DEVELOPMENT OF A METHOD TO MEASURE VLDL SYNTHESIS RATES

Transcription

1 DEVELOPMENT OF A METHOD TO MEASURE VLDL SYNTHESIS RATES WRITTEN BY DEWI VAN HARSKAMP MASTER THESIS SUPERVISORS: M. T. ACKERMANS (SLVE, AMC) AND W. TH. KOK (HIMS, UVA) JUNE 10, 2011

2 1. ABSTRACT Obesity is becoming a major health problem. It is associated with many diseases, amongst others the metabolic syndrome. A current research project at the AMC aims on elucidating the significance of the brain with respect to lipid metabolism in the liver, as an increased production of lipids is associated with the metabolic syndrome. Lipids synthesized in the liver are secreted within Very Low Density Lipoprotein (VLDL). This protein belongs to a class of proteins, the lipoproteins, that is responsible for the transport of lipids through the blood. For research on the influence from the brain on the liver, a method has to be designed that can measure VLDL synthesis rates. Until now, this rate is measured by administering tyloxapol (triton WR1339), which inhibits LPL. However, this method presents some drawbacks. The method to be developed during this research project will involve the infusion of stable isotope labelled d 5-glycerol into subjects (rats). This labelled glycerol will be incorporated in triglycerides in the liver and secreted within VLDL. For the isolation of VLDL from blood plasma, AF4, FPLC, and ultracentrifugation are looked into. For the subsequent hydrolysis of triglycerides in the obtained VLDL fractions chemical and enzymatic hydrolysis are considered. The resulting glycerol will be analysed by GC-MS to obtain Tracer-to-Tracee Ratios (TTR). These will be plotted against time, and from this graphs, the VLDL synthesis rate can be determined. Unfortunately, the aim for this research project is not achieved. The bottleneck appeared to be the isolation of VLDL in such a way that free glycerol from the rat plasma is not present in the VLDL fraction. This undesired free glycerol will be measured during GC-MS analysis and affects the resulting TTR. However, for human subjects, there is a procedure that isolates VLDL without free glycerol from the plasma present in the fractions. Unfortunately, this procedure needs more plasma than can be obtained from rats at subsequent time points. Human VLDL fractions obtained by this ultracentrifugation procedure were used to prove it is possible to obtain a TTR curve that is similar to those in literature, as to prove the method designed during this project gives the desired results.

3 2. TABLE OF CONTENTS 1. Abstract Table of Contents Introduction Lipoproteins Fate of VLDL Measuring lipogenesis Ultracentrifugation Fast Protein Liquid Chromatography (FPLC) (Asymmetrical) Flow Field-Flow Fractionation ((A)F4) Research question Experimental Instrumentation Chemicals Procedures and Results Isolation of VLDL from blood plasma AF4 applied to the VLDL fraction and rat blood plasma Results of AF4 applied to the VLDL fraction and rat blood plasma FPLC Results obtained by FPLC Isolation of VLDL from human blood by ultracentrifugation Results of the isolation of VLDL from human blood by ultracentrifugation Specific hydrolysis of triglycerides from VLDL Isolation of triglycerides from VLDL without precipitation of apob Results of the isolation of triglycerides from VLDL without precipitation of apob Isolation of triglycerides from VLDL with precipitation of apob Results of the isolation of triglycerides from VLDL with precipitation of apob Isolation of triglycerides from VLDL using silica gel Results for the isolation of triglycerides from VLDL using silica gel Hydrolysis of triglycerides by lipase Results of the hydrolysis of triglycerides by lipase Analysis of glycerol enrichment by GC-MS GC-MS conditions Results of the analysis of glycerol enrichment by GC-MS Overall procedure for the analysis of the isotope enrichment of glycerol from VLDL Sample Isolation of VLDL from plasma Hydrolysis of triglycerides in VLDL to form glycerol Measurement of glycerol enrichment Data handling Further research Conclusion Acknowledgements Abbreviations References

4 3. INTRODUCTION Obesity is more and more becoming a major health problem, as it is associated with the development of many diseases. Diseases such as diabetes, coronary heart failure, hypertension and the metabolic syndrome threaten the lives of many obese persons. The exact relationship between obesity and the development of insulin resistance (diabetes type II) is yet unknown, but it appears to be caused by an excess amount of lipids stemming from dietary intake. There is deposition of lipids on tissues, among others on the liver. A neuronal link between the liver and the hypothalamus has been found. Interventions in the brain can cause alteration of glucose metabolism in the liver. A current research project aims on elucidating the significance of the brain with respect to the lipid metabolism in the liver, as an increased production of lipids is associated with the metabolic syndrome. Lipids synthesized in the liver are secreted within Very Low Density Lipoprotein (VLDL). This protein belongs to a class of proteins (lipoproteins) that is responsible for the transport of lipids. 3.1 LIPOPROTEINS Lipoproteins are globular complexes composed of lipids and apolipoproteins. Their major function is to transport lipids through body fluids. The more polar lipids (phospholipids, free cholesterol) and apolipoproteins are found in the outer part of the lipoprotein. In the centre of the complex, the more hydrophobic lipids (esterified cholesterol, neutral lipids, triglycerides) are found. Traditionally, lipoproteins are classified according to their hydrated densities into different categories: High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL), Intermediate Density Lipoprotein (IDL), Very Low Density Lipoprotein (VLDL) and chylomicrons, as can be seen in table 1. Table 1 Classification of lipoproteins In the enterocytes (absorptive intestinal cells), exogenous lipids and apolipoprotein B48 are combined to form chylomicrons. In the liver, the triglyceride-rich lipoprotein VLDL is secreted. Apolipoprotein B100 is the major structural protein of VLDL. VLDL transports, in contrast to chylomicrons, endogenous products. Density (g/ml) Diameter (nm) Chylomicrons <0.95 >75 VLDL IDL LDL HDL HDL FATE OF VLDL Once in the blood, VLDL acquires other apolipoproteins. Triglycerides (TG) are removed from the core by the enzyme LipoProtein Lipase (LPL), which hydrolysis them into glycerol and free fatty acids (FFA). LPLs are found in blood vessels near tissues that use the released fatty acids as fuel. Free cholesterol from VLDL is transported to HDL, where it undergoes esterification by Lecitin:Cholesterol AcylTransferase (LCAT). In their esterified form, they are transported back 4

