The lipoprotein lipase that is shuttled into capillaries by GPIHBP1 enters the glycocalyx where it mediates lipoprotein processing.

Lipoprotein lipase (LPL), the enzyme that carries out the lipolytic processing of triglyceride-rich lipoproteins (TRLs), is synthesized by adipocytes and myocytes and secreted into the interstitial spaces. The LPL is then bound by GPIHBP1, a GPI-anchored protein of endothelial cells (ECs), and transported across ECs to the capillary lumen. The assumption has been that the LPL that is moved into capillaries remains attached to GPIHBP1 and that GPIHBP1 serves as a platform for TRL processing. In the current studies, we examined the validity of that assumption. We found that an LPL-specific monoclonal antibody (mAb), 88B8, which lacks the ability to detect GPIHBP1-bound LPL, binds avidly to LPL within capillaries. We further demonstrated, by confocal microscopy, immunogold electron microscopy, and nanoscale secondary ion mass spectrometry analyses, that the LPL detected by mAb 88B8 is located within the EC glycocalyx, distant from the GPIHBP1 on the EC plasma membrane. The LPL within the glycocalyx mediates the margination of TRLs along capillaries and is active in TRL processing, resulting in the delivery of lipoprotein-derived lipids to immediately adjacent parenchymal cells. Thus, the LPL that GPIHBP1 transports into capillaries can detach and move into the EC glycocalyx, where it functions in the intravascular processing of TRLs.

Quantification of lipoprotein lipase in mouse plasma with a sandwich enzyme-linked immunosorbent assay.

To support in vivo and in vitro studies of intravascular triglyceride metabolism in mice, we created rat monoclonal antibodies (mAbs) against mouse LPL. Two mAbs, mAbs 23A1 and 31A5, were used to develop a sandwich ELISA for mouse LPL. The detection of mouse LPL by the ELISA was linear in concentrations ranging from 0.31 ng/ml to 20 ng/ml. The sensitivity of the ELISA made it possible to quantify LPL in serum and in both pre-heparin and post-heparin plasma samples (including in grossly lipemic samples). LPL mass and activity levels in the post-heparin plasma were lower in Gpihbp1-/- mice than in wild-type mice. In both groups of mice, LPL mass and activity levels were positively correlated. Our mAb-based sandwich ELISA for mouse LPL will be useful for any investigator who uses mouse models to study LPL-mediated intravascular lipolysis.

Lipoprotein lipase is active as a monomer.

Lipoprotein lipase (LPL), the enzyme that hydrolyzes triglycerides in plasma lipoproteins, is assumed to be active only as a homodimer. In support of this idea, several groups have reported that the size of LPL, as measured by density gradient ultracentrifugation, is ∼110 kDa, twice the size of LPL monomers (∼55 kDa). Of note, however, in those studies the LPL had been incubated with heparin, a polyanionic substance that binds and stabilizes LPL. Here we revisited the assumption that LPL is active only as a homodimer. When freshly secreted human LPL (or purified preparations of LPL) was subjected to density gradient ultracentrifugation (in the absence of heparin), LPL mass and activity peaks exhibited the size expected of monomers (near the 66-kDa albumin standard). GPIHBP1-bound LPL also exhibited the size expected for a monomer. In the presence of heparin, LPL size increased, overlapping with a 97.2-kDa standard. We also used density gradient ultracentrifugation to characterize the LPL within the high-salt and low-salt peaks from a heparin-Sepharose column. The catalytically active LPL within the high-salt peak exhibited the size of monomers, whereas most of the inactive LPL in the low-salt peak was at the bottom of the tube (in aggregates). Consistent with those findings, the LPL in the low-salt peak, but not that in the high-salt peak, was easily detectable with single mAb sandwich ELISAs, in which LPL is captured and detected with the same antibody. We conclude that catalytically active LPL can exist in a monomeric state.

Apolipoprotein CIII Deficiency Protects Against Atherosclerosis in Knockout Rabbits.

