Atsuko Takagi

Purification and characterization of lipoprotein lipase and hepatic triglyceride lipase from human postheparin plasma: production of monospecific antibody to the individual lipase.

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were purified to homogeneity from human postheparin plasma. Molecular, catalytic and immunological properties of the purified enzymes were investigated. The native molecular weights of LPL and HTGL were 67,200 and 65,500, respectively, by gel chromatography. The subunit molecular weights of LPL and HTGL were 60,600 and 64,600, respectively, suggesting that these enzymes are catalytically active in a monomeric form. In addition, the purified LPL and HTGL each gave a single protein band when they were detected as glycoproteins with a probe of concanavalin A. The purified enzyme preparations were free of detectable antithrombin III by Western blot analysis. Catalytic properties of the purified enzymes were examined using triolein-gum arabic emulsion and triolein particles stabilized with phospholipid monolayer as substrates. LPL catalyzed the complete hydrolysis of triolein to free oleate and monooleate in the presence of apolipoprotein C-II. Apparent Km values for triolein and apolipoprotein C-II were 1.0 mM and 0.6 microM, and Vmax was 40.7 mmol/h per mg. HTGL hydrolyzed triolein substrate at a rate much slower than LPL, and produced mainly free oleate with little monooleate. Apparent Km and Vmax values were 2.5 mM and 16.1 mmol/h per mg, respectively. Polyclonal antibodies were developed against the purified LPL and HTGL. The purity and specificity of these antisera were ascertained by immunotitration, Ouchterlony double diffusion and Western blot analyses. The anti-human LPL and anti-human HTGL antiserum specifically reacted with the corresponding either native or denaturated enzyme, indicating that two enzymes were immunologically distinct. We developed an assay system for LPL and HTGL in human PHP by selective immunoprecipitation of each enzyme with the corresponding antiserum.

Identification of homozygous lipoprotein lipase gene mutation in a woman with recurrent aggravation of hypertriglyceridaemia induced by pregnancy

Shigeki Suga, Naoki Tamasawa, Ichiro Kinpara, Hiroshi Murakami, Nobuhiko Kasai, Tomio Onuma, Yasuyuki Ikeda, Atsuko Takagi, Toshihiro Suda (Hirosaki University School of Medicine, Aomori; and National Cardiovascular Center Research Institute, Osaka, Japan). Identification of homozygous lipoprotein lipase gene mutation in a woman with recurrent aggravation of hyperglyceridaemia induced by pregnancy (Case Report). J Intern Med 1998; 243: 317–21.

Disruption of Autosomal Recessive Hypercholesterolemia Gene Shows Different Phenotype In Vitro and In Vivo

We previously characterized the patients with autosomal recessive hypercholesterolemia (ARH) as having severe hypercholesterolemia and retarded plasma low-density lipoprotein (LDL) clearance despite normal LDL receptor (LDLR) function in their cultured fibroblasts, and we identified a mutation in the ARH locus in these patients. ARH protein is an adaptor protein of the LDL and reportedly modulates its internalization. We developed ARH knockout mice (ARH−/−) to study the function of this protein. Plasma total cholesterol level was higher in ARH−/− mice than that in wild-type mice (ARH+/+), being attributed to a 6-fold increase of LDL, whereas plasma lipoprotein was normal in the heterozygotes (ARH+/−). Clearance of 125I-LDL from plasma was retarded in ARH−/− mice, as much as that found in LDLR−/− mice. Fluorescence activity of the intravenously injected 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)-LDL was recovered in the cytosol of the hepatocytes of ARH+/+ mice, but not in those of ARH−/− or LDLR−/− mice. Also, less radioactivity was recovered in the liver of ARH−/− or LDLR−/− mice when [3H]cholesteryl oleyl ether (CE)-labeled LDL was injected. In contrast, uptakes of [3H]CE-labeled LDL, 125I-LDL, and DiI-LDL were all normal or slightly subnormal when the ARH−/− hepatocytes were cultured. We thus concluded that the function of the hepatic LDLR is impaired in the ARH−/− mice in vivo, despite its normal function in vitro. These findings were consistent with the observations with the ARH homozygous patients and suggested that certain cellular environmental factors modulate the requirement of ARH for the LDLR function.

