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Patient-Oriented and Epidemiological Research| Volume 60, ISSUE 1, P168-175, January 2019

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Serum paraoxonase 1 activity is paradoxically maintained in nonalcoholic fatty liver disease despite low HDL cholesterol[S]

Open AccessPublished:November 19, 2018DOI:https://doi.org/10.1194/jlr.P088997
      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 (
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      ). The spectrum of NAFLD comprises simple hepatic steatosis, nonalcoholic steatohepatitis, fibrosis, and eventually cirrhosis (
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      ). NAFLD is commonly seen as the liver manifestation of metabolic syndrome (MetS) (
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      ) but in itself may represent a risk marker for the development of MetS and T2D (
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      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.
      ). Elevations in apoB-containing lipoproteins are frequently found in NAFLD together with decreased levels of HDL cholesterol (
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      Hepatic steatosis and insulin resistance, but not steatohepatitis, promote atherogenic dyslipidemia in NAFLD.
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      Prevalence and determinants of non-alcoholic fatty liver disease in lifelines: a large Dutch population cohort.
      ), which probably at least in part explains why patients with NAFLD may also be predisposed to atherosclerotic CVD (
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      Nonalcoholic fatty liver disease is a novel predictor of cardiovascular disease.
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      • Blokzijl H.
      • Dullaart R.P.F.
      Associations of the fatty liver and hepatic steatosis indices with risk of cardiovascular disease: Interrelationship with age.
      ).
      Paraoxonase-1 (PON-1) is a calcium-dependent esterase enzyme that can hydrolyze lipid peroxides (
      • Deakin S.P.
      • James R.W.
      Genetic and environmental factors modulating serum concentrations and activities of the antioxidant enzyme paraoxonase-1.
      ,
      • Mackness M.
      • Mackness B.
      Human paraoxonase-1 (PON1): gene structure and expression, promiscuous activities and multiple physiological roles.
      ). PON-1 has important antioxidative and anti-inflammatory properties (
      • Deakin S.P.
      • James R.W.
      Genetic and environmental factors modulating serum concentrations and activities of the antioxidant enzyme paraoxonase-1.
      ,
      • Mackness M.
      • Mackness B.
      Human paraoxonase-1 (PON1): gene structure and expression, promiscuous activities and multiple physiological roles.
      ,
      • Dullaart 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.
      ,
      • Kappelle 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.
      ,
      • Ebtehaj S.
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      • 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.
      ) that likely contribute to its alleged protection against atherosclerotic vascular damage (
      • Mackness B.
      • Quarck R.
      • Verreth W.
      • Mackness M.
      • Holvoet P.
      Human paraoxonase-1 overexpression inhibits atherosclerosis in a mouse model of metabolic syndrome.
      ,
      • Kunutsor 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.
      ). PON-1 is primarily synthesized in the liver (
      • Hassett C.
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      • Humbert R.
      • Chapline C.
      • Crabb J.W.
      • Omiecinski C.J.
      • Furlong C.E.
      Characterization of cDNA clones encoding rabbit and human serum paraoxonase: the mature protein retains its signal sequence.
      ,
      • Rodrigo L.
      • Gil F.
      • Hernandez A.F.
      • Marina A.
      • Vazquez J.
      • Pla A.
      Purification and characterization of paraoxon hydrolase from rat liver.
