Phase 1 study of DS-1205c combined with gefitinib for EGFR mutation-positive non-small cell lung cancer.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) are effective for the treatment of non-small cell lung cancer (NSCLC) patients with activating mutations of the epidermal growth factor receptor (EGFR), but responses are not durable as tumors develop resistance. DS-1205c is a novel, specific, orally bioavailable, small-molecule AXL receptor TKI. In preclinical studies, DS-1205c restored TKI antitumor activity in a TKI acquired-resistance EGFR-mutant NSCLC tumor xenograft model. METHODS: This first-in-human, multicenter, open-label Phase 1 study (registered at ClinicalTrials.gov: NCT03599518) primarily evaluated the safety and tolerability of combination therapy with DS-1205c and gefitinib in Japanese patients with metastatic or unresectable EGFR-mutant NSCLC and tumor progression during treatment with EGFR-TKIs. Patients (n = 20) received DS-1205c monotherapy (200-1200 mg twice daily [BID]) in a 7-day safety monitoring period before combination DS-1205c/gefitinib (250 mg once daily) in 21-day cycles. RESULTS: The observed common treatment-emergent adverse events (TEAEs) were increased aspartate aminotransferase (35%), increased alanine aminotransferase (30%), rash maculo-papular (30%), and diarrhea (25%). No serious TEAEs were reported. Plasma concentrations and pharmacokinetic parameters of DS-1205a (free form of DS-1205c) were unaffected by concomitant administration of gefitinib. No patient achieved a complete or partial response and 5 patients (25%) had stable disease. CONCLUSION: DS-1205c was generally safe and well tolerated at all dose levels, but the safety profile of ≤800 mg BID was more favorable than 1200 mg BID. The recommended dose for dose-expansion cohorts of DS-1205c in combination therapy with gefitinib was 800 mg BID.

Novel Antibody Exerts Antitumor Effect through Downregulation of CD147 and Activation of Multiple Stress Signals.

CD147 is an immunoglobulin-like receptor that is highly expressed in various cancers and involved in the growth, metastasis, and activation of inflammatory pathways via interactions with various functional molecules, such as integrins, CD44, and monocarboxylate transporters. Through screening of CD147-targeting antibodies with antitumor efficacy, we discovered a novel rat monoclonal antibody #147D. This humanized IgG4-formatted antibody, h4#147D, showed potent antitumor efficacy in xenograft mouse models harboring the human PDAC cell line MIA PaCa-2, HCC cell line Hep G2, and CML cell line KU812, which featured low sensitivity to the corresponding standard-of-care drugs (gemcitabine, sorafenib, and imatinib, respectively). An analysis of tumor cells derived from MIA PaCa-2 xenograft mice treated with h4#147D revealed that cell surface expression of CD147 and its binding partners, including CD44 and integrin α3ß1/α6ß1, was significantly reduced by h4#147D. Inhibition of focal adhesion kinase (FAK), activation of multiple stress responsible signal proteins such as c-JunN-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38MAPK), and expression of SMAD4, as well as activation of caspase-3 were obviously observed in the tumor cells, suggesting that h4#147D induced tumor shrinkage by inducing multiple stress responsible signals. These results suggest that the anti-CD147 antibody h4#147D offers promise as a new antibody drug candidate.

Prospective analysis of the expression status of FGFR2 and HER2 in colorectal and gastric cancer populations: DS-Screen Study.

PURPOSE: Fibroblast growth factor receptor 2 (FGFR2) and human epidermal growth factor receptor 2 (HER2) proteins are both molecular targets for cancer therapy. The objective of this study was to evaluate the expression status of FGFR2 and HER2 in patients with gastric cancer (GC) or colorectal cancer (CRC). METHODS: Archived tumor tissue samples from patients with histologically-confirmed GC or CRC suitable for chemotherapy were analyzed for FGFR2 and HER2 expression using immunohistochemistry and fluorescence in situ hybridization (HER2 in CRC only). RESULTS: A total of 176 GC patients and 389 CRC patients were enrolled. Among patients with GC, 25.6% were FGFR2-positive and 26.1% were HER2-positive. Among patients with CRC, 2.9% were FGFR2-positive and 16.2% were HER2-positive. No clear relationship was found between FGFR2 and HER2 status in either GC or CRC. In GC, FGFR2 and HER2 statuses did not differ between different primary cancer locations, whereas there were some differences between histological types. Based on FGFR2- and/or HER2-positive status, 117 patients were identified as potentially suitable for inclusion in clinical trials of therapeutic agents targeting the relevant protein (GC = 45, CRC = 72; FGFR = 56, HER2 = 62), of whom 7 were eventually enrolled into such clinical trials. CONCLUSIONS: This study indicated the prevalence of FGFR2 and HER2 in GC and CRC in the Japanese population. The screening performed in this study could be useful for identifying eligible patients for future clinical trials of agents targeting these proteins. TRIAL REGISTRATION: Clinical trial registration Japic CTI No.: JapicCTI-163380.  https://www. CLINICALTRIALS: jp/cti-user/trial/ShowDirect.jsp?directLink=RNlzx1PPCuT.PrVNPxPRwA .

