development of a method to measure vldl synthesis rates · 2020-05-15 · development of a method...

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

1. ABSTRACTObesity 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 d5-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.

Development of a method to measure VLDL synthesis rates – D. van Harskamp

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2. TABLE OF CONTENTS1. Abstract .............................................................................................................................................................................22. Table of Contents...........................................................................................................................................................33. Introduction.....................................................................................................................................................................4

3.1 Lipoproteins.............................................................................................................................................................43.2 Fate of VLDL.............................................................................................................................................................43.3 Measuring lipogenesis .........................................................................................................................................5

3.3.1 Ultracentrifugation.......................................................................................................................................63.3.2 Fast Protein Liquid Chromatography (FPLC)....................................................................................63.3.3 (Asymmetrical) Flow Field-Flow Fractionation ((A)F4) ..............................................................7

3.4 Research question .................................................................................................................................................74. Experimental...................................................................................................................................................................8

4.1 Instrumentation .....................................................................................................................................................84.2 Chemicals ..................................................................................................................................................................9

5. Procedures and Results ..............................................................................................................................................95.1 Isolation of VLDL from blood plasma............................................................................................................9

5.1.1 AF4 applied to the VLDL fraction and rat blood plasma...............................................................95.1.2 Results of AF4 applied to the VLDL fraction and rat blood plasma.......................................105.1.3 FPLC.................................................................................................................................................................125.1.4 Results obtained by FPLC .......................................................................................................................125.1.5 Isolation of VLDL from human blood by ultracentrifugation ..................................................125.1.6 Results of the isolation of VLDL from human blood by ultracentrifugation......................13

5.2 Specific hydrolysis of triglycerides from VLDL ......................................................................................185.2.1 Isolation of triglycerides from VLDL without precipitation of apoB ....................................185.2.2 Results of the isolation of triglycerides from VLDL without precipitation of apoB........185.2.3 Isolation of triglycerides from VLDL with precipitation of apoB...........................................205.2.4 Results of the isolation of triglycerides from VLDL with precipitation of apoB ..............205.2.5 Isolation of triglycerides from VLDL using silica gel ...................................................................215.2.6 Results for the isolation of triglycerides from VLDL using silica gel ....................................215.2.7 Hydrolysis of triglycerides by lipase ..................................................................................................215.2.8 Results of the hydrolysis of triglycerides by lipase......................................................................22

5.3 Analysis of glycerol enrichment by GC-MS...............................................................................................235.3.1 GC-MS conditions .......................................................................................................................................235.3.2 Results of the analysis of glycerol enrichment by GC-MS..........................................................24

6. Overall procedure for the analysis of the isotope enrichment of glycerol from VLDL..................296.1 Sample .....................................................................................................................................................................306.2 Isolation of VLDL from plasma......................................................................................................................306.3 Hydrolysis of triglycerides in VLDL to form glycerol...........................................................................306.4 Measurement of glycerol enrichment ........................................................................................................306.5 Data handling........................................................................................................................................................31

7. Further research.........................................................................................................................................................338. Conclusion .....................................................................................................................................................................349. Acknowledgements ...................................................................................................................................................3410. Abbreviations............................................................................................................................................................3511. References ..................................................................................................................................................................36


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3. INTRODUCTIONObesity 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 LIPOPROTEINSLipoproteins 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.

3.2 FATE OF VLDLOnce 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

Density (g/ml) Diameter (nm)

Chylomicrons <0.95 >75

VLDL 0.95-1.006 30-80

IDL 1.006-1.019 25-35

LDL 1.019-1.063 20-30

HDL2 1.063-1.1255-12

HDL3 1.125-1.210


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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 it1, 2. Figure 1 is an schematic representation of this process.

Figure 1 Fate of lipoproteins in the body

3.3 MEASURING LIPOGENESISFor 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 d5-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.


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3.3.1 ULTRACENTRIFUGATIONUltracentrifugation 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 than24 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 used3.

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 glycerol4. 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 lipogenesis5. 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 before6.

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 up-regulation 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 particles7.Cholesterol and lipoprotein contents are commonly analyzed by using commercially available kits.

3.3.2 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. Itoffers 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 fractions8. FPLC tends to overestimate the concentration of LDL and underestimate the concentration of HDL9.

