OFICINA DE INFORMACIÓN TURÍSTICA DE DIRCETUR PUNO

2.3.1 COLLECTION OF BLOOD

Venous blood was taken from healthy volunteers using sterile polypropylene syringes (Plastipak, Becton Dickinson Ltd) and 19 gauge butterfly needles (Venisystems). Blood was immediately transferred into sterile plastic universal tubes (Sterilin) containing ACD anticoagulant (5:1 v/v) and centrifuged at 1800 g for 20 mins at 20°C in a Centra-7R bench centrifuge (International equipment Co., USA) to separate the blood cells from the plasma. Normally 120 mis o f whole blood was taken from each donor which gave approximately 80 mis o f plasma (plus ACD).

2.3.2 PREPARATION OF LDL

LDL (density 1.019 - 1.063 g/ml) was isolated separately from the plasma o f each donor by discontinuous gradient ultracentrifugation in the presence o f 0.3 mM EDTA to avoid autoxidation (Chung et al, 1980). The density o f the plasma was adjusted to 1.3 g/ml by the addition o f solid NaBr (31 g per 70 ml plasma). 10 - 15 mis o f plasma were carefully layered underneath 0.9% (w/v) saline in centrifuge tubes (Beckman). The tubes were then capped, placed in a fixed angle rotor (Beckman 70-Ti) and spun at 200,000 g for 2.5 hrs at 16°C in a Beckman XL-70 ultracentrifuge.

Following centrifugation, the lipoproteins were banded in the tubes with VLDL and chylomicrons at the top, LDL in the middle and HDL at the bottom o f the tubes. The middle LDL band was carefully removed using a syringe and needle and evenly distributed between clean centrifuge tubes containing 6.5 mis o f density solution 1.151 g/ml. The tubes were filled with a solution o f density 1.063 g/ml, capped and gently mixed before being

placed in a fixed angle rotor and spun at 200,000 g for 16 hrs at 16°C. This produced a yellow band at the top o f the tubes which was carefully removed.

2.3.3 CONCENTRATION OF LDL

LDL samples were concentrated by centrifugation in a Sorval RC-5B refiigerated centrifuge (Dupont Instruments, USA) at 8000 rpm at a precooled temperature o f 4°C in tubes containing nitro-cellulose ultrafiltration membranes (Diaflo, Amicon Corp., USA) which retained LDL but allowed the buffer to pass through. LDL samples were concentrated until the volume was reduced to 2 - 4 mis and were then transferred into 2 cm wide dialysis tubing (Scientific Industries International Incorporated) and dialysed against 5 changes o f Tris buffer containing 0.3 mM EDTA for 18 hrs at 4°C. The LDL was finally filtered through a sterile 0.2 pm filter (Acrodisc, Gelman Sciences, UK) to remove any impurities. LDL prepared in this way is referred to as native LDL and was stored in the dark at 4°C and used within 2 weeks.

2.3.4 PROTEIN ASSAY

The protein concentration o f the LDL samples was estimated using a modification (Markwell et al, 1978) o f the Lowry method (Lowry et al,

1951) using Folin-Ciocalteu's pholin reagent. Bovine serum albumin (BSA) was used to prepare a standard curve. The exact concentration o f the BSA stock solution was determined by measuring the UV-absorbance at 279 nm against distilled water. The concentration of the solution was then calculated using the equation below:

E c o CD CD (/)

<

0.6

0.2

0.0

100

0

20

Bovine serum albumin (BSA) standard curve for protein assay.

The protein concentration of all LDL samples was determined as described in section 2.3.4 and expressed as mg LDL protein/ml.

Standards and samples were prepared in triplicate and the absorbance read at 660 nm against distilled water on a Beckman DU-70 spectrophotometer.

A typical standard curve is shown in Figure 4 . Normally LDL samples were prepared at a concentration of between 5 and 8 mg protein/ml.

2.3.5 MODIFICATION OF LDL

Prior to modification, LDL was dialysed against 5 changes o f Tris buffer for 18 hrs at 4°C to remove EDTA from the LDL sample. LDL was dialysed at a ratio o f 1 part LDL to 1000 parts dialysis buffer.

(a) Cu2+-oxidised LDL fOXLDD

Oxidised LDL was prepared by incubating LDL ( 5 - 8 mg protein/ml) with CUSO4 at a concentration o f 1 nmol per mg o f LDL for 24 hrs at room

temperature. The OXLDL was then extensively dialysed against Tris buffer containing 0.3 mM EDTA to remove the excess Cu^"*" ions and filtered using a sterile filter as described previously (section 2.3.3).

(b) Lipoxvgenase-treated LDL (LO-LDL)

LDL modified by treatment with lipoxygenase was prepared by incubating LDL (5 mg protein/ml) with 200,000 U/mg o f affinity purified soybean lipoxygenase in 50 mM borate buffer pH 9.0 for 24 hrs at room temperature (Cathcart et al, 1991). In order to remove the enzyme following oxidation, the LO-LDL was transferred into centrifuge tubes containing density solutions as described in section 2.3.2 and spun at 200,000 g for 4 hrs in a Beckman XL-70 ultracentrifuge. The yellow band o f LO-LDL could be seen at the top of the tubes. Lipoxygenase alone spun following the same procedure and stained with Coomassie Brilliant Blue R-250 protein

stain (Demacker et al, 1983) was located in the bottom part o f the tubes. Following recentrifugation of LO-LDL which had been stained with Coomassie Blue, a band o f protein could be seen at the top o f the tube and also in the bottom part o f the tube showing the separation o f the enzyme from LO-LDL. The LO-LDL was then removed from the tubes and extensively dialysed against Tris buffer containing 0.3 mM EDTA and filtered as described previously (section 2.3.3)