OBJECTIVE: Apo (apolipoprotein) CIII mediates the metabolism of triglyceride (TG)-rich lipoproteins. High levels of plasma apoCIII are positively correlated with the plasma TG levels and increase the cardiovascular risk. However, whether apoCIII is directly involved in the development of atherosclerosis has not been fully elucidated. Approach and Results: To examine the possible roles of apoCIII in lipoprotein metabolism and atherosclerosis, we generated apoCIII KO (knockout) rabbits using ZFN (zinc finger nuclease) technique. On a normal standard diet, apoCIII KO rabbits exhibited significantly lower plasma levels of TG than those of WT (wild type) rabbits while total cholesterol and HDL (high-density lipoprotein) cholesterol levels were unchanged. Analysis of lipoproteins isolated by sequential ultracentrifugation revealed that reduced plasma TG levels in KO rabbits were accompanied by prominent reduction of VLDLs (very-low-density lipoproteins) and IDLs (intermediate-density lipoproteins). In addition, KO rabbits showed faster TG clearance rate after intravenous fat load than WT rabbits. On a cholesterol-rich diet, KO rabbits exhibited constantly and significantly lower levels of plasma total cholesterol and TG than WT rabbits, which was caused by a remarkable reduction of ß-VLDLs-the major atherogenic lipoproteins. ß-VLDLs of KO rabbits showed higher uptake by cultured hepatocytes and were cleared faster from the circulation than ß-VLDLs isolated from WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. CONCLUSIONS: These results indicate that apoCIII deficiency facilitates TG-rich lipoprotein catabolism, and therapeutic inhibition of apoCIII expression may become a novel means not only for the treatment of hyperlipidemia but also for atherosclerosis.

Chylomicronemia from GPIHBP1 autoantibodies.

Some cases of chylomicronemia are caused by autoantibodies against glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1), an endothelial cell protein that shuttles LPL to the capillary lumen. GPIHBP1 autoantibodies prevent binding and transport of LPL by GPIHBP1, thereby disrupting the lipolytic processing of triglyceride-rich lipoproteins. Here, we review the "GPIHBP1 autoantibody syndrome" and summarize clinical and laboratory findings in 22 patients. All patients had GPIHBP1 autoantibodies and chylomicronemia, but we did not find a correlation between triglyceride levels and autoantibody levels. Many of the patients had a history of pancreatitis, and most had clinical and/or serological evidence of autoimmune disease. IgA autoantibodies were present in all patients, and IgG4 autoantibodies were present in 19 of 22 patients. Patients with GPIHBP1 autoantibodies had low plasma LPL levels, consistent with impaired delivery of LPL into capillaries. Plasma levels of GPIHBP1, measured with a monoclonal antibody-based ELISA, were very low in 17 patients, reflecting the inability of the ELISA to detect GPIHBP1 in the presence of autoantibodies (immunoassay interference). However, GPIHBP1 levels were very high in five patients, indicating little capacity of their autoantibodies to interfere with the ELISA. Recently, several GPIHBP1 autoantibody syndrome patients were treated successfully with rituximab, resulting in the disappearance of GPIHBP1 autoantibodies and normalization of both plasma triglyceride and LPL levels. The GPIHBP1 autoantibody syndrome should be considered in any patient with newly acquired and unexplained chylomicronemia.

Autoantibodies against GPIHBP1 as a Cause of Hypertriglyceridemia.

