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Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, 'distorted-chair' form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the 'radiation damage-free' structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation consistent with that proposed previously

The immunoaffinity separation method was developed to isolate and detect VLDL and CM remnants as remnant lipoprotein (RLP), but it can also detect abnormal lipoproteins in RLP fraction such as LpX and apoE-rich HDL as a kind of false positive phenomenon. Because those abnormal, unbound lipoproteins to the immunoaffinity gel have no apoB100 and apoA1, those lipoproteins are fractionated as RLP. However, it has become an interesting diagnostic marker for screening of LpX or apoE-rich HDL. By using the RLP-cholesterol/ RLP-TG ratio of lipoprotein structure, extremely high ratio were found when compared to the conventional RLP ratio because of the lack of TG in those lipoproteins.

Background The association of plasma cardiovascular risk markers and metabolic syndrome (MetS) with non-alcoholic fatty liver disease (NAFLD) has not been well defined. Methods Japanese men ( n = 809) had standard anthropometric measurements done, and had their liver fat quantitated by ultrasound. Three groups were identified: (1) normal controls without significant disease, (2) preliminary-metabolic syndrome (pre-MetS) cases and (3) MetS cases. Plasma adiponectin, high sensitivity-C reactive protein (hs-CRP), HOMA-IR, lipids, lipoproteins and liver enzymes were evaluated among the three groups. Results The prevalence of fatty liver was 13% in controls, 39% in pre-MetS and 62% in MetS. Plasma adiponectin and high density lipoprotein cholesterol (HDL-C) were significantly decreased, and HOMA-IR, hs-CRP, TG, remnant lipoproteins (RLPs) and small dense-LDL-C (sd LDL-C) were significantly increased in subjects with fatty liver compared to those without fatty liver. Multivariate analyses of serum parameters associated with fatty liver revealed that adiponectin and hs-CRP were more strongly associated with the presence of fatty liver than waist circumference. However, HOMA-IR, HDL-C, TG, RLP-C, RLP-TG and sd LDL-C were more strongly associated with waist circumference than with fatty liver. Factor analysis revealed that adiponectin and HDL-C were linked to liver enzymes, lipoproteins and HOMA-IR associated with fatty liver, but not with waist circumference. Conclusions Adiponectin was found to be a more specific diagnostic marker for the presence of fatty liver regardless of MetS status, and was inversely correlated with liver enzyme concentrations. However, RLPs were found to be more specifically associated with the presence of MetS.

Eur J Clin Invest 2012; 42 (10): 1094–1099 Abstract Background Phenotype of autosomal recessive hypercholesterolaemia (ARH), a rare lipid disorder, is known to be milder than that of homozygous familial hypercholesterolaemia (FH) with LDL receptor gene mutation. However, few data exist regarding the functional differences in ARH and FH particularly in terms of remnant-like particles’ (RLP) metabolism. Materials and methods Blood sampling was performed up to 6 h after OFTT cream loading (50 g/body surface area) with 2-h intervals in a single ARH proband, four heterozygous FH patients with LDL receptor gene mutation and four normal controls. Plasma lipoprotein and RLP fraction were determined by HPLC system. The area under curve (AUC) of each lipoprotein including RLP fractions was evaluated. Results The AUC of TG, RLP cholesterol (RLP-C) and RLP triglyceride (RLP-TG) levels of heterozygous FH subjects was significantly higher than those of controls (466 ± 71 mg/dL × h vs. 303 ± 111 mg/dL × h, P

Context: The American Heart Association Nutrition Committee recommends women and men consume no more than 100 and 150 kcal of added sugar per day, respectively, whereas the Dietary Guidelines for Americans, 2010, suggests a maximal added sugar intake of 25% or less of total energy. Objective: To address this discrepancy, we compared the effects of consuming glucose, fructose, or high-fructose corn syrup (HFCS) at 25% of energy requirements (E) on risk factors for cardiovascular disease. Participants, Design and Setting, and Intervention: Forty-eight adults (aged 18–40 yr; body mass index 18–35 kg/m2) resided at the Clinical Research Center for 3.5 d of baseline testing while consuming energy-balanced diets containing 55% E complex carbohydrate. For 12 outpatient days, they consumed usual ad libitum diets along with three servings per day of glucose, fructose, or HFCS-sweetened beverages (n = 16/group), which provided 25% E requirements. Subjects then consumed energy-balanced diets containing 25% E sugar-sweetened beverages/30% E complex carbohydrate during 3.5 d of inpatient intervention testing. Main Outcome Measures: Twenty-four-hour triglyceride area under the curve, fasting plasma low-density lipoprotein (LDL), and apolipoprotein B (apoB) concentrations were measured. Results: Twenty-four-hour triglyceride area under the curve was increased compared with baseline during consumption of fructose (+4.7 ± 1.2 mmol/liter × 24 h, P = 0.0032) and HFCS (+1.8 ± 1.4 mmol/liter × 24 h, P = 0.035) but not glucose (−1.9 ± 0.9 mmol/liter × 24 h, P = 0.14). Fasting LDL and apoB concentrations were increased during consumption of fructose (LDL: +0.29 ± 0.082 mmol/liter, P = 0.0023; apoB: +0.093 ± 0.022 g/liter, P = 0.0005) and HFCS (LDL: +0.42 ± 0.11 mmol/liter, P < 0.0001; apoB: +0.12 ± 0.031 g/liter, P < 0.0001) but not glucose (LDL: +0.012 ± 0.071 mmol/liter, P = 0.86; apoB: +0.0097 ± 0.019 g/liter, P = 0.90). Conclusions: Consumption of HFCS-sweetened beverages for 2 wk at 25% E increased risk factors for cardiovascular disease comparably with fructose and more than glucose in young adults.

金沢大学医学系研究科, Background: The relationship between plasma angiopoietin-like protein 3 (ANGPTL3), and lipoprotein lipase (LPL) activity and hepatic triglyceride lipase (HTGL) activity has not been investigated in the metabolism of remnant lipoproteins (RLPs) and high-density lipoprotein (HDL) in human plasma. Methods: ANGPTL3, LPL activity, HTGL activity, RLP-C and RLP-TG and small, dense LDL-cholesterol (sd LDL-C) were measured in 20 overweight and obese subjects in the fasting and postprandial states. Results: Plasma TG, RLP-C, RLP-TG and sd LDL-C were inversely correlated with LPL activity both in the fasting and postprandial states, but not correlated with HTGL activity and ANGPTL3. However, plasma HDL-C was positively correlated with LPL activity both in the fasting and postprandial states, while inversely correlated with HTGL activity. ANGPTL3 was inversely correlated with HTGL activity both in the fasting and postprandial states, but not correlated with LPL activity. Conclusion: HTGL plays a major role in HDL metabolism, but not RLP metabolism. These findings suggest that ANGPTL3 is strongly associated with the inhibition of HTGL activity and regulates HDL metabolism, but not associated with the inhibition of LPL activity for the metabolism of RLPs in human plasma.

