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• W2553163325 abstract "LPL hydrolyzes triglycerides in plasma lipoproteins. Due to the complex regulation mechanism, it has been difficult to mimic the physiological conditions under which LPL acts in vitro. We demonstrate that isothermal titration calorimetry (ITC), using human plasma as substrate, overcomes several limitations of previously used techniques. The high sensitivity of ITC allows continuous recording of the heat released during hydrolysis. Both initial rates and kinetics for complete hydrolysis of plasma lipids can be studied. The heat rate was shown to correspond to the release of fatty acids and was linearly related to the amount of added enzyme, either purified LPL or postheparin plasma. Addition of apoC-III reduced the initial rate of hydrolysis by LPL, but the inhibition became less prominent with time when the lipoproteins were triglyceride poor. Addition of angiopoietin-like protein (ANGPTL)3 or ANGPTL4 caused reduction of the activity of LPL via a two-step mechanism. We conclude that ITC can be used for quantitative measurements of LPL activity and interactions under in vivo-like conditions, for comparisons of the properties of plasma samples from patients and control subjects as substrates for LPL, as well as for testing of drug candidates developed with the aim to affect the LPL system. LPL hydrolyzes triglycerides in plasma lipoproteins. Due to the complex regulation mechanism, it has been difficult to mimic the physiological conditions under which LPL acts in vitro. We demonstrate that isothermal titration calorimetry (ITC), using human plasma as substrate, overcomes several limitations of previously used techniques. The high sensitivity of ITC allows continuous recording of the heat released during hydrolysis. Both initial rates and kinetics for complete hydrolysis of plasma lipids can be studied. The heat rate was shown to correspond to the release of fatty acids and was linearly related to the amount of added enzyme, either purified LPL or postheparin plasma. Addition of apoC-III reduced the initial rate of hydrolysis by LPL, but the inhibition became less prominent with time when the lipoproteins were triglyceride poor. Addition of angiopoietin-like protein (ANGPTL)3 or ANGPTL4 caused reduction of the activity of LPL via a two-step mechanism. We conclude that ITC can be used for quantitative measurements of LPL activity and interactions under in vivo-like conditions, for comparisons of the properties of plasma samples from patients and control subjects as substrates for LPL, as well as for testing of drug candidates developed with the aim to affect the LPL system. The hydrolytic breakdown of plasma triglycerides by LPL at the capillary endothelium is a crucial event that contributes to control of the levels of triglycerides in plasma (1Olivecrona G. Role of lipoprotein lipase in lipid metabolism.Curr. Opin. Lipidol. 2016; 27: 233-241Crossref PubMed Scopus (129) Google Scholar, 2Young S.G. Zechner R. Biochemistry and pathophysiology of intravascular and intracellular lipolysis.Genes Dev. 2013; 27: 459-484Crossref PubMed Scopus (244) Google Scholar). Many recent studies support the view that an elevated level of triglycerides in plasma is an independent risk factor for development of atherosclerosis (3Nordestgaard B.G. Triglyceride-rich lipoproteins and atherosclerotic cardiovascular disease: new insights from epidemiology, genetics, and biology.Circ. Res. 2016; 118: 547-563Crossref PubMed Scopus (530) Google Scholar, 4Musunuru K. Kathiresan S. Surprises from genetic analyses of lipid risk factors for atherosclerosis.Circ. Res. 2016; 118: 579-585Crossref PubMed Scopus (102) Google Scholar, 5Khetarpal S.A. Rader D.J. Triglyceride-rich lipoproteins and coronary artery disease risk: new insights from human genetics.Arterioscler. Thromb. Vasc. Biol. 2015; 35: e3-e9Crossref PubMed Scopus (49) Google Scholar). Therefore, the LPL system is considered to be an interesting target for drug design (6Rader D.J. New therapeutic approaches to the treatment of dyslipidemia.Cell Metab. 