Development and evaluation of a direct sandwich-enzyme-linked immunosorbent assay for the quantification of human hepatic triglyceride lipase mass in human plasma

Abstract We have developed a direct sandwich-enzyme-linked immunosorbent assay (ELISA) for quantification of the hepatic triglyceride lipase (HTGL) immunoreactive mass in human plasma. This direct sandwich-ELISA uses a combination of two distinct monoclonal antibodies (MAbs), which recognize different epitopes on the HTGL molecule: a horseradish peroxidase (HRP)-labeled anti-human HTGL MAb (2(4)F12C12) as an enzyme-linked MAb, and an anti-human HTGL MAb (1(11)A3H3) coated on a microtiter plate as a solid-phase MAb. Purified human post-heparin plasma (PHP)-HTGL was used as the standard material. The detection range of the sandwich-ELISA was 40–800 ng of HTGL protein per ml of plasma. The intra- and inter-assay coefficients of variation were less than 2.0% and 2.3%, respectively. The recovery tests resulted in variation only between 97.7% and 103.5%. No significant assay interference was caused by a high concentration of triglyceride, hemoglobin, bilirubin, uric acid, or creatinine. The reliability of the HTGL mass values obtained with the direct sandwich-ELISA was assessed by comparison with the HTGL mass values determined by our earlier one-step sandwich-enzyme immunoassay (EIA). The two sets of values showed a highly significant correlation (r=+0.952, n=64). Strong correlation (r=+0.959, n=50) was also found between the HTGL masses with the direct sandwich-ELISA and the HTGL activities determined with a selective immunoinactivation assay. The HTGL mass concentrations in PHP from 64 healthy subjects were 1916±841 ng/ml by the direct sandwich-ELISA and 1925±785 ng/ml (mean±standard deviation (SD)) by the one-step sandwich-EIA. The present direct sandwich-ELISA permits rapid identification of certain HTGL abnormalities in PHP samples from patients with hypertriglyceridemia or diseases such as hypothyroidism or renal failure, which affect HTGL.

Development of a direct DNA sequencing method for detecting heterozygous mutations of the human lipoprotein lipase gene

OBJECTIVE The purpose of this study was to develop an improved method of direct DNA sequencing, which makes it possible to identify heterozygous mutations of the lipoprotein lipase (LPL) gene in order to understand the underlying genetic disorder of type IV hyperlipoproteinemia. METHODS AND RESULTS The direct sequencing method was improved by devising primers for amplifying the LPL gene and for sequencing DNA amplified by the polymerase chain reaction (PCR)T since the reported base sequences of the introns flanking exons of the LPL gene were limited to 40 bases. Improvement was achieved by attaching nine additional bases to both the PCR amplification primer and sequencing primer, and by optimizing the Tm value of the sequencing primers by adjusting the sequence of the nine extra bases. Use of the sequencing primers having suitable Tm values (48 degrees C-58 degrees C) made it possible to reduce nonspecific bands on the sequence ladder pattern and to identify heterozygous mutation sites in LPL gene exons 5 and 6 as model cases. CONCLUSION Our improved direct sequencing method is useful for identifying heterozygous mutation sites in human LPL gene exons and splicing consensus regions.

Development and evaluation of a direct sandwich enzyme-linked immunosorbent assay for the quantification of lipoprotein lipase mass in human plasma

Abstract Objective: The purpose of this study was to develop and evaluate a direct sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the lipoprotein lipase (LPL) immunoreactive mass in human plasma using monoclonal antibodies (MAbs) directed against LPL purified from human postheparin plasma (PHP). Design: The DNA fragment containing exon 1 through 7 of the LD-H gene were amplified by PCR and directly sequenced. Total RNA was prepared from venous blood and the proportion of LD-H cDNA to total LD cDNA was semiquantified. Methods and results: The direct sandwich-ELISA was performed using a combination of two distinct types of MAbs that recognize different epitopes on the LPL molecule. The immunoreactive mass of human LPL was specifically measured using a horseradish peroxidase-labeled anti-human LPL MAb [1(1)D2B2] as an enzyme-linked MAb, and an anti-human LPL MAb [2(10)F8F9] coated on a polystyrene microtiter plate as a solid-phase MAb. Purified human PHP-LPL was used as a standard material. The detection range of the sandwich-ELISA was 3.6–460 ng of LPL protein per mL of plasma. The intra- and interassay coefficients of variation were less than 5.9% and 3.3%, respectively. The validity of this method was additionally assured by the recovery test, which resulted in the variation only between 97.5% and 105.1%, and also by the interference test, which resulted in noninterference of LPL assay with a high concentration of triglyceride, hemoglobin, bilirubin, uric acid, or creatinine. To assess the reliability of the LPL mass values obtained with the direct sandwich-ELISA, they were compared with LPL mass values determined by the one-step sandwich-EIA (MARKIT-F LPL EIA kit) previously established by us. This comparison showed a highly significant correlation ( r = +0.990) between the two sets of values. The LPL mass concentrations in PHP from 33 healthy subjects were 267 ± 53 and 257 ± 59 ng/mL (mean ± SD), respectively. Conclusion: T he present direct sandwich-ELISA is useful for rapidly identifying certain abnormalities of LPL in PHP samples from patients with hypertriglyceridemia.

