A simple and useful method for evaluation of oxidative stress in vivo by spectrofluorometric estimation of urinary pteridines.

Pteridine derivatives are intermediate metabolites of folic acid and its cofactors. Oxidized-form pteridines, but not reduced-form pteridines, are fluorescent substances. The purpose of this study was to clarify whether oxidized-form pteridine level in urine, estimated by spectrofluorometry, reflects oxidative stress in vivo. The subjects were healthy middle-aged men (n = 258). Urinary pteridine level was estimated by spectrofluorometry with an excitation wavelength of 360 nm and an emission wavelength of 450 nm. Relationships of urinary pteridines with oxidative stress markers (urinary DNA/RNA oxidation products and 15-isoprostane F2t) and with smoking were analyzed. Concentrations of pteridines, DNA/RNA oxidation products and 15-isoprostane F2t were used after logarithmic transformation in linear analyses. Pteridine levels were significantly correlated with levels of DNA/RNA oxidation products (Pearson's correlation coefficient: 0.626, p < 0.01) and 15-isoprostane F2t (Pearson's correlation coefficient: 0.695, p < 0.01). These correlations were not confounded by age, body mass index, history of smoking and estimated glomerular filtration rate in multivariate analysis. The mean urinary pteridine level was significantly higher in heavy smokers (16 cigarettes or more per day) than in nonsmokers and light smokers (less than 16 cigarettes per day) and was higher in light smokers than in nonsmokers. Thus, urinary fluorometric pteridine levels were shown to be associated with known biomarkers of oxidative stress as well as smoking, which causes oxidative stress in vivo. We propose spectrofluorometrical estimation of urinary pteridines as a simple and useful method for evaluation of oxidative stress in vivo.

Relationships of oxidized HDL with blood coagulation and fibrinolysis in patients with type 2 diabetes mellitus.

Although oxidization of LDL is known to be a crucial step for atherosclerotic progression, the significance of oxidized HDL remains to be clarified. The purpose of this study was to determine the relationships of oxidized HDL with blood coagulation and fibrinolysis in patients with diabetes. The subjects were outpatients with type 2 diabetes (n = 163; median hemoglobin A1c, 6.9%). Activities of blood coagulation and fibrinolysis were evaluated by levels of thrombin-anti-thrombin complex (TAT) and plasmin-α2 plasmin inhibitor complex (PIC), respectively. Relationships of oxidized HDL with TAT and PIC were investigated by using linear regression analysis and logistic regression analysis. Oxidized HDL showed a significant inverse correlation with TAT and a marginally significant correlation with PIC (Spearman's rank correlation coefficient: TAT, - 0.205 [p < 0.01]; PIC, - 0.135 [p = 0.087]). Prevalence of high TAT was significantly lower in the 3rd tertile group for oxidized HDL than in its 1st tertile (20.4 vs. 5.6%, p < 0.05), and prevalence of high PIC was marginally significantly lower in the 3rd tertile group for oxidized HDL than in its 1st tertile (40.7 vs. 24.1%, p = 0.099). In multivariate logistic regression analysis using age, gender, smoking, alcohol drinking, BMI, hemoglobin A1c, therapy for dyslipidemia, therapy for diabetes and anti-coagulation therapy as explanatory variables, odds ratios for high TAT and high PIC in the 3rd tertile group for oxidized HDL versus its 1st tertile group were significantly lower than the reference level of 1.00 (high TAT: 0.19 [0.04-0.99], p < 0.05; high PIC: 0.33 [0.12-0.95], p < 0.05). The frequency of high TAT or high PIC was lower in the higher tertile group for oxidized HDL than in its lower tertile group. Thus, oxidized HDL is thought to be inversely associated with both blood coagulation and fibrinolysis in patients with type 2 diabetes.

Cysteinylated transthyretin as a discriminator of cardiovascular risk in patients with diabetes mellitus.

