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.

Clinical Evaluation of Cerebrospinal Fluid p217tau and Neurofilament Light Chain Levels in Patients with Alzheimer's Disease or Other Neurological Diseases.

BACKGROUND: The cerebrospinal fluid (CSF) levels of tau phosphorylated at threonine 217 (p217tau) or 181 (p181tau), and neurofilament light chain (NfL) are definite biomarkers of tauopathy and neurodegeneration in Alzheimer's disease (AD). OBJECTIVE: To validate their utility in excluding other neurological diseases and age-related changes in clinical settings. METHODS: We developed monoclonal antibodies against p217tau and NfL, established novel ELISAs, and analyzed 170 CSF samples from patients with AD or other neurological diseases. RESULTS: In AD, p217tau is a more specific and abundant CSF component than p181tau. However, CSF NfL levels increase age-dependently and to a greater extent in central and peripheral nervous diseases than in AD. CONCLUSIONS: CSF p217tau correlates better with AD neurodegeneration than other tau-related biomarkers and the major phosphorylated tau species. The clinical usage of NfL as a neurodegeneration biomarker in AD requires exclusion of various central and peripheral neurological diseases.

Novel ELISAs to measure total and phosphorylated tau in cerebrospinal fluid.

Cerebrospinal fluid (CSF) total tau (t-tau) and tau protein phosphorylated at threonine 181 (p181tau) are established biomarkers for Alzheimer's disease (AD). Herein, we measured t-tau and p181tau to evaluate novel enzyme-linked immunosorbent assays (ELISAs) using 72 CSF samples including from patients with AD with dementia (ADD) and various neurodegenerative diseases. Our assay system showed good correlations with widely used ELISA systems for t-tau and p181tau and showed that serum and hemoglobin contamination in CSF samples did not decrease sensitivity. Significant increases in both t-tau and p181tau levels were observed in ADD. These findings suggested that our ELISAs were reliable assays for CSF t-tau and p181tau similar to commonly used ELISAs.

GPIHBP1 autoantibody syndrome during interferon ß1a treatment.

BACKGROUND: Autoantibodies against glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) cause chylomicronemia by blocking the ability of GPIHBP1 to bind lipoprotein lipase (LPL) and transport the enzyme to its site of action in the capillary lumen. OBJECTIVE: A patient with multiple sclerosis developed chylomicronemia during interferon (IFN) ß1a therapy. The chylomicronemia resolved when the IFN ß1a therapy was discontinued. Here, we sought to determine whether the drug-induced chylomicronemia was caused by GPIHBP1 autoantibodies. METHODS: We tested plasma samples collected during and after IFN ß1a therapy for GPIHBP1 autoantibodies (by western blotting and with enzyme-linked immunosorbent assays). We also tested whether the patient's plasma blocked the binding of LPL to GPIHBP1 on GPIHBP1-expressing cells. RESULTS: During IFN ß1a therapy, the plasma contained GPIHBP1 autoantibodies, and those autoantibodies blocked GPIHBP1's ability to bind LPL. Thus, the chylomicronemia was because of the GPIHBP1 autoantibody syndrome. Consistent with that diagnosis, the plasma levels of GPIHBP1 and LPL were very low. After IFN ß1a therapy was stopped, the plasma triglyceride levels returned to normal, and GPIHBP1 autoantibodies were undetectable. CONCLUSION: The appearance of GPIHBP1 autoantibodies during IFN ß1a therapy caused chylomicronemia. The GPIHBP1 autoantibodies disappeared when the IFN ß1a therapy was stopped, and the plasma triglyceride levels fell within the normal range.

An ELISA for quantifying GPIHBP1 autoantibodies and making a diagnosis of the GPIHBP1 autoantibody syndrome.