2016; 23: 405-412Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 7Dallinga-Thie G.M. Kroon J. Borén J. Chapman M.J. Triglyceride-rich lipoproteins and remnants: targets for therapy?.Curr. Cardiol. Rep. 2016; 18: 67Crossref PubMed Scopus (62) Google Scholar). LPL is produced and secreted from parenchymal cells like adipocytes and myocytes for transport to the luminal side of the endothelium via interaction with the glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) (8Davies B.S.J. Beigneux A.P. Barnes R.H. Tu Y. Gin P. Weinstein M.M. Nobumori C. Nyrén R. Goldberg I. Olivecrona G. et al.GPIHBP1 is responsible for the entry of lipoprotein lipase into capillaries.Cell Metab. 2010; 12: 42-52Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Several plasma components have been shown to directly or indirectly modulate the activity of LPL. apoC-II and apoA-V increase the activity of LPL, while apoC-I, apoC-III, angiopoietin-like protein (ANGPTL)3, ANGPTL4, and ANGPTL8 decrease the activity (1Olivecrona G. Role of lipoprotein lipase in lipid metabolism.Curr. Opin. Lipidol. 2016; 27: 233-241Crossref PubMed Scopus (129) Google Scholar, 9Dijk W. Kersten S. Regulation of lipid metabolism by angiopoietin-like proteins.Curr. Opin. Lipidol. 2016; 27: 249-256Crossref PubMed Scopus (128) Google Scholar). The expression of each of these proteins depends on nutritional and hormonal factors, so that lipid uptake in tissues to a large extent is regulated by posttranslational effects on LPL (1Olivecrona G. Role of lipoprotein lipase in lipid metabolism.Curr. Opin. Lipidol. 2016; 27: 233-241Crossref PubMed Scopus (129) Google Scholar, 9Dijk W. Kersten S. Regulation of lipid metabolism by angiopoietin-like proteins.Curr. Opin. Lipidol. 2016; 27: 249-256Crossref PubMed Scopus (128) Google Scholar). It is possible that the macromolecular environment in plasma itself may be an influence on the interaction of LPL with its ligands. The protein concentration of plasma (80 g/l) has been shown to cause significant crowding effects (10Ellis R.J. Macromolecular crowding: obvious but underappreciated.Trends Biochem. Sci. 2001; 26: 597-604Abstract Full Text Full Text PDF PubMed Scopus (1726) Google Scholar). It is also possible that some plasma regulators of LPL activity have not been identified yet. LPL activity can be measured in vitro by using artificial, usually emulsified, systems of radiolabeled, fluorogenic, or chromogenic substrates, or isolated triglyceride-rich lipoproteins (TRLs). The reaction products are detected at certain time points by chemical quantification or by determination of radioactivity or fluorescence. These methods have been used to unravel important aspects of the action of LPL and also to quantitate the levels of LPL activity in cells and tissues. Only small amounts of LPL activity are normally present in the circulating blood (11Karpe F. Olivecrona T. Walldius G. Hamsten A. Lipoprotein lipase in plasma after an oral fat load: relation to free fatty acids.J. Lipid Res. 1992; 33: 975-984Abstract Full Text PDF PubMed Google Scholar). Therefore, intravenous injections of heparin are made to release LPL from its endothelial binding sites. Determination of LPL activity in postheparin plasma, using artificial substrate systems, is considered to give an estimation of the amount of active LPL at the vascular endothelium (12Tornvall P. Olivecrona G. Karpe F. Hamsten A. Olivecrona T. Lipoprotein lipase mass and activity in plasma and their increase after heparin are separate parameters with different relations to plasma lipoproteins.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1086-1093Crossref PubMed Scopus (150) Google Scholar). Lack of a suitable technique for continuous monitoring of triglyceride hydrolysis in plasma has hampered the understanding of the action of LPL under physiological conditions. The properties of the substrate lipoproteins are likely to change during the lipolysis. When core triglycerides are removed, lipolysis products like monoglycerides and fatty acids may accumulate on the surface, the particle size will decrease, the surface pressure will increase and there will be an exchange of apolipoproteins between TRLs and other lipoproteins in plasma (13Eisenberg S. Olivecrona T. Very low density lipoprotein. Fate of phospholipids, cholesterol, and apolipoprotein C during lipolysis in vitro.J. Lipid Res. 1979; 20: 614-623Abstract Full Text PDF PubMed Google Scholar). Fatty acids can affect