A novel method for measuring human hepatic lipase activity in postheparin plasma
2006; Elsevier BV; Volume: 48; Issue: 2 Linguagem: Inglês
10.1194/jlr.d600022-jlr200
ISSN1539-7262
AutoresShigeyuki Imamura, Junji Kobayashi, Shin‐ichi Sakasegawa, Atsushi Nohara, Katsuyuki Nakajima, Masa‐aki Kawashiri, Akihiro Inazu, Masakazu Yamagishi, Jun‐ichi Koizumi, Hiroshi Mabuchi,
Tópico(s)Pancreatic function and diabetes
ResumoThe objective of this study was to establish a hepatic lipase (HL) assay method that can be applied to automatic clinical analyzers. Seventy-four hyperlipidemic subjects (men/women 45/29) were recruited. Lipase activity was assayed measuring the increase in absorbance at 546 nm due to quinonediimine dye production. Reaction mixture R-1 contained 50 mM Tris-HCl (pH 9.5), 0.5 mM glycerol-1,2-dioleate, 0.4% (unless otherwise noted) polyoxyethylene-nonylphenylether, 3 mM ATP, 3 mM MgCl2, 1.5 mM CaCl2, monoacylglycerol-specific lipase, glycerol kinase, glycerol-3-phosphate oxidase, 0.075% N,N-bis-(4-sulfobutyl)-3-methylaniline-2 Na, peroxidase, ascorbic acid oxidase. Reaction mixture R-2 contained 50 mM Tris-HCl (pH9.5), 0.15% 4-aminoantypirine. Automated assay for activity was performed with a Model 7080 Hitachi analyzer. In the lipase assay, 160 μl of R-1 was incubated at 37°C with 3 μl of samples for 5 min, and 80 μl of R-2 was added. Within-run coefficient of variations was 0.9–1.0%. Calibration curve of lipase activity was linear (r = 0.999) between 0 and 320 U/l. Analytical recoveries of purified HL added to plasma were 96.6–99.8%. HL activity in postheparin plasma measured in this method had a closer correlation with HL mass by a sandwich ELISA (r = 0.888, P < 0.0001) than those in the conventional method using [14C-]triolein (r = 0.730, P < 0.0001). This assay method for HL activity can be applied to an automatic clinical analyzer. The objective of this study was to establish a hepatic lipase (HL) assay method that can be applied to automatic clinical analyzers. Seventy-four hyperlipidemic subjects (men/women 45/29) were recruited. Lipase activity was assayed measuring the increase in absorbance at 546 nm due to quinonediimine dye production. Reaction mixture R-1 contained 50 mM Tris-HCl (pH 9.5), 0.5 mM glycerol-1,2-dioleate, 0.4% (unless otherwise noted) polyoxyethylene-nonylphenylether, 3 mM ATP, 3 mM MgCl2, 1.5 mM CaCl2, monoacylglycerol-specific lipase, glycerol kinase, glycerol-3-phosphate oxidase, 0.075% N,N-bis-(4-sulfobutyl)-3-methylaniline-2 Na, peroxidase, ascorbic acid oxidase. Reaction mixture R-2 contained 50 mM Tris-HCl (pH9.5), 0.15% 4-aminoantypirine. Automated assay for activity was performed with a Model 7080 Hitachi analyzer. In the lipase assay, 160 μl of R-1 was incubated at 37°C with 3 μl of samples for 5 min, and 80 μl of R-2 was added. Within-run coefficient of variations was 0.9–1.0%. Calibration curve of lipase activity was linear (r = 0.999) between 0 and 320 U/l. Analytical recoveries of purified HL added to plasma were 96.6–99.8%. HL activity in postheparin plasma measured in this method had a closer correlation with HL mass by a sandwich ELISA (r = 0.888, P < 0.0001) than those in the conventional method using [14C-]triolein (r = 0.730, P < 0.0001). This assay method for HL activity can be applied to an automatic clinical analyzer. Hepatic lipase (HL), a lipolytic enzyme that is a secreted glycoprotein, is synthesized by hepatocytes and bound to heparin sulfate proteoglycans at the surface of liver sinusoidal capillaries. HL plays a major role in lipoprotein metabolism as a lipolytic enzyme that hydrolyzes triglycerides (TGs) and phospholipids in