Morphological, cytological and genetic analyzes of the 'sango' mutant with the defects in basidiocarp development in edible mushroom Pleurotus pulmonarius.

A spontaneous, morphological variation 'sango' was observed in the progeny of a Pleurotus pulmonarius (Fr.) Quél. wild-type basidiocarp (also known as fruiting body) collected from the field. This variant developed wart- and coral-like structures instead of normal basidiocarps. Microscopic analysis showed that the sango phenotype had defects in the differentiation of the pileus and hymenium. Basidiocarp phenotypic data analysis in the progenies revealed that the sango trait is a heritable mutation character controlled by a single recessive gene. This mutation locus was mapped on linkage group III of a previously constructed genetic linkage map by amplified fragment length polymorphism (AFLP) technique in P. pulmonarius. Four AFLP markers identified by bulked segregant analysis showed linkage to the sango mutation locus, with the genetic distance ranging from 0 to 2.1 cM. Of these markers, one marker was co-segregated with the sango mutation locus. This knowledge will be a useful foundation for practical breeding as well as for elucidating molecular mechanisms in basidiocarp development of main edible mushrooms.

Temporary sequestration of cholesterol and phosphatidylcholine within extracellular domains of ABCA1 during nascent HDL generation.

The quality and quantity of high-density lipoprotein (HDL) in blood plasma are important for preventing coronary artery disease. ATP-binding cassette protein A1 (ABCA1) and apolipoprotein A-I (apoA-I) play essential roles in nascent HDL formation, but controversy persists regarding the mechanism by which nascent HDL is generated. In the "direct loading model", apoA-I acquires lipids directly from ABCA1 while it is bound to the transporter. By contrast, in the "indirect model", apoA-I acquires lipids from the specific membrane domains created by ABCA1. In this study, we found that trypsin treatment causes rapid release of phosphatidylcholine (PC) and cholesterol from BHK/ABCA1 cells, and that the time course of lipid release coincides with those of trypsin digestion of extracellular domains (ECDs) of surface ABCA1 and of release of ECD fragments into the medium. This trypsin-dependent lipid release was dependent on ABCA1 ATPase activity, and did not occur in cells that express ABCG1, which exports lipids like ABCA1 but does not have large ECDs. These results suggest that the trypsin-sensitive sites on the cell surface are the large ECDs of ABCA1, and that lipids transported by ABCA1 are temporarily sequestered within the ECDs during nascent HDL formation.

Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum.

Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process.

ABCB4 exports phosphatidylcholine in a sphingomyelin-dependent manner.

ABCB4, which is specifically expressed on the canalicular membrane of hepatocytes, exports phosphatidylcholine (PC) into bile. Because SM depletion increases cellular PC content and stimulates PC and cholesterol efflux by ABCA1, a key transporter involved in generation of HDL, we predicted that SM depletion also stimulates PC efflux through ABCB4. To test this prediction, we compared the lipid efflux activity of ABCB4 and ABCA1 under SM depletion induced by two different types of inhibitors for SM synthesis, myriocin and (1R,3S)-N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide, in human embryonic kidney 293 and baby hamster kidney cells. Unexpectedly, SM depletion exerted opposite effects on ABCB4 and ABCA1, suppressing PC efflux through ABCB4 while stimulating efflux through ABCA1. Both ABCB4 and ABCA1 were recovered from Triton-X-100-soluble membranes, but ABCB4 was mainly recovered from CHAPS-insoluble SM-rich membranes, whereas ABCA1 was recovered from CHAPS-soluble membranes. These results suggest that a SM-rich membrane environment is required for ABCB4 to function. ABCB4 must have evolved to exert its maximum activity in the SM-rich membrane environment of the canalicular membrane, where it transports PC as the physiological substrate.

