12 A NNEX
12.1 Experimental Part
12.1.3 Desaturation and elongation of CLA and CLA metabolites
12.1.3.1 Dilution of substrates
The radiolabeled fatty acids were diluted with the corresponding unlabeled fatty acids to obtain a final activity of 370mBq/mmol. The radiolabeled fatty acid was withdrawn and the quantity of unlabeled fatty acid was calculated using the specific activity of the radiolabeled fatty acid. The diluted compound was dissolved in ethanol and used for incubation.
As an example the dilution of 9c11t-C18:2 was shown.
The specific activity of the radiolabeled fatty acid was 1968 MBq/mmol. To obtain a radioactivity of 370MBq/mmol in the substrate solution. The radiolabeled fatty acid has to be diluted 5.32-times with unlabeled fatty acid.
To obtain 1.5µmol of diluted substrate (quantity to perform the ∆6-desaturation assay) 0.28µmol radiolabeled 9c11t-C18:2 was mixed with 1.22µmol of unlabeled compound.
12.1.3.2 Isolation of rat liver microsomes (BERDEAUX et al., 1998b)
! Solutions:
Sucrose/Phosphate buffer Na2HPO4•2H2O 17,9g
KH2PO4 13.7g
Sucrose 171,0g
1.8l water were added, the solution was adjusted to pH 7.4 with sodium hydroxide and filled up with water to 2l.
! Protocol:
Livers were removed immediately after sacrifice and sucrose/phosphate buffer was added (about 3 fold quantity of buffer by liver weight), the livers were pooled and homogenized. The homogenate was centrifuged at 400g for 5min at 4°C. The supernatant was separated and centrifuged at 15000 g for 15 min at 4°C. Finally, the supernatant was ultracentrifuged at 105000 g for 60 min at 4°C. The supernatant (cytosol) was withdrawn and the pellet was resuspended into sucrose-phosphate buffer. Aliquots of the suspension were taken to determine the microsomal protein content. The suspension was diluted 0.5 fold with the cytosol. The microsome preparation was stored for 1h at 4°C, the time to perform the quantification of the proteins.
12.1.3.3 Quantification of microsomal protein (LOWRY et al., 1951)
! Solutions:
Tartrate solution Na/K tartrate distilled water
1g ad 50ml Copper sulfate solution CuSO4•5H20
distilled water
0.5g ad 50ml Sodium carbonate solution Na2CO3
NaOH
distilled water
10g 2g
ad 500ml
Reagent 1 Tartrate solution
Copper sulfate solution Sodium carbonate solution
0.5ml 0.5ml 50ml
Reagent 2 Folin-reagent
distilled water
3ml 3ml Standard solution Lipid free Albumin 1mg/ml
The photometric determination of the protein content was carried out using a one-point calibration. The standard solution was diluted 1:20 before use. The microsome suspension was diluted 1:500. Distilled water was used as control.
! Protocol:
The determination of the protein content of control, standard and microsme suspensions was carried out in triplicate. 1ml of the sample solution was mixed with 5ml of reagent 1, the solution was shaken and was left 10min in the darkness. 0.5ml of reagent 2 was added and the mixture was reacted for 30min in the dark. The absorption of the sample solutions were measured photometrically at 730nm against the control, and the protein content of the microsome suspension was calculated.
12.1.3.4 Incubation I: Desaturation conditions (BERDEAUX et al., 1998b)
! Solutions:
Phosphate buffer (desaturation) Na2HPO4•2H2O 17,9g
KH2PO4 13.7g
Sucrose 171,0g
450ml distilled water were added, the solution was adjusted to pH 7.4 with sodium hydroxide and filled up with water to 500ml.
Incubation buffer (desaturation) ATP 225mg
CoA 44.6mg
NADPH 114.2mg
MgCl2•6H2O 102.8mg adjusted with phosphate buffer (desaturation) to 100ml
! Protocol:
The desaturation studies were performed at 37°C for 15 min in a shaking water bath.
Microsomal suspensions containing 5mg protein and 2ml of incubation buffer were incubated in an open flask with the substrate (dissolved in 40µl ethanol) The incubations were stopped by addition of 5ml of potassium hydroxide (12%) in ethanol.
For the ∆6-desaturation assay 9c12c-C18:2, 9c11t-C18:2 or [1-14C]-10t12c-C18:2 were used as substrates at concentrations of 30, 60, 90, and 120 nmol.