chylomicron remnants, intermediate-density lipoproteins (IDLs), and high-density lipoproteins (HDLs). Patients with HL deficiency present with hypercholesterolemia or hypertriglyceridemia and accumulate β-very-low-density lipoproteins (VLDLs), chylomicron remnants, IDLs, TG-rich low-density lipoproteins (LDLs), and HDLs (1Jansen H. Verhoeven A.J. Weeks L. Kastelein J.J. Halley D.J. van den Ouweland A. Jukema J.W. Seidell J.C. Birkenhaer J.C. Common C-to-T substitution at position -480 of the hepatic lipase promoter associated with a lowered lipase activity in coronary artery disease patients. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2837-2842Google Scholar, 2Breckenridge W.C. Little J.A. Alaupovic P. Wang C.S. Kuksis A. Kakis G. Lindgren F. Gardiner G. Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase. Atherosclerosis. 1982; 45: 161-179Google Scholar, 3Hegele R.A. Little J.A. Vezina C. Maguire G.F. Tu L. Wolever T.S. Jenkins D. J.A. Connelly P.W. Hepatic lipase deficiency. Clinical, biochemical, and molecular genetic characteristics. Arterioscler. Thromb. 1993; 13: 720-728Google Scholar, 4Carlson L.A. Holmquist L Nilsson-Ehle P. Deficiency of hepatic lipase activity in post-heparin plasma in familial hyper-[alpha]-triglyceridemia. Acta Med. Scand. 1986; 219: 435-447Google Scholar, 5Brand K. Dugi K.A. Brunzell J.D. Nevin D.N. Brewer Jr., H.B. Santamarina-Fojo S. A novel A→G mutation in intron I of the hepatic lipase gene leads to alternative splicing resulting in enzyme deficiency. J. Lipid Res. 1996; 37: 1213-1223Google Scholar, 6Knudsen P. Antikainen M. Ehnholm S. Uusi-Oukari M. Tenkanen H. Kahri S. Lahdenpera J. Tilly-Kiesi M. Bensadoun A. Taskinen M.R. A compound heterozygote for hepatic lipase gene mutations Leu334→Phe and Thr383→Met: correlation between hepatic lipase activity and phenotypic expression. J. Lipid Res. 1996; 37 (et al.): 825-834Google Scholar, 7Brunzell J.D. Deeb S.S. Scriver C.R. Beaudet A.L. Sly W.S. Familial lipoprotein lipase deficiency, apoC-II deficiency, and hepatic lipase deficiency. In The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 2789-2816Google Scholar).To date, the only available methods for measuring HL activity in postheparin plasma (PHP) have used 3H- or 14C-labeled trioleoyl glycerol as substrates in the presence of 1 M NaCl, because lipoprotein lipase (LPL) activity is known to be completely inhibited and remaining activities are considered to correspond to HL activity under these conditions. This assay procedure is complicated and does not appear to be suitable for routine work (8Kobayashi J. Nishida T. Ameis D. Stahnke G. Schotz M.C. Hashimoto H. Fukamachi I. Shirai K. Saito Y. Yoshida S. 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. Biochem. Biophys. Res. Commun. 1992; 182: 70-77Google Scholar, 9Kobayashi J. Shirai K. Saito Y. Yoshida S. Lipoprotein lipase with a defect in lipid interface recognition in a case with type I hyperlipidaemia. Eur. J. Clin. Invest. 1989; 19: 424-432Google Scholar). Several years ago, a method for measuring HL protein mass by ELISA in PHP was developed (10Nishimura M. Ohkaru Y. Ishii H. Sunahara N. Takagi A. Ikeda Y. Development and evaluation of a direct sandwich-enzyme-linked immunosorbent assay for the quantification of human hepatic triglyceride lipase mass in human plasma. J. Immunol. Methods. 