Background We found a unique cholesteryl ester transfer protein (CETP) deficient case with markedly elevated serum triglyceride (TG) as well as high-density lipoprotein cholesterol (HDL-C) levels. Most of the CETP deficiency cases were reported to have normal or reduced serum TG with elevated HDL-C. Methods The case subject was a 40-year-old male with a compound heterozygous CETP deficiency. Two heterozygous CETP deficient cases and 10 normal volunteers were also recruited as controls. They underwent an oral fat tolerance test (OFTT) and their blood was taken at fasting and during the OFTT to be used for laboratory tests. Results The case subject had apolipoprotein E (apo-E) phenotype 4/2 with fatty liver but without any cardiovascular disease. His serum TG, HDL-C, apo-AI and apo-B48 levels were significantly higher, but the low-density lipoprotein cholesterol level was lower than controls. Although post-heparin plasma lipoprotein lipase and hepatic lipase (both mass and activity) were nearly normal, the serum level of angiopoietin-like-protein-3 was extremely elevated. While his serum remnant-like particles-TG (RLP-TG) and total TG levels significantly increased after a fat load, the RLP-cholesterol (RLP-C) level did not increase during OFTT. Conclusions The case subject was different from the common CETP deficient cases reported previously. Also, the results indicated that the metabolic pathways of RLP-C and RLP-TG formation in the postprandial state are controlled independently in CETP deficient cases. CETP deficiency itself may not be atherogenic, while one with elevated RLPs may be atherogenic. These cases may have raised the controversy of whether CETP deficiency is atherogenic or not.

BackgroundTwo recent publications report that non-fasting triglycerides concentrations in plasma are more predictive of cardiovascular events than conventional measurements of fasting triglycerides. While these observations are consistent with the previous studies, direct correlations between remnant lipoprotein triglyceride (RLP-TG) and remnant lipoprotein cholesterol (RLP-C), which are also considered to be risk factors for cardiovascular disease, and fasting and postprandial TG have not been investigated.MethodsOn four different days, both fasting and postprandial blood samples were collected from twenty-three overweight to obese men and women at UC Davis and analyzed for plasma concentrations of TG, RLP-C and RLP-TG.ResultsSignificantly higher correlations between plasma TG and RLPs were observed in the postprandial state (RLP-C r2 = 0.85; RLP-TG r2 = 0.92) than in the fasting state (RLP-C r2 = 0.61; RLP-TG r2 = 0.73). The differences in the correlations between the fasting and postprandial TG and RLPs were statistically significant (p < 0.001). The increase of RLP-TG (postprandial RLP-TG minus fasting RLP-TG) consisted of approximately 80% of the total increase of TG (postprandial TG minus fasting TG).ConclusionPostprandial TG vs remnant lipoprotein concentrations were significantly more correlated when compared with fasting TG vs RLP concentrations. The increased TG in the postprandial state mainly consisted of TG in remnant lipoproteins. Therefore, the increased sensitivity of non-fasting TG in predicting the risk for cardiovascular events may be directly explained by the increase of remnant lipoproteins in the postprandial state.

The role of CETP in the development of atherosclerosis is debatable, and few data exist regarding the total impact of CETP inhibition on cholesterol efflux.Acceptor capacities of whole serum and HDL subfractions separated by HPLC were compared using 2 different cell systems. Subjects with CETP deficiency (2 homozygous, 1 compound heterozygous, and 5 heterozygous) were analyzed along with 10 normolipidemic controls. The fractional efflux from cholesterol-labeled Fu5AH hepatoma cells was determined to be SR-BI mediated. The efflux difference between control and liver X receptor (LXR) agonist-induced ABCA1-upregulated J774 macrophages was considered as a measure of ABCA1-mediated efflux.For the Fu5AH cell system, the total acceptor capacities of whole serum and HPLC-separated HDL fraction 2 obtained from the homozygous subjects were 38% and 116% higher than the corresponding values for the controls, respectively (p0.05). For the J774 cell system, the total acceptor capacities of whole serum and HPLC-separated HDL fractions were similar among the CETP-deficient subjects and controls.Serum from homozygous subjects with CETP-null defects exhibited enhanced acceptor capacity via an SR-BI dependent pathway, which is regulated by the middle HPLC-separated HDL fraction. Further, the cholesterol acceptor capacity of serum obtained from patients having complete and partial CETP deficiency was preserved via an ABCA1-dependent pathway.

Background Dietary salt restriction has been reported to adversely modify the plasma lipoprotein profile in hypertensive and in normotensive subjects. We investigated the effects of the low sodium intake (LSI) on the plasma lipoprotein profile and on inflammation and thrombosis biomarkers during the fasting and postprandial periods. Methods Non-obese, non-treated hypertensive adults (n = 41) were fed strictly controlled diets. An initial week on a control diet (CD, Na = 160 mmol/day) was followed by 3 weeks on LSI (Na = 60 mmol/day). At admission and on the last day of each period, the 24-h ambulatory blood pressure was monitored and blood was drawn after an overnight fasting period and after a fat-rich test meal. Results The dietary adherence was confirmed by 24-h urinary sodium excretion. Fasting triglyceride (TG), chylomicron-cholesterol, hsC-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) concentrations, renin activity, aldosterone, insulin, and homeostasis model assessment insulin resistance (HOMA-IR) values were higher, but non-esterified fatty acids (NEFA) were lower on LSI than on CD. For LSI, areas under the curve (AUC) of TG, chylomicron-cholesterol, apoB and the cholesterol/apoB ratio were increased, whereas AUC-NEFA was lowered. LSI did not modify body weight, hematocrit, fasting plasma cholesterol, glucose, adiponectin, leptin, fibrinogen and factor VII (FVII), and AUC of lipoprotein lipase and of lipoprotein remnants. Conclusion LSI induced alterations in the plasma lipoproteins and in inflammatory markers that are common features of the metabolic syndrome.