BACKGROUND: A protein that is expressed on capillary endothelial cells, called GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1), binds lipoprotein lipase and shuttles it to its site of action in the capillary lumen. A deficiency in GPIHBP1 prevents lipoprotein lipase from reaching the capillary lumen. Patients with GPIHBP1 deficiency have low plasma levels of lipoprotein lipase, impaired intravascular hydrolysis of triglycerides, and severe hypertriglyceridemia (chylomicronemia). During the characterization of a monoclonal antibody-based immunoassay for GPIHBP1, we encountered two plasma samples (both from patients with chylomicronemia) that contained an interfering substance that made it impossible to measure GPIHBP1. That finding raised the possibility that those samples might contain GPIHBP1 autoantibodies. METHODS: Using a combination of immunoassays, Western blot analyses, and immunocytochemical studies, we tested the two plasma samples (as well as samples from other patients with chylomicronemia) for the presence of GPIHBP1 autoantibodies. We also tested the ability of GPIHBP1 autoantibodies to block the binding of lipoprotein lipase to GPIHBP1. RESULTS: We identified GPIHBP1 autoantibodies in six patients with chylomicronemia and found that these autoantibodies blocked the binding of lipoprotein lipase to GPIHBP1. As in patients with GPIHBP1 deficiency, those with GPIHBP1 autoantibodies had low plasma levels of lipoprotein lipase. Three of the six patients had systemic lupus erythematosus. One of these patients who had GPIHBP1 autoantibodies delivered a baby with plasma containing maternal GPIHBP1 autoantibodies; the infant had severe but transient chylomicronemia. Two of the patients with chylomicronemia and GPIHBP1 autoantibodies had a response to treatment with immunosuppressive agents. CONCLUSIONS: In six patients with chylomicronemia, GPIHBP1 autoantibodies blocked the ability of GPIHBP1 to bind and transport lipoprotein lipase, thereby interfering with lipoprotein lipase-mediated processing of triglyceride-rich lipoproteins and causing severe hypertriglyceridemia. (Funded by the National Heart, Lung, and Blood Institute and the Leducq Foundation.).

Direct Versus Calculated LDL Cholesterol and C-Reactive Protein in Cardiovascular Disease Risk Assessment in the Framingham Offspring Study.

BACKGROUND: Increases in circulating LDL cholesterol (LDL-C) and high-sensitivity C-reactive protein (hsCRP) concentrations are significant risk factors for cardiovascular disease (CVD). We assessed direct LDL-C and hsCRP concentrations compared to standard risk factors in the Framingham Offspring Study. METHODS: We used stored frozen plasma samples (-80 °C) obtained after an overnight fast from 3147 male and female participants (mean age, 58 years) free of CVD at cycle 6 of the Framingham Offspring Study. Overall, 677 participants (21.5%) had a CVD end point over a median of 16.0 years of follow-up. Total cholesterol (TC), triglyceride (TG), HDL cholesterol (HDL-C), direct LDL-C (Denka Seiken and Kyowa Medex methods), and hsCRP (Dade Behring method) concentrations were measured by automated analysis. LDL-C was also calculated by both the Friedewald and Martin methods. RESULTS: Considering all CVD outcomes on univariate analysis, significant factors included standard risk factors (age, hypertension, HDL-C, hypertension treatment, sex, diabetes, smoking, and TC concentration) and nonstandard risk factors (non-HDL-C, direct LDL-C and calculated LDL-C, TG, and hsCRP concentrations). On multivariate analysis, only the Denka Seiken direct LDL-C and the Dade Behring hsCRP were still significant on Cox regression analysis and improved the net risk reclassification index, but with modest effects. Discordance analysis confirmed the benefit of the Denka Seiken direct LDL-C method for prospective hard CVD endpoints (new-onset myocardial infarction, stroke, and/or CVD death). CONCLUSIONS: Our data indicate that the Denka Seiken direct LDL-C and Dade Behring hsCRP measurements add significant, but modest, information about CVD risk, compared to standard risk factors and/or calculated LDL-C.

Association between skeletal muscle mass and serum concentrations of lipoprotein lipase, GPIHBP1, and hepatic triglyceride lipase in young Japanese men.