2000; 235: 41-51Google Scholar).Still, to diagnose HL deficiency, demonstrating the lack of HL activity is considered to be essential. In the present study, therefore, we have developed and established, using dioleoylglycerol as a substrate, a novel and simple assay system for measuring HL activity in PHP that can be applied to an automatic clinical analyzer.MATERIALS AND METHODSSubjectsSeventy-four hyperlipidemic subjects (men/women 45/29) were recruited for this study (Table 1 ). The statement of institutional approval of the study was in accordance with the Declaration of Helsinki, and informed consent was obtained from all participants. Exclusion criteria included: age >75 years, body mass index (BMI) >30 kg/m2, diabetes mellitus, abnormal liver or muscle enzymes, creatinemia, use of antioxidants and lipid regulators, habitual alcohol intake >3 standard drinks/day, or endocrinological disorder. Blood samples were obtained following a fast of ≥12 h. PHP was obtained 10 min after 30 U/kg heparin injection. The monoclonal anti-LPL antibody 5D2 (MAb 5D2) was kindly provided by Dr. John Brunzell of the University of Washington, Seattle.TABLE 1Clinical profile of the study subjectsTotal (n = 74)Men (n = 45)Women (n = 29)Age, years53 ± 1652 ± 1654 ± 16BMI, kg/m223 ± 323 ± 222 ± 4Total cholesterol, mg/dl259 ± 92246 ± 90278 ± 95TGs, mg/dl137 ± 87146 ± 66105 ± 40HDL-C, mg/dl51 ± 1747 ± 1960 ± 12Fasting plasma glucose, mg/dl96 ± 8.3 (n = 64)96 ± 7.4 (n = 40)102 ± 34 (n = 24)HbA1c, %5.1 ± 0.5 (n = 53)5.1 ± 0.5 (n = 32)5.4 ± 1.1 (n = 21)BMI, body mass index; HDL, high-density lipoprotein, TG, triglyceride. Open table in a new tab Method for measuring lipase activity in partially purified HL and LPL fractionsHL and LPL were prepared from PHP using heparin-Sepharose column chromatography (11Ikeda Y. Takagi A. Yamamoto A. Purification and characterization of lipoprotein lipase and hepatic triglyceride lipase from human postheparin plasma: production of monospecific antibody to the individual lipase. Biochim. Biophys. Acta. 1989; 1003: 254-269Google Scholar). HL and LPL were eluted with 0.8 M NaCl and 1.6 M NaCl, respectively. Lipase activity was assayed measuring the increase in absorbance at 546 nm (subwave length; 660 nm) due to the production of quinonediimine dye based on the previously described assay procedure (12Fossati P. Ponti M. Paris P. Berti G. Tarenghi G. Kinetic colorimetric assay of lipase in serum. Clin. Chem. 1992; 38: 211-215Google Scholar, 13Panteghini M. Pagani F. Bonara R. Clinical and analytical evaluation of a continuous enzymatic method for measuring pancreatic lipase activity. Clin. Chem. 1993; 39: 304-308Google Scholar). Reaction mixture R-1 contained 50 mM Tris-HCl (pH 9.5), 0.5 mM dioleoylglycerol, 0.4% polyoxyethylene-nonylphenylether, 3 mM ATP, 3 mM MgCl2, 1.5 mM CaCl2, monoacylglycerol-specific lipase (0.75 U/ml), glycerol kinase (0.75 U/ml), glycerol-3-phosphate oxidase (37.5 U/ml), 0.075% N,N-bis-(4-sulfobutyl)-3-methylaniline-2 Na, peroxidase (10 U/ml), and ascorbic acid oxidase (10 U/ml). Reaction mixture R-2 contained 50 mM Tris-HCl (pH 9.5), and 0.15% 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one. Automated assay of lipase activity was performed with a Model 7080 Hitachi automatic clinical analyzer. In the lipase assay, 160 μl of R-1 was incubated at 37°C with 3 μl of samples for 5 min, and then 80 μl of R-2 was added. This reaction produced a violet quinonediimine dye with a peak absorbance at 546 nm. HL activity generating 1 μmol of monoglyceride from diglyceride per minute was defined as 1 unit.The method for measuring HL activity in PHPWe measured HL activity in PHP using the above-mentioned method either in the presence (HL activity) or absence (total lipase activity) of 1 M NaCl.Separation of HL and LPL by heparin-Sepharose CL-6BPHP (5 ml) was dialyzed against 1 l of 1.6 M NaCl containing 10 mM PIPES-NaOH (pH 7.5) at 4°C for 18 h. The dialysate