BACKGROUND: The cysteine residue on transthyretin (TTR) is susceptible to be oxidized, and serum cysteinylated TTR (Cys-TTR) level is thought to reflect oxidative stress. The purpose of this study was to elucidate the relationship between Cys-TTR and arterial stiffness, a known predictor of cardiovascular disease, in patients with type 2 diabetes. METHODS: The subjects were 105 male outpatients with type 2 diabetes. Arterial stiffness was evaluated by measuring cardio-ankle vascular index (CAVI). The relationship between CAVI and ratio of Cys-TTR to total TTR (Cys-TTR ratio) was analyzed. RESULTS: Cys-TTR ratio was significantly correlated with CAVI (Pearson's correlation coefficient: 0.316, p<0.01), and CAVI was significantly higher in the 3rd tertile group for Cys-TTR ratio than in its 1st tertile group. These relationships were also significant after adjusting for age, smoking, alcohol drinking, body mass index, mean arterial pressure, hemoglobin A1c, LDL cholesterol-to-HDL cholesterol ratio and eGFR. Prevalence of high CAVI (≥10.0) was significantly higher in the 3rd tertile for Cys-TTR ratio than in its 1st tertile and tended to be higher with an increase in tertile (28.6% in the 1st tertile, 42.9% in the 2nd tertile and 60.0% in the 3rd tertile). Odds ratio (OR) for high CAVI of the 3rd vs. 1st tertile groups for Cys-TTR ratio was significantly higher than the reference level of 1.00 both before and after adjustment for the above cardiovascular risk factors (crude OR, 3.75 [1.38-10.17]; adjusted OR, 5.09 [1.39-18.64]). CONCLUSIONS: Cys-TTR ratio is associated with arterial stiffness in patients with diabetes and is proposed as a new discriminator of cardiovascular risk.

Effects of a Japan Diet Intake Program on Metabolic Parameters in Middle-Aged Men.

AIM: We conducted a pilot study to clarify the effects of the Japan Diet nutritional education program on metabolic risk factors for atherosclerotic cardiovascular disease in middle-aged men who were brought up in the westernized dietary environment of modern Japan. METHODS: Thirty-three men, 30-49 years of age, attended a nutrition education class to learn food items and recommended volumes comprising the Japan Diet (more fish, soybeans and soy products, vegetables, seaweed, mushrooms and unrefined cereals, and less animal fat, meat and poultry with fat, sweets, desserts and snacks, and alcoholic drinks), and were encouraged to consume the Japan Diet for 6 weeks. Anthropometric and biochemical parameters were measured and 3-day weighted dietary records were kept before and at completion of the intervention. RESULTS: Ninety-one percent of participants showed improvements in more than one cardiovascular risk factor after 6 weeks. Body weight, serum low density lipoprotein (LDL) cholesterol, malondialdehyde modified (MDA)-LDL and triglyceride concentrations decreased significantly, while high density lipoprotein cholesterol was unchanged. Fish, soy, and sum of seaweed, mushrooms and konjak intakes doubled, and green and yellow vegetable intakes also increased as compared to baseline. Meanwhile, intakes of refined cereals, meat and poultry, sweets, desserts and snacks, and margarine and shortening decreased. Total energy, lipid, and saturated and monounsaturated fatty acid intakes decreased, while n-3 polyunsaturated fatty acid, dietary fiber, beta-carotene, vitamins D and K, potassium, and magnesium increased, with no change in sodium intake. CONCLUSIONS: The Japan Diet is suggested to improve atherosclerotic cardiovascular disease risk factors in middle-aged Japanese men.The clinical trial registration number: UMIN000020639.

Identification of cysteinylated transthyretin, a predictive biomarker of treatment response to partially hydrolyzed guar gum in type 2 diabetes rats, by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.