5 to VLDL by Cholesteryl Ester Transfer Protein (CETP) in exchange for triglycerides. By these actions, VLDL is converted to IDL, which is followed by the conversion into LDL or uptake by the liver for processing. The conversion of VLDL into LDL results in the loss of triglyceride, phospholipids and apolipoproteins other than apo B100 which remains as the major apolipoprotein associated with the resulting LDL particles. LDL is the major supplier of cholesterol to tissues, as it is relatively enriched in cholesterol and small enough to reach it 1, 2. Figure 1 is an schematic representation of this process. Figure 1 Fate of lipoproteins in the body 3.3 MEASURING LIPOGENESIS For research on the influence from the brain on the liver, a method has to be designed that can measure VLDL synthesis rates (lipogenesis). Until now, this rate is measured by administering tyloxapol (triton WR1339), which inhibits LPL. Through the action of tyloxapol, the uptake of triglycerides is inhibited, which results in an increase of triglycerides in the blood. This increase is a measure for the production of triglycerides by the liver. However, the triglycerides measured can originate from other lipoproteins than VLDL, and tyloxapol alters lipid metabolism in vivo. Because of these drawbacks, a new method is to be developed. The enrichment of glycerol from VLDL triglycerides will be measured after infusion of stable isotope labelled d 5-glycerol. The labelled glycerol will be incorporated in triglycerides in the liver and secreted within VLDL. The method will comprise of several steps: the isolation of VLDL from blood plasma, hydrolysis of triglycerides and measurement of the enrichment of glycerol. A search through literature gives several starting points for the development of this method. 5

6 3.3.1 ULTRACENTRIFUGATION Ultracentrifugation is the reference research technique for studying lipoproteins and has many advantages for preparative scale isolation of lipoprotein fractions. Fractions containing specific lipoprotein classes can be isolated, because of the differences in hydrated densities. The common method is to sequentially increase the density of the solution which is added to the plasma, resulting in the floatation of the different lipoprotein classes after each centrifugation run. This, however, is a lengthy and laborious method, as procedures normally take more than 24 hours. In addition, there is a possibility of changes in the structure of the lipoprotein complex, caused by shear. Since the lipoprotein classes are defined by their hydrated densities, this method is still often used 3. A method using stable isotope labelled glycerol and palmitate tracers was used in combination with ultracentrifugation for the isolation of VLDL. This was followed by Thin Layer Chromatography (TLC) for the isolation of triglycerides present in the VLDL (VLDL-TG), chemical hydrolysis to obtain glycerol and derivatization to form fatty acid methyl esters. Gas chromatography coupled with mass spectrometry (GC-MS) in Single Ion Monitoring (SIM) mode is used for the analysis of palmitate methyl ester and glycerol 4. The same strategy was also used in another research, to establish the influence of diet on the assembly, production and clearance of VLDL-TG. However, in this research, only fatty acids were measured to determine lipogenesis 5. In a research on the effects of alcohol consumption on VLDL-TG, isotope labelled glycerol and acetate were incorporated and analyzed by the method described before 6. Ultracentrifugation is also used in combination with commercial kits for lipid analysis and Fast Protein Liquid Chromatography (FPLC) for analysis of apolipoproteins. It is found that an upregulation of VLDL-TG production can be found without an increase in the apob content of the isolated VLDL fractions. This suggests the formation of larger instead of more VLDL particles 7. Cholesterol and lipoprotein contents are commonly analyzed by using commercially available kits FAST PROTEIN LIQUID CHROMATOGRAPHY (FPLC) FPLC is a separation technique that is very similar to Size Exclusion Chromatography (SEC). The column, with larger dimensions than commonly used for HPLC, is filled with an agarose gel. It offers a rapid and reproducible separation of lipoproteins according to their sizes. However, it suffers from a limited selectivity, especially at the high molecular weight side. Also, undesired interactions between the proteins and the stationary phase may occur. It may require a prior procedure, such as ultracentrifugation, for the preparation of the sample to be loaded on the column. In some cases, there is the possibility of pore blockage of packing materials. Moreover, the size-density relationship of lipoproteins is not completely understood, but separation of lipoproteins, based either on size or density, gives practically the same fractions 8. FPLC tends to overestimate the concentration of LDL and underestimate the concentration of HDL 9. The FPLC system can be coupled to tandem MS by ElectroSpray Ionization (ESI) for lipid profiling 10. It can also be used in combination with enzymatic assay kits to determine the concentrations of cholesterol and triglycerides 8, The articles that are cited here all use a Superose 6 column (Pharmacia) for the separation of the lipoproteins. 6