was dialyzed against 500 ml of 0.3 M NaCl containing 10 mM PIPES-NaOH (pH 7.5) at 4°C for 18 h.The dialysate was applied to a heparin-Sepharose CL-6B column (1.6 × 3 cm). The column was washed with 30 ml of 0.3 M NaCl containing 0.1 mM EDTA, 10% sucrose, 0.025% BSA, and 10 mM PIPES-NaOH (pH 7.5).HL and LPL were eluted with 20 ml of 0.8 M NaCl containing 0.1 mM EDTA, 10% sucrose, 0.025% BSA, 10 mM PIPES-NaOH (pH 7.5), 20 ml of 1.6 M NaCl containing 0.1 mM EDTA, 10% sucrose, 0.025% BSA, and 10 mM PIPES-NaOH (pH 7.5) respectively. These HL and LPL fractions were concentrated with an Amicon Ultra membrane (molecular cut; 30 kDa), reconstituted with 2 ml of 10 mM PIPES-NaOH (pH 7.5) containing 10% sucrose and 0.1 mM EDTA. The concentrated HL and LPL was stored at −20°C.Effects of MAb 5D2 on lipase activity in PHP- or NaCl-eluted fraction from heparin-SepharoseTo evaluate the specificities of the lipase activity determined by the present method, we investigated the effects of the addition of MAb 5D2 in the reaction mixture against the enzyme activity in either a PHP- or an 0.8 M NaCl-eluted fraction from heparin-Sepharose.The conventional method for measuring HL activity in PHPIn the conventional method for measuring HL activity in PHP, total lipase activity was measured using Triton X-100-emulsified [14C]triolein, based on the previously reported method (8Kobayashi J. Nishida T. Ameis D. Stahnke G. Schotz M.C. Hashimoto H. Fukamachi I. Shirai K. Saito Y. Yoshida S. 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. Biochem. Biophys. Res. Commun. 1992; 182: 70-77Google Scholar, 9Kobayashi J. Shirai K. Saito Y. Yoshida S. Lipoprotein lipase with a defect in lipid interface recognition in a case with type I hyperlipidaemia. Eur. J. Clin. Invest. 1989; 19: 424-432Google Scholar). The remaining activity in the presence of 1 M NaCl was defined as HL activity.The method for measuring HL protein mass in PHPHL protein mass in PHP was detected using a sandwich ELISA following the previously described method (10Nishimura M. Ohkaru Y. Ishii H. Sunahara N. Takagi A. Ikeda Y. Development and evaluation of a direct sandwich-enzyme-linked immunosorbent assay for the quantification of human hepatic triglyceride lipase mass in human plasma. J. Immunol. Methods. 2000; 235: 41-51Google Scholar).Statistical analysisStatistical evaluation was performed using StatView-J 5.0 software (SAS Institute, Cary NC, on a Macintosh Computer). Pearson's correlation coefficients analysis was carried out. Results were expressed as mean ± SD, and the significance levels were set at P < 0.05.RESULTSEffect on lipase activity of addition of polyoxyethylene-nonylphenylether in the reaction mixtureLipase activity detected in our method increased in a dose-dependent manner with increasing concentrations of polyoxyethylene-nonylphenylether, a nonionic detergent, in the reaction mixture (Fig. 1 ). Lipase activity detected the fractions eluted in 0.3 M, 0.8 M, and 1.6 M NaCl using reaction mixtures containing 0.5 mM dioleoylglycerol (Table 2 ). Lipase activity was detected only in the fractions of heparin-Sepharose chromatography eluted in 0.8 M NaCl, suggesting that this activity corresponds to HL. In contrast, the LPL fraction (1.6 M NaCl) of heparin-Sepharose chromatography showed no lipase activity, even when apolipoprotein C-II (apoC-II) was added as LPL-specific activator.TABLE 2Lipase activity in the fractions eluted in 0.3 M, 0.8 M, and 1.6 M NaCl using dioleoylglycerol as substrateFractionsActivity (U/l)Total Activity (mU)Plasma2521,2600.3 M NaCl000.8 M NaCl eluate387600.8 M NaCl concentrate3206401.6 M NaCl concentrate00 Open