LPL either directly or through binding to some of its ligands, like ANGPTL4 (14Robal T. Larsson M. Martin M. Olivecrona G. Lookene A. Fatty acids bind tightly to the N-terminal domain of angiopoietin-like protein 4 and modulate its interaction with lipoprotein lipase.J. Biol. Chem. 2012; 287: 29739-29752Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 15Peterson J. Bihain B.E. Bengtsson-Olivecrona G. Deckelbaum R.J. Carpentier Y.A. Olivecrona T. Fatty acid control of lipoprotein lipase: a link between energy metabolism and lipid transport.Proc. Natl. Acad. Sci. USA. 1990; 87: 909-913Crossref PubMed Scopus (197) Google Scholar). Because the physical properties of the lipid substrate are sensed by LPL, there are several examples demonstrating that the activity of LPL on nonphysiological substrates is less affected by its regulator proteins (16Olivecrona G. Beisiegel U. Lipid binding of apolipoprotein CII is required for stimulation of lipoprotein lipase activity against apolipoprotein CII-deficient chylomicrons.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1545-1549Crossref PubMed Scopus (52) Google Scholar) and is more resistant to thermal inactivation (17Lookene A. Zhang L. Hultin M. Olivecrona G. Rapid subunit exchange in dimeric lipoprotein lipase and properties of the inactive monomer.J. Biol. Chem. 2004; 279: 49964-49972Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) or proteolytic cleavage (18Lookene A. Bengtsson-Olivecrona G. Chymotryptic cleavage of lipoprotein lipase. Identification of cleavage sites and functional studies of the truncated molecule.Eur. J. Biochem. 1993; 213: 185-194Crossref PubMed Scopus (44) Google Scholar) than when lipoproteins are used as substrates. Therefore, it is unlikely that determination of LPL activity in samples of postheparin plasma using artificial substrate systems will give a sufficiently good image of the lipolysis event in vivo. In the present study, we demonstrate that isothermal titration calorimetry (ITC) overcomes a number of the limitations of other techniques for measurement of LPL activity. ITC provides a continuous assay using the observable heat rate that is directly proportional to the rate of the lipolysis (19Bianconi M.L. Ladbury J.E. Doyle M.L. Titration calorimetry as a tool to determining thermodynamic and kinetic parameters of enzymes. John Wiley & Sons, Ltd., Chichester, UK.2004: 175-185Google Scholar). Raw data from ITC experiments are presented as thermograms in which the changes in the heat rate (also named heat flow, thermal power or heat flux) are monitored at a constant temperature. The method can be easily automated. We demonstrate that ITC can be used for determination of LPL activity on lipoproteins in human plasma. Both initial rates (zero-order kinetics) and kinetics for complete lipolysis can be measured. ITC can also be used for investigations of the effects of activating and inhibiting proteins on LPL activity. The ITC-based approach proposed in this report should be suitable for testing of drug candidates that are developed for targeting LPL activity. Bovine LPL was purified from milk (20Bengtsson-Olivecrona G. Olivecrona T. Phospholipase activity of milk lipoprotein lipase.Methods Enzymol. 1991; 197: 345-356Crossref PubMed Scopus (100) Google Scholar) and dialyzed to buffer containing 10 mM TRIS (pH 8.5, 4°C) and 4 mM sodium deoxycholate. Stock solutions of 0.5 mg LPL per milliliter were stored at −80°C. The N-terminal coiled-coil domain of human ANGPTL4, residues 26-184, was expressed in Escherichia coli and purified as described (14Robal T. Larsson M. Martin M. Olivecrona G. Lookene A. Fatty acids bind tightly to the N-terminal domain of angiopoietin-like protein 4 and modulate its interaction with lipoprotein lipase.J. Biol. Chem. 2012; 287: 29739-29752Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Full-length human ANGPTL3, expressed in Sf 21 cells, was obtained from R&D Systems (USA). apoC-III0 was purified from human plasma (21Bengtsson-Olivecrona G. Sletten K. Primary structure of the bovine analogues to human apolipoproteins CII and CIII. Studies on isoforms and evidence for proteolytic processing.Eur. J. Biochem. 