7 3.3.3 (ASYMMETRICAL) FLOW FIELD-FLOW FRACTIONATION ((A)F4) (A)F4 is a useful technique to separate the different classes of lipoproteins according to size. Actually, the diffusion against the force field (asymmetrical flow) determines the rate of migration. A carrier liquid is pumped through a channel. This channel can have either one (AF4) or two (F4) porous walls, which results in a cross flow. The cross flow causes the analytes to be pressed against one of the walls. The carrier flow shows a parabolic flow profile. Diffusion rates against the cross flow differ between the analytes, which causes differences in migration rates, as more diffused analytes spend more time in the faster parts of the parabolic flow. In contrast to FPLC, in AF4 the smallest component elutes first, because their diffusion is larger. This results in a faster average migration rate. Because there is no second phase used for the separation with AF4, there is no mechanical or shear stress on the native conformational structures of the proteins, so there is a minimal possibility that they will be altered or denatured by the interaction with a surface 14. A balance has to be found between the dilution factor and the time required to get baseline separation between two peaks 15. An advantage of AF4 is that it is much faster than ultracentrifugation, which requires very long centrifugation times. It also has a larger molecular-size range than FPLC, as during the latter technique the column might clog. However, it is important to avoid overloading of the sample, because if the compounds become to concentrated, there might be undesired interactions between analyte molecules. AF4 has been used in combination with enzymatic assays for selective detection of the distribution of cholesterol and triglycerides over lipoprotein fractions 16. Frit-inlet AF4 has been applied with staining of the lipoproteins by Sudan Black B. A membrane having large pores was used, so that albumin (diameter 3,6 nm) can be removed from the sample during injection, which prevents interferences caused by albumin 17. The sizes of the lipoproteins can be calculated based on retention times in F RESEARCH QUESTION A method is to be developed that enables the measurement of the enrichment of glycerol in VLDL triglycerides after infusion of stable isotope labelled glycerol (d 5-glycerol), by first isolating VLDL from the plasma, then hydrolysis of the triglycerides and finally the measurement of the enrichment of glycerol in the samples. When the enrichment of glycerol is determined for blood samples withdrawn at subsequent time points, Tracer-to-Tracee Ratios (TTRs) can be calculated (concentration of d 5-glycerol divided by the concentration of glycerol). When plotted against time, these data result in a TTRcurve. From this curve, the VLDL synthesis rate can be determined. For the accuracy of the measured TTRs, it is important that all possible interferences are eliminated from the sample. The procedure can be divided into three steps: First, VLDL has to be isolated from the blood plasma, to prevent interference of other lipid containing proteins. Second, the triglycerides have to be hydrolysed specifically. Since glycerol is the compound of interest in the final analysis, it is important that no other glycerolcontaining compounds, such as phospholipids, are hydrolysed. Also free glycerol in 7

8 plasma should be removed from the sample. If glycerol from another source than VLDL triglycerides is measured, the analysis will result false TTRs. Last, the glycerol will be measured by GC-MS to establish the isotopic enrichment. These three steps are worked on independently at first, and after a satisfactory procedure is found for each step, they will be combined and optimized to form a procedure to measure the isotopic enrichment of glycerol in VLDL. 4. EXPERIMENTAL 4.1 INSTRUMENTATION AF4 Agilent 1100 series degasser 1200 HPLC series isocratic pump Eclipse2 AF4 separation system (Wyatt Technology Europe GmbH) FPLC Liquid Chromatography Controller LCC-500 (Pharmacia) Fraction collector Frac-100 (Amersham Biosciences) Single path monitor UV-1 Control Unit (Pharmacia) Single path monitor UV-1 Optical Unit (Pharmacia) Waters 510 HPLC pump Recorder (Pharmacia Fine Chemicals) Superose 6 10/300 GL Columns (GE Healthcare) Ultracentrifuge Beckman, 70.1 Ti rotor Airfuge A-100/30,30 fixed angle rotor (blue) 8

9 GC-MS HP 6890 series GC system 5973 Mass Selective Detector (Agilent Technologies) 4.2 CHEMICALS GPO (Pasteurized Plasma Protein Solution) containing 40 g/l human proteins, Sanquin 0.9% NaCl, Baxter Lipase from hog pancreas, lyophilized (Sigma-Aldrich) Colipase from porcine pancreas (Sigma-Aldrich) TG assay, Roche Diagnostics GmbH (Mannheim, Germany) FFA assay, NEFA-HR2 kit, Wako (Neuss, Germany) Glycerol assay, Randox Laboratories Ltd. (Crumlin, UK) Phospholipids assay, Biolabo Reagents (Maizy, France) All other chemicals are of analytical grade. 5. PROCEDURES AND RESULTS 5.1 ISOLATION OF VLDL FROM BLOOD PLASMA AF4 APPLIED TO THE VLDL FRACTION AND RAT BLOOD PLASMA AF4 is a technique that separates compounds according to their size. Because of the size differences between the various lipoproteins, a separation is possible using this technique 16. The separation performed in this article is repeated, but the enzymatic assays are omitted and the channel is directly connected to the UV-detector. The absorption was measured at 214 nm, a wavelength that is absorbed by a number of amino acids. The channel is 12 cm long, the spacer used 350 µm thick, and the channel flow is constant (0.6 ml/min). The injected sample volume was 20 µl and the carrier solution was PBS (138 mm NaCl, 2.7 mm KCl, 10 mm phosphate buffer salts) at ph 7.4. The cross flow varied during the separation, the settings can be seen below in table 2 and figure 2. 9

10 Table 2 Trend of the cross flow during AF4 analysis At (min) During min Action Cross flow at start (ml/min) Cross flow at end (ml/min) Focus flow (ml/min) 0 1 Focus Focus and inject Focus Elution Elution Elution Cross flow AF4 1,4 1,2 flow rate (ml/min) 1 0,8 0,6 0,4 0, time (min) Figure 2 Trend of the cross flow during AF4 analysis A human VLDL sample obtained by ultracentrifugation (see section 5.1.5) and rat plasma are analyzed by AF RESULTS OF AF4 APPLIED TO THE VLDL FRACTION AND RAT BLOOD PLASMA The next chromatogram is obtained for the VLDL sample (1:9 VLDL fraction-pbs). 10