table in a new tab Effect of MAb 5D2 on lipase activity in the PHP- or NaCl-eluted fractionTo determine whether the activity measured in the present method is affected by anti-LPL MAb, we studied the additive effect of the MAb 5D2 on lipase activity in the PHP- or 0.8 M NaCl-eluted fraction from heparin-Sepharose (Fig. 2 ). MAb 5D2 did not affect lipase activity at all in either in the PHP- or the 0.8 M NaCl-eluted fraction, whereas lipase activity in PHP was inhibited by 40% in the presence of the MAb 5D2 in an assay system for LPL activity (data not shown).Fig. 2Effect of anti-lipoprotein lipase monoclonal antibody 5D2 on lipase activity in postheparin plasma (PHP)- or 0.8 NaCl-eluted fraction from heparin-Sepharose. The reaction condition for the measurement of lipase activity was described in Materials and Methods, with the exception that increasing amounts of 5D2 were added in the reaction mixture. PHP was obtained from a normal volunteer 10 min after 30 U/kg heparin injection.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Correlation of HL activity with HL mass in PHPHL activity in PHP measured in the present method was highly correlated with HL mass by a sandwich ELISA in 74 hyperlipidemic Japanese subjects (Fig. 3 ). By comparison, HL activity measured in the conventional method using Triton X-100-emulsified [14C]triolein as a substrate [conventional HL (cHL) activity] had a weaker correlation with HL mass.Fig. 3Relationship of HL mass to HL activity and total lipase activity from PHP in 74 hyperlipidemic Japanese subjects (men/women 45/29). Lipase activity in PHP was measured using the method described in Materials and Methods in either the presence (HL activity) or absence (total lipase activity) of 1 M NaCl. HL activity generating 1 μmol of monoglyceride from diglyceride per minute was defined as 1 unit. HL mass is shown in ng/ml.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also analyzed the correlation of HL activity with HL mass in PHP in men (n = 45). HL activity in PHP measured in the present method was highly correlated with HL mass by a sandwich ELISA (r = 0.886, P < 0.001). By comparison, cHL activity had a weaker correlation with HL mass (r = 0.676, P < 0.001).Furthermore, we analyzed correlation of HL activity with HL mass in PHP in women (n = 29). HL activity in PHP measured in the present method was highly correlated with HL mass by a sandwich ELISA (r = 0.901, P < 0.001). By comparison, cHL activity had a slightly weaker correlation with HL mass (r = 0.852, P < 0.001).HL activity in PHP measured in the presence or absence of NaClThe remaining activity in the presence of 1 M NaCl using Triton X-100-emulsified [14C]triolein as a substrate is usually interpreted as HL activity. Unexpectedly, the correlation coefficient of HL activity in the absence of 1 M NaCl with HL mass was almost equal to that in the presence of 1 M NaCl with HL mass (0.892 vs. 0.901 in women; 0.895 vs. 0.886 in men) (Table 3 ).TABLE 3Correlation of HL activity and mass with several metabolic parameters in men and women combined (N = 74)T-Lipase ActivityHL ActivityHL MasscHL ActivityBMI−0.035−0.0590.024−0.057Total cholesterol−0.075−0.042−0.0470.240aP < 0.05.TGs0.1440.1670.290aP < 0.05.0.119HDL-C−0.330bP < 0.01.−0.310bP < 0.01.−0.356bP < 0.01.−0.183HDL2-C−0.317bP < 0.01.−0.300aP < 0.05.−0.348bP < 0.01.