BACKGROUND: Two important regulators for circulating lipid metabolisms are lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL). In relation to this, glycosylphosphatidylinositol anchored high-density lipoprotein binding protein 1 (GPIHBP1) has been shown to have a vital role in LPL lipolytic processing. However, the relationships between skeletal muscle mass and lipid metabolism, including LPL, GPIHBP1, and HTGL, remain to be elucidated. Demonstration of these relationships may lead to clarification of the metabolic dysfunctions caused by sarcopenia. In this study, these relationships were investigated in young Japanese men who had no age-related factors; participants included wrestling athletes with abundant skeletal muscle. METHODS: A total of 111 young Japanese men who were not taking medications were enrolled; 70 wrestling athletes and 41 control students were included. The participants' body compositions, serum concentrations of lipoprotein, LPL, GPIHBP1 and HTGL and thyroid function test results were determined under conditions of no extreme dietary restrictions and exercises. RESULTS: Compared with the control participants, wrestling athletes had significantly higher skeletal muscle index (SMI) (p < 0.001), higher serum concentrations of LPL (p < 0.001) and GPIHBP1 (p < 0.001), and lower fat mass index (p = 0.024). Kruskal-Wallis tests with Bonferroni multiple comparison tests showed that serum LPL and GPIHBP1 concentrations were significantly higher in the participants with higher SMI. Spearman's correlation analyses showed that SMI was positively correlated with LPL (ρ = 0.341, p < 0.001) and GPIHBP1 (ρ = 0.309, p = 0.001) concentration. The serum concentrations of LPL and GPIHBP1 were also inversely correlated with serum concentrations of triglyceride (LPL, ρ = - 0.198, p = 0.037; GPIHBP1, ρ = - 0.249, p = 0.008). Serum HTGL concentration was positively correlated with serum concentrations of total cholesterol (ρ = 0.308, p = 0.001), low-density lipoprotein-cholesterol (ρ = 0.336, p < 0.001), and free 3,5,3'-triiodothyronine (ρ = 0.260, p = 0.006), but not with SMI. CONCLUSIONS: The results suggest that increased skeletal muscle mass leads to improvements in energy metabolism by promoting triglyceride-rich lipoprotein hydrolysis through the increase in circulating LPL and GPIHBP1.

A new enzyme-linked immunosorbent assay system for human serum hepatic triglyceride lipase.

There is no established method for measuring human hepatic triglyceride (TG) lipase (HTGL) concentration in serum. In this study, we developed new monoclonal Abs (MoAbs) (9A1 mouse MoAb and 141A1 rat MoAb) that react with HTGL both in serum and in postheparin plasma (PHP) and established a novel ELISA system for measuring serum HTGL and PHP-HTGL concentrations. To confirm the specificity of MoAbs, we performed immunoprecipitation-immunoblotting analysis. Both 9A1 mouse MoAb and 141A1 rat MoAb were able to immunoprecipitate not only recombinant HTGL and PHP-HTGL but also serum HTGL, demonstrating that HTGL exists in serum obtained without heparin injection. This method yielded intra- and interassay coefficients of variation of <6% and showed no cross-reactivity with LPL or endothelial lipase. In clinical analysis on 42 male subjects with coronary artery disease, there were strong positive correlations of serum HTGL concentration to PHP-HTGL concentration (r = 0.727, P < 0.01). Serum HTGL concentrations showed positive correlations to serum TGs (r = 0.314, P < 0.05) and alanine aminotransferase (r = 0.406, P < 0.01), and tendencies toward positive correlations to LDL cholesterol, small dense LDL, and γGTP. These results suggest that this new ELISA method for measuring serum HTGL is applicable in daily clinical practice.

Monoclonal antibodies that bind to the Ly6 domain of GPIHBP1 abolish the binding of LPL.

GPIHBP1, an endothelial cell protein, binds LPL in the interstitial spaces and shuttles it to its site of action inside blood vessels. For years, studies of human GPIHBP1 have been hampered by an absence of useful antibodies. We reasoned that monoclonal antibodies (mAbs) against human GPIHBP1 would be useful for 1) defining the functional relevance of GPIHBP1's Ly6 and acidic domains to the binding of LPL; 2) ascertaining whether human GPIHBP1 is expressed exclusively in capillary endothelial cells; and 3) testing whether GPIHBP1 is detectable in human plasma. Here, we report the development of a panel of human GPIHBP1-specific mAbs. Two mAbs against GPIHBP1's Ly6 domain, RE3 and RG3, abolished LPL binding, whereas an antibody against the acidic domain, RF4, did not. Also, mAbs RE3 and RG3 bound with reduced affinity to a mutant GPIHBP1 containing an Ly6 domain mutation (W109S) that abolishes LPL binding. Immunohistochemistry studies with the GPIHBP1 mAbs revealed that human GPIHBP1 is expressed only in capillary endothelial cells. Finally, we created an ELISA that detects GPIHBP1 in human plasma. That ELISA should make it possible for clinical lipidologists to determine whether plasma GPIHBP1 levels are a useful biomarker of metabolic or vascular disease.