Recent evidence has indicated that total fiber intake is inversely related to type 2 diabetes risk. The present study aimed to investigate the effects of chronic administration of partially hydrolyzed guar gum (PHGG), a water-soluble dietary fiber, on the occurrence of diabetes and its complications, fatty liver and nephropathy. We also identified predictive serum biomarkers of treatment response to PHGG by mass spectroscopy-based proteomic analysis using Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a good model of human non-insulin-dependent diabetes mellitus. In this study, at 5 weeks of age, OLETF rats and control strain Long-Evans Tokushima Otsuka (LETO) rats were fed a control diet or a high-fiber diet (5% PHGG) for 57 weeks. Body weight, food intake, oral glucose tolerance test, plasma insulin levels, and urine glucose and protein levels were regularly measured. Oral glucose tolerance tests (OGTT) and storage of serum in a deep freezer were conducted at the beginning of the experiment and every 4 weeks after overnight fasting during the experiments. PHGG treatment affected neither meal patterns nor the body weight of OLETF and LETO rats. Repeated measure analysis of variance revealed significant differences in fasting plasma glucose and plasma glucose at 2 h after OGTT between control OLETF (OLETF-C) rats and OLETF rats treated with PHGG (OLETF-F). The glucose response determined by the area under the curve of OGTT was significantly greater in OLETF-C rats than that in OLETF-F rats at 25 weeks of age. HOMA-IR, an index of insulin resistance, increased at 25 weeks of age in OLETF-C rats, while this increase was significantly inhibited in OLETF-F rats. At 62 weeks of age, PHGG treatment significantly improved hepatic steatosis as well as renal mesangial matrix accumulation in OLETF rats. To identify the risk marker for diabetes mellitus by SELDI-TOF MS, we collected sera from 21-week-old individuals. Among the 12 specific peaks that were risk marker candidates for diabetes mellitus, the m/z 13,720 peak was identified as that of cysteinylated transthyretin by sequencing of four tryptic peptides using tandem mass spectrometry and peak distribution around the m/z 13,720 peak in the SELDI-TOF spectra. In conclusion, we found that chronic treatment with PHGG improved insulin resistance, delayed the onset of diabetes, and inhibited the development of diabetic complications, as well as identified cysteinylated transthyretin as a predictive biomarker of treatment response to PHGG in OLETF rats.

Measurement of urinary advanced glycation end-products (AGEs) using a fluorescence assay for metabolic syndrome-related screening tests.

AIMS: The simple screening test of advanced glycation end-products (AGEs) has not been established yet. We aimed to clarify the usefulness of simple measurement of AGEs for screening tests. METHODS: The subjects were healthy participants and patients with metabolic syndrome. Urine samples were diluted from 1:10 to 1:200 using phosphate-buffered saline, and the fluorescence intensity was measured at 440nm after excitation at 370nm in a 96-well microplate spectrophotometer. The obtained intensities were adjusted according to the urinary creatinine levels. RESULTS: In patients with metabolic syndrome, urinary AGE levels were significantly higher than in healthy individuals (median [range], 168.25 [82.51-1276.15] AU/g creatinine [n=37] versus 134.67 [37.86-776.31] AU/g creatinine [n=350], respectively; p=0.0066). We found significant positive correlations between urinary AGEs and systolic and diastolic blood pressures (Spearman's correlation r=0.119 [p=0.019] and r=0.128 [p=0.012], respectively). There was no significant correlation between estimated glomerular filtration rate and urinary AGEs (r=0.018 [p=0.744]), confirming that renal dysfunction did not influence results of urinary AGE measurements. When all of the participants in the study were classified into four groups according to the numbers of components of metabolic syndrome, we found a significant tendency (p=0.0127) for urinary AGE levels to be higher with the increasing number of metabolic syndrome components. CONCLUSION: These results suggested that measurement of urinary AGE levels may be useful for evaluating the risk of metabolic syndrome.

Elevation of circulating LOX-1 ligand levels in Zucker obese and diabetic rats.

LOX-1 ligands containing apolipoprotein B (LAB) reflect ligand activity of LOX-1, which is a key molecule for initiation of atherosclerosis. The Zucker rat is a well-known model used for research on obesity and diabetes. Blood levels of LAB were compared among Zucker fatty (ZF), Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats. Log-transformed LAB was significantly higher in ZF and ZDF rats than in control ZL rats, while no significant difference was found in log-transformed LAB of ZF and ZDF rats. This study for the first time demonstrated that circulating LOX-1 ligands were elevated in obesity and diabetes model rats.

Titanium-treated surroundings attenuate psychological stress associated with autonomic nerve regulation in office workers with daily emotional stress.

Housing mice in the presence of small particles of titanium has been shown to reduce stress-responsive behavior via the autonomic nervous system. Here, we examined the effects of nighttime titanium exposure on stress parameters and autonomic nerve activity in office workers with emotional stress. A randomized double-blind, placebo controlled trial was performed in 24 male subjects with desk jobs, who were randomly allocated to spend 5 nights in rooms with or without titanium. The serum concentrations of stress-responsive hormones (cortisol, adrenocorticotropin, and catecholamine) were measured, and profiles of emotional stress were collected to subjectively assess relaxation. Autonomic nerve activity was examined by power spectra analysis of heart rate variability. In psychological tests, factors related to tension (-14.5%, 95% CI=-15.7--2.1), anger (-11.3%, 95% CI=-13.9--0.7), and emotional stress (-28.5%, 95% CI=-30.0--5.3) were significantly decreased by exposure to titanium. The serum level of adrenocorticotropin was gradually elevated throughout the experimental period in the placebo group (day 4, 80.5%, 95% CI=7.1-37.5 vs. before trial) but not the titanium group. Power spectral analysis of R-R interval data showed a significant elevation in the high-frequency power spectral ratio in subjects housed in titanium rooms (days 1-2, 14.3%, 95% CI=4.7-21.9; days 3-4, 26.8%, 95% CI=4.9-38.7; and days 5-6, 24.1%, 95% CI=5.8-34.0 vs. before trial). These results suggest that sleeping in a room containing titanium lowers physiological and psychological stress.