−0.231HDL3-C−0.070−0.051−0.0310.159T-lipase activityna0.978cP < 0.001.0.896cP < 0.001.0.743cP < 0.001.HL activity0.978cP < 0.001.na0.888cP < 0.001.0.747cP < 0.001.HL mass0.896cP < 0.001.0.888cP < 0.001.na0.730cP < 0.001.cHL activity0743cP < 0.001.0.747cP < 0.001.0.730cP < 0.001.naT-lipase, total lipase; HL, hepatic lipase; na, not available; cHL activity, HL activity measured in conventional method using Triton X-100-emulsified [14C]triolein.Lipase activities measured in the absence and the presence of 1 M NaCl were referred to as T-lipase activity and HL activity, respectively.a P < 0.05.b P < 0.01.c P < 0.001. Open table in a new tab Correlation of HL activity with age and BMIHL activity did not show a significant correlation with age or BMI in the study subjects (Table 3). Even measured separately for each gender, no correlation existed (data not shown).Correlation of HL activity with lipids and lipoproteinsHL activity in the present method did not have significant associations with total cholesterol or TG, and had an inverse association with HDL-C and HDL2-C (Table 3). Measured separately for each gender, the observed associations between HL activity and HDL-C did not reach statistical significance, partly because of the small sample size (data not shown).Other findingsThe within-run (n = 20) coefficient of variation was 0.9–1.0%. The calibration curve of lipase activity was linear (r = 0.999) between 0 and 500 U/l. Analytical recoveries of purified HL added to plasma were 96.6–99.8%. This method was free of interference by bilirubin C, bilirubin F, ascorbic acid, and intra-lipid. Weak interference by hemoglobin was observed. High activity of human pancreatic lipase (1,000 U/l) showed no lipase activity.DISCUSSIONIn the present study, we have developed a novel method for measuring HL activity in PHP. Instead of radioisotope-labeled substrate, we have used 0.2 mM dioleoylglycerol as a substrate for this new lipase assay.The finding shown in Fig. 1 suggests that the existence of polyoxyethylene-nonylphenylether, a nonionic detergent, in the reaction mixture appeared to be a critical factor in the expression of HL enzyme activity, although at this time, the precise mechanisms have not been elucidated. Glycerol-1,2-dioleate became clear by adding nonionic detergent, suggesting that glycerol-1,2-dioleate came to exist in the reaction mixture as water-soluble mixed micelles with this nonionic detergent.The result shown in Table 2 that lipase activity was detected only in the 0.8 M NaCl-eluted fraction of the heparin-Sepharose indicated that lipase activity detected in the present method corresponds to that of HL. Also, the finding that the MAb 5D2 did not affect lipase activity in the 0.8 M NaCl-eluted fraction and PHP confirmed that this method specifically detected HL activity (Fig. 2). HL activity obtained using this method showed stronger associations with HL mass by sandwich ELISA than did cHL activity measured using Triton X-100-emulsified [14C]triolein (8Kobayashi J. Nishida T. Ameis D. Stahnke G. Schotz M.C. Hashimoto H. Fukamachi I. Shirai K. Saito Y. Yoshida S. 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. Biochem. Biophys. Res. Commun. 1992; 182: 70-77Google Scholar, 9Kobayashi J. Shirai K. Saito Y. Yoshida S. Lipoprotein lipase with a defect in lipid interface recognition in a case with type I hyperlipidaemia. Eur. J. Clin. Invest. 1989; 19: 424-432Google Scholar). This suggests that the present method could be more reliable for measuring HL activity in PHP than the conventional method using Triton X-100-emulsified [14C]triolein as a substrate.A previous report has shown extremely high correlation between HL mass and activity in PHP (10Nishimura M. Ohkaru Y. Ishii H. Sunahara N. Takagi A. Ikeda Y. Development and evaluation of a direct sandwich-enzyme-linked immunosorbent assay for the quantification of human hepatic triglyceride lipase mass in human plasma. J. Immunol. Methods. 