The FPLC system can be coupled to tandem MS by ElectroSpray Ionization (ESI) for lipid profiling10. It can also be used in combination with enzymatic assay kits to determine the concentrations of cholesterol and triglycerides8, 11-13. The articles that are cited here all use aSuperose 6 column (Pharmacia) for the separation of the lipoproteins.


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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 ratesagainst 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 surface14. A balance has to be found between the dilution factor and the time required to get baseline separation between two peaks15. 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 becometo 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 fractions16. 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 albumin17. The sizes of the lipoproteins can becalculated based on retention times in F418.

3.4 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 (d5-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 d5-glycerol divided by the concentration of glycerol). When plotted against time, these data result in a TTR-curve. 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 glycerol-containing compounds, such as phospholipids, are hydrolysed. Also free glycerol in


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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 INSTRUMENTATIONAF4

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)


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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

5.1.1 AF4 APPLIED TO THE VLDL FRACTION AND RAT BLOOD PLASMAAF4 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 technique16. 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.


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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 - - 1.20

1 3 Focus and inject - - 1.20

4 1 Focus - - 1.20

5 7 Elution 1.20 1.20 -

12 20 Elution 1.20 0.01 -

32 10 Elution 0.01 0.00 -

Cross flow AF4

0

0,2

0,4

0,6

0,8

1

1,2

1,4

0 5 10 15 20 25 30 35 40 45

time (min)

flo

w r

ate

(ml/m

in)

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 AF4

5.1.2 RESULTS OF AF4 APPLIED TO THE VLDL FRACTION AND RAT BLOOD PLASMAThe next chromatogram is obtained for the VLDL sample (1:9 VLDL fraction-PBS).


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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 16th to the 23rd minute, fraction 2 from the 23rd to the 30th minute, and fraction 3 from the 30th to the 37th. 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


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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.

5.1.3 FPLCUsing 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.

5.1.4 RESULTS OBTAINED BY FPLCAn 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.

5.1.5 ISOLATION OF VLDL FROM HUMAN BLOOD BY ULTRACENTRIFUGATION500 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 (40 000 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 35-40 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 100 000 rpm at 10°C for 2 hours, the VLDL


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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 tube19. Since blood plasma has a density of 1.006 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 1.019 g/ml) and added to the tube20. For the third procedure, 100 µl of plasma was over layered with 75 µl of 0.9% NaCl solution21. 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 (142 000 g). Afterwards, per tube three fractions were collected by inserting a needle through the wall of the tube and removal ofthe fraction using a syringe.

5.1.6 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.


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Table 3 Triglyceride concentrations in the different samples obtained by the intermediate sized ultracentrifugation procedure

Sample Concentration (mmol/l) Average (mmol/l)

1 0.130.09

2 0.04

3 0.100.09

4 0.07

5 0.080.07

6 0.06

7 0.080.12

8 0.15

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


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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 overlayered 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).


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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 d5-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.


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Table 4 Results for the determination of the origin of the free glycerol in the VLDL fraction

Concentration (µM) TTR

Free glycerol in plasma 78.4 0.009

Free glycerol in VLDL fraction 26.8 ND

Free glycerol and TG-glycerol in VLDL fraction 864.5 0.002

The concentration of d5-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 79.4 0.003

Free glycerol in VLDL fraction 34.7 ND

Free glycerol and TG-glycerol in VLDL fraction 853.4 0.003

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 protocolrequiring much more plasma than is possible to obtain from a rat at multiple subsequent time points.


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5.2 SPECIFIC HYDROLYSIS OF TRIGLYCERIDES FROM VLDL

5.2.1 ISOLATION OF TRIGLYCERIDES FROM VLDL WITHOUT PRECIPITATION OF APOB0.2 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 phospholipids22.

5.2.2 RESULTS OF THE ISOLATION OF TRIGLYCERIDES FROM VLDL WITHOUT PRECIPITATION

OF APOBThe supposed triglycerides and phospholipids fractions (two for each category) were dissolvedin 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.


19

Table 6 concentrations of TG in the different samples

Concentration TG (mmol/l) Recovery

VLDL fraction 2.09

2.17

100% (by definition)

TG fraction 1* 1.02

1.18

48%

55%

TG fraction 2* 1.53

2.28

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 <0.02

2 Aqueous layer <0.02

3 Chloroform-methanol (3:1 v/v) layer <0.02

4 First extraction with isooctane-ethyl acetate (80:1 v/v)

0.03

5 Second extraction with isooctane-ethyl acetate (80:1 v/v)

<0.02

6 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.