      ,
      • Mackness B.
      • Beltran-Debon R.
      • Aragones G.
      • Joven J.
      • Camps J.
      • Mackness M.
      Human tissue distribution of paraoxonases 1 and 2 mRNA.
      ) and secreted in the circulation (
      • Deakin S.P.
      • James R.W.
      Genetic and environmental factors modulating serum concentrations and activities of the antioxidant enzyme paraoxonase-1.
      ). In the liver, PON-1 is believed to counteract oxidative stress-mediated hepatocyte injury (
      • Ferré 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.
      ,
      • García-Heredia A.
      • Kensicki E.
      • Mohney R.P.
      • Rull A.
      • Triguero I.
      • Marsillach J.
      • Tormos C.
      • Mackness B.
      • Mackness M.
      • Shih D.M.
      • et al.
      Paraoxonase-1 deficiency is associated with severe liver steatosis in mice fed a high-fat high-cholesterol diet: a metabolomic approach.
      ,
      • Wang 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.
      ). In serum, PON-1 is primarily bound to HDL and to some extent to VLDLs and protects LDLs from oxidative modification (
      • Deakin S.P.
      • James R.W.
      Genetic and environmental factors modulating serum concentrations and activities of the antioxidant enzyme paraoxonase-1.
      ,
      • Mackness M.
      • Mackness B.
      Human paraoxonase-1 (PON1): gene structure and expression, promiscuous activities and multiple physiological roles.
      ,
      • Dullaart 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.
      ,
      • Deakin S.
      • Moren X.
      • James R.W.
      Very low density lipoproteins provide a vector for secretion of paraoxonase-1 from cells.
      ). The transfer of circulating HDL-associated PON-1 to cell membranes may also contribute to the oxidative damage protective properties of the enzyme (
      • Deakin 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.
      ).
      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 (
      • Ferré 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.
      ,
      • Kilic S.S.
      • Aydin S.
      • Kilic N.
      • Erman F.
      • Aydin S.
      • Celik I.
      Serum arylesterase and paraoxonase activity in patients with chronic hepatitis.
      ). Furthermore, it has been suggested that PON-1 measurement could improve the assessment of impaired liver function (
      • Camps J.
      • Marsillach J.
      • Joven J.
      Measurement of serum paraoxonase-1 activity in the evaluation of liver function.
      ). 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 (
      • Deakin S.P.
      • James R.W.
      Genetic and environmental factors modulating serum concentrations and activities of the antioxidant enzyme paraoxonase-1.
      ,
      • Dullaart 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.
      ,
      • Abbott 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.
      ,
      • Dullaart 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.
      ). In rat and sheep models, diet-induced (e.g., a high-fat diet) hepatic steatosis may reduce serum PON-1 activity (
      • Wang 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.
      ,
      • Cao 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.
      ). 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 (
      • Atamer A.
      • Bilici A.
      • Yenice N.
      • Selek S.
      • Ilhan N.
      • Atamer Y.
      The importance of paraoxonase 1 activity, nitric oxide and lipid peroxidation in hepatosteatosis.
      ). Likewise, serum PON-1 activity toward paraoxon was impaired in 50 Egyptian subjects with NAFLD compared with 20 healthy subjects (
      • Samy W.
      • Hassanian M.A.
      Paraoxonase-1 activity, malondialdehyde and glutathione peroxidase in non-alcoholic fatty liver disease and the effect of atorvastatin.
      ). 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 (
      • Hashemi 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.
      ).
      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.