11 Figure 3 Chromatogram obtained with AF4, VLDL sample (1:9 VLDL fraction-pbs) A human VLDL sample prepared by ultracentrifugation was injected. Therefore, it was expected to see less peaks than are present in the above chromatogram (figure 3). As can be seen, there is no resolution between the peaks. To assign a fraction in which VLDL should be present, a nanosphere 50 nm standard was run with the same method, as the 50 nm particle falls well within the size range of VLDL (30-80 nm). In the chromatogram, the proposed cut-offs of the fractions to be collected are represented by the vertical lines. Fraction 1 will be the eluent from the 16 th to the 23 rd minute, fraction 2 from the 23 rd to the 30 th minute, and fraction 3 from the 30 th to the 37 th. All fractions will have a volume of 4.2 ml. From the experiment with the nanosphere 50 nm standard, it is expected that VLDL is present in fraction 2. Then the method was repeated to collect the fractions as proposed earlier. The non-diluted VLDL fraction was injected, to achieve the highest possible concentration in the fractions. The chromatogram is shown below (figure 4). Figure 4 Chromatogram obtained with AF4, VLDL fraction (non-diluted) As can be seen, the chromatogram looks quite different than before. Maybe this is caused by overloading of the channel. Also rat blood plasma is separated using this method. Fewer, and very low peaks appear in the part of interest. This is due to the lower concentration of VLDL in rat plasma. Because of this, the collected fractions will be too diluted for the purposes of this project. For this reason, and the 11

12 fact that the Laboratory for Endocrinology does not own an AF4 system, it is decided to focus this part of the project on the other two techniques FPLC Using FPLC, proteins will be separated according to size. The biggest protein will elute first, the smallest last. Since VLDL is a big protein, the first peaks are important for the isolation of VLDL. These peaks should have enough resolution, so that complete separation is ensured. A method for the separation of lipoproteins was already known from earlier experiments. TBS (Tris Buffered Saline) was used at a flow rate of 0.31 ml/min to elute the compounds. The injection volume was 100 µl of 1:1 diluted sample, and the UV-detector was set to 280 nm. This method was used as the starting point for the optimization of the method for the isolation of VLDL RESULTS OBTAINED BY FPLC An enzymatic assay on 1 minute fractions was performed. The concentrations measured in these fractions were too low (0.02 mm) to use this method for isolation of VLDL with subsequent analysis of the enrichment. Then 2 minute fractions were collected, which were dried in a speedvac and dissolved in 10 µl GPO. These fractions showed a maximum concentration of 0.74 mm. Collected fractions with this concentration were incubated with lipase and analysed by GC- MS. On average, only 4 µm was measured in these fractions, probably due to interferences caused by the elution buffer. Due to many problems with multiple FPLC columns and the very low recovery upon analysis of fractions by GC-MS (caused by dilution during analysis and a high salt content upon concentrating the fractions), it is decided to focus on ultracentrifugation for isolation of VLDL from plasma. However, to confirm the presence of VLDL in the fractions collected using ultracentrifuge, FPLC still was used. Mister H. Levels from the Laboratory of Experimental Vascular Medicine was so kind to analyse the obtained fractions with his working apparatus ISOLATION OF VLDL FROM HUMAN BLOOD BY ULTRACENTRIFUGATION 500 ml human blood (from female donor, age 49, non-fasting condition, diagnosed with haemochromatosis) is centrifuged (3 000 rpm, 20 min, 4 C) immediately after blood sampling and addition of 740 µl 15% EDTA to each 50 ml tube. KBr was added to the resulting plasma to obtain the density required for this procedure (1.025 g/ml). The plasma and a KBr density solution were added in equal amounts to the ultracentrifugation tubes. First the KBr solution was added to the tubes, and the plasma was under layered by using an intestine biopsy needle. Then the VLDL was isolated by ultracentrifugation ( rpm, 20 h, 12 C). The VLDL fraction was obtained by tube slicing, the upper 4-5 ml of every tube were pooled to obtain ml of VLDL solution. This is stored in the refrigerator and used for the development of the rest of the procedure and during AF4 analysis. Because of the large amount of blood needed for the above procedure, it needs to be downsized. Therefore, smaller tubes are used. Per tube (so per sample) it is possible to isolate the VLDL from 0.9 ml sample. To the plasma, KBr was added (500mg per 900µl) and a 0.9% NaCl solution was used for the layering. First, 4 ml 0.9% NaCl was added to each tube. Then, using needles, 0.9 ml plasma was under layered. After centrifugation at rpm at 10 C for 2 hours, the VLDL 12

13 layer was removed. There is no observable layer indicating the volume of VLDL in the tube. Therefore, four different volumes were removed from the eight tubes in duplo (see figure 5). The samples were stored in the refrigerator. Figure 5 Ultracentrifugation tube. The different lines represent the top of the fluid that remained in the tube. The numbers indicate the sample names (four different volumes in duplo) Still, this procedure requires a relatively large amount of blood plasma, as the method is to be used for rat plasma. Preferably, the amount of plasma needed is as low as possible, but high enough to isolate sufficient VLDL for further analysis. The Airfuge is an ultracentrifuge that can hold tubes with volumes as small as 175 µl. For the isolation of VLDL using this apparatus, three protocols are found in literature and adapted. For the first procedure, 175 µl plasma was added to the tube 19. Since blood plasma has a density of g/ml, VLDL should be found in the top layer after centrifugation. The second procedure makes use of a CsCl density solution. 100 µl plasma was mixed with 75 µl CsCl (d g/ml) and added to the tube 20. For the third procedure, 100 µl of plasma was over layered with 75 µl of 0.9% NaCl solution 21. As for two of these three protocols a centrifugation time of 2.5 h is used, these 6 tubes were centrifuged for 2.5 h at maximum speed ( g). Afterwards, per tube three fractions were collected by inserting a needle through the wall of the tube and removal of the fraction using a syringe RESULTS OF THE ISOLATION OF VLDL FROM HUMAN BLOOD BY ULTRACENTRIFUGATION The concentration of triglycerides in the VLDL fraction recovered during the first ultracentrifugation experiment was 2.13 mmol/l. This fraction is used to set up the rest of the procedure (specific hydrolysis of triglyceride and determination of isotopic enrichment of glycerol). The triglyceride concentrations in the fractions obtained by the intermediate sized ultracentrifugation (0.9 ml plasma per tube) were analysed. The results are represented in table 3. 13

