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Title:
METHOD FOR CALORIMETRIC DETERMINATION OF THE LIPOPROTEIN LIPASE ACTIVITY IN HUMAN PLASMA ENVIRONMENT
Document Type and Number:
WIPO Patent Application WO/2017/216381
Kind Code:
A1
Abstract:
A method for calorimetric determination of the lipoprotein lipase activity in human plasma comprising steps of preparing a reagent, obtaining and preparing human plasma sample, filling the isothermal titration calorimetry cell withe prepared human plasma sample, injecting LPL to the cell from the syringe-stirrer, changing the heat rate as a result of injection of LPL into ITC cell with human plasma, increasing LPL concentration in the ITC cell by at least one injection, performing isothermal titration calorimetry measurements, recording total hydrolysis of plasma lipids by LPL, recording differences in how well LPL can get access to the lipids, determing the influence of added regulators on the LPL reaction in plasma.

Inventors:
LÕOKENE, Aivar (Tallinna Tehnikaülikool, Ehitajate tee 5, Tallinn, 19086, EE)
REIMUND, Mart (Tallinna Tehnikaülikool, Ehitajate tee 5, Tallinn, 19086, EE)
KOVROV, Oleg (Umea University, Umea, Umea, SE-901 87, SE)
OLIVERCONA, Gunilla (Umea University, Umea, Umea, SE-901 87, SE)
Application Number:
EP2017/064850
Publication Date:
December 21, 2017
Filing Date:
June 16, 2017
Export Citation:
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Assignee:
TALLINN UNIVERSITY OF TECHNOLOGY (Ehitajate tee 5, Tallinn, 19086, EE)
International Classes:
C12Q1/44; G01K17/04; G01N33/92
Foreign References:
US20120064556A12012-03-15
Other References:
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Attorney, Agent or Firm:
SARAP, Margus et al. (Sarap and Putk Patent Agency, Kompanii 1C, Tartu, 51004, EE)
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Claims:
Claims

1 . A method for calorimetric determination of the lipoprotein lipase activity in human plasma comprising steps of:

- preparing at least one reagent,

- obtaining at least one human plasma sample,

- preparing the human plasma sample,

- filling the isothermal titration calorimetry cell withe prepared human plasma sample,

- injecting LPL to the cell from the syringe-stirrer,

- changing the heat rate as a result of injection of LPL into ITC cell with human plasma; - increasing LPL concentration in the ITC cell by at least one injection;

- performing isothermal titration calorimetry measurements, wherein ITC measurements are used for measuring LPL activity in human plasma,

- recording the results.

2. The method according to claim 1 , wherein the heat rate is changed as a result of single injection of LPL into ITC cell with human plasma.

3. The method according to claim 1 , wherein the heat rate is changed as a result of sequential injections of LPL into ITC cell with human plasma.

4. The method according to claim 1 , wherein the heat rate is changed proportionally to the reaction rate. 5. The method according to claim 1 , wherein the heat rate is changed as a result of injection of LPL into ITC cell with 1 ml human plasma.

6. The method according to claim 1 , wherein the heat rate is changed as a result of injection of LPL into ITC cell with human plasma substrate mixture containing human VLDL 7. The method according to claim 1 , wherein the heat rate is changed as a result of injection of LPL into ITC cell with human plasma substrate mixture containing Intralipid.

8. The method according to claim 1 , wherein the heat rate is changed as a result of injection of LPL into ITC cell with human post-heparin plasma mixture containing Anti- HL IgG in 1 ml of human pre-heparin plasma.

9. The method according to claim 1 , wherein the heat rate is changed as a result of injection of LPL into ITC cell with human post-heparin plasma mixture in 1 ml of human pre-heparin plasma.

10. The method according to claim 1 , wherein the concentration of LPL in plasma is 0.6 nM -2 2 nM.

1 1 . The method according to claim 1 , wherein the concentration of triglycerides in plasma is 1 mM - 2.7 mM.

12. The method according to claim 1 , wherein LPL concentration is increased in the ITC cell by at least one injection by 50 pM - 230 pM. 13. The method according to claim 1 , wherein total hydrolysis of plasma lipids by LPL is recorded as the results.

14. The method according to claim 1 , wherein differences in how well LPL can get access to the lipids is recorded as the results.

15. The method according to claim 1 , wherein how LPL can catalyse hydrolysis of lipids in plasma samples is recorded as the results.15. The method according to claim 1 , wherein the influence of added regulators on the LPL reaction in plasma is recorded as the results.

Description:
METHOD FOR CALORIMETRIC DETERMINATION OF THE LIPOPROTEIN

LIPASE ACTIVITY IN HUMAN PLASMA ENVIRONMENT

Field of the invention

The present invention relates to the field of calorimetric drug discovery. Background of the invention