Background ApoB-48 is a major apolipoprotein secreted by the small intestine and is the main constitutive apolipoprotein in chylomicrons (CM). In the past, presence of apoB-48 in human aortic atherosclerotic plaques has not been detected. Methods A newly developed apoB-48 ELISA together with an HPLC fractionation technique, were applied to investigate the presence of apoB-48 (CM) in aortic atherosclerotic plaques. The atherosclerotic plaques were obtained from aortae of sudden cardiac death cases. Total cholesterol, triglycerides (TG), apoB-100 and apoB-48 were measured in the aortic plaques extracts. Results HPLC analysis of plaques extracts monitored by cholesterol revealed mainly particle sizes of CM and very low density lipoproteins (VLDL) in the d > 1.006 fractions. The plaques extracts were monitored by apoB-48 and apoB-100 ELISA. There were no TG peaks in any lipoprotein fraction extracted from the plaques except as free glycerol. ApoB-100 was detected in VLDL particles and in LDL sizes. In contrast, apoB-48 was detected in particles of CM, VLDL and LDL sizes. Further, in postmortem plasma, apo B-48 was detected in particles sizes of HDL or smaller and the Western blot analysis could not show any 250 kDa molecular weight (MW) protein in the plaque extracts, but smaller and broader MW staining were observed at 20–150 kDa. Conclusion Hitherto there has been lack of an appropriate assay to measure apoB-48 in plaques. Our investigations show that apoB-48 is present in atherosclerotic plaques with denatured or degraded structure. This is the first report describing presence of apoB-48 in human atherosclerotic plaques.

Background Hypertriglyceridemia was recently shown to be a risk factor for prostate cancer; however, there are only a few reports about the relationship between prostate cancer and TG (triglycerides) rich lipoproteins. Remnant lipoproteins (RLP) are TG-rich lipoproteins, which are produced by the hydrolysis of very low density lipoproteins and chylomicrons. We examined the direct effect of RLP on the proliferation and signal transduction of prostate cancer cells. Methods RLP were isolated from human serum with an immunoaffinity mixed gel containing anti-apoA-1 and anti-apoB-100. We evaluated RLP-induced cell proliferation by using MTS assay. Moreover we examined the direct effect of RLP on the MAPK and Akt signal transductions which are reported to be correlated with prostate cancer by using Western blotting. Results Incubation in the presence of RLP for 48 h induced the proliferation of prostate cancer PC-3 cells more significantly than prostate cancer LNCaP cells and human prostate stromal cells. In PC-3 cells, RLP also induced the phosphorylation of MEK/ERK via a G protein-coupled receptor-protein kinase C dependent pathway. Moreover, activation of Akt pathway was observed after RLP treatment of PC-3. Conclusion These findings suggested that hypertriglyceridemia, especially remnant hyperlipoproteinemia, might be one of the progressive factors for prostate cancer.

Neurodevelopmental abnormalities have been reported in studies on the pathogenesis of schizophrenia. The Wnt-signaling pathway has been implicated in a variety of processes in neurodevelopment, and the frizzled proteins have been identified as receptors for Wnt ligands. Of the frizzled proteins, frizzled-3 (FZD3) is required for formation of the neural crest and for development of major fiber tracts in the CNS. The human FZD3 gene is located on chromosome 8p21, a positive linkage locus for schizophrenia. We analyzed polymorphisms of the FZD3 gene in patients with schizophrenia and control subjects in the Japanese population. We found a significant association between schizophrenia and the FZD3 gene in single nucleotide polymorphisms and haplotype analyses. Our data suggest that dysregulation of the Wnt-signaling pathway may be involved in the susceptibility to schizophrenia.

Familial hypobetalipoproteinemia (FHBL) and abetalipoproteinemia (ABL) are rare inherited forms of hypolipidemia. Their differential diagnosis is important for predicting of the prognosis and selecting appropriate therapy.Genetic analysis was performed in two patients with primary hypocholesterolemia born from consanguineous parents. The oral fat tolerance test (OFTT) was performed in one patient with FHBL (apoB-87.77) and one with ABL as well as in four normal control subjects. After overnight fasting, blood samples were drawn. Serum lipoprotein and remnant-like particle (RLP) fractions were determined by HPLC analysis.Both patients with homozygous FHBL were asymptomatic probably because of preserved levels of fat-soluble vitamins, especially vitamin E. The patients with FHBL were homozygous because of novel apoB-83.52 and apoB-87.77 mutations, and although one of them (apoB-87.77) had fatty liver disease, microscopic findings suggesting nonalcoholic steatohepatitis were absent. Fasting apoB-48 and RLP-triglyceride levels in the patient with homozygous FHBL, which were similar to those in normal control subjects, increased after OFTT both in normal control subjects and the patient with FHBL but not in the patient with ABL, suggesting that the fat load administered was absorbed only in the patient with FHBL.Although lipid levels in the patients with homozygous FHBL and ABL were comparable, fasting, postoral fat loading of apoB-48, as well as RLP-triglyceride levels, may help in the differential diagnosis of FHBL and ABL and provide a prompt diagnosis using genetic analysis in the future.

Background: Estrogen administration is known to increase serum triglyceride concentrations. This study measured changes in lipoproteins of patients with prostate cancer treated with estrogen to determine whether the increased triglyceride concentrations are associated with atherogenic lipoprotein patterns. Methods: Fifteen patients (52–87 years) with histologically diagnosed prostate cancer received diethylstilbestrol diphosphate (250 mg/day). Serum samples were collected before and after 1 and 2 weeks of treatment. Cholesterol and triglyceride profiles of major lipoproteins were determined by HPLC, remnant-like particle cholesterol and triglyceride concentrations by an immunoseparation technique, and apolipoproteins by immunologic methods. Results: Estrogen treatment induced a 63.3% increase in total triglyceride concentrations, which occurred in all major lipoprotein classes with significant increases in HDL-triglycerides (130.4%), LDL-triglycerides (60.7%) and VLDL-triglycerides (56.2%). HDL-cholesterol increased significantly by 26.8%, while LDL-cholesterol decreased (15.6%). Remnant-like particle triglyceride concentrations also increased significantly by 77%, whereas remnant-like particle cholesterol concentrations remained unchanged. Apolipoproteins A-I and A-II increased; apolipoprotein E and Lp(a) decreased. Conclusions: The techniques used here conveniently demonstrated that short-term estrogen treatment in prostate cancer patients resulted in triglyceride enrichment of all major lipoprotein classes but did not induce changes in the lipoprotein profiles generally recognized as increasing risk for cardiovascular disease, except for the elevation of plasma triglyceride and remnant-like particle triglyceride.

To assess the usefulness of remnant-like particles (RLPs), which represent potentially atherogenic lipoprotein remnants, as an index of postprandial hyperlipidemia, RLPs and other lipids were measured in 19 healthy adults (23-44 years old) after ingestion of a test meal (fat content, 32.9%). The lipoprotein derivatives, with a marked increase after fat-loading, were triglycerides, retinyl palmitate and RLPs. The retinyl palmitate peak lagged behind the RLP and triglyceride peaks, indicating that it may have different kinetics than RLP and triglyceride. While triglycerides are considered as an index of atherosclerotic risk factor, RLPs also may be a suitable index for evaluating postprandial hyperlipidemia. RLPs remained high even 8 h after fat-loading, suggesting that the postprandial state persists for almost the whole day. Accordingly, it may be important to assess postprandial remnant concentrations.