Impaired spontaneous thrombolytic activity measured by global thrombosis test in males with metabolic syndrome.

INTRODUCTION: In patients with metabolic syndrome (MetS), activity of the fibrinolytic system is generally surmised to be decreased through increased plasminogen activator inhibitor-1 (PAI-1) generation. However, there have been no detailed reports describing whether the clot lysis activity is more dominant than increased clot formation activity for production of the thrombotic state in MetS. METHODS: The global thrombosis test (GTT) is a novel method designed to test both clot formation and clot lysis activities under physiological conditions by using non-anticoagulated blood samples in vitro. We used the GTT to examine the thrombotic or thrombolytic states in males with MetS. RESULTS: Lysis time, which reflects spontaneous clot lysis activity, was significantly longer in MetS subjects (median, 1494s; range, 865-3596s; n=30) than in control subjects (median 1246s; range, 667-2239s; n=53). There was no significant difference between the two groups in occlusion time, which reflects platelet function. The mean level of PAI-1 was significantly higher in MetS subjects than in controls (mean ± SE, 8.7 ± 1.1 and 5.0 ± 0.5 ng/mL, respectively). PAI-1 level and lysis time were significantly correlated (r=0.400, P<0.01). CONCLUSION: These results suggest that male patients with MetS are more likely than controls to experience a thrombotic state through decreased fibrinolytic activity due to increased PAI-1 generation, and that the GTT is useful for evaluating fibrinolytic activity in vitro.

Associations of atherosclerotic risk factors with oxidized low-density lipoprotein evaluated by LOX-1 ligand activity in healthy men.

BACKGROUND: The aim of this study was to determine the relationships of risk factors for atherosclerosis with oxidized low-density lipoprotein (OxLDL) evaluated by a new enzyme immunoassay for measurement of LOX-1 (lectin-like OxLDL receptor) ligand. METHODS: Subjects were 236 healthy men aged 33-62 years. LOX-1 ligand containing apoB (LAB) was measured by an enzyme-immunoassay using immobilized recombinant LOX-1 and anti-ApoB monoclonal antibody. RESULTS: In simple regression analysis, log-converted LAB showed significant positive correlations with history of smoking, waist circumference, diastolic blood pressure, LDL cholesterol, log-converted triglycerides, uric acid and white blood cell count and showed a significant negative correlation with HDL cholesterol. In multiple regression analysis using history of smoking, history of drinking, waist circumference, diastolic blood pressure, HDL cholesterol, log-converted triglycerides and uric acid as explanatory variables, log-converted LAB showed significant correlations only with history of smoking and log-converted triglycerides. Log-converted LAB was significantly higher in heavy smokers (≥20 cigarettes per day) than in nonsmokers and light smokers (<20 cigarettes per day), while no difference in log-converted LAB was found between nonsmokers and light smokers. Log-converted LAB was significantly higher in subjects with hypertriglyceridemia (≥150 mg/dl), large waist circumference (≥85 cm), high diastolic blood pressure (≥85 mmHg), or metabolic syndrome defined by the NCEP-ATP III criteria than in subjects without each risk factor or metabolic syndrome. CONCLUSIONS: Hypertriglyceridemia and smoking are determinants of LOX-1 ligand activity in healthy men and are thus thought to be crucial risk factors for initiation of atherosclerotic progression through generation of OxLDL.

Pitavastatin reduces lectin-like oxidized low-density lipoprotein receptor-1 ligands in hypercholesterolemic humans.

