Hillege H.L. Wolffenbuttel B.H.R. de Jong P.E. Bakker S.J.L. van der Steege G. van Tol A. Dullaart R.P.F. The effect of cholesteryl ester transfer protein -629C->A promoter polymorphism on high-density lipoprotein cholesterol is dependent on serum triglycerides.J. Clin. Endocrinol. Metab. 2005; 90: 4198-4204Crossref PubMed Scopus (56) Google Scholar). Non-HDL cholesterol was calculated as the difference between total cholesterol and HDL cholesterol. LDL cholesterol was calculated by the Friedewald formula if triglycerides were <4.5 mmol/l. Serum ALT and AST were measured using the standardized kinetic method with pyridoxal phosphate activation (Roche Modular P; Roche Diagnostics, Mannheim, Germany). Serum GGT was assayed by an enzymatic colorimetric method (Roche Modular P). ALT, AST, and GGT were standardized according to International Federation of Clinical Chemistry guidelines (42Schumann G. Bonora R. Ceriotti F. Férard G. Ferrero C.A. Franck P.F.H. Gella F.J. Hoelzel W. Jørgensen P.J. Kanno T. et al.IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase.Clin. Chem. Lab. Med. 2002; 40: 718-724Crossref PubMed Google Scholar, 43Schumann G. Bonora R. Ceriotti F. Férard G. Ferrero C.A. Franck P.F.H. Gella F.J. Hoelzel W. Jørgensen P.J. Kanno T. et al.IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase.Clin. Chem. Lab. Med. 2002; 40: 725-733PubMed Google Scholar, 44Schumann G. Bonora R. Ceriotti F. Férard G. Ferrero C.A. Franck P.F.H. Gella F.J. Hoelzel W. Jørgensen P.J. Kanno T. et al.IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 6. Reference procedure for the measurement of catalytic concentration of gamma-glutamyltransferase.Clin. Chem. Lab. Med. 2002; 40: 734-738Crossref PubMed Google Scholar). High-sensitivity C-reactive protein (hsCRP) was assayed by nephelometry. Serum creatinine was measured by an enzymatic method on a Roche modular analyzer. Serum cystatin C was measured by a cystatin C immunoassay (Gentian AS, Moss, Norway) on a Roche modular analyzer. Urinary albumin was measured by nephelometry (Dade Behring Diagnostics, Marburg, Germany). Serum PON-1 enzymatic activity was measured as its arylesterase activity, i.e., as the rate of hydrolysis of phenyl acetate into phenol, as described previously (13Dullaart R.P.F. de Vries R. Sluiter W.J. Voorbij H.A.M. High plasma C-reactive protein (CRP) is related to low paraoxonase-I (PON-I) activity independently of high leptin and low adiponectin in type 2 diabetes mellitus.Clin. Endocrinol. (Oxf.). 2009; 70: 221-226Crossref PubMed Scopus (51) Google Scholar, 17Kunutsor S.K. Bakker S.J.L. James R.W. Dullaart R.P.F. Serum paraoxonase-1 activity and risk of incident cardiovascular disease: the PREVEND study and meta-analysis of prospective population studies.Atherosclerosis. 2016; 245: 143-154Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The interassay coefficient of variation was 8%. Arylesterase activity, as measured with this assay, is positively correlated with PON-1 enzymatic activity toward paraoxon as well as with PON-1 mass (45van Himbergen T.M. Roest M. de Graaf J. Jansen E.H.J.M. Hattori H. Kastelein J.J.P. Voorbij H.A.M. Stalenhoef A.F.H. van Tits L.J.H. Indications that paraoxonase-1 contributes to plasma high density lipoprotein levels in familial hypercholesterolemia.J. Lipid Res. 2005; 46: 445-451Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). SPSS version 23.0 (IBM, Armonk, NY) was used for data analysis. Results are expressed as mean ± SDs or medians (interquartile ranges) unless otherwise stated. Between-group differences in variables were determined by unpaired t-tests or by Chi-square tests where appropriate. Triglycerides, transaminases, GGT, hsCRP, UAE, and PON-1 activity values were loge-transformed for analysis to achieve approximately normal distributions. Multivariable linear regression analyses were carried out to disclose the independent associations of PON-1 activity with an elevated FLI and HSI when taking account of clinical covariates and laboratory parameters. P < 0.05 was considered significant. The study population consisted of 7,622 subjects, of which 2,083 (27.3%) were categorized with an FLI ≥60. Table 1 shows the clinical characteristics and laboratory data of the participants according to the FLI categorization. Subjects with an FLI ≥60 were older and more likely to be men (men: 68.2%; women: 42.8%), were more likely to be classified with MetS and T2D, and had a positive cardiovascular history more frequently. Subjects with an el