1990; 192: 515-521Crossref PubMed Scopus (19) Google Scholar). Human apoC-II and human apoA-V were expressed in E. coli and purified as described (22Shen Y. Lookene A. Zhang L. Olivecrona G. Site-directed mutagenesis of apolipoprotein CII to probe the role of its secondary structure for activation of lipoprotein lipase.J. Biol. Chem. 2010; 285: 7484-7492Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 23Beckstead J.A. Oda M.N. Martin D.D.O. Forte T.M. Bielicki J.K. Berger T. Luty R. Kay C.M. Ryan R.O. Structure-function studies of human apolipoprotein A-V: a regulator of plasma lipid homeostasis.Biochemistry. 2003; 42: 9416-9423Crossref PubMed Scopus (71) Google Scholar). A synthetic peptide corresponding to the N-terminal domain of human GPIHBP1, residues 23-51, was purchased from Caslo (Denmark). The sequence of the peptide was as follows: QQEE­EEEDEDHGPDDYDEEDEDEVEEEET. Antibodies to human HL were raised in a goat against HL isolated from human postheparin plasma (24Olivecrona T. Olivecrona G. Determination and clinical significance of lipoprotein lipase and hepatic lipase..in: Rifai N. Warnick G.R. Dominiczak M.H. In Handbook of Lipoprotein Testing. AACC Press, Washington, DC2000: 479-498Google Scholar). The IgG fraction was isolated using Protein-A columns and the final preparation contained 5 mg protein per milliliter in 20 mM Na-phosphate buffer and 0.15 M NaCl (pH 7.4). Decoded samples of human plasma (treated with EDTA) were obtained from the Tallinn Blood Centrum. Blood was taken by forearm vein puncture from healthy 20- to 30-year-old volunteers 2 h after they had eaten a normal meal. Cells were removed from plasma by centrifugation for 30 min at 2,000 g at 4°C. The plasma samples were aliquoted and stored at −80°C and were only thawed once. EnzyChrom triglyceride assay kit (BioAssay Systems, USA) or Triglyceride Colorimetric assay kit (Cayman, USA) were used for determination of triglyceride concentrations. Fatty acids were quantified by the NEFA-HR (2Young S.G. Zechner R. Biochemistry and pathophysiology of intravascular and intracellular lipolysis.Genes Dev. 2013; 27: 459-484Crossref PubMed Scopus (244) Google Scholar) kit (Wako Chemicals). A sample of human postheparin plasma (used as LPL standard for many years in the Olivecrona group at Umeå University) was from a male volunteer that had received 100 IU heparin per kilogram body weight by intravenous injection in one forearm. After 15 min, blood was collected from the other arm into heparinized tubes and plasma was collected by centrifugation (11Karpe F. Olivecrona T. Walldius G. Hamsten A. Lipoprotein lipase in plasma after an oral fat load: relation to free fatty acids.J. Lipid Res. 1992; 33: 975-984Abstract Full Text PDF PubMed Google Scholar, 12Tornvall P. Olivecrona G. Karpe F. Hamsten A. Olivecrona T. Lipoprotein lipase mass and activity in plasma and their increase after heparin are separate parameters with different relations to plasma lipoproteins.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1086-1093Crossref PubMed Scopus (150) Google Scholar). Human VLDLs were purified from normal plasma by floatation in the ultracentrifuge at d = 1.006 g/ml (25Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6485) Google Scholar). The final preparation contained 1.3 mg protein per milliliter and 2.5 mM triglycerides. Commercial human VLDL was purchased from Kalen Biomedical (USA). This preparation contained 1 mg protein per milliliter and 4.2 mM triglycerides. Goat serum was from Invitrogen (product code 10000C). Intralipid (a 20% phospholipid-stabilized emulsion of soy bean triglycerides used for parenteral nutrition of patients) was obtained from Sigma. Heparin was purchased from LEO Pharma (Denmark). Before the experiments, the plasma samples were diluted 1.2 times with TRIS buffer (pH 7.4) or with the additions specified for each experiment. The final concentration of TRIS was 20 mM in all cases. The stock solution of bovine LPL was diluted in cold 10 mM TRIS (pH 8.5) containing 4 mM sodium deoxycholate. In this buffer, LPL is stable for a long period of time, even at low protein concentrations. The final concentration of deoxycholate during incubations with plasma or lipoproteins was 10 to 100 times lower than the initial. Control experiments showed that these levels had no influence on the enzymatic reaction. For inhibition of HL activity in postheparin plasma, 