The hydrolytic breakdown of plasma triglycerides by lipoprotein lipase (LPL) at the vascular endothelium is a crucial factor that determines the triglyceride level in plasma (Olivecrona 2016). Numerous recent studies suggest that elevated levels of triglyceride-rich lipoproteins in plasma are an independent risk factor of atherosclerosis (Nordestgaard 201 6; Musunuru & Kathiresan 201 6; Khetarpal & Rader 2015). Therefore, LPL has regarded as a potential drug target in lowering triglyceride levels (Rader 201 6; Dallinga-Thie et al. 201 6). The rate of lipolysis does not only depend on the production of LPL - several plasma components have been shown to directly or indirectly modulate the activity or stability of LPL. Apolipoproteins C-ll (apoC-ll) and apolipoprotein A-V (apoA-V) increase the rate of LPL while apolipoproteins C-l, C-lll (apoC-l, apoC-lll) and angiopoietin-like proteins 3, 4, 8 (ANGPTL 3, 4, 8) behave as inhibitors of the lipolysis (Olivecrona 201 6; Dijk & Kersten 201 6). Due to polymorphisms and posttranslational modifications, LPL and its regulator proteins can exist in multiple forms. Expression of these proteins depends on physiological status, such as fasting/feeding, exercises or temperature (Olivecrona 201 6; Dijk & Kersten 201 6). Apart from the substrate lipoproteins, triglyceride-rich VLDL and chylomicrons, LPL binds cholesterol-rich LDL with appreciable affinity (Lookene et al. 1997). Both genetic and environmental factors influence concentration and proportion of these lipoproteins. Free fatty acids, products of the lipolysis, can affect LPL activity directly or via its ligands (Robal et al. 2012; Peterson et al. 1990). Thus, it is reasonable to consider that the efficiency of the hydrolytic degradation of triglycerides by LPL depends on coaction of numerous different extracellular compounds whose concentrations can vary. Moreover, since plasma is a complex fluid containing thousands of proteins, and other components, the majority of them are present in very low concentrations, it is possible that some plasma regulators of LPL have not been identified yet. Due to the crowding effect, even macromolecular environment of plasma itself may influence LPL interactions with its ligands. The protein concentration of plasma 80 g/liter has been shown to be sufficient to cause significant crowding effect (Ellis 2001 ). There is another aspect that is specific and important for lipases - these enzymes act at the surface of aggregated substrates that is continuously changing during the lipolysis, resulting to complex reaction kinetics (Eisenberg & Olivecrona 1979). It is likely that the influence of the ligands on LPL can change during the lipolysis.

Lack of a suitable technique for continuous monitoring of lipolysis in blood plasma has hampered the understanding of the action of LPL under physiological conditions. Typically, LPL activity is measured using artificial systems of radiolabeled (Briquet- Laugier et al. 1999), fluorogenic (Basu et al. 201 1 ) or chromogenic (Panteghini et al. 2001 ) substrates, and the reaction products are detected in stopped assays by chemical quantification or by determination of radioactivity. As a rule, only the initial rates are determined in these assays. Those studies have provided essential information about several aspects of the LPL action. For example, determination of LPL activity in post-heparin plasma, using artificial substrate systems, is used for estimation of amount of active LPL molecules at the vascular endothelium (Tornvall et al. 1995). However, those measurements are not sufficient to entirely evaluate the rate of lipolysis in plasma. First, the activity of LPL on non-physiological substrates can be less affected by its ligands (Olivecrona & Beisiegel 1997) and is more resistant to thermal inactivation (Lookene et al. 2004) or proteolytic cleavage (Lookene & Bengtsson-Olivecrona 1993) than its activity on lipoproteins. Second, as discussed above, the activity of LPL in vivo is influenced by numerous plasma components (Olivecrona 201 6). In summary, determination of LPL activity in postheparin plasma using artificial substrate systems is likely not to be sufficient for estimation of the real lipolysis rate in vivo. Thus, the problem of known methods is that there is no sufficient method to evaluate the rate of lipolysis and measure lipoprotein lipase activity in human blood plasma to determine risk factors of atherosclerosis, to test how added regulators influence the lipoprotein lipase reaction in plasma, to find out the causes of hypertriglyceridemia.

Brief description of the invention The aim of the method for calorimetric determination of the lipoprotein lipase activity in human plasma environment according to present invention is to provide an effective method and tool for finding out causes of hypertriglyceridemia and assay for testing potential drug candidates against hypertriglyceridemia under in-vivo like conditions, to evaluate the rate of lipolysis and measure lipoprotein lipase activity in human blood plasma to determine risk factors of atherosclerosis, to test how added regulators influence the lipoprotein lipase reaction in plasma.

The aim is achieved by isothermal titration calorimetry. Lipoprotein lipase (LPL) is a key enzyme in the hydrolytic degradation of triglyceride-rich lipoproteins in blood plasma. Activity and stability of LPL is regulated by numerous plasma components (Olivecrona 201 6). Previous studies performed under artificial conditions have provided essential information about several aspects of the LPL action. However, the prior art of how the ligands affect LPL under native conditions that is plasma environment is still limited.

The present invention demonstrates that isothermal titration calorimetry (ITC) according to the present invention overcomes several limits of prior art techniques. First, ITC measurements can be successfully performed in minimally diluted human plasma. Second, the ITC based assay of LPL is highly sensitive, ITC can be used for determination of LPL activity in postheparin plasma. The estimated detection limit for LPL is as low as 50 pM. Third, ITC enables quantitative comparison of the properties of plasma samples from patients and control subjects as substrates for LPL. This can be done by recording kinetics of total substrate hydrolysis by LPL. Fourth, ITC provides new and detailed information about how ligands influence LPL in plasma. Using ITC, it is demonstrated that 1 ) increasing of apoC-lllconcentration in plasma reduces LPL activity on triglyceride rich lipoproteins but not on triglyceride poor fraction of lipoproteins 2) ANGPTL3 and 4 suppress the activity of LPL in plasma via analogous mechanism- in both cases a fast phase inhibition and a slow phase inactivationcan be clearly distinguished. The method according to the present inventions demonstrates that isothermal titration calorimetry (ITC) overcomes several limits of other techniques by providing a continuous assay using the observable thermal power that is directly proportional to the rate of the lipolysis. Raw data of ITC experiments are presented as thermograms in which the changes in the heat rate (heat flow, thermal power, heat flux) are monitored at constant temperature (Bianconi 2005). ITC can be used for determination of LPL activity in plasma, for registration of total lipolysis kinetics or for investigations of ligand effects on LPL in plasma. Considering that increasing of LPL activity through blocking inhibitory effect of its ligands is one possible way for lowering plasma triglyceride levels (Rader 2016; Dallinga-Thie et al. 201 6), the ITC-based approaches proposed in this report can be used for testing of drug candidates in human plasma.