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 2.17 0.58 -

TG fractions (average) 1.50 0.80 0

PL fractions (average) <0.10 0.08 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.

5.2.3 ISOLATION OF TRIGLYCERIDES FROM VLDL WITH PRECIPITATION OF APOB0.5 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 1 000 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 3 000 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 3 000 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 particles23.

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 dryness24.

5.2.4 RESULTS OF THE ISOLATION OF TRIGLYCERIDES FROM VLDL WITH PRECIPITATION OF

APOBThe washing step is performed twice with 0.5 ml isopropanol/water each time. The pellet wasvery loose, which gave problems during the pipetting of the supernatant. Therefore, from this step forward, centrifugation was performed at 3 000 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.


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 3 000 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.

5.2.5 ISOLATION OF TRIGLYCERIDES FROM VLDL USING SILICA GELFor 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 succession22.

5.2.6 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.

5.2.7 HYDROLYSIS OF TRIGLYCERIDES BY LIPASELipase 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


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 0.125 mg/ml. The colipase was added to this solution at a ratio of 0.21 µg colipase/µg lipase25. 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°C26.

5.2.8 RESULTS OF THE HYDROLYSIS OF TRIGLYCERIDES BY LIPASEThe 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 10.


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

5.3.1 GC-MS CONDITIONSAfter 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 (1,2,3-13C3-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


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 1,2,3-13C3-glycerol and m/z 472 for d5-glycerol.

Derivatization with acetic anhydrideAnother GC-MS method uses acetic anhydride to derivatize glycerol. First, ion exchange columns are made. Two resins are used (AG50W-X8 (H+, 200-400 mesh) and AG1-X8 (HCOO-, 200-400 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 (1,2,3-13C3-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 10 000 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) - 4.4 (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 ion-exchange 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, df 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 1,2,3-13C3-glycerol; m/z 164 for d5-glycerol)27.

5.3.2 RESULTS OF THE ANALYSIS OF GLYCEROL ENRICHMENT BY GC-MSUsing the GC-MS procedure that uses HFBA for the derivatization of glycerol, typical mass spectra are recorded as represented in figure 10.


25

4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.800

20000

40000

60000

Time-->

Abundance

Ion 470.00 (469.70 to 470.70): 08.D

4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.800

20000

40000

60000

Time-->

Abundance

Ion 467.00 (466.70 to 467.70): 08.D

4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.800

20000

40000

60000

Time-->

Abundance

Ion 472.00 (471.70 to 472.70): 08.D

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. GPOcontains 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 d5-glycerol to measure VLDL synthesis rates in humans. Because of the expiry date of the tracer, it was decided

internal standard (13C3-glycerol)

glycerol

d5-glycerol


26

to measure the enrichment in these samples using the procedure, although it is not completely optimized yet. It turned out that the enrichment can be measured and the TTR can be determined. Results can be found in table 11. Triglyceride concentrations are determined by an enzymatic assay, the glycerol concentrations by GC-MS.

Table 11 Results from pilot study lipogenesis in humans

Sample TG (mM) glycerol (mM)

d5-glycerol (µM)

TTR Recovery

00:30 Chylomicrons 0.18 0.181 101%

VLDL1 1.32 1.207 91%

VLDL2 0.39 0.361 93%

03:30 Chylomicrons 0.06 0.079 132%

VLDL1 0.70 0.591 1.74 0.0029 85%

VLDL2 0.59 0.512 1.85 0.0036 87%

06:30 Chylomicrons 0.17 0.180 106%

VLDL1 1.00 0.929 1.65 0.0018 93%

VLDL2 0.49 0.442 90%

To make the method more sensitive to d5-glycerol, the amount of VLDL fraction should be as high as possible, so that small amounts of d5-glycerol still can be measured (i.e. have concentrations above the detection limit). To achieve this, the amount of VLDL fraction was raised with a constant amount of incubation buffer (40 µl 7:1 0.125 mg/ml lipase and 0.21 µg colipase/µg lipase in GPO – 4% sodium deoxycholate in water), and the effects on recovery weremeasured by GC-MS. Results are shown in figure 11.


27

hydrolysis of triglycerides by 40 µl lipase incubation buffer

0%

10%

20%

30%

40%

50%

60%

10 20 30 40 50 60 70 80 90 100

volume VLDL fraction added to the incubation buffer (µl)

reco

very

Figure 11 To find the optimum conditions for hydrolysis, the volume VLDL fraction added was varied. However, the recoveries are much lower than during previous measurements.

