Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL.

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.

Post-prandial remnant lipoprotein metabolism in autosomal recessive hypercholesterolaemia.

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 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. 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<0·05; 35±7 mg/dL×h vs. 21±8 mg/dL×h, P<0·05; 124±57 mg/dL×h vs. 51±13 mg/dL×h, P<0·05, 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). CONCLUSION: 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.

Detection of apolipoproteins B-48 and B-100 carrying particles in lipoprotein fractions extracted from human aortic atherosclerotic plaques in sudden cardiac death cases.

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.

Remnant lipoproteins stimulate proliferation and activate MAPK and Akt signaling pathways via G protein-coupled receptor in PC-3 prostate cancer cells.

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.

Cholesteryl ester transfer protein promotes the formation of cholesterol-rich remnant like lipoprotein particles in human plasma.

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 degrees C in vitro. RESULTS: The RLP-cholesterol (RLP-C) level increased time-dependently and the amount of RLP-C increase (DeltaRLP-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 degrees C incubation, and the amount of [(3)H]CE transferred to RLP correlated significantly with DeltaRLP-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.

The oxidative modification hypothesis of atherosclerosis: the comparison of atherogenic effects on oxidized LDL and remnant lipoproteins in plasma.

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.

Effect of remnant-like particle on shear-induced platelet activation and its inhibition by antiplatelet agents.

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.

Extreme Contrast of Postprandial Remnant-Like Particles Formed in Abetalipoproteinemia and Homozygous Familial Hypobetalipoproteinemia.

BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

The human frizzled-3 (FZD3) gene on chromosome 8p21, a receptor gene for Wnt ligands, is associated with the susceptibility to schizophrenia.

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.

Hypertriglyceridemia in pregnancy does not contribute to the enhanced formation of remnant lipoprotein particles.

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.

Estrogen treatment of prostate cancer increases triglycerides in lipoproteins as demonstrated by HPLC and immunoseparation techniques.

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.

Remnant-like particles from subjects who died of coronary artery disease suppress NO synthase activity and attenuate endothelium-dependent vasorelaxation.

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 microg triglyceride/ml inhibited vasorelaxation, and at 5-160 microg 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.

Validity of plasma remnant lipoproteins as surrogate markers of antemortem level in cases of sudden coronary death.

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.

Plasma adiponectin is a more specific marker of fatty liver than a marker of metabolic syndrome in Japanese men.

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.

Metallothionein (MT) 1/2 expression in MT 1/2 and MT 3 knock-out mice and Long-Evans Cinnamon (LEC) rats.

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.

Comparative reactivity of remnant-like lipoprotein particles (RLP) and low-density lipoprotein (LDL) to LDL receptor and VLDL receptor: effect of a high-dose statin on VLDL receptor expression.

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 ß-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, ß-VLDL and LDL all bound to LDL receptor. Cold RLP competed efficiently with DiI-ß-VLDL; however, cold LDL competed weakly. In VLDL receptor transfectants, RLP and ß-VLDL bound to VLDL receptor, but not LDL. RLP bound to VLDL receptor with higher affinity than ß-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.

The characteristics of remnant lipoproteins in the fasting and postprandial plasma.

BACKGROUND: 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 concentrations <150 mg/dl. METHOD: 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. RESULTS: When fasting TG concentrations were <150 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 concentration <150 mg/dl was significantly higher in the metabolic syndrome cases than in the controls. RLP-TG increased significantly in plasma to >20mg/dl after an oral fat load in cases with TG concentrations >80 mg/dl. Further, RLP apoB100, but not RLP apoB48 was highly correlated with the increase of TG in the postprandial plasma. CONCLUSION: RLP-TG and RLP-C were increased significantly above the cut-off values in the postprandial plasma in healthy volunteers from a TG concentration >80 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.

Particle size of apoB-48 carrying lipoproteins in remnant lipoproteins isolated from postprandial plasma.

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.

Measurement of serum remnant-like lipoprotein particle-triglyceride (RLP-TG) and RLP-TG/total TG ratio using highly sensitive triglyceride assay reagent.

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 2.0mg/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 13.1mg/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.

Postprandial lipoprotein metabolism: VLDL vs chylomicrons.

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.