The method according to the present invention does not need chromogenic, fluorogenic or radioisotope labelled ligands that are used in known enzyme assay methods and is used to obtain data that are not attainable by prior art techniques.

The method according to the present invention provides reliable and reproducible data about the action of LPL in plasma that is the physiologic environment for this enzyme. The advantage of ITC stems from possibility to perform measurements directly in plasma, using observable changes in heat flow for determination of reaction rate. However, in the examination of the ITC experiments of plasma lipolysis, it is important to take into account that determined heat rate is a complex parameter which includes heat effects of four different processes: hydrolysis of ester bonds, structural changes of lipoproteins, fatty acid neutralization by the buffer and binding of fatty acids to albumin. Several observations of this study suggest that the heat rate is a suitable quantitative parameter for measurement of catalytic activity of LPL in plasma. First, LPL when added to plasma was stable in the ITC cell for a long time period. Second, the heat produced by the action of LPL was proportional to the amount of released fatty acids. Third, there was a linear dependence between LPL concentration and heat flow. Fourth, in the case of complete hydrolysis, the total heat production was linearly dependent on the initial triglyceride concentration. Fifth, the heat effect was nearly the same regardless of whether used substrate for LPL was synthetic triglyceride emulsion Intralipid, VLDL or triglyceride rich plasma. Based on this, it is reasonable to assume that the heat effect accompanying with structural changes of lipoproteins or due to other interactions established in the system are not significant in comparison with those due to hydrolysis of ester bonds. In principle, activity of LPL in plasma can be measured by detecting produced fatty acid after stopping the reaction. However, since plasma contains fatty acids at high concentrations (0.3 - 1 mM) (Dole 1956), these assays are applicable only when significant amount of fatty acids have been produced by added LPL. Thus, detection of produced fatty acids has limited usage in investigation of LPL action in plasma. In addition, these measurements do not provide continuous data and are more laborious and time consuming than the ITC measurements. The ITC assay for measurement of initial rates was sufficiently sensitive to determine LPL activity in postheparin plasma. Thus, the ITC assay is a promising alternative to assays that use radiolabeled or fluorogenic substrates or measure released fatty acids by NEFA kit. The advantage of ITC - no additional reagents are needed to perform the experiments and minimally diluted human plasma can be used. The possibility to use plasma as a source of triglycerides is also advantage, since both catalytic activity and ligand interactions of LPL are dependent on the used substrate system (Lookene et al. 2004; Olivecrona & Beisiegel 1997). In humans, LPL and hepatic lipase (HL)are bound to the vascular endothelium, and their activities have been analyzed in plasma obtained from blood samples collected after intravenous injection of heparin, a procedure to release LPL and HL from cellular proteoglycans (Tornvall et al. 1995). By using inhibitory antibodies of HL the detected activity in post-heparin plasma can be almost completely attributed to LPL. An ITC based assay for determination of LPL activity in plasma using commercially available VLDL or Intralipid is devised for general use.

In addition to determination of initial rate of hydrolysis, ITC allows continuous monitoring of total lipolysis in plasma. This is important since the effect of different plasma components on LPL may depend on the concentration of triglycerides or composition of the lipoprotein fraction. Determination of the initial activity might not be sufficient to evaluate how efficiently triglycerides are hydrolysed by LPL. Transformation of a single lipolysis time course into the plot of heat rate versus remaining substrateconcentration (hydrolysable ester bonds) revealed how the reaction rate was changing during degradation of triglyceride rich lipoproteins to remnant particles. This kind of analysis gives proper evaluation for ability of LPL to degrade lipoproteins of a certain plasma sample. Although triglyceride concentrations vary between people, recordings of total hydrolysis curves make lipolysis comparable. The comparison of total hydrolysis of two randomly selected plasma samples clearly demonstrated that there are individual differences in efficiency of lipolysis of plasma lipoproteins. Thus, the analysis of total hydrolysis curves can be used to ascertain whether hypertriglyceridemia is caused by plasma composition.