2000; 235: 41-51Google Scholar). However, their method for measuring HL activity requires 3H-labeled triolein and is labor intensive and time consuming. HL is a lipolytic enzyme catalyzing the hydrolysis of TG and PL in IDL and HDL2. Whether HL is atherogenic or anti-atherogenic is still the subject of debate (14Jansen H. Verhoeven A.J. Sijbrands E.J. Hepatic lipase: a pro- or anti-atherogenic protein? J. Lipid Res. 2002; 43: 1352-1362Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 15Jansen H. Hepatic lipase: friend or foe and under what circumstances? Curr. Atheroscler. Rep. 2004; 6: 343-347Crossref PubMed Scopus (19) Google Scholar, 16Zambon A. Deeb S.S. Pauletto P. Crepaldi G. Brunzell J.D. Hepatic lipase: a marker for cardiovascular disease risk and response to therapy. Curr. Opin. Lipidol. 2003; 14: 179-189Google Scholar, 17Santamarina-Fojo S. Haudenschild C. Amar M. The role of hepatic lipase in lipoprotein metabolism and atherosclerosis. Curr. Opin. Lipidol. 1998; 9: 211-219Crossref PubMed Scopus (211) Google Scholar). Because HL lowers plasma concentrations of the pro-atherogenic apoB-containing lipoproteins as well as the anti-atherogenic HDL, the net effect of these HL-induced alterations in plasma lipoproteins on coronary artery disease is not easily predictable. Patients with HL deficiency present with hypercholesterolemia or hypertriglyceridemia and accumulate β-VLDLs, chylomicron remnants, IDLs, TG-rich LDLs, and HDLs (1Jansen H. Verhoeven A.J. Weeks L. Kastelein J.J. Halley D.J. van den Ouweland A. Jukema J.W. Seidell J.C. Birkenhaer J.C. Common C-to-T substitution at position -480 of the hepatic lipase promoter associated with a lowered lipase activity in coronary artery disease patients. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2837-2842Google Scholar, 2Breckenridge W.C. Little J.A. Alaupovic P. Wang C.S. Kuksis A. Kakis G. Lindgren F. Gardiner G. Lipoprotein abnormalities associated with a familial deficiency of hepatic lipase. Atherosclerosis. 1982; 45: 161-179Google Scholar, 3Hegele R.A. Little J.A. Vezina C. Maguire G.F. Tu L. Wolever T.S. Jenkins D. J.A. Connelly P.W. Hepatic lipase deficiency. Clinical, biochemical, and molecular genetic characteristics. Arterioscler. Thromb. 1993; 13: 720-728Google Scholar, 4Carlson L.A. Holmquist L Nilsson-Ehle P. Deficiency of hepatic lipase activity in post-heparin plasma in familial hyper-[alpha]-triglyceridemia. Acta Med. Scand. 1986; 219: 435-447Google Scholar, 5Brand K. Dugi K.A. Brunzell J.D. Nevin D.N. Brewer Jr., H.B. Santamarina-Fojo S. A novel A→G mutation in intron I of the hepatic lipase gene leads to alternative splicing resulting in enzyme deficiency. J. Lipid Res. 1996; 37: 1213-1223Google Scholar, 6Knudsen P. Antikainen M. Ehnholm S. Uusi-Oukari M. Tenkanen H. Kahri S. Lahdenpera J. Tilly-Kiesi M. Bensadoun A. Taskinen M.R. A compound heterozygote for hepatic lipase gene mutations Leu334→Phe and Thr383→Met: correlation between hepatic lipase activity and phenotypic expression. J. Lipid Res. 1996; 37 (et al.): 825-834Google Scholar, 7Brunzell J.D. Deeb S.S. Scriver C.R. Beaudet A.L. Sly W.S. Familial lipoprotein lipase deficiency, apoC-II deficiency, and hepatic lipase deficiency. In The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 2789-2816Google Scholar). The clinical profile of this lipid disorder could be quite similar to that of type III hyperlipidemia, which makes it difficult to identify this lipid disorder. To identify patients with HL deficiency, it is essential to demonstrate the lack of HL activity in PHP. In contrast, LPL deficiency is relatively easily