      MATERIALS AND METHODS

      Study population

      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 (
      • Kappelle 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.
      ,
      • Borggreve 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.
      ). 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.

      Measurements and definitions

      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 (
      • Inker 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.
      ). 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 (
      • Kunutsor 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.
      ). 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 (
      • Bedogni 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.
      ). 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 (
      • Bedogni 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.
      ). The FLI is currently considered as one of the best-validated steatosis scores (
      • Marchesini 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.
      ) and was validated in a Caucasian population from Italy that conforms to our Western European population. Alternatively, we used the hepatic steatosis index (HSI) (
      • Lee 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.
      ). 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 (
      • Lee 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.
      ). MetS was defined according to the revised National Cholesterol Education Program Adult Treatment Panel III criteria (
      • Grundy 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.
      ). 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).

      Laboratory methods

      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 (
      • Kappelle 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.
      ,
      • Borggreve 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.
      ). 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 (
      • Schumann 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.
      ,
      • Schumann 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.
      ,
      • Schumann 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.
      ). 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 (
      • Dullaart 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.
      ,
      • Kunutsor 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.
      ). 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 (
      • van 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.
      ).

      Statistical analysis

      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.

      RESULTS

      Clinical and laboratory characteristics according to elevated FLI and HSI

      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 elevated FLI also used antihypertensive, glucose-lowering, and lipid-lowering drugs more frequently. BMI, waist circumference, waist-hip ratio, systolic and diastolic blood pressure, plasma glucose, hsCRP, transaminases and GGT, UAE, total cholesterol, non-HDL cholesterol, LDL cholesterol, and triglycerides were higher in subjects with an elevated FLI, but eGFR and HDL cholesterol were lower in subjects with an elevated FLI (Table 1). Alcohol consumption ≥10 g/day was recorded in subjects with an elevated FLI more frequently. Cigarette smoking was not different between subjects with and without an elevated FLI. Remarkably, serum PON-1 activity was not different between subjects with and without an elevated FLI. In a sex-stratified analysis, PON-1 activity was significantly higher in men with an FLI ≥60 compared with men with an FLI <60 (supplemental Table S1). Of all women included, 41.9% were postmenopausal, and 23.5% were using oral contraceptives. PON-1 was significantly higher in women using oral contraceptives (59.61 vs. 52.63 U/L; P < 0.001) and varied according to menopausal status (premenopausal: 54.69 U/l; postmenopausal: 53.42 U/l; P = 0.021).