Continuous monitoring of the LPL catalyzed reactions provided detailed information about how apoC-lll, ANGPTL3 and ANGPTL4 suppress LPL activity in plasma. These proteins are regarded as potential targets for drug development with the aim to lower triglyceride levels in plasma (Rader 201 6; Dallinga-Thie et al. 201 6). It is preferred to investigate their inhibitory mechanism in human plasma. ApoC-lll could only moderately affect LPL activity in plasma - increasing its concentration to 50 μΜ reduced the activity of LPL about by 30%. This is different from the data obtained by isolated chylomicrons - in that case complete inhibition was observed already at 10 μΜ apoC-lll (Larsson et al. 2013). Thus, apoC-lll was less potent LPL inhibitor in plasma than in an assay of isolated chylomicrons. Assuming that apoC-lll displaces LPL from lipoproteins (Larsson et al. 2013), it is reasonable to propose that plasma contains components that reduce apoC-lll ability to inhibit binding of LPL to triglyceride rich lipoproteins. Analysis of the total hydrolysis curves demonstrated that apoC-lll affected LPL activity on triglyceride rich lipoproteins but not on triglyceride poor fraction of lipoproteins. This can be explained by leaving of apoC-lll from the surface of lipoproteins when their triglyceride content is decreasing. Effect of ANGPTL4and ANGPTL3 on LPL in plasma was detectable already at nanomolar concentrations. However, these concentrations were at least one magnitude higher than the estimated plasma concentrations of these proteins (Robciuc et al. 2010). Thus, real plasma concentrations of these proteins are too low to affect activity of LPL. Although ANGPTL4was more efficient in lowering of LPL activity thanANGPTL3, the inhibition time courses of the two proteins were similar - in both cases it was possible to clearly distinguish a fastand a slow phase. This observation suggest analogous inhibition mechanisms. There is no consensus by which mechanisms ANGPTL3 and 4 suppress the activity of LPL. It has been proposed that ANGPTL3and ANGPTL4 inhibit LPL through different mechanisms (Shan et al. 2009) and that only ANGPTL4induces irreversible inactivation of LPL (Sukonina et al. 2006). However, it is also proposed that ANGPTL4acts as a non-competitive inhibitor LPL, forming a reversible complex with LPL (Lafferty et al. 2013). It is important to stress that these two mechanisms are not mutually exclusive - reversible complex formation can precede to the inactivation. It is tempting to speculate that both mechanisms exist in plasma. Increasing of LPL activity via reducing the inhibitory effect of apoC-lll, ANGPTL3 or ANGPTL4 is one possible way to lower triglyceride levels (Rader 201 6; Dallinga-Thie et al. 201 6). ITC allows testing of possible drug candidates that block the action of these proteins in human plasma.

Brief description of the drawings The preferred embodiment of present invention is explained more precisely with following figures added, where

Figures 1 a to Figure 1 d show enzymatic activity of LPL as measured by ITC.

Figure 1 a illustrates the schematic model of the ITC system used in the present method, wherein the ITC cell is filled with human plasma, LPL is injected to the ITC cell from the syringe-stirrer.

Figure 1 b shows the heat rate change as a result of a single injection of LPL into ITC cell with 1 ml of human plasma. TG and LPL concentration in the ITC cell is for example 2.7 mM and 500 pM, respectively. The change of heat rate dQ/dt is proportional to the reaction rate v.

Figure 1 c shows sequential injections of LPL into the same aliquot of plasma as in figure 1 b. Each injection increased LPL concentration in the ITC cell by 230 pM.

Figure 1 d illustrates the data obtained from figure 1 c presented as relationship of heat rate versus LPL concentration. Figure 2. illustrates ITC-based assays for determination of LPL activity using different substrate systems. Sequential injections of LPL into human plasma, substrate mixture containing human VLDL or in alternative embodiment of present invention substrate mixture containing Intralipid. In all measurements, triglyceride concentration was 1 mM and each injection increased LPL concentration by 50 pM. The slopes of the lines were as follows: human plasma 0.0092, Intralipid 0.0102, and VLDL 0.0106. Human plasma contained 20 mM TRIS, pH 7.4. VLDL substrate mixture contained 10 mg/ml BSA, 10 IE/ml heparin, 20 mM TRIS pH 7.4, 0.15 M NaCI. Intralipid substrate mixture contained 10 mg/ml BSA, 10 IE/ml heparin, 63 % goat inactivated serum (source for apoC-ll), 20 mM TRIS pH 7.4, 0.15 M NaCI. The values are mean ± S.D. of three different measurements.

Figure 3a and figure 3b illustrate post-heparin plasma LPL activity as measured by ITC.

Figure 3a illustrates the activity, wherein five sequential injections of human post- heparin plasma (1 .5 times diluted) with (+Anti-HL IgG) or in alternative embodiment of present invention without (- Anti-HL IgG) hepatic lipase inhibitor (antibodies to HL) were made into 1 ml of human pre-heparin plasma. Final concentration of triglycerides of pre-heparin plasma was 1 mM. Antibodies to HL were added to the post-heparin plasma to evaluate the contribution of hepatic lipase on the total activity. Concentration of anti-HL IgG was 1 .5 mg/ml.

Figure 3b illustrates the heat rate, wherein three sequential injections of 20 μΙ of human post-heparin plasma were made into 1 ml of substrate mixture containing VLDL (0.42 mM triclyceride and 0.1 mg/ml protein), 10 mg/ml BSA, 10 IE/ml heparin in 10 mM TRIS, 0.15 M NaCI, pH 7.4 buffer. The concentration of LPL in the post-heparin plasma was 7.3 nM based on Figures 3A and 2, human plasma. The values are mean ± S.D. of three different determinations.

Figure 4a to figure 4c illustrate total hydrolysis of human plasma lipids by LPL as measured by ITC.

Figure 4a illustrates raw thermograms of total hydrolysis of human plasma lipids by LPL. Experiments were performed with plasma sample that contained 1 mM or 2.7 mM triglycerides. Concentration of LPL was 22 nM. Three measurements with both plasma samples are presented. Figure 4b shows relationship between total heat production and concentration of released fatty acids. Heat rates were obtained from full hydrolysis of 10 different human plasma samples on MicroCal Auto-ITC200. In these experiments 200 μΙ of plasma was hydrolyzed by 8 nM LPL for one hour. The surface areas of the curves for full hydrolysis were calculated in the MicroCal Origin and compared with the release of free fatty acids (final concentration minus initial concentration) measured using NEFA kit.

Figure 4c presents transformation of total hydrolysis data presented in figure 4a into relationship between heat rate and remaining substrate concentration (expressed as concentration of hydrolysable ester bonds).