The recoveries found during the analysis were much lower than measured during previous analyses. When repeating the experiment, about the same results were found. A fresh lipase incubation buffer also did not improve the diminished recovery. Testing the triglyceride concentration in the VLDL fraction that was used for optimization of the procedure with an enzymatic assay did not show a decreased concentration. Measuring the recovery of the lipase incubation with an enzymatic assay for glycerol showed a recovery of 100%, so the problem must be during the sample preparation for the GC-MS or the analysis itself.

Therefore, an other GC-MS analysis for the enrichment of glycerol (using perchloric acid for denaturation of proteins, acetic anhydride for derivatization and ion exchange columns to clean up the sample) was performed. Typical mass spectra obtained by using this method are represented in figure 12.


28

4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.900

100000

200000

300000

400000

500000

Time-->

AbundanceIon 162.00 (161.70 to 162.70): 13.D

4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.900

100000

200000

300000

400000

500000

Time-->


4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.900

100000

200000

300000

400000

500000

Time-->


Figure 12 Typical mass spectra recorded with the GC-MS method involving derivatization with acetic anhydride.

During the first attempt, an average recovery of 100% was measured. Now the optimization of the ratio VLDL fraction-incubation mixture can be continued. Multiple experiments were performed, the results of one of them is shown in figure 13.

internal standard (13C3-glycerol)

glycerol

d5-glycerol


29

hydrolysis of triglycerides by 40 µl incubation buffer

0%

20%

40%

60%

80%

100%

120%

10 50 75 100

125

150

175

200

volume of VLDL fraction added to the incubation buffer (µl)

reco

very

Figure 13 To find the optimum conditions for hydrolysis, the volume VLDL fraction added was varied.

From the experiments, it can be concluded that a ratio of 1:1 VLDL-fraction-incubation buffer gives highest recovery.

For the last few analyses, the control samples gave varying results. Because TTRs are calculated and the short time left for this internship, it is decided to neglect this, as no concentrations are measured, but TTRs. However, the absolute values of the TTRs might deviate from the true value.

6. OVERALL PROCEDURE FOR THE ANALYSIS OF THE ISOTOPE

ENRICHMENT OF GLYCEROL FROM VLDL

Unfortunately, during this research project, I did not succeed in setting up a procedure that can measure VLDL synthesis rates in rat plasma. The problem is that free glycerol in plasma is not separated from the VLDL during the isolation of VLDL from plasma. Other steps in the procedure work fine, and can be used to obtain a TTR curve.

To obtain a TTR curve, some samples from a pilot study with human subjects by the Experimental Vascular Medicine group are analysed. The procedure and results are discussed below.


30

6.1 SAMPLEHuman plasma. Humans were given a bolus injection (500 mg) of d5-glycerol. Blood samples are obtained at distinct time points and stabilized with EDTA. The plasma samples are placed in a -80°C freezer until analysis.

6.2 ISOLATION OF VLDL FROM PLASMAVLDL fractions are obtained by ultracentrifugation. Density solutions are prepared as follows:

Solution 1 (1,006 g/ml) contains 31.5 g NaCl and 1.30 g EDTA in 3500 ml destilled water; Solution 2 (1.020 g/ml) contains 14 g KBr dissolved in 700 ml of solution 1; Solution 3 (1.065 g/ml) contains 60.47 g KBr dissolved in 700 ml of solution 1.

Add 0.56 g KBr to the ultracentrifugation tube. Add 4 ml of plasma and stir until the KBr is dissolved in the plasma. Carefully layer 3 ml of solution 3 on top of the plasma, then 3 ml of solution 2 on top of solution 3 and 2.2 ml of solution 1 on top of solution 2.Put the tubes in the ultracentrifuge and centrifugate at 36 000 rpm for 32 min (during every centrifugation step, the temperature is set to 4°C and acceleration and deceleration are set to slow). After this, remove the top 1 ml from each tube (this is the chylomicron fraction). Replace this with 1 ml of solution 1. Now, centrifugate the tubes at 30 000 rpm for 3 h 28 min. Again, remove the top 1 ml, which is the VLDL-1 fraction. Replace it with 1 ml of solution 1. Centrifugate at 40 000 rpm for 17 h. The top 1 ml is the VLDL-2 fraction.