A 41-year-old woman and her 2 sons had Type V hyperlipoproteinemia. Interestingly, the patient and her younger son had higher levels of remnant-like particle (RLP)-cholesterol than her elder son; the former two had apolipoprotein E phenotype E3/E4, while the latter had E3/E3. Hyper-RLP-ch levels may be associated with the apolipoprotein E4 phenotype. Moreover, after administration of bezafibrate, the patient's triglyceride, RLP-cholesterol and RLP-triglyceride levels decreased markedly and the chylomicron fraction disappeared. Further studies may be necessary to determine if hypertriglyceridemic patients with or without apolipoprotein E4 show a greater reduction in serum TG levels with lipid-lowering agents.

Phenotype of autosomal recessive hypercholesterolaemia (ARH), a rare lipid disorder, is known to be milder than that of homozygous familial hypercholesterolaemia (FH) with LDL receptor gene mutation. However, few data exist regarding the functional differences in ARH and FH particularly in terms of remnant-like particles' (RLP) metabolism.Blood sampling was performed up to 6h after OFTT cream loading (50 g/body surface area) with 2-h intervals in a single ARH proband, four heterozygous FH patients with LDL receptor gene mutation and four normal controls. Plasma lipoprotein and RLP fraction were determined by HPLC system. The area under curve (AUC) of each lipoprotein including RLP fractions was evaluated.The AUC of TG, RLP cholesterol (RLP-C) and RLP triglyceride (RLP-TG) levels of heterozygous FH subjects was significantly higher than those of controls (466±71 mg/dL×h vs. 303±111 mg/dL×h, P0·05; 35±7 mg/dL×h vs. 21±8 mg/dL×h, P0·05; 124±57 mg/dL×h vs. 51±13 mg/dL×h, P0·05, respectively). Under these conditions, those values of ARH were close to those of controls (310 mg/dL×h, 22 mg/dL×h, 23 mg/dL×h, respectively).These data demonstrate that unlike in FH, RLP clearance is preserved in ARH. The preservation of post-prandial RLP clearance may contribute to the mild phenotype of ARH compared with FH.

Remnant-like lipoprotein particles (RLP) have been measured by cholesterol as RLP-C for CHD risk assessment in the fasting plasma. However, RLP-triglyceride (TG) is a better marker of the characteristics of remnant lipoproteins in the postprandial plasma, especially in plasma with TG concentrations150 mg/dl.The RLP-TG and RLP-C concentrations in subjects undergoing a health check-up and in volunteers receiving an oral fat load were determined in the fasting and postprandial plasma. TC, TG, HDL-C, LDL-C, apoB 100, apoB48, RLP apoB-100 and RLP apoB48 were also determined.When fasting TG concentrations were150 mg/dl, the 95th percentile of RLP-TG was 20mg/dl and the RLP-C 7.5 mg/dl in healthy subjects. The prevalence of RLP-TG and RLP-C above the cut-off values with a TG concentration150 mg/dl was significantly higher in the metabolic syndrome cases than in the controls. RLP-TG increased significantly in plasma to20mg/dl after an oral fat load in cases with TG concentrations80 mg/dl. Further, RLP apoB100, but not RLP apoB48 was highly correlated with the increase of TG in the postprandial plasma.RLP-TG and RLP-C were increased significantly above the cut-off values in the postprandial plasma in healthy volunteers from a TG concentration80 mg/dl. RLP apoB100, but not RLP apoB48, increased significantly when the plasma TG increased after an oral fat load despite the increase of plasma apoB48. The results show that the major lipoproteins which were increased in postprandial plasma were VLDL remnants, not CM remnants.

Metallothionein (MT) is known to be involved in various physiological roles and diseases. However, a standard method for MT measurement has not been established until recently. Therefore, we have developed an easy and specific enzyme-linked immunosorbent assays (ELISA) to determine MT-1 and MT-2. In order to evaluate the method we developed, MT-1/2 in liver, kidney and brain was determined in wild type (WT), MT-1/2 knockout (KO) and MT-3 KO mice, with and without Cd treatment. MT 1/2 in urine was determined in genetically disordered LEC rats (an animal model of Wilson disease). MT-1/2 concentrations in the liver, kidney and brain in MT-1/2 KO mice were significantly lower compared to those of WT and MT-3 KO mice. MT-1/2 concentrations in the livers of WT mice significantly increased with Cd administration, but not in MT-1/2 KO mice. Similar results were observed by immunohistochemical staining. To confirm the molecular weight (MW) of MT detected in organs by the ELISA, analysis with a Sephadex G-75 was performed. Two peaks of MT-1/2 (MW small and large) were detected in WT and MT-3 KO mice. The small MT peak was mostly depleted in MT 1/2-KO mice, while a large MT peak remained. A significant increase in MT-1/2 concentration was detected in the urine of LEC rats with age and especially at the hepatitis stage. In conclusion, MT-1/2 ELISA and immunohistochemical staining was highly correlated with MT-1/2 determination in experimental animal specimens and could be a robust analytical tool for physiological and toxicological studies.

Type III is a remnant hyperlipoproteinemia identified by the presence of beta-VLDL (remnant lipoprotein) as well as a genetic variant of apo E (apo E2/2). The RLP isolated from the serum of Type III patients by a new method we have developed, the RLPcholesterol assay, was identified as chylomicron and VLDL remnant. In addition, the RLP-C levels of the Type III patients were significantly higher than other hyperlipidemic patients with similar serum TG levels, while the ratio of TC/TG in RLP-C of both groups was not significantly different. The RLP-cholesterol assay appears to be useful for the screening and monitoring of Type III hyperlipoproteinemia when used in conjunction with the assays of serum TG level and genetic apo E isoform analysis.

Background Comparison of the reactivity of remnant-like lipoprotein particles (RLP) and LDL particles to LDL receptor and VLDL receptor has not been investigated. Methods LDL receptor- or VLDL receptor-transfected ldlA-7, HepG2 and L6 cells were used. Human LDL and rabbit β-VLDL were isolated by ultracentrifugation. Human RLP was isolated using an immunoaffinity mixed gel. The effect of statin on lipoprotein receptors was examined. Results Both LDL receptor and VLDL receptor recognized RLP. In LDL receptor transfectants, RLP, β-VLDL and LDL all bound to LDL receptor. Cold RLP competed efficiently with DiI-β-VLDL; however, cold LDL competed weakly. In VLDL receptor transfectants, RLP and β-VLDL bound to VLDL receptor, but not LDL. RLP bound to VLDL receptor with higher affinity than β-VLDL because of higher apolipoprotein E in RLP. LDL receptor expression was induced in HepG2 by the low concentration of statin while VLDL receptor expression was induced in L6 myoblasts at higher concentration. Conclusions RLP are bound to hepatic LDL receptor more efficiently than LDL, which may explain the mechanism by which statins prevent cardiovascular risk by primarily reducing plasma RLP rather than by reducing LDL. Additionally, a high-dose of statins also may reduce plasma RLP through muscular VLDL receptor.