0.5 vol of goat anti-human HL IgG or the corresponding volume of PBS were added to the plasma. The mixture was then incubated for 2 h on ice prior to the experiments (24Olivecrona T. Olivecrona G. Determination and clinical significance of lipoprotein lipase and hepatic lipase..in: Rifai N. Warnick G.R. Dominiczak M.H. In Handbook of Lipoprotein Testing. AACC Press, Washington, DC2000: 479-498Google Scholar). All samples were degassed under vacuum for 15 min before the ITC experiments. Most of the experiments were performed on a Nano ITC model 5300 (TA Instruments, USA) at 25°C. A MicroCal Auto-iTC200 (GE Healthcare) instrument was used for experiments on the relationship between total heat production and released fatty acids. In a typical experiment performed by Nano ITC, the lipase substrate (plasma, Intralipid 20%, or VLDL) with or without added ligands was placed in the calorimetric cell (1,035 μl) and the syringe (250 μl) was filled with LPL-containing solution (bovine LPL or postheparin plasma) (see Fig. 1A). The reference cell contained MilliQ water (1,032 μl). The stirring speed in the sample cell was 400 rpm. The baseline was left to stabilize for at least 1 h before LPL or postheparin plasma was injected. The first injection was 2 μl and after that single or sequential injections of 10 to 25 μl were made. The interval between the injections was from 200 to 500 s. In experiments performed by MicroCal Auto-iTC200, 400 μl of lipase substrate (plasma), 400 μl of equilibration buffer (20 mM TRIS, pH 7,4), and a sufficient volume of LPL (0.8 μM) were placed on the loading plate. The calorimetric cell (200 μl) was rinsed with equilibration buffer and loaded with 200 μl of substrate in an automated fashion. The syringe was loaded with 40 μl of LPL-containing solution. The stirring speed in the sample cell was 600 rpm and the baseline was stabilized automatically. The first injection of LPL was 0.2 μl and the second injection was 2 μl. The experiment time was 1 h to allow full hydrolysis of plasma triglycerides. After each experiment the Nano ITC sample cell was washed with the following solutions, one after the other: MilliQ water, 2% SDS, 40 mM NaOH, and finally 95% ethanol. The MicroCal Auto-iTC200 was washed with MilliQ water, 10% Decon90 (Decon Laboratories Ltd.), and 100% methanol. Raw ITC data were analyzed using the NanoAnalyze (TA Instruments) or MicroCal Origin (GE Healthcare). Measurements of HL activity were performed using a gum arabic-stabilized emulsion of 3H-triolein and soy bean triglycerides (24Olivecrona T. Olivecrona G. Determination and clinical significance of lipoprotein lipase and hepatic lipase..in: Rifai N. Warnick G.R. Dominiczak M.H. In Handbook of Lipoprotein Testing. AACC Press, Washington, DC2000: 479-498Google Scholar). For measurement of HL activity, samples corresponding to 10 μl of postheparin plasma were incubated (in triplicates) in a total volume of 200 μl emulsion mixture for 40 min at 25°C. One unit of enzyme activity corresponds to release of 1 μmol fatty acid per minute. To evaluate whether ITC could be used for studies of LPL action, we first tested the stability of LPL during the ITC experiments. The ITC cell was filled with human plasma and LPL was injected (Fig. 1A). As can be seen in Fig. 1B, this resulted in an increase of the heat rate, which then remained constant for the duration of the experiment (5,500 s). This demonstrated, as expected, that the reaction was exothermic, and also that the catalytic activity of LPL was unchanged during the whole experiment. The reaction followed zero-order kinetics, meaning that consumption of the substrate (presumably triglycerides and phospholipids in plasma lipoproteins) or changes of the physical properties of the substrate due to lipolysis did not influence the reaction rate. Sequential injections of LPL into the ITC cell led to a step-wise increase of the heat rate (Fig. 1C). The heat rate level increased almost equally with each injection, indicating a proportional relationship between the concentration of LPL and the reaction rate (Fig. 1D). Next we compared the activity of LPL in plasma to that recorded with isolated human VLDL or with the synthetic lipid emulsion, Intralipid (with goat serum as a source of apoC-II), as substrate (Fig. 