Novel combined GPIHBP1 mutations in a patient with hypertriglyceridemia associated with CAD.

AIM: Lipoprotein lipase (LPL) deficiency is a rare autosomal recessive disorder characterized by severe hypertriglyceridemia. Similar clinical phenotypes have been reported with respect to defects in several LPL-associated proteins. However, it remains controversial whether severe hypertriglyceridemia itself is atherogenic. We herein present a case of LPL deficiency due to novel combined mutations of glycosylphosphatidylinositol (GPI)-anchored high-density lipoprotein (HDL)-binding protein 1 (GPIHBP1) in a patient with coronary artery disease (CAD). PATIENT: We evaluated a 54-year-old woman with severe hypertriglyceridemia and double vessel CAD. Although the LPL mass and activity in the postheparin plasma were extremely low, no mutations were detected in the LPL gene itself. RESULTS: Genetic analyses revealed that the patient had double homozygous mutations at 41 bp (c.41 G > T) and 202 bp (c.202 T > C) in the GPIHBP1 gene, resulting in C14F and C68R, respectively. Although the C14F/C68R GPIHBP1 exhibited a normal LPL-binding activity, the levels of mutant proteins were extremely reduced compared to those of the wild-type proteins in vitro. CONCLUSION: We found novel combined mutations of GPIHBP1 in a patient with hypertriglyceridemia and severe CAD. The present case provides important insight into the pathogenesis of severe hypertriglyceridemia associated with atherosclerosis.

ATPase activity of nucleotide binding domains of human MDR3 in the context of MDR1.

Although human MDR1 and MDR3 share 86% similarity in their amino acid sequences and are predicted to share conserved domains for drug recognition, their physiological transport substrates are quite different: MDR1 transports xenobiotics and confers multidrug resistance, while MDR3 exports phosphatidylcholine into bile. Although MDR1 shows high ATPase activity, attempts to demonstrate the ATPase activity of human MDR3 have not succeeded. Therefore, it is possible that the difference in the functions of these proteins is caused by their different ATPase activities. To test this hypothesis, a chimera protein containing the transmembrane domains (TMDs) of MDR1 and the nucleotide binding domains (NBDs) of MDR3 was constructed and analyzed. The chimera protein was expressed on the plasma membrane and conferred resistance against vinblastine and paclitaxel, indicating that MDR3 NBDs can support drug transport. Vanadate-induced ADP trapping of MDR3 NBDs in the chimera protein was stimulated by verapamil as was MDR1 NBDs. The purified chimera protein showed drug-stimulated ATPase activity like MDR1, while its Vmax was more than 10-times lower than MDR1. These results demonstrate that the low ATPase activity of human MDR3 cannot account for the difference in the functions of these proteins, and furthermore, that TMDs determine the features of NBDs. To our knowledge, this is the first study analyzing the features of human MDR3 NBDs.

Apolipoprotein B-48 to triglyceride ratio is a novel and useful marker for detection of type III hyperlipidemia after antihyperlipidemic intervention.

AIM: Remnant lipoproteins are atherogenic and are accumulated in patients with type III hyperlipidemia (HL). Although type III HL is diagnosed by phenotyping apolipoprotein (apo) E, this procedure is time-consuming and inconvenient for routine clinical use. Clinical indices for screening type III HL in untreated HL patients have been proposed; however, in clinical settings, HL patients are promptly treated with lipid-lowering agents without diagnosing the underlying cause. We investigated whether existing clinical indices for screening type III HL as well as the apo B-48/triglyceride (TG) ratio, which was suggested to be related to the accumulation of small chylomicron (CM) remnants, are useful after the initiation of lipid-lowering therapies. METHODS: In 25 normolipidemic subjects and 191 treated HL patients (type I, n =6; IIa, 62; IIb, 66; III, 12; IV, 22; and V, 23) from Osaka University Hospital and related hospitals, fasting low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), TG, and apolipoproteins were measured and clinical indices were evaluated statistically. RESULTS: Apo B-48 levels were significantly higher in patients with type I, III, and V HL, and TG levels were significantly higher in patients with type I and V HL. The apo B-48/TG ratio was significantly higher only in patients with type III HL compared with other types of HL (p<0.001), and was statistically significant among the other clinical indices (AUC-ROC value, 0.895; cut-off value, 0.110). CONCLUSION: The apo B-48/TG ratio is a novel and useful marker for detecting type III HL even after the initiation of lipid-lowering interventions.