Figure 5a and figure 5b illustrate Activation of LPL by apoC-ll. Figure 5a shows raw ITC thermograms of Intralipid hydrolysis by LPL in the absence or in alternative embodiment of present invention presence of different concentrations of apoC-ll. The substrate mixture composition was as follows: 1 mg triglyceride/ml Intralipid, 10 mg/ml BSA, 10 lU/ml heparin, 0.15 M NaCI, 10 mM TRIS pH 7.4. Single injection of LPL were made into the substrate mixture that contained or in alternative embodiment of present invention did not contained apoC-ll. Final concentration of LPL was 1 .2 nM in all experiments. Figure 5b shows hydrolysis heat rate as a function of apoC-ll concentration. Heat rate points at 200 s were obtained from figure 5a. The data were fitted using the following equation (Quinn et al. 1983) where v is the reaction rate, kcat

is catalytic rate constant, L is the concentration of LPL, C is the concentration of apoC- II, β is the activation factor which indicates how much more active the LPL/apoC-ll complex is compared to LPL alone, Kd is the equilibrium dissociation constant.

Figure 6a and figure 6b illustrate the effect of apoC-lll and apoA-V on LPL activity in plasma.

Figure 6a illustrates effect of apoC-lll on LPL activity under zero-order reaction conditions. Sequential injections of LPL were made into plasma samples in which apoC-lll concentration was increased by 15 μΜ, 50 μΜ or in alternative embodiment of present invention not increased. Each injection increased LPL concentration by 1 nM. Concentration of triglycerides in plasma was 2 mM.

Figure 6b illustrates total hydrolysis thermograms of plasma triglycerides by LPL in the case when plasma concentration of apoC-lll was not increased or in alternative embodiment of present invention was increased by 15 μΜ. Concentration of LPL was 22 nM and concentration of triglycerides was 2.7 mM. An example of one measurement from three different measurements is presented for both cases.

Figure 6c illustrates dependence of heat rate on triglyceride concentration as calculated from the total hydrolysis curves presented infigure 6b.

Figure 6d illustrates LPL activity, which was measured in the absence or in alternative embodiment of present invention in the presesnce of 10 nM or 100 nM apoA-V. Single injection of LPL into the plasma was made. TG and LPL concentration in the ITC cell were 1 mM and 0.6 nM, respectively.

Figure 7a to figure 7c illustrate effect of ANGPTL4 or ANGPTL3 on LPL activity.

Figure la and figure 7c illustrates LPL activity, which was measured in the absence or in alternative embodiment of present invention presence of different concentrations of ANGPTL4 (A) or ANGPTL3 (C). Figure 7b illustrates ANGPTL4 effect on LPL, which was tested in the presence of the N-terminal peptide of GPIHBP1 . In all experiments single injection of LPL into plasma sample was made; resulting concentration of LPL in the ITC cell was 0.6 nM. Triglyceride concentration was 1 mM. Figure 8 illustrates effect of tetrahydrolipstatin (THL) on LPL activity, wherein single injection of 10 μΙ of 100 nM LPL into 1 ml of human plasma that contained or in alternative embodiment of present invention did not contain THL. Final concentration of LPL was 1 nM.

Detailed description of the invention The method for calorimetric determination of the lipoprotein lipase activity in human plasma according to present invention comprises steps of preparing at least one reagent, obtaining at least one decoded human plasma sample, preparing the sample, performing isothermal titration calorimetry (ITC) measurements, wherein ITC measurements are used for measuring LPL activity in plasma, recording total hydrolysis of plasma lipids by LPL and to recording differences in how well LPL can get access to the lipids, determing the influence of added regulators on the LPL reaction in plasma.

More specifically, the method according to present invention for calorimetric determination of the lipoprotein lipase activity in human plasma comprising steps of: - preparing at least one reagent,

- obtaining at least one human plasma sample,

- preparing the human plasma sample,

- filling the isothermal titration calorimetry cell withe prepared human plasma sample,

- injecting LPL to the cell from the syringe-stirrer,

- changing the heat rate as a result of injection of LPL into ITC cell with human plasma;

- increasing LPL concentration in the ITC cell by at least one injection;

- performing isothermal titration calorimetry measurements, wherein ITC measurements are used for measuring LPL activity in human plasma,

- recording the results. The heat rate is changed as a result of single injection or in an alternative embodiment as a result of sequential injection of LPL into ITC cell with human plasma. The heat rate is changed proportionally to the reaction rate, as a result of injection of LPL into ITC cell with 1 ml human plasma, as a result of injection of LPL into ITC cell with human plasma substrate mixture containing human VLDL, as a result of injection of LPL into ITC cell with human plasma substrate mixture containing Intralipid, as a result of injection of LPL into ITC cell with human post-heparin plasma mixture containing Anti- HL IgG in 1 ml of human pre-heparin plasma, as a result of injection of LPL into ITC cell with human post-heparin plasma mixture in 1 ml of human pre-heparin plasma.

The concentration of LPL in plasma is 0.6 nM -2 2 nM and the concentration of triglycerides in plasma is 1 mM - 2.7 mM. The LPL concentration is increased in the ITC cell by at least one injection by 50 pM - 230 pM.

By recording the results, total hydrolysis of plasma lipids by LPL, differences in how well LPL can get access to the lipids, how LPL can catalyse hydrolysis of lipids in plasma samples is recorded as the results or the influence of added regulators on the LPL reaction in plasma is recorded as the results.