      TABLE 1Clinical and laboratory characteristics of serum paraoxonase-1 activity in 5,539 subjects with an FLI <60 and 2,083 subjects with an FLI ≥60
      FLI <60FLI ≥60P
      Age (years)47.8 ± 12.554.8 ± 11.5<0.001
      Sex [men/women (n)]2,375/3,1641,422/661<0.001
      T2D (n)92177<0.001
      MetS (n)4761,310<0.001
      History of CVD (n)398344<0.001
      Current smokers (n)1,8736950.77
      Alcohol intake ≥10 g/day (n)1,301616<0.001
      Antihypertensive medication (n)576600<0.001
      Glucose-lowering drugs (n)5974<0.001
      Lipid-lowering drugs (n)247236<0.001
      Systolic blood pressure (mm Hg)125 ± 19140 ± 19<0.001
      Diastolic blood pressure (mm Hg)72 ± 979 ± 9<0.001
      BMI (kg/m2)24.4 ± 2.930.5 ± 4.1<0.001
      Waist circumference (cm)83 ± 10103 ± 9<0.001
      Waist-hip ratio0.85 ± 0.080.96 ± 0.07<0.001
      Glucose (mmol/l)4.65 ± 0.825.42 ± 1.63<0.001
      hsCRP (mg/l)0.97 (0.44–2.33)2.38 (1.20–4.91)<0.001
      ALT (U/l)18 (14-24)28 (20–39)<0.001
      AST (U/l)23 (20–27)27 (23–32)<0.001
      GGT (U/l)20 (14–28)43 (29–65)<0.001
      eGFR (ml/min/1.73 m2)97.2 ± 16.589.2 ± 17.8<0.001
      UAE (mg/24 h)8.4 (6.0–14.0)14.0 (8.0–33.1)<0.001
      Total cholesterol (mmol/l)5.46 ± 1.086.10 ± 1.11<0.001
      Non-HDL cholesterol (mmol/l)4.04 ± 1.135.01 ± 1.13<0.001
      LDL cholesterol (mmol/l)3.54 ± 1.024.05 ± 1.01<0.001
      HDL cholesterol (mmol/l)1.41 ± 0.401.09 ± 0.30<0.001
      Triglycerides (mmol/l)1.00 (0.76–1.33)1.90 (1.38–2.65)<0.001
      PON-1 activity (U/l)53.1 (43.2–65.2)53.3 (43.7–64.7)0.98
      Data are means ± SDs or medians (interquartile ranges) unless otherwise stated. LDL cholesterol was calculated by the Friedewald formula in 5,509 subjects with an FLI <60 and in 1,973 subjects with an FLI ≥60.
      In subjects with an HSI >36 versus subjects with an HSI ≤36, essentially similar differences in glucose, lipid levels, hsCRP, and UAE were also found (Table 2). PON-1 activity was also not decreased in subjects with an elevated HSI, also when subdivided into men and women separately (supplemental Table S2).
      TABLE 2Clinical and laboratory characteristics of serum paraoxonase-1 activity in 5,117 subjects with an HSI ≤36 and 2,505 subjects with an HSI >36
      HSI ≤36HSI >36P
      Age (years)48.4 ± 12.852.4 ± 11.9<0.001
      Sex [men/women (n)]2,499/2,6181,298/1,2070.016
      T2D (n)70199<0.001
      MetS (n)5311,255<0.001
      History of CVD (n)428314<0.001
      Current smokers (n)1,847721<0.001
      Alcohol intake ≥10 g/day (n)1,3185990.093
      Antihypertensive medication (n)572604<0.001
      Glucose-lowering drugs (n)4291<0.001
      Lipid-lowering drugs (n)268215<0.001
      Systolic blood pressure (mm Hg)125 ± 19137 ± 20<0.001
      Diastolic blood pressure (mm Hg)72 ± 1077 ± 9<0.001
      BMI (kg/m2)24.0 ± 2.530.3 ± 3.9<0.001
      Waist circumference (cm)83 ± 1099 ± 11<0.001
      Waist-hip ratio0.86 ± 0.090.93 ± 0.09<0.001
      Glucose (mmol/l)4.64 ± 0.805.30 ± 1.57<0.001
      hsCRP (mg/l)0.97 (0.43–2.35)2.07 (1.00–4.41)<0.001
      ALT (U/l)18 (14–23)28 (20–39)<0.001
      AST (U/l)23 (20–27)25 (22–31)<0.001
      GGT (U/l)20 (14–30)33 (23–53)<0.001
      eGFR (ml/min/1.73 m2)96.3 ± 17.092.3 ± 17.4<0.001
      UAE (mg/24 h)8.7 (6.1–15.2)11.3 (7.0–24.1)<0.001
      Total cholesterol (mmol/l)5.49 ± 1.115.92 ± 1.11<0.001
      Non-HDL cholesterol (mmol/l)4.09 ± 1.184.75 ± 1.15<0.001
      LDL cholesterol (mmol/l)3.54 ± 1.043.95 ± 1.02<0.001
      HDL cholesterol (mmol/l)1.41 ± 0.411.17 ± 0.34<0.001
      Triglycerides (mmol/l)1.03 (0.77–1.431.51 (1.08–2.20)<0.001
      PON-1 activity (U/l)53.1 (43.1–65.2)53.5 (43.7–64.9)0.74
      Data are means ± SDs or medians (interquartile ranges) unless otherwise stated. LDL cholesterol was calculated by the Friedewald formula in 565 subjects with an HSI ≤36 and in 2,417 subjects with an HSI >36.
      Subjects with an FLI ≥60 and HSI >36 showed 78.5% overlap. In this overlap group serum PON-1 activity was not different between subjects with and without suspected NAFLD (supplemental Table S3).