Thyroid function influences serum apolipoprotein B-48 levels in patients with thyroid disease.

AIM: Apolipoprotein B-48 (apoB-48) is a major apolipoprotein of intestine-derived chylomicrons (CM) and CM remnants (CMR). Clinically overt hypothyroidism (OH) has been associated with premature and accelerated coronary atherosclerosis. To clarify the clinical significance of apoB-48 measurement in patients with thyroid disease, we investigated the correlations between the serum apoB-48 level and thyroid hormones. METHODS: From outpatients of Osaka University Hospital, patients with OH, subjects with subclinical hypothyroidism (SH) and subjects with normal thyroid function were collected and analyzed by measuring serum TSH, FT4 and FT3 levels. Serum apoB-48 levels were measured by a chemiluminescence enzyme immunoassay and the correlations with thyroid hormone levels or lipid profiles were assessed. These levels were compared among subjects with OH, SH and healthy controls. RESULTS: Serum apoB-48 level was correlated with TSH, total cholesterol (TC) and triglycerides (TG), but negatively with FT4 and FT3 level. LDL-C and HDL-C levels were not correlated with serum apoB-48 levels. Serum apoB-48 in patients with OH (7.4 ± 5.9 µg/mL) was significantly higher than in those with hyperthyroidism (5.1 ± 3.5 µg/mL; p<0.01) and normal subjects (4.7 ± 3.7 µg/mL; p<0.01), but decreased after levo-thyroxine replacement. ApoB-48, TG and TSH were significantly higher in SH subjects than normal subjects, suggesting that serum apoB-48 level depends on the thyroid function status, similar to TC, LDL-C and TG. CONCLUSION: Increased serum apoB-48 concentrations and CMR may contribute to the increased risk of atherosclerosis and premature coronary artery disease in the hypothyroid state.

Effect of probucol on antioxidant properties of HDL in patients with heterozygous familial hypercholesterolemia.

AIM: High density lipoprotein (HDL) has multi-antiatherogenic effects such as antioxidation and anti-inflammation, in addition to being a key mediator of reverse cholesterol transport. Probucol, known as a lipid lowering drug, is also a potent antioxidant, but it decreases serum HDL cholesterol (HDL-C) levels. To elucidate the effect of probucol on antioxidant properties of HDL, we investigated the function of HDL derived from patients with heterozygous familial hypercholesterolemia (FH) who have been treated with probucol. METHODS AND RESULTS: Probucol-treated FH patients (n=21) showed a 47% reduction of serum HDL-C levels compared to probucol-untreated FH patients (n=15). High performance liquid chromatography (HPLC) analysis revealed that probucol diminished HDL particle size compared to the non-treated group. Antioxidant capacity of HDL was evaluated by its effect to protect reference LDL from oxidation induced in the presence of an oxidizing agent, AAPH. The HDL derived from the probucol-treated group demonstrated a significantly prolonged time to start oxidation by 112%, decreased the maximum oxidation rate by 14%, and lowered the maximum concentration of conjugated dienes formation by 15%. Furthermore, HDL-associated paraoxonase 1 (PON1) activity, but not platelet-activating factor acetyl-hydrolase (PAF-AH) correlated with these measurements of HDL anti-oxidative activity. Treatment with probucol in vitro and inhibition of PON1 activity demonstrated that probucol in HDL particles and increase of PON1 activity might largely contribute to the increase of HDL anti-oxidative activity. CONCLUSION: Probucol reduced HDL-C levels and HDL particle size in patients with heterozygous FH, while it concomitantly enhanced HDL anti-oxidative properties, possibly through increasing PON1 activity.

Serum adiponectin level is correlated with the size of HDL and LDL particles determined by high performance liquid chromatography.