An LPL-specific monoclonal antibody, 88B8, that abolishes the binding of LPL to GPIHBP1.

LPL contains two principal domains: an amino-terminal catalytic domain (residues 1-297) and a carboxyl-terminal domain (residues 298-448) that is important for binding lipids and binding glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) (an endothelial cell protein that shuttles LPL to the capillary lumen). The LPL sequences required for GPIHBP1 binding have not been examined in detail, but one study suggested that sequences near LPL's carboxyl terminus (residues ∼403-438) were crucial. Here, we tested the ability of LPL-specific monoclonal antibodies (mAbs) to block the binding of LPL to GPIHBP1. One antibody, 88B8, abolished LPL binding to GPIHBP1. Consistent with those results, antibody 88B8 could not bind to GPIHBP1-bound LPL on cultured cells. Antibody 88B8 bound poorly to LPL proteins with amino acid substitutions that interfered with GPIHBP1 binding (e.g., C418Y, E421K). However, the sequences near LPL's carboxyl terminus (residues ∼403-438) were not sufficient for 88B8 binding; upstream sequences (residues 298-400) were also required. Additional studies showed that these same sequences are required for LPL binding to GPIHBP1. In conclusion, we identified an LPL mAb that binds to LPL's GPIHBP1-binding domain. The binding of both antibody 88B8 and GPIHBP1 to LPL depends on large segments of LPL's carboxyl-terminal domain.

Changes in lipoprotein lipase and endothelial lipase mass in familial hypercholesterolemia during three-drug lipid-lowering combination therapy.

BACKGROUND: This study was performed to compare the effects of three different lipid-lowering therapies (statins, ezetimibe, and colestimide) on lipoprotein lipase and endothelial lipase masses in pre-heparin plasma (pre-heparin LPL and EL mass, respectively) from patients with familial hypercholesterolemia (FH). FH is usually treated by coadministration of these three drugs. METHODS: The pre-heparin LPL and EL masses were measured in fresh frozen plasma drawn and stored at various time points during coadministration of the three drugs from patients with heterozygous FH harboring a single mutation in the LDL receptor (n = 16, mean age 63 years). The patients were randomly divided into two groups based on the timing when ezetimibe was added. RESULTS: Plasma LPL mass concentration was significantly reduced by rosuvastatin at 20 mg/day (median = 87.4 [IQR: 71.4-124.7] to 67.5 [IQR: 62.1-114.3] ng/ml, P < 0.05). In contrast, ezetimibe at 10 mg/day as well as colestimide at 3.62 g/day did not alter its level substantially (median = 67.5 [IQR: 62.1-114.3] to 70.2 [IQR: 58.3-106.2], and to 74.9 [IQR: 55.6-101.3] ng/ml, respectively) in the group starting with rosuvastatin followed by the addition of ezetimibe and colestimide. On the other hand, the magnitude in LPL mass reduction was lower in the group starting with ezetimibe at 10 mg/day before reaching the maximum dose of 20 mg/day of rosuvastatin. Plasma EL mass concentration was significantly increased by rosuvastatin at 20 mg/day (median = 278.8 [IQR: 186.7-288.7] to 297.0 [IQR: 266.2-300.2] ng/ml, P < 0.05), whereas other drugs did not significantly alter its level. CONCLUSION: The effects on changes of LPL and EL mass differed depending on the lipid-lowering therapy, which may impact the prevention of atherosclerosis differently.

Biological Antioxidant Potential Negatively Correlates With Carotid Artery Intima-Media Thickness.