BACKGROUND: Autoantibodies against GPIHBP1, the endothelial cell transporter for lipoprotein lipase (LPL), cause severe hypertriglyceridemia ("GPIHBP1 autoantibody syndrome"). Affected patients have low serum GPIHBP1 and LPL levels. We report the development of a sensitive and specific ELISA, suitable for routine clinical use, to detect GPIHBP1 autoantibodies in serum and plasma. METHODS: Serum and plasma samples were added to wells of an ELISA plate that had been coated with recombinant human GPIHBP1. GPIHBP1 autoantibodies bound to GPIHBP1 were detected with an HRP-labeled antibody against human immunoglobulin. Sensitivity, specificity, and reproducibility of the ELISA was evaluated with plasma or serum samples from patients with the GPIHBP1 autoantibody syndrome. RESULTS: A solid-phase ELISA to detect and quantify GPIHBP1 autoantibodies in human plasma and serum was developed. Spiking recombinant human GPIHBP1 into the samples reduced the ability of the ELISA to detect GPIHBP1 autoantibodies. The ELISA is reproducible and sensitive; it can detect GPIHBP1 autoantibodies in samples diluted by >1000-fold. CONCLUSION: We have developed a sensitive and specific ELISA for detecting GPIHBP1 autoantibodies in human serum and plasma; this assay will make it possible to rapidly diagnose the GPIHBP1 autoantibody syndrome.

An automated method for measuring lipoprotein lipase and hepatic triglyceride lipase activities in post-heparin plasma.

BACKGROUND: Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) play a central role in triglyceride-rich lipoprotein metabolism by catalyzing the hydrolysis of triglycerides. Quantification of LPL and HTGL activity is useful for diagnosing lipid disorders, but there has been no automated method for measuring these lipase activities. METHODS: The automated kinetic colorimetric method was used for assaying LPL and HTGL activity in the post-heparin plasma using the natural long-chain fatty acid 2-diglyceride as a substrate. LPL activity was determined with apoCII and HTGL activity was determined without apoCII with 2 channel of auto-analyzer. RESULTS: The calibration curve for dilution tests of the LPL and HTGL activity assay ranged from 0.0 to 500 U/L. Within-run CV was obtained within a range of 5%. No interference was observed in the testing of specimens containing potentially interfering substances. The measurement range of LPL activity in the post-heparin plasma was 30-153 U/L, while HTGL activity was 135-431 U/L in normal controls. CONCLUSIONS: The L PL and HTGL activity assays are applicable to quantitating the LPL and HTGL activity in the post-heparin plasma. This assay is more convenient and faster than radiochemical assay and highly suitable for the detection of lipid disorders.

An enzyme-linked immunosorbent assay for measuring GPIHBP1 levels in human plasma or serum.

BACKGROUND: Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), a glycosylphosphatidylinositol (GPI)-anchored protein of capillary endothelial cells, transports lipoprotein lipase to the capillary lumen and is essential for the lipolytic processing of triglyceride-rich lipoproteins. OBJECTIVE: Because some GPI-anchored proteins have been detected in plasma, we tested whether GPIHBP1 is present in human blood and whether GPIHBP1 deficiency or a history of cardiovascular disease affected GPIHBP1 circulating levels. METHODS: We developed 2 monoclonal antibodies against GPIHBP1 and used the antibodies to establish a sandwich enzyme-linked immunosorbent assay (ELISA) to measure GPIHBP1 levels in human blood. RESULTS: The GPIHBP1 ELISA was linear in the 8 to 500 pg/mL range and allowed the quantification of GPIHBP1 in serum and in pre- and post-heparin plasma (including lipemic samples). GPIHBP1 was undetectable in the plasma of subjects with null mutations in GPIHBP1. Serum GPIHBP1 median levels were 849 pg/mL (range: 740-1014) in healthy volunteers (n = 28) and 1087 pg/mL (range: 877-1371) in patients with a history of cardiovascular or metabolic disease (n = 415). There was an extremely small inverse correlation between GPIHBP1 and triglyceride levels (r = 0.109; P < .0275). GPIHBP1 levels tended to be slightly higher in patients who had a major cardiovascular event after revascularization. CONCLUSION: We developed an ELISA for quantifying GPIHBP1 in human blood. This assay will be useful to identify patients with GPIHBP1 deficiency and patients with GPIHBP1 autoantibodies. The potential of plasma GPIHBP1 as a biomarker for metabolic or cardiovascular disease is yet questionable but needs additional testing.