Preparing the reagents - Bovine LPL was purified from milk (Bengtsson-Olivecrona & Olivecrona 1991 ) and dialysed to buffer containing 10 mM TRIS, pH 8.5 (4Ό), 4 mM sodium deoxycholate. Stock solutions of 0.5 mg/ml LPL were stored at -80Ό. The N- terminal coiled-coil domain of human angiopoietin-like protein 4 (ANGPTL4), residues 26-184, was expressed in E. col i and purified as described (Robal et al. 2012). Full- length human angiopoietin-like protein 3 (ANGPTL3) expressed in Sf 2λ cells was obtained from R&D Systems (USA). Apolipoprotein C-lllo was from human blood plasma (Bengtsson-Olivecrona & Sletten 1990). Apolipoprotein C-ll and apolipoprotein A-V were expressed in E. coli and purified as described (Shen et al. 2010; Beckstead et al. 2003). A synthetic peptide corresponding to the N-terminal domain of human GPIHBP1 , residues 23-51 , was bought from Casio (Denmark). The sequence of the peptide was as follows: QQEEEEEDEDHGPDDYDEEDEDEVEEEET. Antibodies (goat IgG) to human hepatic lipase were raised in a goat against hepatic lipase isolated from human post-heparin plasma (Olivecrona & Olivecrona 2000). 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 NaCI (pH 7.4).

Decoded human plasma samples (treated by EDTA) were obtained from the Blood Centrum or were obtained from blood taken by forearm vein puncture from healty 20- 30 years old volunteers 2 hours after they had eaten a normal meal. Cells were removed from plasma by centrifugation for 30 minutes at 2000 x g at 4Ό. The plasma samples were aliquoted and stored at -80Ό and were generally only used once. EnzyChrom Triglyceride Assay Kit (BioAssay Systems, USA) or Triglyceride Colorimetric Assay Kit (Cayman, USA) were used for determination of triglyceride concentrations. Free fatty acids were quantified by the NEFA-HR(2) kit (Wako Chemicals). A standard sample of human post-heparin plasma (used for many years in the gorup at Umea University) was from a male volunteer that had recieved 100 IU heparin/kg body weight by i.v. injection in one forearm. After 15 min blood was collected into heparinized tubes from the other arm and plasma was collected by centrifugation (Human PHPL st 23) (Karpe et al. 1992; Tornvall et al. 1995). Human very low density lipoproteins (VLDL) were purified from normal human plasma by ultracentrifugation (Havel et al. 1955). The final preparation contained 1 .3 mg protein/ml and 2.5 mM triglycerides. Some human VLDL were purchased from Kalen Biomedical (USA). This preparation contained 1 mg protein/ml 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)

Sample preparation - Before experiments the human 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. Stock solutions of LPL were 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. In experiments with post-heparin plasma, antibodies to hepatic lipase (in PBS) or the same volume of PBS only were added (180 μΙ of IgG or PBS was mixed with 360 μΙ of post-heparin plasma) and the mixture was then incubated for 2 hours on ice prior to experiments (Olivecrona & Olivecrona 2000). All samples were degassed under vacuum for 15 minutes before the ITC experiments.

ITC measurements - Most of the experiments were performed on an Nano ITC model 5300 (TA Instruments, USA) at 25Ό. A MicroCal Auto-iTC200 (GE Healthcare) instrument was used for experiments regarding 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 into the calorimetric cell (1035 μΙ) and the syringe (250 μΙ) was filled with LPL-containing solution (bovine LPL or post-heparin plasma) (see Figure 1 A). MilliQ water was loaded into the reference cell (1032 μΙ). The stirring speed in the sample cell was 400 rpm. The baseline was left to stabilize for at least 1 hour before injections of LPL or post-heparin plasma. In all experiments, the first injection was 2 μΙ and after that single or sequential injections of 10 to 25 μΙ were made. The interval between the injections was from 200 s to 500 s. In experiments performed by MicroCal Auto- iTC200, 400 μΙ of lipase substrate (plasma), 400 μΙ of equilibration buffer (20 mM TRIS, pH 7,4) and a sufficient volume of LPL (0.8 μΜ) were placed on the loading plate. The calorimetric cell (200 μΙ) was rinsed with equilibration buffer and loaded with 200 μΙ of substrate in an automated fashion. The syringe was loaded with 40 μΙ 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 μΙ and the second injection was 2 μΙ. The experiment time was 1 hour 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 analysed using the NanoAnalyze (TA Instruments) Or MicroCal Origin (GE Healthcare).

ITC can be used for measurements of LPL activity in plasma To evaluate whether ITC can be used for studies of LPL, we first tested the stability of LPL during the ITC experiments. The ITC cell was filled with normal human plasma and a diluted sample of LPL was injected from the syringe (Figure 1 A). As can be seen in Figure 1 B, this resulted in an increase of the heat rate that remained constant for the duration of the experiment (5500 seconds). This demonstrated, as expected, that the reaction was exothermic, but also, and less expected, 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, did not influence on the reaction rate. Sequential injections of LPL into the ITC cell with plasma led to a step-wise increase of the heat rate (Figure 1 C). The heat rate level increased almost equally by each injection of LPL, indicating a proportional relationship between the concentration of LPL and the reaction rate (Figure 1 D). As expected, the standard deviations of the slopes calculated from sequential injections were smaller than that of increments of heat rate level after single injections. The difference of the two standard deviations was about two times. Thus, it was preferable to measure LPL activity using the sequential injection mode.