      Independent positive association of PON-1 activity with elevated FLI and HSI

      Multivariable linear regression analyses were subsequently performed to establish any association of PON-1 activity with an elevated FLI (Table 3). Remarkably, in age- and sex-adjusted analyses, PON-1 activity was positively associated with an elevated FLI (Table 3; model 1). This positive association was also demonstrated after additionally adjusting for the presence of T2D, MetS, alcohol consumption, and current smoking (Table 3; model 2), as well as after further adjusting for individual MetS components (Table 3; model 3). The association of PON-1 activity with an elevated FLI also remained present after additionally adjusting for eGFR, UAE, a positive cardiovascular history, and the use of antihypertensive medication, glucose-lowering drugs, and lipid-lowering drugs (Table 3; model 2: #x03B2; = 0.081, P < 0.001; model 3: #x03B2; = 0.055, P < 0.001). In an alternative analysis, with an elevated HSI instead of an elevated FLI, a similar positive association of PON-1 activity with an elevated HSI was found (Table 4). This association also remained present in a fully adjusted analysis (Table 4; model 2: #x03B2; = 0.037, P < 0.001; model 3: #x03B2; = 0.036, P = 0.01). In these analyses, there were positive associations of alcohol consumption and inverse associations with smoking, as well as inverse associations of PON-1 activity with low HDL cholesterol and elevated triglycerides. When adjusted for oral contraceptives and menopausal status in a multivariable regression analysis, a positive association of PON-1 activity with an elevated FLI (data not shown) remained.
      TABLE 3.Multivariable regression analysis demonstrating the positive association of serum paraoxonase-1 activity with an elevated FLI (≥60) after adjusting for clinical and laboratory covariates in 7,622 subjects
      Model 1
      Adjusted for age and sex.
      Model 2
      Adjusted for age, sex, T2D, MetS, alcohol consumption, and current smoking.
      Model 3
      Adjusted for age, sex, waist circumference, and individual MetS criteria (elevated blood pressure, enlarged waist, elevated glucose, low HDL cholesterol).
      βPβPβP
      Age−0.106<0.001−0.097<0.001−0.124<0.001
      Sex (men vs. women)−0.074<0.001−0.094<0.001−0.100<0.001
      FLI ≥60 vs. <600.043<0.0010.083<0.0010.064<0.001
      T2D (yes/no)−0.0320.007
      MetS (yes/no)−0.066<0.001
      Alcoholic intake ≥10 g/day0.088<0.0010.069<0.001
      Current smoking (yes/no)−0.078<0.001−0.059<0.001
      Elevated blood pressure0.0230.083
      Enlarged waist−0.0060.67
      Elevated glucose−0.0370.002
      Low HDL cholesterol−0.183<0.001
      Elevated triglycerides0.088<0.001
      a Adjusted for age and sex.
      b Adjusted for age, sex, T2D, MetS, alcohol consumption, and current smoking.
      c Adjusted for age, sex, waist circumference, and individual MetS criteria (elevated blood pressure, enlarged waist, elevated glucose, low HDL cholesterol).
      TABLE 4.Multivariable regression analysis demonstrating the positive association of serum paraoxonase-1 activity with an elevated HSI (>36) after adjusting for clinical and laboratory covariates in 7,622 subjects
      Model 1
      Adjusted for age and sex.
      Model 2
      Adjusted for age, sex, T2D, MetS, alcohol consumption, and current smoking.
      Model 3
      Adjusted for age, sex, waist circumference, and individual MetS criteria (elevated blood pressure, enlarged waist, elevated glucose, low HDL cholesterol).
      βPβPβP
      Age−0.009<0.001−0.091<0.001−0.122<0.001
      Sex (men vs. women)−0.066<0.001−0.079<0.001−0.089<0.001
      HSI ≥36 vs. <360.0200.0750.0380.0030.0380.007
      T2D (yes/no)−0.0340.005
      MetS (yes/no)−0.0380.005
      Alcoholic intake ≥10 g/day0.091<0.0010.072<0.001
      Current smoking (yes/no)−0.075<0.001−0.057<0.001
      Elevated blood pressure0.0250.058
      Enlarged waist0.0050.73
      Elevated glucose−0.0360.003
      Low HDL cholesterol−0.183<0.001
      Elevated triglycerides0.1020.003
      a Adjusted for age and sex.
      b Adjusted for age, sex, T2D, MetS, alcohol consumption, and current smoking.
      c Adjusted for age, sex, waist circumference, and individual MetS criteria (elevated blood pressure, enlarged waist, elevated glucose, low HDL cholesterol).
      A sensitivity analysis of 5,126 subjects, which was performed after excluding subjects with a positive cardiovascular history, eGFR <60 ml/min/1.73 m2, and UAE >30 mg/24 h and those using antihypertensive, glucose-lowering, and lipid-lowering drugs, also demonstrated an independent positive association of PON-1 activity with an elevated FLI independent of the presence of T2D, MetS, individual MetS components, alcohol consumption, and smoking (Table 5).
      TABLE 5.Multivariable regression analysis demonstrating the positive association of serum paraoxonase-1 activity with an elevated FLI (≥60) after adjusting for clinical and laboratory covariates in 5,126 subjects, excluding subjects with a positive cardiovascular history, impaired estimated glomerular filtration rate, and elevated UAE and those using antihypertensive, glucose-lowering, and lipid-lowering drugs
      Model 1
      Adjusted for age and sex.
      Model 2
      Adjusted for age, sex, T2D, MetS, alcohol consumption, and current smoking.
      Model 3
      Adjusted for age, sex, waist circumference, and individual MetS criteria (elevated blood pressure, enlarged waist, elevated glucose, low HDL cholesterol).
      βPβPβP
      Age−0.062<0.001−0064<0.001−0.085<0.001
      Sex (men vs. women)−0.078<0.001−0.093<0.001−0.106<0.001
      FLI ≥60 vs. <600.0310.0300.0500.0030.0450.017
      T2D (yes/no)−0.0120.38
      MetS (yes/no)−0.0320.057
      Alcoholic intake ≥10 g/day0.074<0.0010.051<0.001
      Current smoking (yes/no)−0.067<0.001−0.0460.001
      Elevated blood pressure0.0370.013
      Enlarged waist−0.0200.23
      Elevated glucose−0.0010.97
      Low HDL cholesterol−0.185<0.001
      Elevated triglycerides0.076<0.001
      a Adjusted for age and sex.
      b Adjusted for age, sex, T2D, MetS, alcohol consumption, and current smoking.
      c Adjusted for age, sex, waist circumference, and individual MetS criteria (elevated blood pressure, enlarged waist, elevated glucose, low HDL cholesterol).