6.3 HYDROLYSIS OF TRIGLYCERIDES IN VLDL TO FORM GLYCEROLDefrost the VLDL fractions. An incubation buffer is prepared by dissolving 0.125 mg/ml porcine pancreatic lipase and 0.21 µg colipase / µg lipase in GPO. Also, a 4% sodium deoxycholate solution is made. These solutions are mixed 7:1 lipase-deoxycholate (v/v). 110 µl of this mixture is added to 110 µl of each VLDL fraction and incubated overnight at 37°C.

6.4 MEASUREMENT OF GLYCEROL ENRICHMENTDefrost standards, controls and internal standard for glycerol analysis.

Ion exchange columns are prepared for the sample preparation prior to GC-MS analysis. Two resins are used (AG50W-X8 (H+, 200-400 mesh) and AG1-X8 (HCOO-, 200-400 mesh)). To both 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 water and allow it to drip through.

100 µl of the samples, standards and controls are added to 1.5 ml Eppendorf vials (each sampleand control in duplo, single for standards). Add 10 µl internal standard (concentration circa 1 mM). Then, add 250 µl 7% PCA and vortex. Centrifuge for 5 min at 10 000 g. Transfer 300 µl of the supernatant into an other Eppendorf vial and add 25 µl methyl orange solution (0.04 g in 100 ml water). Neutralize the solutions with 5 M KOH (solution turns yellow). Add 1% PCA until solution is pink again. Centrifuge for 5 min at 10 000 g. Transfer the supernatants onto the top of the ion-exchange columns.

Wash three times with 1 ml water, and collect all eluate. Dry overnight in speedvac.

The following morning, dissolve the dry residue in 100 µl pyridine:acetic anhydride (1:1, v/v). Allow it to react for 30 min at room temperature. Evaporate to dryness under N2 at room


31

temperature and dissolve in 100 µl ethyl acetate. Transfer this solution to GC-vials filled with inserts.

1 µl is introduced into the GC-MS using splitless injection at 250°C. A DB-1701 capillary column (30 m x 0.25 mm x 0.2 µm, df 0.25 µm) is installed. The column temperature is 70°C at injection and is increased after 1 min with 30˚C/min to 220°C. Positive chemical ionization takes place using methane gas (pressure in source 0.6 Torr) and an electron energy of 200 eV. The MS is operating in SIM mode monitoring at m/z 159 (glycerol), m/z 162 (internal standard, 1,2,3-13C3-glycerol) and m/z 164 (d5-glycerol). The temperature in the MS source is set to 250°C and in the quadrupole T=150°C.

6.5 DATA HANDLINGPeak data are transferred to Excel. Concentrations and TTRs are calculated from the results. A graph is made in which the TTR is plotted against time (figures 14 to 16).

Figure 14 Average TTRs of VLDL-1 and VLDL-2 fractions per time point plotted against time


32

Figure 15 TTRs of VLDL-1 fractions plotted against time

Figure 16 TTRs of VLDL-2 fractions plotted against time

From figure 15 can be seen that the incorporation of d5-glycerol in VLDL-1-TG sets in after about half an hour, and is fast, but the removal of isotope labelled triglycerides follows rapidly. During the removal of isotope labelled TG from VLDL-1, there is a rise in its concentration in VLDL-2.


33

These TTR curves look very similar to those found in literature (Adiels et al. 28, figure 2). This shows that the procedure developed gives the desired results.

7. FURTHER RESEARCHAs the isolation of VLDL from rat plasma is not completed, here are two examples of further research for the development of this method.

Orlistat is a lipase inhibitor. When added to the blood during withdrawal, it prevents in vitro lipolysis. The effect of orlistat was tested on human blood samples. 3 blood samples were obtained: stabilized with heparine (to make a comparison with the rat samples, as they are stabilized with heparine), with EDTA and orlistat (100 µl 1 mg/ml in ethanol per 5 ml blood), and with EDTA and ethanol to serve as a blanco for the orlistat sample. VLDL fractions were obtained using the 0.9% NaCl Airfuge protocol. Free glycerol in the plasma and these fractions was determined by the GC-MS protocol involving derivatization with acetic anhydride. Measured concentrations are represented in table 12.