GPIHBP1 autoantibodies in a patient with unexplained chylomicronemia.

BACKGROUND: GPIHBP1, a glycolipid-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) in the interstitial spaces and transports it to the capillary lumen. GPIHBP1 deficiency prevents LPL from reaching the capillary lumen, resulting in low intravascular LPL levels, impaired intravascular triglyceride processing, and severe hypertriglyceridemia (chylomicronemia). A recent study showed that some cases of hypertriglyceridemia are caused by autoantibodies against GPIHBP1 ("GPIHBP1 autoantibody syndrome"). OBJECTIVE: Our objective was to gain additional insights into the frequency of the GPIHBP1 autoantibody syndrome in patients with unexplained chylomicronemia. METHODS: We used enzyme-linked immunosorbent assays to screen for GPIHBP1 autoantibodies in 33 patients with unexplained chylomicronemia and then used Western blots and immunocytochemistry studies to characterize the GPIHBP1 autoantibodies. RESULTS: The plasma of 1 patient, a 36-year-old man with severe hypertriglyceridemia, contained GPIHBP1 autoantibodies. The autoantibodies, which were easily detectable by Western blot, blocked the ability of GPIHBP1 to bind LPL. The plasma levels of LPL mass and activity were low. The patient had no history of autoimmune disease, but his plasma was positive for antinuclear antibodies. CONCLUSIONS: One of 33 patients with unexplained chylomicronemia had the GPIHBP1 autoantibody syndrome. Additional studies in large lipid clinics will be helpful for better defining the frequency of this syndrome and for exploring the best strategies for treatment.

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.

Soluble LR11/SorLA represses thermogenesis in adipose tissue and correlates with BMI in humans.

Thermogenesis in brown adipose tissue (BAT) is an important component of energy expenditure in mammals. Recent studies have confirmed its presence and metabolic role in humans. Defining the physiological regulation of BAT is therefore of great importance for developing strategies to treat metabolic diseases. Here we show that the soluble form of the low-density lipoprotein receptor relative, LR11/SorLA (sLR11), suppresses thermogenesis in adipose tissue in a cell-autonomous manner. Mice lacking LR11 are protected from diet-induced obesity associated with an increased browning of white adipose tissue and hypermetabolism. Treatment of adipocytes with sLR11 inhibits thermogenesis via the bone morphogenetic protein/TGFß signalling pathway and reduces Smad phosphorylation. In addition, sLR11 levels in humans are shown to positively correlate with body mass index and adiposity. Given the need for tight regulation of a tissue with a high capacity for energy wastage, we propose that LR11 plays an energy conserving role that is exaggerated in states of obesity.

Circulating LR11 is a novel soluble-receptor marker for early-stage clinical conditions in patients with non-Hodgkin's lymphoma.

BACKGROUND: We reported that the soluble LDL receptor relative with 11 ligand-binding repeats (sLR11) is a promising biomarker for follicular lymphoma (FL). In this study, we evaluated the fluctuations in serum sLR11 levels compared with those of serum soluble urokinase-type plasminogen activator receptor (suPAR) and soluble interleukin-2 receptor (sIL-2R) in patients with non-Hodgkin's lymphoma (NHL). METHODS: Serum sLR11, suPAR, and sIL-2R levels were measured using ELISA in 175 NHL patients and 57 healthy controls. The levels at diagnosis and at remission were evaluated in 64 paired samples. RESULTS: Serum sLR11 levels were significantly increased in FL, diffuse large B-cell lymphoma (DLBCL), and peripheral T-cell lymphoma patients compared with healthy controls. Serum sLR11 levels revealed significant positive correlations with serum suPAR and sIL-2R levels. Serum sLR11 levels at remission were decreased compared with those at diagnosis, and the declines at remission expressed a slope of approximately -1 with an intercept near that of controls. The receiver operating characteristic-area under the curve of serum sLR11 concentrations was equivalent to that of serum suPAR and sIL-2R concentrations in an early-stage DLBCL and FL. CONCLUSIONS: sLR11 may be a novel soluble receptor indicative of early-stage NHL, with potential use for evaluating therapeutic efficacy.