identified, because patients with this lipid disorder usually have drastic hyperlipidemia due to marked accumulation of chylomicrons in the serum, causing acute pancreatitis (7Brunzell J.D. Deeb S.S. Scriver C.R. Beaudet A.L. Sly W.S. Familial lipoprotein lipase deficiency, apoC-II deficiency, and hepatic lipase deficiency. In The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 2789-2816Google Scholar). To date, as mentioned above, the available method for measuring HL activity in PHP has used 3H- or 14C-labeled trioleoyl glycerol as substrates in the presence of 1 M NaCl (8Kobayashi J. Nishida T. Ameis D. Stahnke G. Schotz M.C. Hashimoto H. Fukamachi I. Shirai K. Saito Y. Yoshida S. 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. Biochem. Biophys. Res. Commun. 1992; 182: 70-77Google Scholar, 9Kobayashi J. Shirai K. Saito Y. Yoshida S. Lipoprotein lipase with a defect in lipid interface recognition in a case with type I hyperlipidaemia. Eur. J. Clin. Invest. 1989; 19: 424-432Google Scholar). However, these assay procedures are complicated and require the use of radioisotopes. In the present study, therefore, we have developed and established a novel assay system for measuring HL activity in PHP using dioleoylglycerol as a substrate, without requiring radioisotope labeling. The results presented in Table 3, showing that total lipase activity (measured in the absence of 1 M NaCl) was similar to HL activity (measured in the presence of 1 M NaCl) in terms of correlation coefficients with HL mass, suggested that this assay could be suitable for measuring HL activity in the presence or absence 1 M NaCl.With regard to the correlation of HL activity with lipid and lipoproteins, there were inverse correlations with HDL-C (HDL2-C) in the whole subjects, but there was no correlation with BMI, total cholesterol, TGs, or HDL3-C. These findings might be compatible with the fact that HL is involved in catalyzing hydrolysis of TG in HDL2-C (7Brunzell J.D. Deeb S.S. Scriver C.R. Beaudet A.L. Sly W.S. Familial lipoprotein lipase deficiency, apoC-II deficiency, and hepatic lipase deficiency. In The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 2789-2816Google Scholar, 18Shirai K. Barnhart R.L. Jackson R.L. Hydrolysis of human plasma high density lipoprotein 2- phospholipids and triglycerides by hepatic lipase. Biochem. Biophys. Res. Commun. 1981; 100: 591-599Google Scholar).In addition to LPL and HL, in the past decade, considerable attention has been paid to the physiological role of endothelial lipase (EL) (19Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. A novel endothelial-derived lipase that modulates HDL metabolism. Nat. Genet. 1999; 21: 424-428Google Scholar), which is known to have higher phospholipase activity and lower activity in TG hydrolysis, compared with the other two lipases (20McCoy M.G. Sun G.S. Marchadier D. Maugeais C. Glick J.M. Rader D.J. Characterization of the lipolytic activity of endothelial lipase. J. Lipid Res. 2002; 43: 921-929Google Scholar). To our knowledge, however, there has been no report on what proportion of lipase activity in PHP is accounted for by EL. Despite this, we presume that there is little or no possibility that our present method for measuring HL activity overlapped the activity of EL, in view of their high positive correlation to HL mass.In summary, we have developed a novel and simple method for the assay of HL activity in PHP, which is suitable for application to an automatic clinical analyzer. Hepatic lipase (HL), a lipolytic enzyme that is a secreted glycoprotein, is synthesized by hep
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