Since Zilversmit first proposed postprandial lipemia as the most common risk of cardiovascular disease, chylomicrons (CM) and CM remnants have been thought to be the major lipoproteins which are increased in the postprandial hyperlipidemia. However, it has been shown over the last two decades that the major increase in the postprandial lipoproteins after food intake occurs in the very low density lipoprotein (VLDL) remnants (apoB-100 particles), not CM or CM remnants (apoB-48 particles). This finding was obtained using the following three analytical methods; isolation of remnant-like lipoprotein particles (RLP) with specific antibodies, separation and detection of lipoprotein subclasses by gel permeation HPLC and determination of apoB-48 in fractionated lipoproteins by a specific ELISA. The amount of the apoB-48 particles in the postprandial RLP is significantly less than the apoB-100 particles, and the particle sizes of apoB-48 and apoB-100 in RLP are very similar when analyzed by HPLC. Moreover, CM or CM remnants having a large amount of TG were not found in the postprandial RLP. Therefore, the major portion of the TG which is increased in the postprandial state is composed of VLDL remnants, which have been recognized as a significant risk for cardiovascular disease.

Background Particle size of apoB-48 carrying lipoproteins in remnant-like lipoprotein particles (RLP) in postprandial plasma has not been well characterized. Methods Plasma lipids, lipoproteins and apolipoproteins in 12 healthy subjects were analysed after an oral fat load. RLP isolated by immunoaffinity gel from plasma of a normolipidaemic and a hyperlipidaemic subject in four hours after an oral fat load was fractionated by high-performance liquid chromatography (HPLC) and monitored by total cholesterol (TC), triglycerides (TG), apoB-48 and apoB-100. Results TC, low-density lipoprotein (LDL)-C and apoB did not change after an oral fat load, while TG, RLP-C, RLP-TG and apoB-48 increased significantly in postprandial plasma. HPLC profiles monitored by TC and TG revealed that major lipoproteins increased in RLP after an oral fat load was VLDL size particles. The percentage of RLP-TG in total TG and the ratio of RLP-TG/RLP-C were significantly increased in four hours after an oral fat load compared with the fasting state ( P < 0.01). RLP in four hours after an oral fat load fractionated by HPLC and monitored by TC, TG, apoB-48 and apoB-100 revealed that VLDL size or smaller particles were the major lipoproteins. Conclusions ApoB-48 carrying lipoproteins in RLP isolated from a normolipidaemic and a hyperlipidaemic subject after an oral fat load showed a similar particle size with apoB-100 carrying VLDL remnants. Therefore, the most apoB-48 carrying particles found in postprandial RLP can be classified as CM remnants. The majority of remnants in the postprandial state were not CM remnants, but VLDL remnants.

Background Serum concentration of remnant-like lipoprotein particles (RLP) have been measured by cholesterol as RLP-C for clinical diagnostic purpose. However, the measurement of TG in RLP and the ratio of RLP-TG/total TG has not been well established. Method Highly sensitive triglyceride assay reagent (TG-EX) was used for RLP-TG assay and compared with the previously used TG reagent (Determiner LTGII). Sera in health check-up populations, cardiovascular disease, diabetes and oral fat load cases were used for the evaluation of the new RLP-TG assay. Serum TC, TG, HDL-C, LDL-C and RLP-C concentrations were also determined in above cases. Results The detection limit of new RLP-TG using TG-EX was 2.0 mg/dl. The within-run imprecision (n = 10) was CV = 3.0% (RLP-TG: 4.1 mg ± 0.7 mg/dl), CV = 1.4% (RLP-TG: 42.0 ± 0.6 mg/dl) and CV = 0.5% (RLP-TG: 100.6 ± 0.6 mg/dl). Cut-off value (75 percentile) of RLP-TG determined in the fasting Japanese population was 13.1 mg/dl in men and 9.9 mg/dl in women. In patients with metabolic syndrome, cardiovascular disease and diabetes, RLP-TG levels were significantly higher than those in normal control subjects. RLP-TG levels increased significantly after an oral fat load and the ratio of RLP-TG/total TG increased > 3-fold compared to the ratio in the fasting state. Approximately 80% of TG increased after an oral fat load was TG derived from remnant lipoproteins. Conclusion Normal range of plasma RLP-TG in the fasting Japanese population was first determined using a highly sensitive TG assay reagent. RLP-TG was shown to be higher in cases with metabolic syndrome, cardiovascular disease, etc and a better marker than RLP-C for the measurement of postprandial remnant lipoproteins, together with total TG for RLP-TG/total TG ratio.

Studies in animals have documented that, compared with glucose, dietary fructose induces dyslipidemia and insulin resistance. To assess the relative effects of these dietary sugars during sustained consumption in humans, overweight and obese subjects consumed glucose- or fructose-sweetened beverages providing 25% of energy requirements for 10 weeks. Although both groups exhibited similar weight gain during the intervention, visceral adipose volume was significantly increased only in subjects consuming fructose. Fasting plasma triglyceride concentrations increased by approximately 10% during 10 weeks of glucose consumption but not after fructose consumption. In contrast, hepatic de novo lipogenesis (DNL) and the 23-hour postprandial triglyceride AUC were increased specifically during fructose consumption. Similarly, markers of altered lipid metabolism and lipoprotein remodeling, including fasting apoB, LDL, small dense LDL, oxidized LDL, and postprandial concentrations of remnant-like particle-triglyceride and -cholesterol significantly increased during fructose but not glucose consumption. In addition, fasting plasma glucose and insulin levels increased and insulin sensitivity decreased in subjects consuming fructose but not in those consuming glucose. These data suggest that dietary fructose specifically increases DNL, promotes dyslipidemia, decreases insulin sensitivity, and increases visceral adiposity in overweight/obese adults.