Next the activity of LPL in plasma is compared to recorded with isolated VLDL or with the synthetic lipid emulsion Intralipid (Figure 2). Using the same initial triglyceride concentration (1 mM), the heat rate level was proportional to the LPL concentration at least up to 400 pM in all tested systems (Figure 2). The observed heat production rate was almost the same in all three systems, suggesting that the total heat production is mainly due to hydrolysis of triglycerides. The lowest concentration of LPL detectable by ITC varied only slightly between the substrate systems. It is generally accepted that a measurement is reliable when determined parameter is at least ten times over the noise level. The noise level calculated as standard deviation of the heat rate level was found to be 24.6±2.1 nJ/s. Hence, for reliable determination 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 (Figure 2, human plasma), it was possible to determine that 50 pM LPL is the lowest concentration 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 LPL activity measurements.

LPL in its active form is normally not present in blood, but is attached to capillary walls in a heparin-releasable manner. Therefore i.v. injection of heparin is often used to release LPL from its binding sites into the circulating blood. The amount of LPL in post- heparin plasma gives an estimate of the total pool of LPL that have access to lipoprotein in plasma and is often designated the functional pool of LPL in contrast to LPL contained within cells and the extracellular matrix (Tornvall et al. 1995). To investigate whether ITC could be used for measurement of LPL activity in post-heparin plasma, we injected small amounts of human post-heparin plasma from the syringe into the ITC cell containing triglyceride-rich, normal human plasma as substrate. Like in the case with purified bovine LPL, a linear relationship between the amountof post- heparin plasma and the heat rate level was observed (Figure 3A). Post-heparin plasma is known to contain another lipase in addition to LPL, named hepatic lipase (HL) based on its tissue origin (Perret et al. 2002). Therefore, a sample of post-heparin plasma is treated with antibodies known to specifically inactivate hepatic lipase (Anti-HL IgG) (Tornvall et al. 1995; Olivecrona & Olivecrona 2000). This only slightly affected the slope of the heat rate versus plasma volume. The f-test analysis revealed that the differences between the slopes of the two lines (with and without the inhibitory antibody) were not significantly different (P=0.13; paired, two-tailed distribution). Thus, the detected activity in post-heparin plasma can be almost completely attributed to LPL. Based on comparison of the activity of purified bovine LPL with that of post- heparin plasma LPL (Figures 2, human plasma and 3A), it is estimated that the sample of post-heparin plasma should contain 7.3 pmol LPL/ml. To devise an ITC assay for LPL for general use, measurements were performed with a commercial preparation of VLDL (Figure 3B). Due to the low triglyceride concentration in this preparation, the activity of LPL was lower than in Figure 3A (about one forth), but still sufficiently high for a linear determination of LPL activity based on increased additions of post-heparin plasma to the ITC cell.

ITC can be used to record total hydrolysis of plasma lipids by LPL and to record differences in how well LPL can get access to the lipids. In the next experiments, ITC was used for monitoring of the total hydrolysis of lipids in plasma by LPL. For practical registration of total hydrolysis in real time, the concentration of LPL used was 40-100 times higher than that used for determination of initial rates (Figure 1 B and 1 C). Examples of total hydrolysis curves (run in triplicates) for two plasma samples that differed in their initial triglyceride concentrations (1 mM and 2.7 mM, respectively) are shown in Figure 4A. The areas under these curves correspond to the total heat production. They differed 2.6 times, indicating a good correlation between total heat production and the initial triglyceride concentration of the plasma samples. The concentration of released fatty acids determined by the NEFA kit at the end of each reaction was approximately two times higher than the initial triglyceride concentration. This is in agreement with that LPL is known to catalyze hydrolysis of the ester bonds only at positions sn1 and sn3 of triglycerides (Olivecrona & Olivecrona 2009), and with that isomerization of acyl groups from the sn 2 position to the sn 1 (3) positions is slow at pH 7.4. Thus, it is reasonable to assume that the real substrate concentration is equal to the concentration of hydrolysable ester bonds which is two times higher than the triglyceride concentration. Using results from several experiments with different plasma samples, a linear correlation was found between the total heat production and the amount of formed fatty acids (Figure 4B). Since this relationship included data from lipolysis of various depth, it was reasonable to consider that the total heat effect is equally determined by hydrolysis of ester bonds of triglycerides and diglycerides. The relationship also suggests that the heat effect of structural changes and reorganization of lipoproteins during the lipolysis is negligible in comparison of that of the hydrolysis of ester bonds. To further analyze the total hydrolysis curves in Figure 4A, the raw data were transformed into a plot of reaction rate versus remaining substrate concentration (Figure 4C). This was obtained by subtracting the hydrolyzed substrate concentration at a chosen time point from the total substrate concentration (the total area). The calculations were based on the assumption that the substrate concentration was equal to concentration of hydrolysable ester bonds that was in turn two times higher than the triglyceride concentration. This transformation enabled us to examine how the reaction rate depended on the substrate concentration using a single hydrolysis curve. As can be seen, the reaction rate for these samples differed when the substrate concentration in plasma was high, but the rates were overlapping in the lower substrate range. This result indicate that there might be detectable differences in how good access LPL has to lipid substrates in different plasma samples during total hydrolysis.

