Isamu Fukamachi

Lipoprotein lipase mass and activity in severe hypertriglyceridemia

To clarify the role of defective lipoprotein lipase (LPL) in hypertriglyceridemia, the LPL masses and LPL activities in post-heparin plasma (PHP) were studied in severe hypertriglyceridemias. The developed sandwich enzyme immunoassay for the LPL was sensitive from 0.5 to 20 ng/ml of LPL in human PHP. The plasma LPL mass increased by heparin injection (30 USP units/kg) and was found to positively correlate with LPL activity. The mean LPL activity from PHP of normal controls was 2,960 +/- 1,057 nmol/ml/h. The mean LPL masses from human pre- and 15-min post-heparin plasma from normal subjects were 25 +/- 5 ng/ml and 224 +/- 60 ng/ml, respectively. Thus the specific activity of LPL from PHP of normal controls was calculated to be 13.3 mumol FFA released/h/microgram LPL. Among hypertriglyceridemic patients with over 1,000 mg/dl of serum triglyceride, the incidence of patients with LPL masses less than -2 standard deviations (S.D.) of those of average normal control subjects was found to be 27%. Seventy percent of patients showed specific activities within + 2 S.D. of those of average control LPL, and 30% showed significantly low specific activities less than -2 S.D. despite the fact that LPL masses were not less than -2 S.D. of the average normal controls. These results suggest that the evaluation of LPL masses in PHP would be useful for finding functionally defective LPL in patients with hypertriglyceridemia, and that up to 30% severe hypertriglyceridemias may have functionally defective LPL.

A heterozygous mutation (the codon for Ser447----a stop codon) in lipoprotein lipase contributes to a defect in lipid interface recognition in a case with type I hyperlipidemia.

Previously, we reported a case with type I hyperlipidemia due to a lipid interface recognition deficiency in lipoprotein lipase (LPL) (1). The LPL from postheparin plasma of this patient did not hydrolyze TritonX-100-triolein or very low density lipoprotein-triolein but did hydrolyze tributyrin and LysoPC-triolein substrates. Sequence analysis of the probands DNA revealed a heterozygous nucleotide change: a C----G transversion at position of 1595, resulting in changing the codon for Ser447 to a stop codon. Expression studies of this mutant LPLcDNA in Cos-1 cells produced and secreted considerable amounts of LPL mass in the culture media. The mutated LPL hydrolyzed much less TritonX-100-triolein than wild type LPL, whereas hydrolysis of tributyrin and LysoPC--triolein was the same with both the mutant and wild type LPL. These results suggest that this mutation might be responsible for the property of the LPL with a defect in lipid interface recognition in the type I patient we reported.

LCAT-Dependent Conversion of Preβ1-HDL into α-Migrating HDL is Severely Delayed in Hemodialysis Patients

ABSTRACT. Preβ1-HDL is a minor HDL subfraction that acts as an efficient initial acceptor of cell-derived free cholesterol. During 37°C incubation, plasma preβ1-HDL decreases over time due to its conversion to α-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 preβ1-HDL to α-migrating HDL is delayed in hemodialysis patients, preβ1-HDL concentrations were determined in 45 hemodialysis patients and 45 gender-matched control subjects before and after 37°C incubation with and without the LCAT inhibitor. It was found that the baseline preβ1-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 versus 97 ± 17% of baseline; P preβ1 ) was the same for samples from hemodialysis patients exhibiting normal (≥1.03 mmol/L) and low HDL-cholesterol levels (32 ± 32 versus 28 ± 23% of baseline; NS). DR preβ1 was positively correlated with LCAT activity ( r = 0.617; P

Analytical performance of a sandwich enzyme immunoassay for preβ1-HDL in stabilized plasma

We have established an immunoassay for preβ1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because preβ1-HDL is unstable during storage, fresh plasma must be used for preβ1-HDL measurements. In this study, we describe a method of stabilizing preβ1-HDL, and evaluate the analytical performance of the immunoassay for preβ1-HDL. Fresh plasma was stored under various conditions with or without a pretreatment consisting of a 21-fold dilution into 50% (v/v) sucrose. Preβ1-HDL concentration was measured by immunoassay. In nonpretreated samples, preβ1-HDL decreased significantly from the baseline after 6 h at room temperature. Although preβ1-HDL was more stable at 0°C than at room temperature, it increased from 30.2 ± 8.5 (SE) to 56.5 ± 5.5 mg/l apolipoprotein A-I (apoA-I) (P < 0.001) in hyperlipidemics, and from 18.4 ± 1.2 to 37.9 ± 3.3 mg/l apoA-I (P < 0.001) in normolipidemics after 5-day storage. After 30-day storage at −80°C, preβ1-HDL increased from 29.0 ± 4.0 to 38.0 ± 5.7 mg/l apoA-I (P < 0.001) in hyperlipidemics, whereas it did not change in normolipidemics. In pretreated samples, preβ1-HDL concentration did not change significantly under any of the above conditions. Moreover, preβ1-HDL concentrations determined by immunoassay correlated with those determined by native two-dimensional gel electrophoresis (n = 24, r = 0.833, P < 0.05). An immunoassay using MAb55201 with pretreated plasma is useful for clinical measurement of preβ1-HDL.

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-β (TGF-β), 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.

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.

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 β-amyloid42 (Aβ42) were determined by sandwich ELISA. Results: The CSF tau level and tau/Aβ42 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-Ε4-positive AD patients have higher sLR11 levels than the APOE-Ε4-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.

Delineation of the Role of Pre-β1-HDL in Cholesterol Efflux Using Isolated Pre-β1-HDL

OBJECTIVE The role of pre-beta1-high density lipoprotein (pre-beta1-HDL) in cholesterol efflux was investigated by separating human plasma into purified pre-beta1-HDL and pre-beta1-HDL-deficient plasma by using a monoclonal antibody specifically reacting with pre-beta1-HDL. METHODS AND RESULTS When compared with whole plasma, pre-beta1-HDL-deficient plasma was equally efficient in promoting cholesterol efflux from human skin fibroblasts and THP-1 human macrophage cells. When added at the same apolipoprotein A-I concentration, pre-beta1-HDL was less effective than whole plasma in promoting cholesterol efflux from fibroblasts but equally effective in promoting cholesterol efflux from THP-1 cells. However, pre-beta1-HDL-deficient plasma reconstituted with 16% pre-beta1-HDL was more active than whole plasma, demonstrating that pre-beta1-HDL does promote cholesterol efflux actively. The amount of cellular cholesterol present in reisolated pre-beta1-HDL was 1.5- to 2-fold greater after incubation of the cells with whole plasma than after incubation of the cells with pre-beta1-HDL-deficient plasma or plasma treated with the anti-pre-beta1-HDL antibody. However, the anti-pre-beta1-HDL antibody did not inhibit cholesterol efflux. CONCLUSIONS We conclude that whereas pre-beta1-HDL is capable of taking up cellular cholesterol, its presence in plasma is not essential for cholesterol efflux, at least in vitro. Instead, pre-beta1-HDL may be the first product of apolipoprotein A-I lipidation during the formation of HDL but may not play a major role in transferring cellular cholesterol to HDL.

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/TGFβ 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.

Formation of preβ1-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.