2). At the same initial triglyceride concentration (1 mM), the heat rate level was proportional to the concentration of LPL in all three systems, at least up to 400 pM (Fig. 2). The observed heat production rate was almost the same, suggesting that the total heat production was mainly due to hydrolysis of triglycerides. The calculated P values (unpaired, two-tailed distribution; pairwise comparison between slopes of the lines) were as follows: plasma/VLDL, P = 0.006; plasma/Intralipid, P = 0.07; VLDL/Intralipid, P = 0.2. The lowest detectable concentration of LPL differed only slightly between the substrate systems. It is generally accepted that a measurement is reliable when the determined parameter is at least ten times over the noise level. The noise level, calculated as the standard deviation of the heat rate level, was found to be 24.6 ± 2.1 nJ/s. Hence, for reliable determinations of LPL activity the change of heat rate must be over 250 nJ/s. Based on the slope of the relationship between heat rate and LPL concentration (Fig. 2, human plasma), it was possible to determine that 50 pM is the lowest concentration of LPL that can be reliably measured by the ITC instrument when the triglyceride concentration is 1 mM. The broad linearity range demonstrates that the ITC technique is suitable for quantitative measurements of LPL activity. To investigate whether ITC could be used for measurement of LPL activity in human postheparin plasma, we injected small amounts of postheparin plasma from the syringe into the ITC cell containing triglyceride-rich normal human plasma. As with purified LPL, a linear relationship between the amount of postheparin plasma and the heat rate level was observed (Fig. 3A). Because postheparin plasma contains another triglyceride lipase in addition to LPL, named HL based on its tissue origin (26Perret B. Mabile L. Martinez L. Tercé F. Barbaras R. Collet X. Hepatic lipase: structure/function relationship, synthesis, and regulation.J. Lipid Res. 2002; 43: 1163-1169Abstract Full Text Full Text PDF PubMed Google Scholar), we pretreated a sample of postheparin plasma with antibodies known to specifically inhibit HL (12Tornvall P. Olivecrona G. Karpe F. Hamsten A. Olivecrona T. Lipoprotein lipase mass and activity in plasma and their increase after heparin are separate parameters with different relations to plasma lipoproteins.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1086-1093Crossref PubMed Scopus (150) Google Scholar, 24Olivecrona T. Olivecrona G. Determination and clinical significance of lipoprotein lipase and hepatic lipase..in: Rifai N. Warnick G.R. Dominiczak M.H. In Handbook of Lipoprotein Testing. AACC Press, Washington, DC2000: 479-498Google Scholar). The antibody concentration used in these experiments (1.5 mg/ml) had been shown to be sufficient to completely inhibit activity of HL (12Tornvall P. Olivecrona G. Karpe F. Hamsten A. Olivecrona T. Lipoprotein lipase mass and activity in plasma and their increase after heparin are separate parameters with different relations to plasma lipoproteins.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1086-1093Crossref PubMed Scopus (150) Google Scholar, 24Olivecrona T. Olivecrona G. Determination and clinical significance of lipoprotein lipase and hepatic lipase..in: Rifai N. Warnick G.R. Dominiczak M.H. In Handbook of Lipoprotein Testing. AACC Press, Washington, DC2000: 479-498Google Scholar). After injection to the ITC cell, the slope of the heat rate versus plasma volume was only slightly decreased with plasma containing the antibodies compared with the original postheparin plasma diluted to the same extent with buffer. Analysis by t-test revealed that the differences between the slopes of the two lines (post-heparin plasma with and without the inhibitory antibody) were not significantly different (P = 0.13; paired, two-tailed distribution). To verify that the anti-HL IgG was able to fully inhibit HL, we made concentration curves with different amounts of IgG using ITC (Fig. 3C). We also used a specific HL assay with radiolabeled substrate to measure the remaining HL activity (Fig. 3D). These results demonstrate that the heat rate detected with injection of postheparin plasma to the ITC cell was almost fully due to LPL. Based on comparison of the