The aim of this study was to determine the impact of pitavastatin on low-density lipoprotein cholesterol (LDL-C) and lectin-like oxidized LDL receptor-1 (LOX-1) in patients with hypercholesterolemia. Twenty-five hypercholesterolemic patients (8 male, 17 female; age 66 +/- 13, 21-80 years) who had not received anti-dyslipidemic agents and had LDL-C levels of more than 160 mg/dL were examined. Biochemical factors were measured at baseline and after treatment with pitavastatin (2 mg/day) for 6 months. Serum levels of LOX-1 with apolipoprotein B-100 particle ligand and a soluble form of LOX-1 (sLOX-1) were measured by ELISA. All subjects completed the study with no adverse side effects. Total-C (268 +/- 26 vs. 176 +/- 17 mg/dL), LDL-C (182 +/- 21 vs. 96 +/- 14 mg/dL), and LOX-1 ligand (867 +/- 452 vs. 435 +/- 262 ng/mL) were reduced with pitavastatin treatment (P < 0.0001 for each). Significant decreases in triacylglycerols were noted (P < 0.0001), but there were no changes in high-density lipoprotein cholesterol. After 6 months, there were no significant changes in high-sensitivity CRP or soluble LOX-1. At baseline, there were no significant correlations between LOX-1 ligand and either LDL-C or sLOX-1. The decrease in LOX-1 ligand was not correlated with the decrease in LDL-C, but was correlated with the decrease in sLOX-1 (r = 0.47, P < 0.05). In conclusion, pitavastatin therapy had beneficial effects on markers of oxidative stress in hypercholesterolemic subjects. Serum levels of LOX-1 ligand may be a useful biomarker of the pleiotropic effects of statins.

LOX index, a novel predictive biochemical marker for coronary heart disease and stroke.

BACKGROUND: Lectin-like oxidized LDL receptor 1 (LOX-1) is implicated in atherothrombotic diseases. Activation of LOX-1 in humans can be evaluated by use of the LOX index, obtained by multiplying the circulating concentration of LOX-1 ligands containing apolipoprotein B (LAB) times that of the soluble form of LOX-1 (sLOX-1) [LOX index = LAB x sLOX-1]. This study aimed to establish the prognostic value of the LOX index for coronary heart disease (CHD) and stroke in a community-based cohort. METHODS: An 11-year cohort study of 2437 residents age 30-79 years was performed in an urban area located in Japan. Of these, we included in the analysis 1094 men and 1201 women without history of stroke and CHD. We measured LAB and sLOX-1 using ELISAs with recombinant LOX-1 and monoclonal anti-apolipoprotein B antibody and with 2 monoclonal antibodies against LOX-1, respectively. RESULTS: During the follow-up period, there were 68 incident cases of CHD and 91 cases of stroke (with 60 ischemic strokes). Compared with the bottom quartile, the hazard ratio (HR) of the top quartile of LOX index was 1.74 (95% CI 0.92-3.30) for stroke and 2.09 (1.00-4.35) for CHD after adjusting for sex, age, body mass index, drinking, smoking, hypertension, diabetes, non-HDL cholesterol, and use of lipid-lowering agents. Compared with the bottom quartile of LOX index, the fully adjusted HRs for ischemic stroke were consistently high from the second to the top quartile: 3.39 (95% CI 1.34-8.53), 3.15 (1.22-8.13) and 3.23 (1.24-8.37), respectively. CONCLUSIONS: Higher LOX index values were associated with an increased risk of CHD. Low LOX index values may be protective against ischemic stroke.

[Significance of urinary uracil measurement following administration of DPD inhibitory fluoropyrimidine(DIF)products].

Individual differences in 5-FU metabolism are mainly attributed to individual differences in the activity of DPD, an enzyme that can metabolize more than 85% of 5-FU. Because urinary uracil is a reflection of DPD activity, it is measured to predict and prevent the occurrence of side effects caused by pyrimidine-type chemotherapeutic agents. From urinary uracil values measured in 84 gastrointestinal cancer patients, 0-60 mmol/g.creatinine was set as a standard. In patients whose urinary uracil values exceeded the standard, 5-FU tended to be accumulated when S-1, a DIF product, was administered and side effects, such as anorexia, vomiting and diarrhea occurred immediately after the start of S-1 administration. If an appropriate DIF product is selected and its dosage set based on the patient's urinary uracil value, the occurrence of side effects would be reduced. Subsequently, a continuation of medication would be possible.

