Circadian Rhythm of Subspecies of Low-Density Lipoprotein-Cholesterol and High-Density Lipoprotein-Cholesterol in Healthy Subjects and Patients with Type 2 Diabetes.

AIMS: We established automated assay kits for quantifying small dense low-density lipoprotein (sdLDL)-cholesterol (C), LDL-triglyceride (TG), and high-density lipoprotein (HDL)3-C, and apolipoprotein (apo)E-rich HDL-C, and these have been recognized as sensitive biomarkers for predicting coronary artery disease. We investigated the circadian rhythms of these novel lipids to determine if fasting is required to determine basal levels. METHODS: Forty-eight inpatients with type 2 diabetes and 19 healthy volunteers were studied. Blood samples were collected at seven time points, which were obtained after an overnight fast, before and 2 h after each meal, and before the next breakfast. sdLDL-C, LDL-TG, remnant-like particle (RLP)-C, TG-rich lipoprotein (TRL-C), HDL3-C, and apoE-rich HDL-C were measured by the homogeneous methods. NonHDL-C, large buoyant (lb)LDL-C and HDL2-C were calculated by subtracting sdLDL-C from LDL-C or HDL3-C from HDL-C, respectively. RESULTS: Serum TG levels were significantly increased after meals in both healthy participants and patients with diabetes. RLP-C and TRL-C were also increased postprandially. LDL-TG, LDL-C, nonHDL-C, HDL2,3-C, and apoE-rich HDL-C did not exhibit significant fluctuation during the day in healthy participants and patients with diabetes. sdLDL-C was slightly increased postprandially in subjects with diabetes (1-2 mg/dl, 3%-9%), though its increase was not significant compared to the baseline (fasting) level. Significant postprandial reduction was observed with LDL-C and lbLDL-C. There was no influence of statin therapy or oral anti-diabetes drugs on the circadian rhythm of LDL-C subspecies. CONCLUSIONS: Subtle postprandial increase in sdLDL-C is considered a negligible level in general clinical practice. Fasting is not mandatory to measure basal concentrations of LDL and HDL subspecies.

Metabolic Properties of Lowdensity Lipoprotein (LDL) Triglycerides in Patients with Type 2 Diabetes, Comparison with Small Dense LDL-Cholesterol.

AIMS: Abnormal compositional changes in low-density lipoprotein (LDL) particles, such as triglyceride (TG) enrichment and size reduction, are common in patients with diabetes. Several cohort studies have demonstrated that LDL-TG and sdLDL-cholesterol (C) are sensitive biomarkers for predicting atherosclerotic cardiovascular diseases beyond LDL-C. Although sdLDL has been extensively studied, little is known about the properties of LDL-TG. We investigated similarities or differences between LDL-TG and sdLDL-C. METHODS: Fasting plasma was obtained from 1,085 patients with type 2 diabetes who were enrolled in the diabetes regional cohort study (ViNA Cohort). LDL-TG and sdLDL-C concentrations were measured using a homogeneous assay established by us. In a subset of subjects, LDL-TG and sdLDL-C levels were measured postprandially or after treatment with lipid-lowering drugs. RESULTS: In a quartile analysis, higher LDL-TG quartiles were associated with higher frequency of female and fibrate users, whereas sdLDL-C quartiles were associated with frequency of men, drinking, and metabolic syndrome-related measurements. Higher quartiles of LDL-TG/LDL-C were associated with smoking, drinking, fibrate users, and statin users. LDL-TG was significantly correlated with TG, LDL-C, sdLDL-C, and apolipoprotein (apo) B, with apoB being the primary determinant. LDL-TG correlated to high sensitive C-reactive protein (CRP) independently of other lipids. Mean LDL-TG did not change with fasting/non-fasting. Statin treatment reduced LDL-TG, whereas fibrates increased it, but these drugs reduced sdLDL-C equally. CONCLUSIONS: LDL-TG levels were more tightly regulated by the number of LDL particles than plasma TG levels were. SdLDL-C was closely associated with metabolic syndrome-related factors, whereas LDL-TG was associated with low-grade systemic inflammation.

Development of a homogeneous assay for measurement of small dense LDL cholesterol.

BACKGROUND: Plasma concentrations of small dense (sd)-LDL are associated with the prevalence of cardiovascular events. However, the special equipment and long assay times required for sd-LDL measurement have hindered its clinical application. Herein, we report development of a simple homogeneous assay for sd-LDL-cholesterol (C) adaptable to autoanalyzers. MATERIALS AND METHODS: We identified suitable surfactants and phospholipases by screening for those selective for the sd-LDL fraction (d 1.044-1.063 kg/L) and for the dissociation of other lipoproteins, including large buoyant LDL (lb-LDL). Principal characteristics of this assay were compared with ultracentrifugal isolation of LDL subfractions and with our previous heparin-magnesium precipitation assay for sd-LDL. We measured sd-LDL-C concentrations in 460 healthy, normolipidemic individuals. RESULTS: We used a polyoxyethylene benzylphenyl ether derivative to dissociate triglyceride-rich lipoproteins and HDLs, whereas sphingomyelinase proved most effective for dissociation of lb-LDL from LDL owing to the higher sphingomyelin content in the lb-LDL subfractions. A polyoxyethylene styrenephenyl ether derivative protected sd-LDL against the dissociative actions of sphingomyelinase and cholesterol oxidase/esterase during an initial incubation step. Next, polyoxyethylene alkyl ether dissociated sd-LDL-C and the cholesterol released from sd-LDL were subsequently measured by using cholesterol oxidase/esterase. The homogeneous method correlated excellently with ultracentrifugation for sd-LDL-C (y = 0.99x-0.09, R(2) = 0.91, n = 60) and exhibited within-run precision CVs <1.1%. The distribution of sd-LDL-C was skewed, and the central 95% of sd-LDL-C concentrations ranged from 0.24 to 0.88 mmol/L (9.4-34.0 mg/dL). CONCLUSIONS: The homogeneous assay allows reproducible measurement of sd-LDL-C within 10 min and appears promising in further investigations of the clinical significance of sd-LDL-C.