Table 12 Comparison of different stabilizers

Sample glycerol (µM)

Plasma

Orlistat 4.5

Blanco 3.5

Heparine 5.9

UC fraction

Orlistat ND

Blanco ND

Heparine ND

Free glycerol in plasma is normal, so these results are good. During this experiment, glycerol is not detected in any of the human VLDL fractions. In previous experiments involving the Airfuge protocol, it was detected in both human and rat VLDL fractions. The next step is to perform the above experiment on rat plasma, to look into the influence of orlistat on these samples. However, the lipase inhibiting effect of orlistat has to be considered, as the following step in sample preparation is hydrolysis with porcine lipase. It is expected that orlistat does not influence this, because it may be lost during ultracentrifugation (as VLDL is specifically floated, not other proteins to which orlistat should be attached). If not, an excess amount of lipase is added during overnight incubation, which may overrule the effects of orlistat)

Also, it can be tried to downsize the procedure used by the Experimental Vascular Medicine group (as described in section 6.2), as this procedure gives good results.


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8. CONCLUSIONUnfortunately, 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 plasma is not present in the VLDL fraction. Using AF4, only very small peaks appeared in the VLDL region after injection of rat plasma. With FPLC, multiple columns gave similar problems, as the pressure raised after a few uses. Furthermore, the mobile phase was a cause of interferences during the rest of the procedure. Also, because the VLDL-TG concentration was very low in the collected fractions, it would be hard to measure an isotopic ratio. Drying larger fractions in the speedvac to increasethe concentration is also causing a higher salt concentration, so does not circumvent this problem. Therefore, it is decided to work with ultracentrifugation. Many protocols work with relative low salt concentrations, and fractions obtained with ultracentrifugation are more concentrated than those resulting from other techniques.

The Beckman Airfuge is an ultracentrifuge that can hold 175 µl tubes. These tubes offer the possibility of isolating VLDL from small amounts of plasma. A good procedure that prevents free glycerol from the plasma to be present in the VLDL fraction is still to be found. Some ideas for future work are mentioned in this thesis.

It turned out that separation of triglycerides and phospholipids is not that easy, after trying some procedures from literature. An insufficient separation makes chemical hydrolysis inadequate, as glycerol originating from phospholipids will cause incorrect TTRs. Therefore, it was decided to design an enzymatic step that specifically cleaves triglycerides and leaves phospholipids intact. This step was coupled to a GC-MS procedure for the measurement of isotopic ratios of glycerol. Two GC-MS procedures were used during this project. It turned out that the procedure with a more extensive sample preparation gave the best results.

As stated earlier, the procedure still does not work for the measurement of VLDL synthesis rates in rats, because the VLDL isolation needs improvement. However, for human plasma, there is a procedure that isolates VLDL from plasma without free glycerol from plasma present in the fractions. Unfortunately, this procedure needs more plasma than can be obtained from rats at subsequent time points. Nonetheless, human VLDL fractions obtained by this procedure wereused 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.

9. ACKNOWLEDGEMENTS I want to thank my supervisors Mariëtte Ackermans and Wim Kok. Thanks to Eveline Bruinstroop, for whom I attempted to design this method. I want to thank Yvette Kettelarij, An Ruiter and Miriam Bouali for helping me find my way in the Laboratory of Endocrinology. Furthermore, I would like to thank Geesje Dallinga, Shreyas de Jong and Dave Speijer for their help with the ultracentrifugation procedures and Han Levels for analysis of the fractionsobtained with new procedures. I want to thank the Experimental Vascular Medicine group for providing me the samples from the human pilot study. Also thanks to Rudy Vonk for his help with AF4. And last but not least, my special thanks go to everyone from the Laboratory of Endocrinology for giving me such a good time during this internship.


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10. ABBREVIATIONSTable 13 Abbreviations used in this thesis

(A)F4 (Asymmetrical) Flow Field-Flow Fractionation

CETP Cholesterylester Transfer Protein

ESI Electrospray Ionization

FFA Free Fatty Acid

FPLC Fast Protein Liquid Chromatography

GC-MS Gas Chromatography-Mass Spectrometry

HDL High Density Lipoprotein

IDL Intermediate Density Lipoprotein

LCAT Lecitin:Cholesterol Acyl Transferase

LDL Low Density Lipoprotein

LPL Lipoprotein Lipase

PBS Phosphate Buffered Saline

PL Phospholipid

SEC Size Exclusion Chromatography

SIM Single Ion Monitoring

TBS Tris Buffered Saline

TG Triglyceride

TLC Thin Layer Chromatography

TTR Tracer-to-Tracee Ratio

VLDL Very Low Density Lipoprotein

VLDL-TG Triglycerides present in VLDL particle


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