For the ∆5-desaturation assay [1-14C]-8c11c14c-C20:3 and [1-14C]-8c11c13t-C20:3 were used as substrates at concentrations of 10, 20, 40, 60, 80 and 100 nmol .
12.1.3.5 Incubation II: Elongation conditions (BERDEAUX et al., 1998b)
! Solutions:
Phosphate buffer (elongation) Na2HPO4•2H2O 8.95g
KH2PO4 6.85g
Sucrose 171,0g
450ml water were added, the solution was adjusted to pH 7.0 with sodium hydroxide and filled up with water to 500ml.
Incubation buffer (elongation) ATP 367mg
Malonyl CoA 11.4mg
NADPH 111mg
MgCl2•6H2O 67.8mg
Gluthatione 40.3mg
KCN 6.53mg
KF 232.4mg
CoA 5.12mg
Nicotinamide 4.1mg
phosphate buffer (elongation)
80ml adjusted with distilled water to 200ml
! Protocol:
The elongation study was carried out at 37°C for 30min in a shaking water bath.
Incubations flasks were filled with argon as protection gas. 5mg of microsomal protein and 3ml of incubation buffer (elongation) were incubated with the substrate (dissolved in 40µl ethanol). The incubations were stopped by addition of 5ml of potassium hydroxide (12%) in ethanol.
For the elongation assay [1-14C]-6c9c12c-C18:3 and [1-14C]-6c9c11t-C18:3 were used as substrates at concentrations of 10, 20, 40, 60, 80 and 100 nmol .
12.1.3.6 Lipid extraction and methylation
Lipids were saponified by heating for 1h at 75°C. The samples were cooled to room temperature. 3ml of distilled water and 3ml of acetic acid were added to the sample mixture. The lipids were extracted twice, each time with 4ml of hexane. The solvent was evaporated under nitrogen. For methylation, 1ml of BF3/MeOH (14%) was added, the mixture was shaken and left at room temperature for 15min. To neutralize, 5ml of a saturated solution of sodium hydrogen carbonate was added.
The FAME were extracted twice with 2ml of hexane. Hexane was evaporated and the FAME were dissolved in 600µL of acetone for radio-HPLC analysis.
12.1.3.7 Radio-HPLC-Analysis
Instrument: Waters 600
Detector: radiochromatographic Flo-One β detector (Series A-100, Radiomatic Instruments); effluent was mixed with a Uniscint BD scintillation cocktail (National Diagnostic) in ratio of 1:1.2 (effluent/scintillation cocktail)
Column: C-18 Nucleosil, Interchim, 250mm × 4.6 mm I.D, 5µm particle size
mobile phase : Acetonitrile
Flow: 0.5ml/min
Operating system: Millenium Software (Waters)
12.1.3.8 Radio-GC-Analysis
Instrument: Hewlett Packard 5890, equipped with an autosampler
Injector: splitless
1.Detector: radio-GC-detector
2.Detector: FID
Column: Stabilwax wide bore column, Restek, 60m × 0.53mm I.D., 0.5µm film thickness
Carrier gas: helium, 1ml/min
Temperature program: 70°C (4min), 13°C/min to 175°C (27min), 4°C/min to 215°C (31min)
Operating system: Laura software
The output flow from the column was splitted between the FID and the radio-GC-detector in a ratio 10:90.
12.1.3.9 Analytical GC
Instrument: Hewlett Packard 5890,
Injector: splitless
Detector: FID
Column: BPX-70 capillary column, 50m x 0.25mm I.D., 0.25µm, SGE
Carrier gas: helium, 1ml/min
Temperature program: 60°C (1min), 10°C/min to 170°C (60min), 20°C/min to 220°C (15min)
Operating system: Borwin-Software, JMBS Developments
12.1.3.10 GC-MS-Analysis
Instrument: Hewlett-Packard 6890 gas chromatograph coupled to an HP model 5973
Injector: splitless
Detector: electron impact mode at 70eV
1. Column: HP5, fused silica capillary column, Hewlett Packard, 30m × 0.25mm I.D., 0.25µm film thickness
Temperature program: 60°C to 190°C at 20°C/min for 60min
2. Column: BPX-70, fused silica capillary column, SGE, 30m × 0.25mm I.D., film thickness 0.25µm
Temperature program: 60°C to 300°C at 18°C/min for 60min
Carrier gas: helium, 1ml/min
Operating system: HPChem-Station-Software
12.1.3.11 Calculation of results
Results were calculated as % conversion of substrate into product. From this value the amount of transformed substrate in nmol was calculated.