Aplicación del principio de no confiscatoriedad a las detracciones

Isotonie lOmM phosphate buffered saline (PBS) pH7.4, Img/ml bovine serum albumin (BSA) protein standard, bicinchoninic acid (BCA), 1,6-diphenyl-1,3,5-hexatriene (DPH), 5a-cholestane, cholesterol, campesterol, stigmasterol, sitosterol, fucosterol and tétraméthylammonium hydroxide in methanol were all purchased from the Sigma Chemical Company (Poole, UK). Di-sodium hydrogen orthophosphate 2-hydrate, sodium di-hydrogen orthophosphate 1-hydrate and copper sulphate 5-hydrate were purchased from BDH Laboratory Supplies (Poole, UK). HPLC grade propan-2-ol and tetrahydrofuran were bought from Merck Chemical Ltd., (Lutterworth, UK).

Tetrachloroethylene and methylbutyrate were purchased from the Aldrich Chemical Company, (Gillingham, UK). Tri-sil was purchased from Pierce (Luton, UK) and the phospholipid kit from Boehringer Mannheim (Lewes, UK).

2.6 Methods

2.6.1 Blood Samples from Patients Receiving Total Parenteral Nutrition

Eight patients, two male and six female, aged between 4 and 27 months were studied (see Table 2-1 for details). All the patients were hospitalised and were receiving 20% Intralipid. Three of the eight patients showed biochemical evidence of severe cholestatic liver disease (aspartate aminotransferase (AST), >200Units/L [normal: 20-60]; total bilirubin >100pM [normal: <17]). These patients were being considered for gut-liver transplantation. Another four patients had only mild elevations of liver function tests. One control patient was studied who was receiving TPN but who appeared to show no signs o f liver disease. Three other controls used for the study were healthy adult

volunteers from the Biochemistry Unit, Institute of Child Health. Samples of l-2ml of fresh venous blood were drawn from patients and control subjects into lithium heparin tubes.

Table 2-1. Details of Patients Receiving TPN

Patient Number

Sex Age AST

(Months) (Units/L)

Bilirubin (^M)

Presenting Condition Severe Liver Dysfunction

1 F 20 656 534 Gut failure

2 F 9 627 148 Short gut

3 F 16 403 482 Gut failure

Mild Liver Dysfunction

4 F 17 167 2 2 Enteropathy 5 M 27 62 8 Bone marrow transplant 6 F 4 60 65 Necrotising enterocolitis 7 F 10 81 7 Gastrectomy Controls 8 M 23 42 12 Bone marrow transplant 9 M

10 M Normal Adult Controls

2.6.2 Preparation of Erythrocyte Membrane Ghosts

Erythrocyte ghosts were prepared by a modification of the method of Burton et al.

C ^ - G O a )

(1981). Whole blood samples were centrifuged at 2000fpm for 5 minutes and the plasma removed. The erythrocytes were then washed three times with 2ml isotonic

lOmM phosphate buffered saline (PBS) pH7.4. (Note: PBS was isotonic to body fluids and consisted of lOmM Na2HP0 4/NaH2P0 4 buffer; 138mM NaCl and 2.7mM KCl at pH7.4). Membrane ghosts were prepared by the lysis of 250-1 OOOjiil RBC. The erythrocytes were diluted 1:5 volume/volume (v/v) with chilled 2.5mM phosphate buffer pH8.0 and stirred for 15 minutes. The erythrocyte ghost pellet obtained after centrifuging at 18000rpm for 20 minutes at 4°C was then washed and centrifuged twice more with 5ml 1.25mM phosphate buffer pH8.0 as above. This removed the

haemoglobin i.e. pellet was creamy white to the naked eye. The red cell ghosts were then resuspended in 500pl isotonic lOmM PBS pH7.4 gassed under nitrogen gas and stored at -20“C. To minimise any potential effects of storage and inter-assay variation all samples were studied on the same occasion and within 2 months of preparation.

2.6.3 Protein Determination of Erythrocyte Membrane Ghosts

This method was based on the bicinchoninic acid (BCA) method of Smith et al. (1985). The method combines the biuret reaction with unique characteristics of BCA. In the reaction proteins react with alkaline Cu^^ to produce Cu^. Two molecules of BCA then react with one cuprous ion to produce an intense purple colour from which the

spectrophotometric quantitation of protein in the aqueous solution can be determined.

All assays were carried out in duplicate. A standard curve was prepared containing 5- 50|ig of 1 mg/ml BSA standard in 50p,l distilled water. For samples 5pi RBC membrane preparation was diluted to 50pl with distilled water. After the addition of 1ml BCA the samples and standards were incubated at 37”C for 10 minutes. 20pl o f copper sulphate solution was added and the samples incubated at 37°C for at least 20 minutes. The absorbance was then read at 562nm and the protein concentration of the sample in pg/pl was calculated from the standard curve.

2.6.4 Determination of Membrane Fluidity

A 2mM stock solution of DPH in tetrahydrofuran was stored in the dark at -20°C. A fresh 0.5fj,M working solution was made by diluting the stock DPH solution 1:4000 (v/v) with isotonic lOmM PBS pH7.4. This was kept in the dark at 4°C and stirred for at least 2 hours prior to use to ensure adequate mixing. A 50pl aliquot of the red cell ghost suspension was incubated with 2ml O.SpM DPH at 37°C for Ihour. Fluorescence was then measured in a 1cm cuvette heated to 37“C in a Perkin-Elmer LS3 fluorimeter using an excitation wavelength of 360nm and an emission wavelength of 430nm. Four measurements were made with each sample i.e.vertical (0°) and horizontal (90°) polarised excitation with either vertical (0°) or horizontal (90°) polarisation of the emission filter. Steady-state anisotropy was then determined using the equation:

0 90) (9 0 0

Ô" W I V %

rs

0 90/ I 0 90

Blank readings of the fluorescence 5pM DPH in isotonic lOmM PBS pH7.4 without membranes were made in an identical manner and subtracted from readings given by DPH and membranes for each polariser setting. Due to a limited amount of material it was not possible to take blank readings of membranes incubated in PBS without 5pM DPH. However, validation studies previously undertaken in the Biochemistry Unit, showed that the fluorescence produced by these samples was negligible.

2.6.5 Gas Chromatography-Mass Spectrometry of Phytosterols and Cholesterol

Plasma total (free and esterified) phytosterol and cholesterol concentrations were measured by gas chromatography-mass spectrometry (GC-MS) by the method of Clayton et al. (1993). The procedure which used 5a-cholestane as the internal standard (ISTD) is shown schematically in Figure 2-3.

Sterol Extraction

1ml of a saponification mixture containing 0.0372mg 5a-cholestane, mixed with 0.25ml tétraméthylammonium hydroxide in methanol and made up to volume with propan-2-ol

Plasma