OBJECTIVE: Adiponectin (APN) improves insulin resistance and prevents atherosclerosis, and HDL removes cholesterol from atherosclerotic lesions. We have demonstrated that serum HDL-cholesterol (HDL-C) and APN concentrations are positively correlated and that APN accelerates reverse cholesterol transport (RCT) by increasing HDL synthesis in the liver and cholesterol efflux from macrophages. We previously reported that APN reduced apolipoprotein (apo) B secretion from the liver. It is well-known that insulin resistance influences the lipoprotein profile. In this study, we investigated the clinical significance of APN levels and insulin resistance in lipoprotein metabolism. MATERIAL/METHOD: We investigated the correlation between serum APN concentration, HOMA-R, the lipid concentrations and lipoprotein particle size by high-performance liquid chromatography (HPLC) in 245 Japanese men during an annual health checkup. RESULTS: Serum APN level was positively correlated with the cholesterol content in large LDL and HDL particles, but inversely correlated with the cholesterol content in large VLDL and small LDL particles. HOMA-R was negatively correlated with the cholesterol content in large LDL and HDL particles and positively correlated with the cholesterol content in large VLDL and small LDL particles. By multivariate analysis, APN was correlated with the particle size of LDL-C and HDL-C independently of age, BMI and HOMA-R. CONCLUSIONS: APN may be associated with the formation of both HDL and LDL particles, reflecting the enhancement of RCT and the improvement in TG-rich lipoprotein metabolism and insulin resistance.

Correlation of fasting serum apolipoprotein B-48 with coronary artery disease prevalence.

BACKGROUND: Postprandial hyperlipidemia partially refers to the postprandial accumulation of chylomicrons and chylomicron remnants (CM-R). Many in vitro studies have shown that CM-R has highly atherogenic properties, but consensus is lacking on whether CM-R accumulation correlates with the development of atherosclerotic cardiovascular diseases. We investigated the correlation between CM-R accumulation and the prevalence of coronary artery disease (CAD). DESIGN: Subjects who received a coronary angiography and did not take any lipid-lowering drugs (n = 189) were enrolled. Subjects with coronary artery stenosis (≥ 75%) were diagnosed as CAD. Biochemical markers for glucose and lipid metabolism including fasting apolipoprotein (apo) B-48 concentration were compared between CAD patients (n = 96) and age-, sex-, and body mass index (BMI)-matched non-CAD subjects without overt coronary stenosis (< 75%) (n = 67). We tried to determine which metabolic parameters were correlated with the prevalence of CAD by multiple logistic regression analysis, and whether or not the combination of high apo B-48 and other coronary risk factors (high triglyceride, low HDL-C, high HbA1c or low adiponectin levels) increased the prevalence of CAD. RESULTS: Fasting serum apo B-48 levels were significantly higher in CAD patients than in non-CAD subjects (3·9 ± 2·4 vs. 6·9 ± 2·6 µg/mL, P < 0·0001) and had the most significant correlation with the existence of CAD. The clustering of high fasting apo B-48 levels (> 4·34 µg/mL, the cut-off value) and other coronary risk factors were found to be associated with a stronger risk of CAD compared with single high fasting apo B-48 levels. CONCLUSION: Fasting serum apo B-48 levels significantly correlated with the prevalence of CAD.

Establishment of chemiluminescence enzyme immunoassay for apolipoprotein B-48 and its clinical applications for evaluation of impaired chylomicron remnant metabolism.

BACKGROUND: Apolipoprotein B-48 (apoB-48) is a constituent of chylomicron remnants synthesized in the small intestines. The serum concentration of apoB-48 at fasting has been reported to be a marker of postprandial hyperlipidemia, a presumed risk factor for atherosclerosis. METHODS: We evaluated the basal performance of a recently developed chemiluminescent enzyme immunoassay (CLEIA). We also examined the correlations between serum apoB-48 concentrations and other lipid concentrations or life style patterns, including smoking and drinking. We analyzed the data of 273 clinical samples by multiple regression analysis to examine the influence of other serum lipid values, age, sex, smoking, drinking status and BMI on serum apoB-48 values. RESULTS: Within-run and between-run precision was obtained with 1.7-2.7% and 1.2-7.3%, respectively. The correlativity of enzyme-linked immunosorbent assay was correlation coefficient r=0.953, and regression y=1.02×-1.59. Serum apoB-48 concentrations were higher in males than in females, and were correlated with the status of smoking as well as with remnant-like particle-cholesterol (RLP-C) concentrations. Patients with the metabolic syndrome showed higher values of serum apoB-48 compared with control subjects. CONCLUSION: Serum apoB-48 measurement by CLEIA was satisfactory for clinical use to assess abnormalities in the chylomicron remnant metabolism.