14 Table 3 Triglyceride concentrations in the different samples obtained by the intermediate sized ultracentrifugation procedure Sample Concentration (mmol/l) Average (mmol/l) The results are not very consistent, and for rats, the large amount of plasma needed per tube is still quite large. Therefore, ultracentrifugation using the Airfuge is explored. To find a procedure for the Airfuge that gives the desired results, human pool plasma was used. The fractions collected from the three protocols for ultracentrifugation were analysed by FPLC at the Laboratory of Experimental Vascular Medicine. The chromatograms obtained for the top fractions are shown in figures 6 to 8. Figure 6 FPLC chromatogram for the VLDL fraction obtained by ultracentrifugation of the plasma without additions 14

15 Figure 7 FPLC chromatogram for the VLDL fraction obtained by ultracentrifugation with a CsCl density solution Figure 8 FPLC chromatogram for the VLDL fraction obtained by ultracentrifugation of plasma over layered with a 0.9% NaCl solution As can be seen, the procedure in which plasma was over layered by 0.9% NaCl solution shows the best separation of VLDL from the other lipoproteins. This procedure was then used for the ultracentrifugation of rat plasma, and top fractions varying in volume were collected and analyzed by FPLC (Figure 9). 15

16 Figure 9 Overlay of FPLC chromatograms from the different top fractions (rat plasma) Unfortunately, there is a big free glycerol peak. Depending on its origin, this might be a problem. If the peak is caused by free glycerol from the plasma or lipolysis (hydrolysis of triglycerides) prior to ultracentrifugation, it will cause inaccurate results for the measurement of the TTRs. If it is caused by free glycerol resulting from lipolysis after ultracentrifugation, it will make no difference for the isotopic ratio of glycerol, as the next step in the procedure is also lipolysis. To determine the origin of the free glycerol, free glycerol in plasma, free glycerol in the VLDL fraction and glycerol in the VLDL fraction after lipolysis were measured by using a human plasma sample from a pilot study from the Experimental Vascular Medicine group, in which d 5- glycerol was infused to measure VLDL synthesis rates. The results were obtained as described in sections 6.3 and 6.4. The concentrations and TTRs of free glycerol in plasma, free glycerol in the VLDL fraction, and the combination of free glycerol and the glycerol that is product of hydrolysis by lipase are compared in table 4. If the TTR of free glycerol in the VLDL fraction is identical to one of the other measured TTRs, the origin is determined. 16

17 Table 4 Results for the determination of the origin of the free glycerol in the VLDL fraction Concentration (µm) TTR Free glycerol in plasma Free glycerol in VLDL fraction 26.8 ND Free glycerol and TG-glycerol in VLDL fraction The concentration of d 5-glycerol in the free glycerol in VLDL fraction samples was too low to be determined. Therefore this experiment was repeated with pooled fractions so that less dilution of the samples is needed, and the isotope labelled glycerol is more likely to be measured. Results are shown in table 5. Table 5 Results for the determination of the origin of the free glycerol in the pooled VLDL fractions Concentration (µm) TTR Free glycerol in plasma Free glycerol in VLDL fraction 34.7 ND Free glycerol and TG-glycerol in VLDL fraction These TTRs should be the same as in the previous experiment, as the same plasma was used. However, they differ significantly. To exclude a source of glycerol contamination during ultracentrifugation or further sample preparation, the procedure was performed by adding 0.9% NaCl to the tube without plasma. This results in the measurement of glycerol concentrations below the limit of detection. The possibility of eliminating free glycerol by varying centrifugation times was also explored. To get a plasma-like environment, GPO was spiked with a high concentration of glycerol (1mM), so that any fluctuations in concentration are likely to be measured. The procedure was repeated for different lengths of time. Unfortunately, it turned out that the concentration glycerol was almost equally distributed across the tube. Extending the centrifugation time does not influence this. From the same human plasma sample, VLDL fractions were isolated by the Laboratory of Experimental Vascular Medicine, using a different protocol than those described above. Two of these fractions were measured without the lipase step, and turned out to contain no free glycerol. This shows that it should be possible to optimize the ultracentrifugation method in such a way that no free glycerol is present in the VLDL fraction. However, they use a protocol requiring much more plasma than is possible to obtain from a rat at multiple subsequent time points. 17

18 5.2 SPECIFIC HYDROLYSIS OF TRIGLYCERIDES FROM VLDL ISOLATION OF TRIGLYCERIDES FROM VLDL WITHOUT PRECIPITATION OF APOB 0.2 ml VLDL fraction obtained by the first ultracentrifugation procedure was mixed with chloroform-methanol (3:1 v/v) and vortexed for two minutes, after which it was centrifuged for 5 minutes at 1200 g. The upper layer (aqueous) was removed, and the organic (lower) layer was transferred to another glass tube and evaporated to dryness under a stream of nitrogen. The remainder was extracted twice in succession with 0.5 ml isooctane-ethyl acetate (80:1 v/v). The isooctane-ethyl acetate solution was again evaporated to dryness under a stream of nitrogen. This tube should hold the triglycerides, whereas the remainder in the extracted tube should contain the phospholipids RESULTS OF THE ISOLATION OF TRIGLYCERIDES FROM VLDL WITHOUT PRECIPITATION OF APOB The supposed triglycerides and phospholipids fractions (two for each category) were dissolved in 200 µl GPO and tested for triglycerides using an assay for plasma triglycerides. Also the triglyceride concentration in the VLDL solution without further sample preparation was determined using this assay. For the phospholipid fractions, a concentration of <0.10 mmol/l was found, for the triglyceride fractions 1.02 and 1.18 mmol/l. However, the concentration of triglycerides in VLDL without further sample preparation was 2.17 mmol/l. This means that somewhere in the isolation of triglycerides from VLDL a part of the triglycerides is lost (the recoveries are 48% and 55%). The transfer of the organic layer to a new glass tube might be the cause of the loss of triglycerides. Also, the removal of the aqueous layer might also be a cause for this loss, when accidentally to much of the organic layer is pipetted to ensure the complete removal of the aqueous layer. To find the cause of the low recovery, the procedure was started again, this time without the transfer of the organic layer to another glass tube. Again, the final fractions were evaporated to dryness, and the triglyceride concentration in the phospholipid fraction again was below the detection limit. 500 µl GPO was added this time, to avoid incomplete dissolving of the triglycerides. After the removal of the aqueous layer, differences in the volumes of the duplos were obvious, so in one vial the concentration inevitably came out higher than in the other after evaporation and dissolving in GPO. The concentrations can be found in table 6, as well as the previous obtained data. 18