      DISCUSSION

      This cross-sectional study was carried out in a large, predominantly Caucasian population. We used an elevated FLI (
      • Bedogni 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.
      ), and in alternative analyses an elevated HSI (
      • Lee 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.
      ), as a proxy of NAFLD, thereby following recent international guidelines that recommend the use of biomarker-derived algorithms to categorize subjects with probable NAFLD in large-scale studies (
      • Marchesini 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.
      ). Unexpectedly, in a univariate analysis, serum PON-1 activity was neither decreased in subjects with an elevated FLI nor in those with an elevated HSI (Tables 1, 2) despite increased prevalence of T2D and MetS as well as low levels of HDL cholesterol (
      • Bril F.
      • Sninsky J.J.
      • Baca A.M.
      • Superko H.R.
      • Portillo Sanchez P.
      • Biernacki D.
      • Maximos M.
      • Lomonaco R.
      • Orsak B.
      • Suman A.
      • et al.
      Hepatic steatosis and insulin resistance, but not steatohepatitis, promote atherogenic dyslipidemia in NAFLD.
      ,
      • Nass K.J.
      • van den Berg E.H.
      • Faber K.N.
      • Schreuder T.C.M.A.
      • Blokzijl H.
      • Dullaart R.P.F.
      High prevalence of apolipoprotein B dyslipoproteinemias in non-alcoholic fatty liver disease: the lifelines cohort study.
      ,
      • van den Berg E.H.
      • Amini M.
      • Schreuder T.C.M.A.
      • Dullaart R.P.F.
      • Faber K.N.
      • Alizadeh B.Z.
      • Blokzijl H.
      Prevalence and determinants of non-alcoholic fatty liver disease in lifelines: a large Dutch population cohort.
      ,
      • Nass K.J.
      • van den Berg E.H.
      • Gruppen E.G.
      • Dullaart R.P.F.
      Plasma lecithin:cholesterol acyltransferase and phospholipid transfer protein activity independently associate with nonalcoholic fatty liver disease.
      ). In multivariable linear regression analyses, serum PON-1 activity was positively and independently associated with an elevated FLI (Table 3) in addition to anticipated inverse associations of PON-1 activity with T2D, MetS, current smoking, and alcohol consumption ≥10 g/day (
      • van den Berg E.H.
      • Amini M.
      • Schreuder T.C.M.A.
      • Dullaart R.P.F.
      • Faber K.N.
      • Alizadeh B.Z.
      • Blokzijl H.
      Prevalence and determinants of non-alcoholic fatty liver disease in lifelines: a large Dutch population cohort.
      ,
      • Kunutsor 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.
      ,
      • van der Gaag M.S.
      • van Tol A.
      • Scheek L.M.
      • James R.W.
      • Urgert R.
      • Schaafsma G.
      • Hendriks H.F.
      Daily moderate alcohol consumption increases serum paraoxonase activity; a diet-controlled, randomised intervention study in middle-aged men.
      ,
      • James R.W.
      • Leviev I.
      • Righetti A.
      Smoking is associated with reduced serum paraoxonase activity and concentration in patients with coronary artery disease.
      ). Analyses with an elevated HSI instead of an elevated FLI (Table 4) confirmed these findings, whereas a sensitivity analysis (Table 5), in which subjects with a cardiovascular history, impaired renal function, and elevated UAE and those using medication were excluded, also demonstrated an independent positive association of serum PON-1 activity with an elevated FLI. Taken together, this large-scale study thus paradoxically shows that an elevated FLI per se, as a proxy of NALFD, is not associated with impaired serum PON-1 activity but instead may relate to higher circulating PON-1 activity.
      In two previous studies demonstrating reduced serum PON-1 activity in the context of NAFLD, paraoxon was used as a substrate (
      • Atamer A.
      • Bilici A.
      • Yenice N.
      • Selek S.
      • Ilhan N.
      • Atamer Y.
      The importance of paraoxonase 1 activity, nitric oxide and lipid peroxidation in hepatosteatosis.