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma.

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preß1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preß1-HDL ELISA. Here, we purified preß1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preß1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preß1-HDL and original plasma preß1-HDL, or between the preß1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preß1-HDL MAbs, reactivity with intact plasma preß1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preß1-HDL is lipid-free monomolecular apoA-1.

Circulating soluble LR11/SorLA levels are highly increased and ameliorated by chemotherapy in acute leukemias.

BACKGROUND: LR11/SorLA, a receptor interacting with CD87 on monocytes and macrophages, is highly expressed on human immature hematopoietic stem cells. However, it is unknown whether LR11 is expressed on premature leukemic cells, and whether the levels of circulating soluble LR11 (sLR11) shed from leukemic cells correlate with disease state. METHODS: The expression of LR11 on leucocytes and leukemic cells was examined by flow cytometry. Serum sLR11 levels were measured by ELISA in patients with various hematological diseases, including 43 acute myeloid leukemia (AML) and 23 acute lymphoblastic leukemia (ALL) patients. Data were subjected to statistical analysis for validation of sLR11 levels and patients' clinical data. RESULTS: LR11 is specifically expressed in monocytes, and surface levels on leukemic cells are highly induced in both AML and ALL. sLR11 levels of acute leukemia patients were significantly increased (P<0.001) (ALL, 73.5±93.5 ng/ml; AML, 26.8±29.1 ng/ml) in comparison to controls (9.2±3.3 ng/ml). Patients with AML and ALL in remission showed significantly decreased sLR11 levels to below 20 ng/ml. CONCLUSIONS: LR11 and its released soluble form are strongly elevated in acute leukemias. Remarkably, this increase in circulating sLR11 levels is ameliorated at complete remission.

Plasma CCN2 (connective tissue growth factor; CTGF) is a potential biomarker in idiopathic pulmonary fibrosis (IPF).

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal pulmonary fibrotic disease and useful biomarkers are required to diagnose and predict disease activity. CCN2 (connective tissue growth factor; CTGF) has been reported as one of the key profibrotic factors associated with transforming growth factor-ß (TGF-ß), and its assay has potential as a non-invasive measure in various fibrotic diseases. Recently, we developed a new subtraction method for determination of plasma CCN2 levels. We examined the utility of plasma CCN2 levels as a surrogate marker in IPF. METHODS: Plasma CCN2 levels were calculated in 33 patients with IPF, 14 patients with non-IPF idiopathic interstitial pneumonias (IIPs) and 101 healthy volunteers by sandwich enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibodies for two distinct epitopes of human CCN2. We evaluated the utility of plasma CCN2 levels by comparison with clinical parameters. RESULTS: Plasma CCN2 levels were significantly higher in patients with IPF than in those with non-IPF IIPs and healthy volunteers. Importantly, plasma CCN2 levels showed significantly negative correlation with 6-month change of forced vital capacity (FVC) in patients with IPF. CONCLUSIONS: Plasma CCN2 is a potential biomarker for IPF.

Increased levels of soluble LR11 in cerebrospinal fluid of patients with Alzheimer disease.

BACKGROUND: Recent genetic and pathological studies have suggested that a lipoprotein receptor, LR11, is intricately implicated in the pathogenesis of Alzheimer disease (AD). We have recently established a novel sandwich ELISA, which enabled the sensitive quantification of a soluble LR11 (sLR11). By this ELISA, we attempted to determine the difference in the levels of CSF sLR11 in AD patients. METHODS: We examined CSF from 29 AD patients, 20 frontotemporal lobar degeneration patients and 27 age-matched control subjects. The CSF sLR11 level as well as the levels of tau and beta-amyloid42 (Abeta42) were determined by sandwich ELISA. RESULTS: The CSF tau level and tau/Abeta42 ratio were significantly increased (p < 0.01) in the AD patients. The CSF sLR11 level in the AD patients was significantly higher (p < 0.01) than that of the frontotemporal lobar degeneration patients and the controls. The APOE-epsilon4-positive AD patients have higher sLR11 levels than the APOE-epsilon4-negative patients (p < 0.01). CONCLUSIONS: These results suggest that the quantification of CSF sLR11 may serve as a biomarker of AD, although the diagnostic value for individual patients is limited. An elevated CSF sLR11 level in AD patients may be relevant to AD pathogenesis.