19 Table 6 concentrations of TG in the different samples VLDL fraction 2.09 Concentration TG (mmol/l) Recovery 100% (by definition) 2.17 TG fraction 1* TG fraction 2* % 55% 72% 107% * fraction 1 was obtained using the procedure with transfer of the organic layer to another glass tube, fraction 2 without the transfer. As can be seen, the differences between the procedure with and without transfer are huge. Obviously, the procedure will be performed without transfer from now on. The big difference between the recoveries of the duplos of TG fraction 2 is caused during the removal of the aqueous layer, because in one of the duplos too much was removed. To ensure no triglycerides are lost due to splicing into fatty acids and glycerol, from every step in the procedure without precipitation 100 µl was taken aside for the determination of free fatty acid concentrations using an enzymatic assay. If the concentrations remain constant during the procedure, the conclusion can be drawn that no triglycerides are hydrolysed during sample handling. Results of the free fatty acid assay are shown in table 7. Table 7 FFA concentrations in the different samples Sample Origin Concentration 1 VLDL fraction < Aqueous layer < Chloroform-methanol (3:1 v/v) layer < First extraction with isooctane-ethyl acetate (80:1 v/v) 5 Second extraction with isooctane-ethyl acetate (80:1 v/v) 0.03 < Residue <0.02 In most of the fractions, the concentration is below the limit of detection. It can be concluded that (almost) no hydrolysis takes place during this procedure. 19

20 To determine the concentrations of the phospholipids (PLs) in the fractions obtained during the isolation of triglycerides from VLDL without precipitation of apob, a colometric enzymatic method for phospholipids was used. Unfortunately, in the triglyceride fractions (where no phospholipids should appear), relatively high concentrations of phospholipids were found in comparison to the concentrations in the phospholipid fractions (where all phospholipids should be), as can be seen from table 8. Table 8 TG and PL concentrations in the different samples Sample Concentration TG (mmol/l) Concentration PL (mmol/l) Expected concentration PL (mmol/l) VLDL fraction TG fractions (average) PL fractions (average) < Same as VLDL fraction Because phospholipids are also present in the triglyceride fractions, this method cannot be used during this research. We are interested in glycerol, which is a building block of both phospholipids and triglycerides. Upon chemical hydrolysis of triglycerides, it is very likely for phospholipids to be hydrolysed too. When phospholipids are present in the fractions that will be used for the determination of the TTRs of glycerol, the results will be incorrect. Therefore, it is necessary to find another procedure that does separate the triglycerides and phospholipids ISOLATION OF TRIGLYCERIDES FROM VLDL WITH PRECIPITATION OF APOB 0.5 ml VLDL fraction and 0.5 ml isopropanol were mixed and vortexed for 1 min. It was incubated overnight at room temperature. In the morning, it was centrifuged at g for 30 minutes. The supernatant of this and every following centrifuge step were collected. After this, the pellet was washed with 0.5 ml isopropanol-water (1:1 v/v) and centrifuged at rpm for 15 minutes twice. Then, 1 ml isopropanol was added to the pellet, and it was incubated for two hours at room temperature. The sample was again centrifuged for 20 minutes at rpm, and the collected supernatant was filtered using a syringe filter (Gelman Nylon acrodisc, 13 mm, 0.45 µm) to remove the accidental transferred pellet particles 23. The remainder containing the lipids was extracted with an equal volume diethyl ether-ethanol (3:2 v/v). This extraction was repeated 5-6 times with diethyl ether-ethanol (3:1 v/v). The supernatants from every step are combined and evaporated to dryness RESULTS OF THE ISOLATION OF TRIGLYCERIDES FROM VLDL WITH PRECIPITATION OF APOB The washing step is performed twice with 0.5 ml isopropanol/water each time. The pellet was very loose, which gave problems during the pipetting of the supernatant. Therefore, from this step forward, centrifugation was performed at rpm for 15 minutes. In addition to this precaution, a syringe filter was used to filter the solution before the extraction steps, to make sure no pellet particles that were accidentally pipetted with the supernatant will be extracted. 20