      ,
      • Samy W.
      • Hassanian M.A.
      Paraoxonase-1 activity, malondialdehyde and glutathione peroxidase in non-alcoholic fatty liver disease and the effect of atorvastatin.
      ). In a third report, serum PON-1 arylesterase activity was elevated, whereas PON-1 paraoxonase activity was not different in patients with NAFLD (
      • Hashemi 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.
      ). In the current study, we assayed PON-1 enzymatic activity as its arylesterase activity with phenyl acetate as a substrate. PON-1 arylesterase activity has an approximately normal distribution after logarithmic transformation and has been previously reported to be less variable between subjects compared with its activity toward paraoxon (
      • Dullaart 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.
      ,
      • Kunutsor 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.
      ,
      • van 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.
      ). Therefore, we preferred to measure serum PON-1 arylesterase activity, considering that this assay method would be more sensitive compared with paraoxonase activity in testing PON-1 differences between subjects with and without an elevated FLI, as well as in multivariable linear analysis. Thus, it is possible that the difference in the substrate used to assay PON-1 activity could in part explain the discrepancy with earlier reports (
      • Atamer A.
      • Bilici A.
      • Yenice N.
      • Selek S.
      • Ilhan N.
      • Atamer Y.
      The importance of paraoxonase 1 activity, nitric oxide and lipid peroxidation in hepatosteatosis.
      ,
      • Samy W.
      • Hassanian M.A.
      Paraoxonase-1 activity, malondialdehyde and glutathione peroxidase in non-alcoholic fatty liver disease and the effect of atorvastatin.
      ,
      • Hashemi 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.
      ). Another consideration is the number of study participants, which was more than 30-fold higher in this study that included PREVEND study participants compared with earlier reports (
      • Atamer A.
      • Bilici A.
      • Yenice N.
      • Selek S.
      • Ilhan N.
      • Atamer Y.
      The importance of paraoxonase 1 activity, nitric oxide and lipid peroxidation in hepatosteatosis.
      ,
      • Samy W.
      • Hassanian M.A.
      Paraoxonase-1 activity, malondialdehyde and glutathione peroxidase in non-alcoholic fatty liver disease and the effect of atorvastatin.
      ,
      • Hashemi 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.
      ). Furthermore, it cannot be excluded that differences in ethnical background, i.e., Caucasian subjects versus subjects of Turkish or Egyptian descent, could to some extent be responsible for the apparent discrepancies.
      The robust relationship of circulating PON-1 with HDL cholesterol and the concentration of HDL particles is well established (
      • Deakin S.P.
      • James R.W.
      Genetic and environmental factors modulating serum concentrations and activities of the antioxidant enzyme paraoxonase-1.
      ,
      • Dullaart 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.
      ,
      • Dullaart 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.
      ). Accordingly, in multivariable linear regression analyses serum PON-1 activity was strongly associated with HDL cholesterol independent of alcohol consumption, smoking, and other MetS components. Notably, serum PON-1 activity was also positively associated with high triglycerides (Tables 3–5). Circulating PON-1 is to some extent bound to VLDL (
      • Deakin S.
      • Moren X.
      • James R.W.
      Very low density lipoproteins provide a vector for secretion of paraoxonase-1 from cells.
      ), and it is known that enhanced VLDL secretion is a feature of NAFLD (
      • Adiels M.
      • Taskinen M-R.
      • Packard C.
      • Caslake M.J.
      • Soro-Paavonen A.
      • Westerbacka J.
      • Vehkavaara S.
      • Häkkinen A.