Patients with CD36 deficiency are associated with enhanced atherosclerotic cardiovascular diseases.

AIM: The clustering of dyslipidemia, impaired glucose tolerance and hypertension increases the morbidity and mortality from cardiovascular events. A class B scavenger receptor, CD36, is a receptor for oxidized LDL and a transporter of long-chain fatty acids. Because of the impaired uptake of oxidized LDL in CD36-deficient macrophages and from the results of CD36 knockout mice, CD36 deficiency (CD36-D) was supposed to be associated with reduced risks for coronary artery disease (CAD); however, CD36-D patients are often accompanied by a clustering of coronary risk factors. The current study aimed to investigate the morbidity and severity of cardiovascular diseases in CD36-D patients. METHODS: By screening for CD36 antigen on platelets and monocytes using FACS or the absent myocardial accumulation of 123I-BMIPP by scintigraphy, 40 patients with type I CD36-D were collected, the morbidity of CAD and their features of atherosclerotic cardiovascular diseases were observed. Screening for CD36-D in both CAD patients (n = 319) and healthy subjects (n = 1,239) were underwent. RESULTS: The morbidity of CAD was significantly higher in CD36-D patients than in the general population; 50% of patients (20 out of 40) had CAD identified by BMIPP scintigraphy and 37.5% (3 out of 8) by FACS screening, respectively. Three representative CD36-D cases demonstrated severe CAD and atherosclerosis. The frequency of CD36-D was three times higher in CAD patients than in healthy subjects (0.9% vs 0.3%, p < 0.0001). CONCLUSION: The morbidity of CAD is significantly higher in CD36-D patients suffering from severe atherosclerosis, implying that the status of CD36-D might be atherogenic.

Fasting serum apolipoprotein B-48 can be a marker of postprandial hyperlipidemia.

AIM: Postprandial hyperlipidemia (PH) is thought to be caused by the impaired postprandial metabolism of triglycerides (TG)-rich lipoproteins in both endogenous and exogenous pathways; however, there is no consensus. It is difficult to estimate the presence of PH without performing a time-consuming oral fat loading (OFL) test, so postprandial lipoprotein metabolism was analyzed by measuring the postprandial levels of apolipoprotein (apo) B-48 and apo B-100, and the correlation between postprandial TG increase and fasting apoB-48 levels was assessed to establish a good marker of PH without performing an OFL test. METHODS: Ten male normolipidemic subjects were loaded with a high-fat (HF, 1045 kcal) or standard (ST, 566 kcal) meal, and the lipids, apolipoproteins and lipoprotein profiles were analyzed after each meal. RESULTS: TG, apo B-48, remnant-like particles (RLP)-cholesterol and RLP-TG levels were increased and their levels were significantly higher after intake of the HF meal than the ST meal; however, there was no postprandial increase in apo B-100 and LDL-C levels. Postprandial increases in TG levels of CM, VLDL, LDL and HDL were significantly higher after intake of the HF meal than the ST meal. Fasting apo B-48 levels were strongly correlated with the incremental area under the curve of TG after intake of the HF meal, but not the ST meal. CONCLUSION: Postprandial TG increase was mainly due to increased CM and CM-R, but not VLDL. Measurement of fasting serum apo B-48 may be a simple and useful method for assessment of the existence of PH.

Remnant lipoprotein-cholesterol is a predictive biomarker for large artery atherosclerosis in apparently healthy women: usefulness as a parameter for annual health examinations.