21 It appeared that no phase separation was obtained after the addition of diethyl ether-ethanol (3:2 v/v). After addition of an excess of salt (0.25 g NaCl), and centrifugation of the solution at rpm for 5 minutes, a phase separation was obtained. The aqueous layer was washed twice with diethyl ether-ethanol (3:1 v/v), then the volume of the aqueous layer was decreased. In the article it was stated that at this point the washing procedure should be stopped. The solvents of the organic layer were evaporated and the residu was dissolved in 500 µl GPO. The concentrations of triglycerides were found to be 1.03 mmol/l and 0.37 mmol/l. This is a difference of a factor 2.8. Also the phospholipid concentrations were found to be 0.48 mmol/l and 0.13 mmol/l. Therefore, it is decided to decline this procedure ISOLATION OF TRIGLYCERIDES FROM VLDL USING SILICA GEL For the preparation of the Pasteur pipette columns, one part silica gel was suspended in two parts of methanol. After it was settled, the methanol was decanted. This was repeated four more times. The methanol was evaporated at 50 C, until the silica behaved as a free flowing powder. The silica was suspended in isooctane (1:1) and transferred into the pipettes, where a glass bead prevents the silica from draining out. The column was washed twice with 1 ml isooctane-ethyl acetate 80:1 (v/v). 0.2 ml VLDL fraction was mixed with chloroform-methanol (3:1 v/v) and vortexed for two minutes, after which it was centrifuged for 5 minutes at 1200 g. The upper layer (aqueous) was removed, and the organic layer was transferred to another glass tube and evaporated to dryness under a stream of nitrogen. The remainder was extracted twice in succession with 0.5 ml isooctane-ethyl acetate (80:1 v/v). The isooctane-ethyl acetate solution was transferred to the column. Seven fractions were collected: the first upon elution of 4.5 ml isooctane-ethyl acetate 80:1 (v/v) and the second after elution of 5 ml isooctane-ethyl acetate 20:1 (v/v). Then the remainder was extracted twice again, with 0.5 ml isooctane 75:25 (v/v), and this extract was transferred to the column. The third fraction was collected during the elution of 4.5 ml isooctane-ethyl acetate 75:25 (v/v) and the fourth using isooctane-ethyl acetate-acetic acid 75:25:2 (v/v/v). The fifth fraction was collected by eluting another 8 ml of isooctane-ethyl acetate-acetic acid 75:25:2 (v/v/v). At last, the remainder was extracted twice with 0.5 ml methanol. These extracts were again transferred to the column and fractions 6 and 7 each were collected by twice eluting 4 ml methanol in succession RESULTS FOR THE ISOLATION OF TRIGLYCERIDES FROM VLDL USING SILICA GEL The obtained fractions were evaporated to dryness and dissolved in 200 µl GPO. A disadvantage of this procedure is that it is very laborious to elute all fractions from the silica column (it takes about 6 to 7 hours and needs constant attention to prevent the column from running dry and breaking). Using a triglyceride assay kit, the concentrations were determined in all fractions. Unfortunately, the fraction that should contain all triglycerides did hold only 23% of the total concentration measured. This method is not specific enough for this project HYDROLYSIS OF TRIGLYCERIDES BY LIPASE Lipase is a protein that hydrolysis triglycerides into glycerol and three free fatty acids. If it is possible to design a method that uses lipase to obtain glycerol from triglycerides, two steps can 21

22 be combined in one. The isolation of triglycerides from VLDL and the hydrolysis of triglycerides to form glycerol are then performed simultaneously. Porcine pancreatic lipase is commercially available, as well as its colipase. Lipase was dissolved in 0.1 M PBS (ph 8) at a concentration of mg/ml. The colipase was added to this solution at a ratio of 0.21 µg colipase/µg lipase 25. Another solution containing 4% sodium deoxycholate was prepared. The incubation mixture is composed of 70% buffer solution containing the lipases, 20% triglyceride-containing sample and 10% sodium deoxycholate solution. 5 µl of the sodium deoxycholate solution, 10 µl VLDL fraction (sample) and 35 µl lipase-buffer were pipetted into a 0.6 ml eppendorftube and incubated during different lengths of time at 37 C RESULTS OF THE HYDROLYSIS OF TRIGLYCERIDES BY LIPASE The recovery of the hydrolysis of triglycerides by lipase was checked by combining the outcomes of a triglyceride assay (for the total concentration in the sample) and free fatty acid assay to determine the amount of triglycerides hydrolyzed. The results can be found in table 9. Table 9 Recoveries of the different hydrolysis samples Incubation time (hours) Recovery 1 85% 2 83% 3 107% The sample was checked for free fatty acids present before hydrolysis, but none were detected. Also in the lipase buffer no fatty acids were present. After incubation at 37 C for three hours without lipase, no hydrolysis is measured. It would be convenient if this step of the procedure can be carried out overnight. Prior to this procedure, the isolation of VLDL from plasma takes place, and afterwards, the enrichment of glycerol will be measured with GC-MS. The latter requires a few hours of sample preparation and with the lipase procedure taking place overnight, this can be started in the morning. To see if this is possible, the reaction mixture was incubated in duplo both at 37 C and at room temperature during two different nights. The recoveries calculated from free fatty acid concentrations are shown in table

23 Table 10 FFA concentrations and recoveries of the overnight hydrolysis samples Temperature Recovery 37 C 110% 107% RT 97% 103% The specificity of the method was tested using a phospholipase standard. A reaction mixture was incubated overnight to prove the specificity for triglycerides. Additional mixtures were measured to rule out any misinterpretations. A control mixture (without addition of lipase and colipase) was incubated overnight, to make sure that if the concentration free fatty acids increases, this is due to lipase activity or degradation. Also the free fatty acid concentration in a solution of the phospholipid standard at the same concentration as the reaction and control mixtures was measured, to determine the free fatty acid concentration present in the standard solution. The glycerol concentrations in these mixtures were also measured. As expected, the concentrations of free fatty acids and glycerol were below detection limit in every fraction. In the phospholipid solution, no hydrolysis takes place during overnight storage at 37 C or due to lipase activity. This shows that this reaction is specific for triglycerides. The glycerol concentration remains the same. 5.3 ANALYSIS OF GLYCEROL ENRICHMENT BY GC-MS GC-MS CONDITIONS After the hydrolysis step, glycerol can be measured with GC-MS after derivatization. As a starting point, the standard operating procedure used by the Laboratory of Endocrinology for the analysis of glycerol in plasma will be followed. The coupling of the enzymatic step and the GC-MS analysis should be optimized. The sample preparation starts with the dilution of the incubation mixture with phosphate buffer to obtain enough volume for the procedure. During each GC-MS analysis, 5 calibration standards (single analysis) and 2 control samples (both in duplo) are measured. The calibration standards make quantification possible, and the control samples make it possible to monitor the quality of every analysis. Derivatization with heptafluorobutyric acid (HFBA) Per sample, 100 µl is transferred to plastic tubes in duplo and 10 µl of internal standard (1,2,3-13C 3-glycerol, ca. 1mM) is added. This is followed by denaturation of the proteins with 400 µl acetonitrile. Now, the glycerol is dissolved in this solvent, so after vortexing and centrifugation the supernatant is transferred to another tube and evaporated at RT. After addition of 50 µl 1:3 (v/v) heptafluorobutyric acid/ethyl acetate, the samples are placed in an oven for 10 minutes to 23