Background: Hypertriglyceridemia has been shown to be one of the risk factors for prostate cancer. In this study, we investigated the effect of remnant lipoproteins on cell growth in prostate cancer cell lines. Methods: Remnant lipoproteins were isolated as remnant like particles (RLP) from human plasma. We used RLP for TG-rich lipoproteins and low density lipoproteins (LDL) for cholesterol-rich lipoproteins respectively and examined the effect of lipoproteins on proliferation of PC-3 and LNCaP cells using MTS assays. Moreover, we studied the effect of RLP and LDL treatment on the regulation of lipoprotein receptors in prostate cancer cells to investigate the relationship between lipoprotein-induced cell proliferation and lipoprotein receptor expression using real-time PCR, Western blotting assays and siRNA. Results: RLP effectively induced PC-3 cell proliferation more than LDL, whereas both RLP and LDL could not induce LNCaP cell proliferation except at a higher concentration of RLP. LDL receptor (LDLr) was expressed in both prostate cancer cells but there was a sharp difference of sterol regulation between two cells. In PC-3 cells, LDL decreased the LDLr expression in some degree, but RLP did not. Meanwhile LDLr expression in LNCaP was easily downregulated by RLP and LDL. Blocking LDLr function significantly inhibited both RLP- and LDL-induced PC-3 cell proliferation. Conclusions: This study demonstrated that RLP-induced PC-3 cell proliferation more than LDL; however, both RLP and LDL hardly induced LNCaP cell proliferation. The differences of proliferation by lipoproteins might be involved in the regulation of LDLr expression.

The effect of glucose concentration and insulin on glucose incorporation was studied in primary cultures of rat hepatocytes. The rate of glucose incorporation into hepatocytes was proportional to the medium glucose concentration from 100 to 800 mg/dl. At 800 mg/dl glucose the rate reached a plateau. Of the glucose taken up by hepatocytes, 16 and 18% was incorporated into glycogen and lipid, respectively, and 58% into the nucleotide fraction after incubation for 4 h. In the medium, lactate was the major product found. Insulin stimulates glucose incorporation by 20–112% into all the above pathways at glucose concentrations between 100 and 800 mg/dl. The insulin effect was noted as early as 2–4 h (early effect) and up to 24 h (delayed effect). This effect of insulin was observed to be dose dependent from 5 to 200 ng/ml insulin. While the delayed insulin effect was abolished by cycloheximide, the early effect of insulin was not affected. With respect to the key enzyme activities of glucose utilization, activation of glycogen synthase (increase of I-activity/total activity) and pyruvate kinase (activation at 0.2 mM phosphoenolpyruvate) was noted 4 h after insulin addition, and these effects were not abolished by cycloheximide. These two enzymes increased in total activity after 24 h. Both glucokinase and glucose-6-phosphate dehydrogenase activities increased by 30–35% and 65–93% at 4 and 24 h, respectively. The results indicate that hepatocytes directly utilize glucose in a dose-dependent manner with respect to glucose and insulin. A major early and delayed effect of insulin appeared due to the activation and induction of the key hepatic enzymes of glucose utilization, respectively.

Background —Remnant lipoproteins are atherogenic, but assays of remnants have not been available in routine clinical laboratories because of the lack of practical and validated methods. A simple and reliable method for such an assay, using an immunochemical approach, has recently been developed. This study prospectively examined whether remnant lipoprotein levels in fasting serum, measured by our method, may have prognostic value in patients with coronary artery disease (CAD). Methods and Results —Remnant lipoprotein levels in fasting serum were measured in 135 patients with CAD by an immunoaffinity mixed gel containing anti-apolipoprotein (apo) A-1 and anti–apoB-100 monoclonal antibodies. Patients were followed up for ≤36 months until occurrence of 1 of the following clinical coronary events: recurrent or refractory angina pectoris requiring coronary revascularization, nonfatal myocardial infarction, or cardiac death. Kaplan-Meier analysis demonstrated a significantly higher probability of developing coronary events in patients with the highest tertile of remnant levels (>5.1 mg cholesterol/dL; 75th percentile of distribution of remnant levels) than in those with the lowest tertile of remnant levels (≤3.3 mg cholesterol/dL; 50th percentile of the distribution). Higher levels of remnants were a significant and independent predictor of developing coronary events in multivariate Cox hazard analysis including the following covariates: extent of coronary artery stenosis, age, sex, smoking, hypertension, diabetes mellitus, hypercholesterolemia, low HDL cholesterol, and hypertriglyceridemia. Conclusions —Higher levels of remnant lipoproteins in fasting serum predict future coronary events in patients with CAD independently of other risk factors. Thus, measurement of fasting remnant levels, assessed by the current immunoseparation method, may be helpful in assessment of CAD risk.

Apolipoprotein (apo) B subspecies were separated by high-performance liquid chromatography (HPLC) to analyze Apo B-48 contents in very-low-density lipoproteins (VLDL) more easily. Apo B-100 and B-48 were eluted through two connected column of Shim-pack Diol-300 at a retention time of 24 min and 32.3 min, respectively. The molecular masses estimated by this method were approximately 600 kDa in apo B-100, 220 kDa in apo B-48. % apo B-48 in total apo B (% B-48) determined by measuring peak area of HPLC-separated apo B in a healthy subject was 76% in chylomicrons, 13% in fasting VLDL, and 20% in 2 h-postprandial VLDL. No peak of apo B-48 was detected in LDL. Recovery of Apo B determined by re-chromatography of HPLC-separated sample was 91 +/- 4.0% and 95 +/- 3.6% in Apo B-100 and apo B-48, respectively. 125I-labeled apo B in VLDL were also analyzed by both HPLC and SDS-PAGE. Percent radioactivity of apo B-48 fraction in the total apo B determined by the HPLC (17.3%) was similar to the value obtained through the measurement of radioactivity separated by the SDS-PAGE (17.6%). Coefficient of variation in % B-48 determined by the peak area was 2.5%. Percent B-48 determined by the HPLC method was significantly correlated with % B-48 by SDS-PAGE, but 6-8 times higher, which might be accounted for in part by the reported difference of chromogenicity between apo B-100 and apo B-48. % B-48 in VLDL separated from fasting plasma were 17.1 +/- 5.6% in 14 healthy subjects, and positively related to VLDL triglyceride and cholesterol concentrations. Apo B isoprotein analysis by the HPLC method is reliable and easy to perform for studying apo B metabolism in triglyceride-rich lipoproteins in physiological as well as pathological conditions.

Plasma cholesteryl ester transfer protein (CETP) mediates CE/TG exchange among various lipoproteins. CETP deficiency results in low LDL and high HDL phenotype including apoE-rich large HDL. Large HDL could provide apoE to chylomicron/VLDL during lipolysis in post-prandial state, accelerating remnant lipoprotein uptake in the liver. To determine the effects of low CETP levels on post-prandial lipoprotein metabolism, lipid levels of plasma remnant-like lipoprotein particles (RLP) fraction were determined in one homozygous and three heterozygous CETP deficiency and controls with apoE3/3 phenotype. After oral fat-load, the area under curve (AUC) of TG levels were remarkably decreased in CETP deficiency as compared to controls (423+/-187 [S.D.] mg/dl x h in three heterozygous CETP deficiency and 926+/-268 [S.D.] in 10 controls, P=0.012). Similarly, the homozygote had a low AUC of TG levels (416 mg/dl x h). Plasma RLP-cholesterol levels were decreased in heterozygotes, but not significantly as compared to controls (P=0.14). HPLC analysis showed that increased RLP-cholesterol level was not due to conventional VLDL-LDL size RLP, but to those in large HDL size in the homozygote. In heterozygotes, bimodal distribution of RLP-cholesterol level was found in lipoprotein sizes of conventional VLDL-LDL and large HDL. Subjects with CETP deficiency appeared to have low levels of TG response and diminished remnant lipoprotein formation after fat-load.