Subtraction method for determination of N-terminal connective tissue growth factor.

BACKGROUND: Connective tissue growth factor (CTGF) may be a potential marker of fibrosis. However, platelet-derived CTGF may be released into the plasma by platelet activation during or after blood collection, thereby interfering with accurate determination of the true plasma CTGF level. Plasma CTGF exists as the N-terminal CTGF fragment (N-fragment), composed of modules 1 and 2, whereas platelet CTGF exists as full-length CTGF (full-length), composed of modules 1-4. We perceived the need to develop a method for distinguishing between the N-fragment and full-length CTGF levels, so that the true plasma and serum CTGF (N-fragment) levels could be accurately determined. METHODS: Full-length levels were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies recognizing modules 1 and 4, respectively (M1/4 ELISA). Total CTGF (full-length CTGF plus N-terminal CTGF) levels were determined by a sandwich ELISA using two monoclonal antibodies recognizing modules 1 and 2, respectively (M1/2 ELISA). N-terminal CTGF levels were determined by subtracting the full-length levels from the total CTGF levels. RESULTS: Both the M1/2 and M1/4 ELISAs showed good analytical performance. When the CTGF levels of plasma and serum collected simultaneously from the same subject were compared, the N-fragment levels determined by the subtraction method were the same, in spite of the fact that full-length CTGF was present in the sample. CONCLUSION: N-fragment levels in plasma and serum can be accurately determined by this subtraction method, even if full-length CTGF in platelets is released during or after blood collection.

Development of an immunoassay for the quantification of soluble LR11, a circulating marker of atherosclerosis.

BACKGROUND: Vascular smooth muscle cells (SMCs) migrate from the arterial media to the intima in the progression of atherosclerosis, and dysfunction of SMCs leads to enhanced atherogenesis. A soluble form of the LDL receptor relative with 11 ligand-binding repeats (sLR11) is produced by the intimal SMCs, and the circulating concentrations of sLR11 likely reflect the pathophysiological condition of intimal SMCs. Furthermore, polymorphism of the LR11 gene has been found to be related to the onset of Alzheimer disease. This study describes the development of a sandwich immunoassay for quantifying sLR11 in human serum and cerebrospinal fluid. METHODS: We used synthetic peptides or DNA immunization to produce monoclonal antibodies (MAbs) A2-2-3, M3, and R14 against different epitopes of LR11. RESULTS: sLR11 was immunologically identified as a 250-kDa protein in human serum and cerebrospinal fluid by SDS-PAGE separation, and was purified from serum by use of a receptor-associated protein and MAb M3. An immunoassay for quantification of sLR11 with a working range of 0.25-4.0 microg/L was developed using the combination of MAbs M3 and R14. Treatment of serum with 5.25% n-nonanoyl-N-methyl-d-glucamine reduced the matrix effects of serum on the absorbance detection in the ELISA system. The linear dynamic range of the ELISA spanned the variation of circulating sLR11 concentrations in individuals with atherosclerosis. CONCLUSIONS: A sandwich ELISA was established for quantifying sLR11 in serum and cerebrospinal fluid. This technique provides a novel means for assessing the pathophysiology of atherosclerosis, and possibly neurodegenerative diseases.

Formation of prebeta1-HDL during lipolysis of triglyceride-rich lipoprotein.