Oxidative stress is a crucial factor in the pathogenesis and development of cardiovascular disease. Recently, simplified methods for the detection of reactive oxygen species (ROS) using the derivatives of reactive oxygen metabolites (d-ROMs) test as an index of ROS products and the biological antioxidant potential (BAP) test as an index of antioxidant potential have been utilized. These methods are easy to perform, quick, inexpensive since they use small equipment, and provide reliable results compared with established oxidative stress and antioxidant markers. Because oxidative stress has been shown to represent the balance of production of ROS and antioxidant capacity, it is more appropriate to evaluate ROS and antioxidant capacity simultaneously. However, no study has examined the associations among d-ROMs, BAP values, and carotid artery intima-media thickness (IMT) concurrently. Therefore, we studied the associations among d-ROMs, BAP values, and the carotid artery IMT. Carotid artery IMT, blood pressure (BP), fasting circulating d-ROMs, BAP, glucose metabolism, lipid, and C-reactive protein levels were measured in 95 subjects (age: 49.5 ± 13.8 years; men: 41; women: 54), including 42 healthy subjects and 53 patients with hypertension, dyslipidemia, and diabetes mellitus who were not on medication. The results of multiple regression analysis revealed that dependent carotid artery IMT determinants remained significantly associated with age, systolic BP, total cholesterol, and BAP, whereas dependent BAP determinants remained significantly associated with body mass index and carotid artery IMT. BAP was strongly correlated with carotid artery IMT in our cohort. Our results suggest that BAP may be a useful risk marker for carotid atherosclerosis.

[Possibility of New Circulating Atherosclerosis-Related Lipid Markers Measurement in Medical and Complete Medical Checkups: Small Dense Low-Density Lipoprotein Cholesterol and Lipoprotein Lipase].

Small dense low-density lipoprotein cholesterol (sdLDL-C) concentrations correlate more strongly with cardiovascular disease (CVD) than other LDL-C and large LDL particle concentrations. Lipoprotein lipase (LPL) plays a central role in triglyceride-rich lipoprotein metabolism by catalyzing the hydrolysis of triglycerides in chylomicrons and very low-density lipoprotein particles and is a useful biomarker in diagnosing Type I, Type IV, and Type V hyperlipidemia. Therefore, the measurement of circulating sdLDL-C and LPL concentrations contributes to the assessment of circulating atherosclerosis-related lipid markers. However, the measurement of these lipids has not been fully adopted in medical and complete medical checkups. Recently, novel automated homogenous assay for measuring sdLDL-C and latex particle-enhanced turbidimetric immunoassay (LTIA) for measuring LPL have been developed, respectively. Using these new assays, sdLDL-C values showed excellent agreement with those obtained by isolation of the d = 1.044 - 1.063 g/mL plasma fraction by sequential ultracentrifugation, and LPL values measured with and without heparin injection were highly correlated with the values measured by the LPL-enzyme-linked immunosorbent assay (ELISA). These assays may be superior to the previous assays for the measurement of sdLDL-C and LPL concentrations due their simplicity and reproducibility. The measurements of sdLDL-C and LPL concentrations may be useful as lipid markers in the assessment of the development and progression of atherosclerosis and the detection of pathological conditions and diseases if these markers are measured in medical and complete medical checkups. We have introduced the possibility of the novel measurement of circulating atherosclerosis-related lipid markers such as sdLDL-C and LPL in medical and complete medical checkups. Further studies are needed to clarify whether sdLDL-C and LPL concentrations are related to the development and progression of atherosclerosis and CVD events.

Difference in bias approach for commutability assessment: application to frozen pools of human serum measured by 8 direct methods for HDL and LDL cholesterol.

BACKGROUND: We used a difference in bias approach to evaluate the commutability of 4 frozen serum pools for 8 direct methods for measurement of HDL and LDL cholesterol (HDLC and LDLC). METHODS: Freshly collected nonfrozen sera from 138 diseased and 37 nondiseased patients and 4 frozen pools from the CDC Lipid Standardization Program were measured by direct methods and by the beta-quantification reference measurement procedure of the CDC. We used an error components model to estimate the difference in the bias component of error plus its uncertainty for frozen pools vs patient samples between the direct method and the reference procedure. Frozen pools with bias differences less than a critical value determined by either medical requirements for bias or the random error components of the measurement procedures were considered commutable. RESULTS: On the basis of medical requirement criteria, 1 of the 4 frozen pools was commutable for most of the HDLC methods for both diseased and nondiseased patients, and none was commutable for LDLC methods. On the basis of random error criteria, all of the frozen pools were generally commutable for all of the HDLC methods for both diseased and nondiseased patients, and 1 of the 4 frozen pools was generally commutable for most of the LDLC methods for both diseased and nondiseased patients. CONCLUSIONS: Commutability was assessed as the closeness of agreement of the difference in bias between a reference material and a set of patient samples. Criteria for commutability could be based on fixed medical requirements for bias or on random error components.