A new cookie test was developed for the simultaneous evaluation of multiple risk factors such as glucose intolerance, hyperinsulinemia, insulin resistance and postprandial dyslipidemia. The cookie consisting of 75 g carbohydrate and 25 g fat is ingested and the blood samples are obtained at 0, 1 and 2 hours later. When the two carbohydrate sources, liquid glucose and test cookie, were compared as a glucose load within 3 months, the 2 hr plasma glucose levels were not statistically different, proposing the use of the same criteria at 2 hour glucose level for the diagnosis of diabetes and impaired glucose tolerance (IGT) in subjects without exocrine pancreatic dysfunction. In addition, hyperinsulinemia, insulin resistance (AUC insulin, and/or AUC insulin X AUC glucose), and postprandial hyperlipidemia (DeltaTG, Triglyceride; DeltaRLP, remnant like particles) have been simultaneously uncovered. Reactive hypoglycemia with adverse epigastric discomfort was observed in 26.3% of the control subjects with liquid glucose, while it was observed in only 1 case (5.3%) without any symptom with cookie tests. In fact, one reactive hypoglycemia out of 5 with liquid glucose turned out to be IGT with cookie test. In 64 subjects with lifestyle-related diseases, cookie test revealed hyperinsulinemia and insulin resistance in 56% respectively, postprandial hyperlipidemia in 39%, diabetes and IGT in 22-23% of each of the subjects and all showed at least one abnormal value. In contrast, in university students with exercise habit, all showed normal results with cookie test. In addition, improved insulin sensitivity over non-exercise group was obverved. In summary, the cookie test provided more informations compared with OGTT using liquid glucose and with fewer side effects. Simultaneous evaluation of glucose intolerance, hyperinsulinemia, insulin resistance, and postprandial hyperlipidemia was also possible.

Background Cholesteryl ester transfer protein (CETP) is suggested to be involved in the cholesterol level in remnant like lipoprotein particles (RLP), but there is no direct evidence that CETP increases cholesterol-rich RLP in plasma. Methods Human plasma was incubated with or without HDL containing [ 3 H]-labeled cholesteryl ester ([ 3 H]CE), recombinant CETP or CETP inhibitors at 37 °C in vitro . Results The RLP–cholesterol (RLP–C) level increased time-dependently and the amount of RLP–C increase ( Δ RLP–C) by the incubation was positively correlated with triglyceride (TG) level in plasma ( r = 0.597, P = 0.0070). [ 3 H]CE in HDL was transferred to RLP fraction under 37 °C incubation, and the amount of [ 3 H]CE transferred to RLP correlated significantly with Δ RLP–C in plasma ( r = 0.611, P = 0.0156). Human recombinant CETP enhanced the RLP–C increase, while CETP inhibitor JTT-705 and anti-human CETP monoclonal antibody inhibited both the RLP–C increase and [ 3 H]CE transfer to RLP. On the other hand, an inhibition of lecithin: cholesterol acyltransferase (LCAT) did not affect the RLP–C increase. In triglyceride-rich lipoproteins (TRL) fraction, JTT-705 inhibited [ 3 H]CE transfer to RLP more strongly than that to non-RLP. Conclusions CETP promotes the formation of cholesterol-rich RLP through the transfer of CE from HDL to TRL and CETP inhibitors are useful to reduce RLP–C.

A tremendous number of articles on oxidized LDL (Ox-LDL) and scavenger receptor in macrophage have been published since Steinberg proposed Ox-LDL hypothesis as the major cause of atherosclerosis. This hypothesis has provided strong support for the efficacy of LDL lowering drugs, indicating that lowering LDL means lowering Ox-LDL in vivo. This manuscript proposed a new oxidative modification hypothesis that remnant lipoproteins determined as remnant-like lipoprotein particles (RLP), not LDL are the major oxidized lipoproteins in plasma, resulting from the plasma concentration of these oxidized lipoproteins. Remnant lipoproteins may play a pivotal role for the initiation of atherosclerosis via lectin-like oxidized LDL receptor-1 (LOX-1) in endothelial cells. Isolated remnant lipoproteins were found to be oxidized or susceptible to be oxidized in plasma, not necessary to be further oxidized in vitro as Ox-LDL. High similarity of proatherogenic and proinflammatory properties of isolated Ox-LDL and remnant lipoporteins have been reported and predicted the presence of similar oxidized phospholipids in both lipoproteins as bioactive components. These results suggest the possibility that reducing plasma remnant lipoproteins rather than LDL should be the target for hyperlipidemic therapy especially in patients with metabolic syndrome for the prevention of endothelial dysfunction in the initiation of atherosclerosis.

Remnant-like particles (RLPs) have been reported to promote atherosclerosis and to have effects on platelet function. We studied the effects of RLP on shear-induced platelet activation and their inhibition by antiplatelet agents in vitro. RLP were separated using two monoclonal antibodies, anti apo B-100 and anti apo A-I. These RLP fractions were added to whole blood (WB) or platelet-rich plasma (PRP) in serial dilution of 1, 10 or 100 microg RLP triglyceride (TG) per ml of total sample volume. These samples were incubated, and then stimulated with a high shear stress of 108 dyn/cm(2). Shear-induced platelet aggregation (SIPA) was calculated from the percentage of single platelet loss. P-selectin expression on platelet surface and platelet-derived microparticle (PMP) generation were measured before and after stimulation with shear stress using flow cytometer. SIPA was significantly enhanced by RLP in WB but not in PRP. This enhancing effect was not dose-dependent and was greatest at 10 microg TG/ml. P-selectin expression induced by shear stress was only enhanced by RLP at a concentration of 100 microg TG/ml in both WB and PRP, while generation of PMP induced by shear stress was only enhanced by RLP at a concentration of 100 microg TG/ml in WB. Aspirin inhibited only the enhancement of SIPA by RLPs, while cilostazol inhibited the enhancement of not only SIPA but also p-selectin expression and PMP generation by RLPs.

