Kagehiro Uchida

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.

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.

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.

Automated fluorogenic methods for the evaluation of the extrinsic coagulation reactions in human plasma

Highly sensitive automated methods for the evaluation of the extrinsic coagulation reactions in human plasma were developed by the combination of fluorogenic peptide substrate (MCA) for thrombin and a centrifugal autoanalyzer (Cobas Bio). Prothrombin time (PT) was measured by the reaction time to reach 0.1 relative fluorescence which was caused by the action of thrombin generated after the activation of 3 microliters plasma with human placental tissue factor (Thromborel S). Factors X and VII contents in plasma were measured by the same method after mixing diluted plasma with each factor deficient plasma, tissue factor, calcium and MCA in which 10-800% of each factor was quantitatively measured. Prothrombin content in plasma was quantitated by measuring thrombin activity after the activation with human activated Factor X in the presence of phospholipid and calcium in which 10-160% of prothrombin was measured. By the application of these fluorogenic methods to the patients with cardiovascular diseases, it was demonstrated that these methods are highly sensitive not only to hypocoagulable state, but also to hypercoagulable state, particularly to the increase of Factors X and VII concentrations in plasma.

Clinical usefulness of serum tartrate-resistant fluoride-sensitive acid phosphatase activity in evaluating bone turnover.

Abstract: This study was carried out to evaluate the clinical validity and usefulness of serum tartrate-resistant fluoride-sensitive acid phosphatase (TrFsACP) activity using 2,6-dichloro-4-acetylphenyl phosphate as substrate at pH 6.2 in metabolic bone diseases. The mean Z-scores of TrFsACP activity in patients on hemodialysis were higher than in healthy subjects (male: 2.04 ± 1.98, n = 49, P < .05; female: 1.49 ± 2.43, n = 39, P < .05) and increased with duration of hemodialysis (r = .516, P < .01). Bone alkaline phosphatase also was found to be significantly higher in hemodialysis patients (male: 0.93 ± 1.49, P < .05; female: 1.66 ± 2.42, P < .05) compared with normal subjects; but had lower correlation with duration of hemodialysis than TrFsACP (r = .277, P < .05). Ulcerative colitis (1.37 ± 2.21, n = 15) in males showed a significantly higher Z-score of TrFsACP compared with control subjects (P < .05). The relationship of TrFsACP activity and ultrasound findings (stiffness; speed of sound [SOS]; broadband ultra sound attenuation [BUA]) in healthy women aged 30–75 years (n = 95) were inversely and significantly correlated with stiffness (r = −.465, P < .01), SOS (r = −.484, P < .01), and BUA (r = −.366, P < .01), but were age dependent. TrFsACP activity significantly correlated with stiffness (r = −.521, P < .05) and SOS (r = −.527, P < .05) only in the age group of 46–55 years. BUA (r = −.313, P > .05) did not correlate significantly in any subject in the present study. We conclude that serum TrFsACP activity is useful in the diagnosis and monitoring of bone turnover.

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.

Inverse associations of HDL cholesterol and oxidized HDL with d-dimer in patients with type 2 diabetes mellitus

Please cite this article as: Shigeyuki Ebara, Mikio Marumo, Chika Yamabata, Ikumi Nishibe, Jun-ichi Soneda, Jun Mukai, Makoto Ohki, Kagehiro Uchida, Ichiro Wakabayashi , Inverse associations of HDL cholesterol and oxidized HDL with d-dimer in patients with type 2 diabetes mellitus. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Tr(2017), doi: 10.1016/ j.thromres.2017.04.018

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

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.

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 (Spearmans 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.