activity of the purified LPL with that detected by injection of postheparin plasma (Fig. 2, human plasma and Fig. 3A), we estimated that the sample of postheparin plasma contained 7.3 pmol LPL per milliliter. This equals about 0.8 μg LPL/ml. To demonstrate that the ITC assay for LPL activity could be adopted for general use, measurements were performed with a commercial preparation of human VLDL (Fig. 3B). The triglyceride concentration was lower (0.42 mM) in this preparation than in the previously used plasma samples (1 mM), and the recorded LPL activity was lower than that obtained in Fig. 3A (about one-fourth). The activity was, however, sufficiently high for a linear determination based on sequential injections of postheparin plasma to the ITC cell (Fig. 3B). In the next experiments, ITC was used to monitor the kinetics for complete hydrolysis of available substrate lipids in plasma by LPL. For practical reasons, the amounts of LPL used were 40–100 times higher than those used for determination of initial rates. Examples of hydrolysis curves (run in triplicates) for two plasma samples, that differed in their initial triglyceride concentrations by a factor of 2.7, are shown in Fig. 4A. The areas under the curves correspond to the total heat production. The areas differed by a factor of 2.6, indicating a good correlation between total heat production and the initial triglyceride concentration of the plasma samples. The amounts of fatty acids released, as determined by the NEFA kit at the end of each reaction, were approximately 2-fold higher than the initial triglyceride concentrations in the plasma samples. This is in agreement with LPL being known to catalyze hydrolysis of the ester bonds at positions sn-1 and sn-3 of triglycerides (27Olivecrona T. Olivecrona G. The ins and outs of adipose tissue..in: Ehnholm C. In Cellular Lipid Metabolism. Springer Berlin, Heidelberg2009: 315-369Crossref Scopus (24) Google Scholar) and that isomerization of acyl groups from the sn-2 position to the sn-1(3) positions is slow at pH 7.4. Thus, the actual substrate concentration can be considered to be equal to the concentration of hydrolyzable ester bonds, which is two times higher than the triglyceride concentration. Using results from several experiments, with plasma samples from different individuals, a linear correlation was found between the total heat production and the amounts of fatty acids released (Fig. 4B). The slope of this relationship was used for calculation of the apparent enthalpy ΔH, using the equation: Q = [P]VΔH, where Q is the total heat production, P is the concentration of released fatty acids, and V is the volume of the ITC cell (19Bianconi M.L. Ladbury J.E. Doyle M.L. Titration calorimetry as a tool to determining thermodynamic and kinetic parameters of enzymes. John Wiley & Sons, Ltd., Chichester, UK.2004: 175-185Google Scholar). This calculation resulted in a ΔH value equal to 38.8 kJ/mol. To further analyze the curves for complete hydrolysis in Fig. 4A, the data were transformed into a plot of reaction rate versus remaining substrate concentration (Fig. 4C). This was obtained by subtracting the amount of hydrolyzed substrate at a chosen time point from the concentration of hydrolyzable ester bonds (the total area). This transformation enabled us to examine how the reaction rate depended on the substrate concentration, using data from a single hydrolysis curve. As can be seen, the reaction rate for the samples differed when the substrate concentration was high, but the rates were overlapping in the lower substrate range. This indicates that there might be detectable differences in the properties of plasma samples with regard to their ability to undergo hydrolysis by LPL. In the next set of experiments, we examined how the LPL activity in plasma was influenced by addition of apoC-II, apoC-III, apoA-V, ANGPTL3, or ANGPTL4. Experiment" @default.
• W2553163325 created "2016-11-30" @default.
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• W2553163325 date "2017-01-01" @default.
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• W2553163325 title "Lipoprotein lipase activity and interactions studied in human plasma by isothermal titration calorimetry" @default.
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