BACKGROUND: Recently, remnant lipoprotein is expected to be a new therapeutic target in the age of 'beyond LDL-cholesterol'. The aim of this study was to clarify the clinical significance of remnant lipoprotein cholesterol (RemL-C) determination in annual health examinations with the focus on large artery atherosclerosis. Methods and results Subjects investigated were men (n = 528) and women (n = 318) who underwent annual health examinations at Osaka University. RemL-C was measured with a newly developed homogeneous assay. Carotid and aortic atherosclerosis was estimated by intima-media thickness (IMT) and cardio-ankle vascular index (CAVI), respectively. First, simple regression analysis revealed that the RemL-C levels positively correlated with maximum IMT, mean IMT and CAVI in the whole group (P < 0.05). Next, receiver operating characteristic curve analysis showed that the most effective levels of RemL-C for predicting carotid and aortic atherosclerosis were 0.21 mmol/L (P < 0.05) and 0.22 mmol/L (P < 0.01) or more, respectively. Odds ratios (ORs) of high RemL-C levels (0.21 mmol/L or more) for carotid and aortic atherosclerosis were significantly increased, especially in low-risk, apparently healthy women (OR: 4.20, P < 0.05 and 3.79, P < 0.01, respectively). Five out of 13 female low-risk cases (38%) with carotid atherosclerosis showed high serum RemL-C levels. It should be emphasized that conventional risk factors are still strong predictors for large artery atherosclerosis in the whole group. CONCLUSIONS: Our results indicate that high serum RemL-C level is a predictive hallmark for large artery atherosclerosis in apparently healthy women. Determination of RemL-C should be employed as one of the parameters in annual health examinations.

Serum apolipoprotein B-48 levels are correlated with carotid intima-media thickness in subjects with normal serum triglyceride levels.

BACKGROUND: Postprandial hyperlipidemia (PPHL) is an independent risk factor for coronary heart disease (CHD) which is based on the accumulation of chylomicrons (CM) and CM remnants containing apolipoprotein B-48 (apoB-48). Since atherosclerotic cardiovascular diseases are frequently observed even in subjects with normal serum triglyceride (TG) level, the correlation between fasting apoB-48 containing lipoproteins and carotid intima-media thickness (IMT) was analyzed in subjects with normal TG levels. METHODS: From subjects who took their annual health check at the Osaka Police Hospital (n=245, male), one-hundred and sixty-four male subjects were selected to take part in this study; the excluding factors were: systolic blood pressure ≥ 140 mmHg, intake of antihypertensive or antihyperlipidemic drugs, or age >65 years. The association between biochemical markers and IMT was analyzed and independent predictors of max-IMT were determined by multiple regression analysis in all subjects and in groups N-1 (TG<100mg/dl, n=58), N-2 (100 ≤ TG<150 mg/dl, n=53) and H (150 ≤ TG mg/dl, n=53), respectively. RESULTS: Fasting total cholesterol, LDL-cholesterol, HDL-cholesterol, apoB-100 and lnRemL-C (remnant lipoprotein-cholesterol) levels were not correlated with max-IMT, but lnTG and lnapoB-48 were significantly correlated with max-IMT in all subjects. LnapoB-48 and apoB-48/TG ratio were significantly correlated with max-IMT in group N-2. By multiple regression analysis, age and lnapoB-48 were independent variables associated with max-IMT in group N-2. CONCLUSION: Serum apoB-48 level might be a good marker for the detection of early atherosclerosis in middle-aged subjects with normal-range levels of blood pressure and TG.

A novel apolipoprotein E mutation, ApoE Osaka (Arg158 Pro), in a dyslipidemic patient with lipoprotein glomerulopathy.

Lipoprotein glomerulopathy (LPG) is a rare disease characterized by the presence of thrombuslike deposition in markedly dilated glomerular capillaries and is often accompanied by an increased serum apolipoprotein E (apoE) level. Several gene mutations of apoE have been reported to be associated with LPG. In the current study, we report an LPG patient with a novel apoE mutation, apoE Osaka. The patient was a 45-year-old man who was hospitalized due to nephrotic syndrome. Light and electron microscopic observations of renal biopsy clearly showed characteristic findings of LPG, including lamellate thrombi in the lumen of dilated glomerular capillaries. His apoE phenotype was apoE3/2 and he had mild dyslipidemia with a mid-band on polyacrylamide gel electrophoresis. It is intriguing that the serum apoE level was within normal limits. We determined the sequence of the apoE gene using direct sequencing of the polymerase chain reaction (PCR) products. ApoE gene analysis showed a nucleotide substitution of G to C at codon 158 of exon 4. This mutation denoted an amino acid substitution of arginine residue for the proline residue at position 158 of apoE. The result of PCR associated with restriction fragment length polymorphism analysis also suggested that this mutation is heterozygous. It is possible that apoE Osaka mutation causes a conformational change of apoE protein and affects the interaction between abnormal apoE-containing lipoproteins and the endothelial cells of glomerular capillaries. The precise mechanism of LPG related with apoE Osaka, however, remains to be elucidated.