Low-Density Lipoprotein (LDL)-Triglyceride and Its Ratio to LDL-Cholesterol as Diagnostic Biomarkers for Nonalcoholic Steatohepatitis.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most common type of liver disease, but it is difficult to distinguish its pathogenic phenotype, nonalcoholic steatohepatitis (NASH), from nonalcoholic fatty liver (NAFL) without a liver biopsy. We analyzed serum lipids, including low-density lipoprotein triglyceride (LDL-TG), to elucidate their usefulness for diagnosing NASH. PATIENTS AND METHODS: Serum samples obtained from 35 NASH and 9 NAFL biopsy-confirmed patients and 6 healthy volunteers (HLT) were studied for 13 lipid-related markers and compared between HLT, NAFL, and NASH groups. The relationship between histological findings and the lipid markers was also analyzed. RESULTS: There were significant differences in triglyceride, LDL-TG, the ratio of LDL-TG to the LDL-cholesterol (LDL-TG/LDL-C), small dense LDL-C, and apolipoprotein E between the three groups. Among the 5 lipid components, serum LDL-TG level and the ratio of LDL-TG to the LDL-cholesterol (LDL-TG/LDL-C) were significantly elevated in NASH. The median concentrations of LDL-TG in HLT, NAFL, and NASH were 9, 15, and 20 mg/dL (P < 0.001), and those of LDL-TG/LDL-C were 0.097, 0.102, and 0.173 (P < 0.001), respectively. Although the degree of steatosis was not correlated with the LDL-TG/LDL-C, the ratio was significantly higher in patients with lobular inflammation (P = 0.071), ballooning (P = 0.031), and fibrosis (P < 0.001). The area under the receiver operating characteristic curve of the ratio for distinguishing NASH from NAFL was 0.857. The rest of studied markers showed no significant utility. CONCLUSION: Serum LDL-TG levels and the LDL-TG/LDL-C ratio might serve as simple and noninvasive diagnostic biomarkers for NASH.

Development and Population Results of a Fully Automated Homogeneous Assay for LDL Triglyceride.

BACKGROUND: Low-density lipoprotein (LDL) is measured by its cholesterol content (LDL-C), but it has been suggested that LDL triglyceride (LDL-TG) may also be related to coronary artery disease risk. LDL-TG can be measured after ultracentrifugation or electrophoresis, but these are labor intensive methods, indicating the need for an automated homogeneous assay. METHODS: TG-rich lipoproteins (TRLs), LDL, and HDL were isolated by ultracentrifugation and used to determine optimal characteristics of surfactants and various enzymes for assay development. We analyzed assay precision and linearity, and compared results with those obtained after ultracentrifugation. Serum samples from a large population study (n = 12284 subjects) were used to generate reference intervals for LDL-TG and to determine levels in various types of hyperlipidemia. RESULTS: An assay for LDL-TG has been developed by use of surfactants 1 and 2, and enzymes to measure LDL-TG directly on an automated analyzer. There was an excellent correlation between results obtained with this assay and after isolation of LDL by ultracentrifugation. When the assay was applied to serum samples from normal and hyperlipidemic subjects, median normal values were 0.09 mmol/L, with significant median elevations observed in subjects with increased LDL-C, hypertriglyceridemia, combined hyperlipidemia, and hyperchylomicronemia of 0.19, 0.18, 0.28, and 0.43 mmol/L, respectively, as compared with mean LDL-C values in these subjects of 2.25, 4.01, 2.66, 3.96, and 2.43 mmol/L, respectively. CONCLUSIONS: We have developed an automated homogeneous assay for LDL-TG for potential use in research and clinical laboratories, and documented that the TG molar content of LDL is about 5% of its cholesterol content.

Analysis of acetaminophen glucuronide conjugate accompanied by adduct ion production by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry.

Liquid chromatography-mass spectrometry (LC-MS) is an effective method for the analysis of polar compounds. A coupling of LC-MS, which is used under conventional conditions, and atmospheric-pressure chemical ionization (APCI), which applies mild ionization for the analysis of water-soluble drug conjugates, would offer a very convenient method. The APCI method is effective for ionizing low- and medium-polarized compounds, but not for highly polarized compounds. In this study, we have tried derivatization of carboxyl group of glucuronic acid, to which direct ionization is difficult to apply under the APCI method, was conducted using glucuronides. Methyl ester derivatives were found to be effectively ionized. Furthermore, acetaminophen glucuronide conjugate was investigated in detail. Methyl ester derivatives of acetaminophen glucuronide conjugate (ACEG) were detected at m/z 373 as O(2) adduct ion [M+O(2)](-) in the negative mode, and p-nitrophenyl beta-D-glucuronide (PNPG) demonstrated ionization behaviors very similar to ACEG. Quantitation of ACEG was examined using PNPG as an internal standard, and satisfactory results were obtained for the recovery test and quantification.