Identification of compound heterozygous mutations (G188E/W382X) of lipoprotein lipase gene in a Japanese infant with hyperchylomicronemia : The G188E mutation was newly identified in Japanese

We herein report a case of a 5-month-old Japanese female (patient AN) with fasting hyperchylomicronemia due to a primary lipoprotein lipase (LPL) deficiency. Patient AN was compound heterozygous for a missense mutation (GG818G-->GAG/Gly188-->Glu; G188E) in exon 5 and a nonsense mutation (TGG1401-->TGA/Trp382-->Stop; W382X) in exon 8 of the LPL gene. This resulted in less than 10% of the control levels for both the LPL activity and immunoreactive LPL mass in the postheparin plasma. A G188E mutation was thus identified for the first time in a Japanese, and the haplotype of this G188E allele was different from that of the G188E alleles identified in other ethnic groups. This additional mutation might be useful for early diagnosis of LPL gene aberrations in Japanese patients with fasting hyperchylomicronemia.

A newly identified lipoprotein lipase (LPL) gene mutation (F270L) in a Japanese patient with familial LPL deficiency.

We have systematically investigated the molecular defects resulting in a primary lipoprotein lipase (LPL) deficiency in a Japanese male infant (proband SH) with fasting hyperchylomicronemia. Neither LPL activity nor immunoreactive LPL mass was detected in pre- or postheparin plasma from proband SH. DNA sequence analysis of the LPL gene of proband SH revealed homozygosity for a novel missense mutation of F270L (Phe(270)-->Leu/TTT(1065)-->TTG) in exon 6. The function of the mutant F270L LPL was determined by both biochemical and immunocytochemical studies. In vitro expression experiments on the mutant F270L LPL cDNA in COS-1 cells demonstrated that the mutant LPL protein was synthesized as a catalytically inactive form and its total amount was almost equal to that of the normal LPL. Moreover, the synthesized mutant LPL was non-releasable by heparin because the intracellular transport of the mutant LPL to the cell surface - by which normal LPL becomes heparin-releasable - was impaired due to the abnormal structure of the mutant LPL protein. These findings explain the failure to detect LPL activities and masses in pre- and postheparin plasma of the proband. The mutant F270L allele generated an XcmI restriction enzyme site in exon 6 of the LPL gene. The carrier status of F270L in the probands family members was examined by digestion with XcmI. The proband was ascertained to be homozygous for the F270L mutation and his parents and sister were all heterozygous. The LPL activities and masses of the parents and the sister (carriers) were half or less than half of the control values. Regarding the phenotype of the carriers, the mother with a sign of hyperinsulinemia manifested hypertriglyceridemia (type IV hyperlipoproteinemia), whereas the healthy father and the sister were normolipidemic. Hyperinsulinemia may be a strong determinant of hypertriglyceridemia in subjects with heterozygous LPL deficiency.

Change in Immunoreactive Human Hepatic Triglyceride Lipase (HTGL) Mass and the Shelf‐Life of the HTGL ELISA Kit in Long‐Term Storage

Abstract Objectives of this work are to study changes in the immunoreactive HTGL mass during storage under various conditions. In addition, the shelf‐life of the HTGL ELISA kit was confirmed. The immunological reactivity of HTGL in PHP stored in the liquid, frozen, or lyophilized state was monitored using purified human PHP‐HTGL as the standard material. Furthermore, the long‐term stability of the HTGL ELISA kit was ascertained. The immunoreactive HTGL mass in the lyophilized PHP maintained its initial immunological reactivity for at least 26 months at 4°C or lower. The other reagents included in the HTGL ELISA kit also have a long shelf‐life when they are stored at 4°C or less. HTGL in PHP was stabilized by lyophilization and can be used as the standard material for HTGL ELISA; the HTGL ELISA kit has a long shelf‐life, i.e., more than two years.

Tricaprin Rescues Myocardial Abnormality in a Mouse Model of Triglyceride Deposit Cardiomyovasculopathy

Triglyceride deposit cardiomyovasculopathy (TGCV) is an intractable cardiovascular disease for which a specific treatment is urgently required. In TGCV, adipose triglyceride lipase (ATGL) deficiency results in the abnormal intracellular metabolism of long-chain fatty acid (LCFA) which leads to TG deposition. Medium-chain triglycerides have been used as an important functional food for various human diseases. To address the potential activities of tricaprin, a medium-chain triglyceride, on cardiac dysfunctions of TGCV, we examined the effects of tricaprin diet on Atgl knock out (KO) mice, an animal model for TGCV. Cardiac imaging tests showed that the tricaprin diet reduced TG accumulation, resulting from improvement of LCFA metabolism, and improved left ventricular function in Atgl KO mice compared to that in mice fed the control diet. In conclusion, tricaprin improved myocardial abnormality in the TGCV model, thus, it may be useful for the treatment of patients with TGCV.