Prebeta1-HDL, a putative discoid-shaped high-density lipoprotein (HDL) is known to participate in the retrieval of cholesterol from peripheral tissues. In this study, to clarify potential sources of this lipoprotein, we conducted heparin injection on four Japanese volunteer men and found that serum triglyceride (TG) level decreased in parallel with the increase in serum nonesterified fatty acids and plasma lipoprotein lipase (LPL) protein mass after heparin injection. Plasma prebeta1-HDL showed considerable increases at 15 min after the heparin injection in all of the subjects. In contrast, serum HDL-C levels did not change. Gel filtration with fast protein liquid chromatography system (FPLC) study on lipoprotein profile revealed that in post-heparin plasma, low-density lipoprotein and alphaHDL fractions did not change, whereas there was a considerable decrease in very low-density lipoprotein (VLDL) fraction and an increase in prebeta1-HDL fraction when compared with those in pre-heparin plasma. We also conducted in vitro analysis on whether prebeta1-HDL was produced during VLDL lipolysis by LPL. One hundred microliters of VLDL extracted from pooled serum by ultracentrifugation was incubated with purified bovine milk LPL at 37 degrees C for 0-120 min. Prebeta1-HDL concentration increased in a dose dependent manner with increased concentration of added LPL in the reaction mixture and with increased incubation time, indicating that prebeta1-HDL was produced during lipolysis of VLDL by LPL. Taken these in vivo and in vitro analysis together, we suggest that lipolysis of VLDL particle by LPL is an important source for formation of prebeta1-HDL.

Plasma pre beta1-HDL level is elevated in unstable angina pectoris.

Pre beta1-HDL, a minor HDL subfraction consisting of apolipoprotein A-I (apoA-I), phospholipids and unesterified cholesterol, plays an important role in reverse cholesterol transport. Plasma pre beta1-HDL levels have been reported to be increased in patients with coronary artery disease (CAD) and dyslipidemia. To clarify the clinical significance of measuring plasma pre beta1-HDL levels, we examined those levels in 112 patients with CAD, consisting of 76 patients with stable CAD (sCAD) and 36 patients with unstable angina pectoris (uAP), and in 30 patients without CAD as controls. The pre beta1-HDL levels were determined by immunoassay using a specific monoclonal antibody (Mab55201) that we established earlier. The mean pre beta1-HDL level in the CAD patients was significantly higher than the level in the controls (34.8+/-12.9 mg/L vs. 26.6+/-6.9 mg/L, p<0.001). In addition, the mean pre beta1-HDL level was markedly higher in the uAP subgroup than in the sCAD subgroup (43.1+/-11.5mg/L vs. 30.9+/-11.7 mg/L, p<0.0001). These tendencies remained even after excluding dyslipidemic subjects. These results suggest that elevation of the plasma pre beta1-HDL level is associated with the atherosclerotic phase of CAD and may be useful for identifying patients with uAP.

LCAT-dependent conversion of prebeta1-HDL into alpha-migrating HDL is severely delayed in hemodialysis patients.

Prebeta1-HDL is a minor HDL subfraction that acts as an efficient initial acceptor of cell-derived free cholesterol. During 37 degrees C incubation, plasma prebeta1-HDL decreases over time due to its conversion to alpha-migrating HDL by lecithin:cholesterol acyltransferase (LCAT). This conversion may be delayed in hemodialysis patients who have decreased LCAT activity. To clarify whether LCAT-dependent conversion of prebeta1-HDL to alpha-migrating HDL is delayed in hemodialysis patients, prebeta1-HDL concentrations were determined in 45 hemodialysis patients and 45 gender-matched control subjects before and after 37 degrees C incubation with and without the LCAT inhibitor. It was found that the baseline prebeta1-HDL concentration in hemodialysis patients was more than twice that in the controls (44.9 +/- 21.4 versus 19.8 +/- 6.7 mg/L apoAI; P < 0.001). After 2-h incubation, the LCAT-dependent decrease in prebeta1-HDL in hemodialysis patients was about one-third of that in the controls (30 +/- 27 versus 97 +/- 17% of baseline; P < 0.01). The LCAT-dependent rate of decrease in prebeta1-HDL levels (DR(prebeta1)) was the same for samples from hemodialysis patients exhibiting normal (> or =1.03 mmol/L) and low HDL-cholesterol levels (32 +/- 32 versus 28 +/- 23% of baseline; NS). DR(prebeta1) was positively correlated with LCAT activity (r = 0.617; P < 0.001). In conclusion, the LCAT-dependent conversion of prebeta1-HDL to alpha-migrating HDL is severely delayed in hemodialysis patients. The impaired catabolism of prebeta1-HDL may accelerate atherosclerosis in hemodialysis patients.