Intestinal absorption of fucoidan extracted from the brown seaweed, Cladosiphon okamuranus.

The aim of this study was to examine the absorption of fucoidan through the intestinal tract. Fucoidan (0.1, 0.5, 1.0, 1.5 and 2.0 mg/mL) was added to Transwell inserts containing Caco-2 cells. The transport of fucoidan across Caco-2 cells increased in a dose-dependent manner up to 1.0 mg/mL. It reached a maximum after 1 h and then rapidly decreased. In another experiment, rats were fed standard chow containing 2% fucoidan for one or two weeks. Immunohistochemical staining revealed that fucoidan accumulated in jejunal epithelial cells, mononuclear cells in the jejunal lamina propria and sinusoidal non-parenchymal cells in the liver. Since we previously speculated that nitrosamine may enhance the intestinal absorption of fucoidan, its absorption was estimated in rats administered N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in their drinking water. Rats were fed 0.2% fucoidan chow (BBN + 0.2% fucoidan rats), 2% fucoidan chow (BBN + 2% fucoidan rats) and standard chow for eight weeks. The uptake of fucoidan through the intestinal tract seemed to be low, but was measurable by our ELISA method. Fucoidan-positive cells were abundant in the small intestinal mucosa of BBN + 2% fucoidan rats. Most fucoidan-positive cells also stained positive for ED1, suggesting that fucoidan was incorporated into intestinal macrophages. The uptake of fucoidan by Kupffer cells was observed in the livers of BBN + 2% fucoidan rats. In conclusion, the absorption of fucoidan through the small intestine was demonstrated both in vivo and in vitro.

Quantification of soluble very low-density lipoprotein receptor in human serum using a sandwich enzyme-linked immunosorbent assay.

To investigate the regulation of soluble very low-density lipoprotein receptor (sVLDL-R), which is cleaved mostly from the extracellular domain of VLDL-R II, we generated two rat monoclonal antibodies (mAbs) against human sVLDL-R, and used them to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to measure sVLDL-R levels in human serum or plasma. The ELISA had a linear range from 0.20 ng/mL to 13.02 ng/mL and allowed for the quantification of sVLDL-R in serum and culture cell medium. The coefficient of variation (CV) was less than 10% for both the intra- and inter-assays. The bililubin F, and C, triglyceride (TG), and hemoglobin levels did not affect assay precision. The sVLDL-R concentration was negatively associated with body fat percentage, TG, and HbA1c, suggesting the possibility of obesity and diabetes in middle-aged Japanese women.

Hypertriglyceridemia in Apoa5-/- mice results from reduced amounts of lipoprotein lipase in the capillary lumen.

Why apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia has remained unclear, but we have suspected that the underlying cause is reduced amounts of lipoprotein lipase (LPL) in capillaries. By routine immunohistochemistry, we observed reduced LPL staining of heart and brown adipose tissue (BAT) capillaries in Apoa5-/- mice. Also, after an intravenous injection of LPL-, CD31-, and GPIHBP1-specific mAbs, the binding of LPL Abs to heart and BAT capillaries (relative to CD31 or GPIHBP1 Abs) was reduced in Apoa5-/- mice. LPL levels in the postheparin plasma were also lower in Apoa5-/- mice. We suspected that a recent biochemical observation - that APOA5 binds to the ANGPTL3/8 complex and suppresses its capacity to inhibit LPL catalytic activity - could be related to the low intracapillary LPL levels in Apoa5-/- mice. We showed that an ANGPTL3/8-specific mAb (IBA490) and APOA5 normalized plasma triglyceride (TG) levels and intracapillary LPL levels in Apoa5-/- mice. We also showed that ANGPTL3/8 detached LPL from heparan sulfate proteoglycans and GPIHBP1 on the surface of cells and that the LPL detachment was blocked by IBA490 and APOA5. Our studies explain the hypertriglyceridemia in Apoa5-/- mice and further illuminate the molecular mechanisms that regulate plasma TG metabolism.