Background: During pregnancy, serum cholesterol (TC) and triglyceride (TG) concentrations are known to increase significantly, but whether remnant lipoprotein particles (RLP) increase has not been shown. Methods: We compared lipid profiles in 22 healthy pregnant women to 31 healthy nonpregnant women and 24 patients with diabetes mellitus (DM), by measuring cholesterol and TG concentrations in major lipoprotein classes after HPLC separation and immunoseparation of RLP. Results: Serum TG and TC concentrations were significantly higher in the pregnant group than in the healthy control or DM groups. Cholesterol and TG concentrations of all major lipoprotein classes were also significantly higher in the pregnant group than the control and DM groups, except for VLDL-TG in the DM group. RLP-C and RLP-TG concentrations were significantly higher in the pregnant group (8.7 mg/dl and 25.4 mg/dl on average) than the control group (2.4 mg/dl and 5.7 mg/dl), but not different from the DM group (8.8 mg/dl and 24.1 mg/dl). RLP-TG to RLP-C ratios were similar among the three groups and correlated with the VLDL-TG to VLDL-C ratio. The percentages of RLP-C in VLDL-C and RLP-TG in VLDL-TG in the pregnant group (15.9% and 15.7%) were significantly lower than those of the control (48.5% and 35.6%) and the DM (32.7% and 20.8%) groups. Conclusions: RLP increased moderately during gestation with the increase in VLDL and TG, but the percentage of RLP in VLDL was significantly lower in the pregnant women compared with the control and DM patients, suggesting that hypertriglyceridemia in pregnancy is not primarily due to an increase in the atherogenic RLP.

Background : Plasma levels of remnant-like particles (RLP) is one of the predictive markers for coronary artery disease (CAD), and the inhibition by RLP of endothelium-dependent vasorelaxation has been reported. We attempted to clarify whether or not RLP, which inhibits endothelium-dependent vasorelaxation, affects nitric oxide (NO) production and NO synthase (eNOS) levels in cultured endothelial cells. Methods : RLP were obtained from postmortem blood of subjects who died of CAD. Modification by RLP of acetylcholine-induced relaxation of rabbit aorta, and changes in NO production and (eNOS) in cultured bovine endothelial cells were examined. Results : RLP at 750 and 1500 μg triglyceride/ml inhibited vasorelaxation, and at 5–160 μg triglyceride/ml, concentration-dependently inhibited NO production. However, (eNOS) did not decrease after incubation with RLP. Conclusion : Postmortem RLP from subjects who died of CAD do not change the amount of (eNOS), but rather, inhibits its activity and attenuates endothelium-dependent vasorelaxation.

In our recent report, it remained unclear whether or not triglyceride-rich lipoprotein remnants (RLP) were associated with the risk of sudden coronary death in younger cases without coronary atherosclerosis that were detected in about 10% of all sudden coronary death cases in Japan. These cases were categorized as 'origin unknown, but suspected to be due to coronary spasm', the so called 'pokkuri disease' in Japan. The present study population consisted of 108 sudden death cases without coronary atherosclerosis [(pokkuri disease n=57) and non-cardiac sudden death (control n=51)] aged 20-69 years from Kanagawa prefecture in Japan. All individuals had died suddenly and unexpectedly, most had no significant history of medical conditions including cardiac symptoms and had not taken medications prior to death according to their medical records. All the autopsies were performed within 12 h after death. Plasma total cholesterol (TC), triglycerides (TG), phospholipids, RLP-C and RLP-TG, VLDL-C, LDL-C, HDL-C, apolipoproteins A-I, A-II, B, C-II, C-III, E, Lp (a) and homocysteine were measured in postmortem plasma samples. The TG-rich lipoprotein remnants measured as RLP-C and RLP-TG were significantly higher in pokkuri disease compared with controls both in fasting and postprandial states (P

Hitherto triglycerides (TG) and TG-rich lipoproteins were been of limited value as surrogates for antemortem levels. We measured TG levels in postmortem plasma from sudden coronary death cases (SCD, n=91) by using two TG assays, Dry Chem TG (free glycerol was added) and the Determiner L-TG (without added free glycerol) that measured net TG. TG levels were markedly higher by the Dry Chem TG (y) vs. Determiner L-TG (x), y=1.03x+229 mg/dl. HPLC showed large amounts of free glycerol in postmortem plasma and in TG-rich lipoprotein remnants (RLP). These results were verified in a rabbit model of SCD. Further, RLP from SCD were found to be biophysically similar to those from living patients with coronary artery disease (CAD). In conclusion, postmortem plasma sampled up to 12 h after death is appropriate for measuring lipid and lipoproteins, TG and RLP-TG as surrogates for antemortem levels when a TG assay without added free glycerol is used.

Remnant-Like Particles (RLP) isolated by an immunoseparation method are heterogeneous in their physical and biochemical properties. The objective of this study was to examine the relation between RLP-triglyceride (RLP-TG) to RLP-cholesterol (RLP-C) ratio and particle size distribution in RLP-C profiles from patients with hyperlipoproteinemia by HPLC. RLP were isolated from serum samples from 147 subjects. RLP-C and RLP-TG were quantified by respective enzymatic methods. Particle sizes of the RLP were measured using HPLC with 4 connected TSKgel LipopropakXL columns. Based on HPLC profiles of RLP-C from individual subjects, three different types were classified: predominantly LDL, predominantly VLDL, and mostly VLDL types. All patients with type III hyperlipidemia were mostly VLDL type but with smaller particle size of VLDL (32 nm) than other subjects. Severe hypertriglyceridemic (TG>4.52 mmoll(-1)) subjects were mostly VLDL type with large particle size (41 nm). As for all subjects (n=105) without predominantly LDL type, a significant correlation between RLP particle size and RLP-TG to RLP-C ratio (r=0. 432, P

Among the risk factors for coronary atherosclerosis, elevated LDL-C level is best known. The action of lipoprotein lipase on triglyceride-rich lipoproteins produces remnant lipoprotein particles enriched in cholesterol and apolipoprotein E (apo E). Apo E serves as the ligand for uptake of remnant lipoproteins via the LDL-receptor or the remnant receptor. In this study, postmortem plasma total cholesterol, triglycerides (TG), VLDL-C, HDL-C, lipoprotein (a) [Lp(a)] and remnant-like lipoprotein particles (RLP)-cholesterol, RLP-TG, apolipoproteins B, C III and E were measured, together with LDL-C to assess their potential contribution to the severity of coronary and aortic atherosclerosis of the 197 cases of sudden death (132 cardiac death and 65 non-cardiac death). In all cases, the severity of coronary atherosclerosis was determined at postmortem pathological examination. RLP-cholesterol (RLP-C) and LDL-C concentrations were significantly higher in cases with advanced coronary atherosclerosis compared with those without coronary atherosclerosis; respective median values were 13.5 vs 8.4 mg/dl (P < 0.001) and 140 vs 115 mg/dl (P < 0.05). RLP-C levels were more strongly correlated with the severity score of coronary atherosclerosis than LDL-C.