Changes of midazolam pharmacokinetics in Wistar rats treated with lipopolysaccharide: relationship between total CYP and CYP3A2.

It has been reported that infection interferes with drug metabolism, resulting in changes in pharmacokinetics. In this study, we investigated the effects of lipopolysaccharide (LPS) on hepatic total cytochrome P450 (CYP), CYP3A2, and CYP2C11 contents in a transient, LPS-induced, endotoxemia model of rats. In addition, to assess the effects on CYP3A2 activities, the pharmacokinetics of midazolam (CYP3A2 substrate) and 1-OH-midazolam (metabolite of midazolam) were investigated. Hepatic total CYP contents were significantly low until day 3 (P < 0.05) but returned to the control level on day 5. Hepatic CYP3A2 contents were significantly decreased on day 1 until day 5 (P < 0.05) but returned to the control level on day 7. Hepatic CYP2C11 contents were continuously low until day 7, and lowest on day 3. The AUC of 1-OH-midazolam was significantly decreased on day 1 after LPS administration (P < 0.01). In conclusion, LPS (5 mg/kg) challenge decreased hepatic total CYP, CYP3A2, and CYP2C11 contents and also decreased the activities of hepatic CYP3A2. It took at least 7 days for hepatic total CYP and CYP3A2 to recover to control levels, and it was suggested that the changes of hepatic total CYP contents might correlate with those of hepatic CYP3A2 contents and activities. Additionally, it is shown that their changes might reflect the recovery process from inflammation.

Changes in digoxin pharmacokinetics treated with lipopolysaccharide in Wistar rats.

Lipopolysaccharide (LPS) is a highly bioactive substance that can cause local as well as systemic damage to various organs of both humans and animals, even at very low doses. However, there are a few reports on drug pharmacokinetics during endotoxemia. In this study, we analyzed the pharmacokinetics of digoxin (a therapeutic agent for cardiac insufficiency) as a probe drug for a two-compartment model in a rat model of endotoxemia induced by LPS for 5 d. Digoxin was given to Wistar rats intravenously (i.v.), orally (p.o.), and intra-intestinally using an in situ closed-loop method (loop). The AUCi.v. was significantly increased in the LPS (+) group throughout the experiment (p<0.05). There was significant decrease in V2 (volume of distribution of tissue compartment) on Day 1-3 (p<0.05). On Day 1-2 after LPS administration, the AUCp.o. was significantly increased in the LPS (+) group (p<0.05). The AUCloop was significantly increased throughout the experiment (p<0.05). The elimination rate constant was unchanged. Thus LPS administration affected the absorption but not the excretion of digoxin. The findings of this study suggest that digoxin absorption increased and the volume of distribution of tissue compartment decreased after LPS administration (5 mg/kg, i.p.). It appears that digoxin pharmacokinetics recover over 3 d after LPS administration.

The ratio of serum paraoxonase/arylesterase activity using an improved assay for arylesterase activity to discriminate PON1(R192) from PON1(Q192).

Human serum paraoxonase (PON1) exists in 2 major polymorphic forms: Q (glutamine) or R (arginine) at codon 192. The PON1(192) activity polymorphism is substrate dependent. The PON1(Q192) isoform has a higher rate of in vitro hydrolysis of diazoxon, sarin, and soman, whereas the PON1(R192) isoform has higher activity for the hydrolysis of paraoxon and chlorpyrifos oxon. Both isoforms hydrolyze phenyl acetate at approximately the same rate. The present study described and evaluated a kinetic method of arylesterase activity determination with a modified fixed incubation method that used the oxidative coupling of phenol with 4-aminoantipyrine of phenyl acetate as the substrate. Our improved method shows that arylesterase activity is lower with the PON1(R192) isoform than with the PON1(Q192) isoform. The average activities of serum of individuals of a specific PON1(Q192) genotype showed higher arylesterase and lower paraoxonase activity than the PON1(R192) genotype. The ratio of paraoxonase/arylesterase activity showed a clear separation of all three PON1(192) genotypes with no overlap between the groups (QQ: < 5.0, QR: 5.0-11.0, RR: > 11.0). PCR has suggested that the PON1(192) phenotypes correspond to the PON1(192) genotypes. Therefore, when conducting epidemiological or mechanistic studies that examine the role of PON1 in organophosphorus or lipid metabolism, this ratio is more useful and informative than a PCR-based genotype alone.