      • Olofsson S-O.
      • Yki-Järvinen H.
      • et al.
      Overproduction of large VLDL particles is driven by increased liver fat content in man.
      ), which could explain why serum PON-1 activity may be increased in the context of NAFLD. PON-1 release out of Chinese ovary cells and hepatocytes is stimulated by VLDL in vitro, thereby affecting PON-1 secretion and metabolism (
      • Deakin S.
      • Moren X.
      • James R.W.
      Very low density lipoproteins provide a vector for secretion of paraoxonase-1 from cells.
      ). PON-1 secreted by this pathway is active and retains its antioxidative functionality (
      • Deakin S.
      • Moren X.
      • James R.W.
      Very low density lipoproteins provide a vector for secretion of paraoxonase-1 from cells.
      ). Obviously, the precise mechanisms responsible for the association of higher circulating PON-1 with NAFLD remains to be established. Moreover, while this report essentially rules out reduced serum PON-1 activity as a feature of NAFLD, we cannot exclude a role of disturbed intracellular PON-1 activity in the pathogenesis of NAFLD, as shown in rodent models of hepatic steatosis (
      • García-Heredia A.
      • Kensicki E.
      • Mohney R.P.
      • Rull A.
      • Triguero I.
      • Marsillach J.
      • Tormos C.
      • Mackness B.
      • Mackness M.
      • Shih D.M.
      • et al.
      Paraoxonase-1 deficiency is associated with severe liver steatosis in mice fed a high-fat high-cholesterol diet: a metabolomic approach.
      ,
      • Wang 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.
      ).
      Several other methodological aspects of this study need to be considered. First, we performed a cross-sectional analysis. For this reason neither cause-and-effect relationships can be established with certainty nor can the possibility of reversed causation be excluded. Second, an elevated FLI was chosen to categorize subjects with suspected NAFLD. The FLI is considered to have sufficient accuracy for NAFLD assessment, and its use is in line with international recommendations to apply steatosis scores to characterize NAFLD in larger-sized cohorts (
      • Bedogni 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.
      ,
      • Marchesini 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.
      ). Moreover, the positive association of serum PON-1 activity with suspected NAFLD was confirmed by using the HSI as an alternative proxy for NAFLD. Performing liver ultrasound was not feasible in the setup of the large-scale PREVEND cohort study. Third, we could not differentiate between simple hepatic steatosis and hepatic fibrosis or determine their association with circulating PON-1. Fourth, it is possible that the PREVEND cohort contains a high percentage of individuals with microalbuminuria. For this reason, we adjusted for eGFR and UAE in multivariable linear regression analyses and carried out a sensitivity analysis that excluded subjects with impaired eGFR and elevated UAE. This analysis yielded a similar positive and independent association of serum PON-1 activity with suspected NAFLD. In conclusion, we suggest that the circulating activity of the antioxidative enzyme PON-1 is paradoxically maintained and may even be increased in NAFLD despite inverse associations of PON-1 activity with T2D, MetS, and low HDL cholesterol.

      Acknowledgments

      Dr. J. E. Kootstra-Ros, Laboratory Center, University Medical Center Groningen, supervised the performance of the liver function tests. The Dutch Kidney Foundation supported the infrastructure of the PREVEND program from 1997 to 2003 (Grant E.033). The University Medical Center Groningen supported the infrastructure from 2003 to 2006. Dade Behring, Ausam, Roche, and Abbott financed laboratory equipment and reagents by which various laboratory determinations could be performed. The Dutch Heart Foundation supported studies on lipid metabolism (Grant 2001-005).

      Supplementary Material

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