ITC can be used to study the influence of added regulators on the LPL reaction in plasma In the next set of experiments, it is examined how the LPL activity on plasma lipoproteins was influenced by the regulator proteins apoC-l l, apoC-l l l, apo A-V, ANGPTL3 and ANGPTL4. Experiments with the activator apoC-l l were carried out using Intralipid as substrate, because plasma normally contains sufficient apoC-l l for full activation of LPL (Kei et al. 201 2). In these measurements, the LPL concentration was held constant while the apoC-l l concentration varied from 0 to 1 000 nM. Raw ITC thermograms are presented in Figure 5A. As expected, increased concentrations of apoC-l l caused increased activity of LPL. To estimate the activation factor and the affinity of apoC-l l for LPL, the heat rate values at 200 s were plotted against the apoC- I I concentration (Figure 5B). As can be seen, apoC-l l activated LPL in a saturable fashion. For the analysis of the apoC-l l activation data, we used a model proposed by Quinn, Shirai, and Jackson (Quinn et al. 1 983). The estimated maximal activation factor was 4.2±1 .2 and the Kd value was 22±8 nM. The real time recordings demonstrated that LPL was not stable in the absence of apoC-l l, or when its concentration was lower than 1 00 nM. Thus, in addition to activation apoC-l l also stabilized LPL. The stabilization effect of apoC-l l is usually not detectable in other substrate systems. The continuous monitoring of the reaction rate by ITC provides more detailed information about the first minutes of the reaction. The effect of the inhibiting protein apoC-lll on LPL activity was studied after addition to plasma under two experimental circumstances: 1 ) under zero order conditions when the LPL concentration was so low that the reaction rate was constant during the reaction time (zero-order conditions - depletion of substrates did not influence the reaction rate) 2) in the case of total hydrolysis of the available substrates in plasma with about 20-fold more LPL to allow monitoring in real time. Under the zero-order conditions the reduction of the reaction rate was observed at the starting of the reaction and remained unchanged for more than one hour (data not shown). This suggested that apoC-lll affected the LPL activity, but not the stability of LPL in plasma. Figure 6A presents results of sequential injection of LPL into plasma samples under zero-order conditions without additional apoC-lll and after increasing the apoC-lll concentration by 15 or 50 μΜ. From these data it is evident that apoC-lll somehow restricts the activity of LPL. In Figure 6B the effect of addition of apoC-lll to an additional concentration of 15 μΜ on the time course for total lipolysis in plasma is shown. This means about a doubling of the concentration of apoC-lll in normal human plasma (Fredenrich et al. 1997). The initial inhibiting effect on heat rate production is clearly seen, while with time, when most of the available substrate had been degraded, the rates became similar. After transformation of the raw ITC data into a plot of reaction rate versus remaining triglyceride concentration, it became evident that apoC-lll inhibited LPL only when lipoproteins with high triglyceride concentrations were available (Figure 6C).

Animal experiments (Pennacchio et al. 2001 ) and populations studies in humans (Do et al. 2015; Anon 2010) have demonstrated that ApoA-V is an important regulator of triglyceride levels in plasma. ApoAV lowers triglyceride concentrations in vivo, but its effect has been difficult to reproduce in vitro (Nilsson et al. 201 1 ). Although many evidence point to a stimulation of the activity of LPL by apoA-V, the detailed mechanism is still unknown. In ITC experiments low concentrations of apoAV (10 nM) did not change the activity of LPL (Figure 6D). On the contrary, at higher concentrations of apoA-V (100 nM), some inhibition was observed. It should be noted that the normal concentration of apoA-V in plasma is much lower than for apoC-lll, about 4 nM (O'Brien et al. 2005).

Studies in recent years have identified some of the angiopoietin-like proteins (ANGPTLs) as important regulators of lipid metabolism and in particular as inhibitors/inactivators of LPL (Dijk & Kersten 2016). The normal range for ANGPTL4 in plasma is between 0.04 and 3 nM (Robciuc et al. 2010). In experiments with ITC some suppression of the LPL activity in plasma by ANGPTL4 (the N-terminal fragment) was observed from the start of the lipolysis, immediately after injection of LPL. A 10% drop of LPL activity was detected when the ANGPTL4 concentration was increased in plasma by only 10 nM (Figure 7A). With much higher concentrations of added ANGPTL4 fragment (100 nM or 1000 nM), the activity of LPL initially dropped more and continued to decrease after the first drop (Figure 7A). These observations suggest that ANGPTL4 may affect LPL activity in plasma through two different mechanisms, one immediate that may involve inhibition of activity, but likely not to irreversible inactivation. The other, seen at higher concentrations of ANGPTL4, is likely reflecting continuous, irreversible inactivation of LPL. This occurs even though the enzyme is very stable in plasma under the conditions of ITC and the measurements performed under the zero order conditions, when decreased availability of substrates should not influence the activity of LPL. Others have demonstrated that the endothelial protein GPIHBP1 , a transporter protein for LPL, can stabilize LPL against inactivation by ANGPTL4 (Sonnenburg et al. 2009). The exceptionally acidic N-terminal peptide of GPIHBP1 , known to bind and stabilize LPL under other conditions (Reimund et al. 2015), could not protect LPL from inactivation by 100 nM ANGPTL4 under the conditions for ITC (Figure 7B). The full-length ANGPTL3 had similar effects as the N- terminal fragment of ANGPTL4 (Figure 7C).

To test whether ITC is a suitable technique for investigations of LPL inhibitors, experiments with THL that is an active site inhibitor of LPL and other lipases are conducted (Lookene et al. 1994). THL reduced the activity of LPL in plasma in a concentration dependent manner, while the inhibition was detectable already at 90 nM concentration of THL (Figure 8). This indicates high specificity of the LPL/THL interaction, since the LPL concentration in this plasma sample was very low in comparison to abundant plasma proteins. In the presence of 10 μΜ THL, the activity of LPL dropped immediately 80% but remained after that unchanged. The absence of inhibition kinetics in this case suggests that not all of LPL in plasma is accessible for THL. Surprisingly, poloxamer 407, an inhibitor of LPL in mice (Johnston 2010), did not affect the LPL activity in human plasma even at concentration 50 μΜ.

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