24 derivatize the glycerol. Again, the solvents are evaporated at RT. Then the derivatized glycerol is dissolved in 100 µl ethyl acetate, transferred to GC-vials and ready for analysis. The GC is equipped with a DB-17 column (30 m, 0.25 mm, 0,25 µm). For injection, the split ratio is set to 10:1 (Carrier gas: Helium, gas flow 12.0 ml/min) at 250 C. The oven temperature is 50 C at injection, and kept at this temperature for 1 minute. After this, the oven temperature is raised at a rate of 10 C/min until it reaches 100 C. Then, the temperature is raised at a rate of 60 C/min until it reaches 300 C. The oven is held at this temperature for 2 minutes. The carrier gas helium is set to a constant flow of 1.2 ml/min. The analytes are ionized by electron ionization. The MS is operated in SIM mode. The following m/z values are looked at: m/z 467 for glycerol; m/z 470 for 1,2,3-13 C 3-glycerol and m/z 472 for d 5-glycerol. Derivatization with acetic anhydride Another GC-MS method uses acetic anhydride to derivatize glycerol. First, ion exchange columns are made. Two resins are used (AG50W-X8 (H +, mesh) and AG1-X8 (HCOO -, mesh)). To each of the resins, distilled water is added 1:1 (w/w). Mix for 5 min on a stirring plate and allow it to settle. Decant the water and repeat the washing procedure once more. Resuspend the resins in water (1:1 (w/w)). Place a glass ball in a Pasteur pipette. Load 1 ml of AG50W-X8 into the pipette and wash with 1 ml distilled water. Then apply 1 ml of AG1-X8 on top of the other resin. Wash with 2 ml distilled water and allow it to drip through. 100 µl of the samples is transferred to 1.5 ml eppendorf vials in duplo. 10 µl of the internal standard (1,2,3-13 C 3-glycerol, ca. 1 mm) is added to the samples. Then, 250 µl 7% perchloric acid (PCA) is used to denaturate the proteins. After centrifugation at g for 5 minutes, the supernatant is transferred to a new tube and 25 µl methyl orange solution (ph indicator: transition ph-range 3.1 (pink) (yellow); 0.04 g/100 ml) was added. The solution was neutralized with 5 M KOH (solution turns yellow), after which 1% PCA is added drop wise until the solution turns pink again. After centrifugation, the supernatant is transferred to ionexchange columns that are made by the analist. The columns are washed with three times 1 ml water. All eluates are collected and dried overnight in a speedvac. The next morning, derivatization of the glycerol takes place after addition of acetic anhydride-pyridine 1:1 (v/v) at room temperature for 30 minutes. This mixture is evaporated to dryness, dissolved in ethyl acetate and transferred to a GC-vial for analysis. The GC is equipped with a DB-1701 capillary column (30 m, 0.25 mm, 0.2 µm, d f 0.25 µm). 1 µl is injected splitless at 250 C. The oven temperature is 70 C at the start of the run, which rises at a rate of 30 C/min to 220 C. The carrier gas is helium and set to a constant flow of 1.5 ml/min. The spectra were recorded using positive chemical ionization with methane as reagent gas. The ion source is 250 C and the quadrupole 150 C. The MS was performed in Selected Ion Monitoring (SIM) mode (m/z 159 for glycerol; m/z 162 for 1,2,3-13 C 3-glycerol; m/z 164 for d 5-glycerol) RESULTS OF THE ANALYSIS OF GLYCEROL ENRICHMENT BY GC-MS Using the GC-MS procedure that uses HFBA for the derivatization of glycerol, typical mass spectra are recorded as represented in figure

25 Abundance internal standard ( 13 C 3-glycerol) Ion ( to ): 08.D Time--> Abundance glycerol Ion ( to ): 08.D Time--> Abundance d 5-glycerol Ion ( to ): 08.D Time--> Figure 10 Typical mass spectra recorded with the GC-MS method involving derivatization with HFBA. To start with, the VLDL fraction and phospholipid standard were incubated overnight by lipase in phosphate buffer and analysed by GC-MS using the procedure involving derivatization with HFBA. Glycerol was be measured in the VLDL samples, and not in the phospholipid samples. However, the recovery of glycerol in the VLDL samples was not optimal (on average 30%). This may be caused by the phosphate buffer, in which the incubation was carried out. Differences between dilution of the samples with phosphate buffer and water were determined, and improvement was found upon dilution with water (average recovery 70%). This is not satisfactory, so it was tried to use GPO as buffer solution to replace the phosphate buffer. GPO contains a.o. albumin, which binds the free fatty acids that are released upon hydrolysis. This then acts as a buffer. Also, GPO was used for the dilution of the incubation mixture, so the sample matrix resembles plasma (the GC-MS method was designed for analysis of glycerol in plasma). During the first attempt, a recovery of 90% was measured. The Experimental Vascular Medicine group has performed a pilot study infusing d 5-glycerol to measure VLDL synthesis rates in humans. Because of the expiry date of the tracer, it was decided 25