Lipoprotein centrifugation

Sample Collection and Enzyme Stability. Serum samples are collected with chemically clean, sterile glassware. Blood is allowed to clot at room temperature, the clot is gently separated from the test tube with an applicator stick, and the blood is centrifuged for 10 minutes at 1,000 g. If the red cells are known to contain the enzymes whose activity is being measured, as in the case of LD, even slightly hemolyzed serums must be discarded. When acid phosphatase is to be measured, the serum should be placed immediately in ice and processed as soon as possible, or it should be acidified by the addition of a small amount of sodium citrate. Anticoagulants such as EDTA, fluoride and oxalate inhibit some serum enzymes. However, heparin activates serum lipoprotein lipase. 

A correlation may be established between the concentration of oxidized lipids and the TEARS value, expressed as MDA equivalents, in uM units. Correction is due in some cases for the interference by dyes or other factors. For example, the presence of anthocyanins in red cabbage leaves or turbiditjf causes overestimation of lipid hydroperoxides in plant tissue by the TEARS method. TEARS was used to assert the level of endogenous peroxides in hypo- and hyperthyroidism, both conditions being characterized by low lipid and lipoprotein plasma levels and enhanced oxidative metabolism . In a procedure for determination of TEARS in edible oils, the sample is placed in a centrifuge at 12000 g before measuring at 532 nm (e = 1.56 x 10 M cm ) . A usual procedure for determination of TEARS in certain complex matrices involves steam distillation of the aldehydes responsible for the value, instead of extraction. In nitrite-cured meats, excess nitrite may cause nitrosation of MDA, thus interfering with distillation. To avoid this interference sulfanilamide is added, which is converted to a diazonium salt and... 

Separate yolks of chicken eggs from egg white and discard egg white. Wash the yolks carefully with water to remove adhering egg white. Suspend the yolks in 5 vol. Soln. A by vigorous stirring. Precipitate lipids and lipoproteins by addition of 6 ml Soln. B and 15 ml Soln. C per 100 ml yolk suspension. Stir at RT for 30-60 min and spin at 5000 x g for 10 min. Wash the pellet with a small volume of Soln. A (about 20 ml per yolk) and centrifuge again. Combine the supernatants and filter through a paper filter. Add Soln. D to the clear filtrate to a final concentration of 30 mM EDTA. 

A method for determination of TBARS in the plasma of a 50 p,L blood sample is based on FLD of 169, instead of the usual UVD, with Xex =515 nm and Xn = 553 nm455. The lipoprotein in human serum can be separated into three fractions, VLDL, LDL and HDL, by a series of precipitation and centrifugation operations, carried out in the presence of EDTA and GSH (28) to avoid oxidation. In TBARS measurements carried out for serum by the fluorescence method (Xex =515 nm, Xa = 548 nm) it was found that 43% was bound to the lipoproteins, distributed as follows VLDL + LDL 27% and HDL 16%456. 

Cells Lymphoblasts or primary skin fibroblasts cultured for 4-7 days in lipoprotein-depleted medium, harvested by centrifugation and used fresh or after storage as pellets at - 18°C or lower. The minimum amount required for analysis is = 1.10 cells. 

Milk is clarified by high-speed centrifugation to remove extraneous matter held in suspension. Clarification occurs prior to heat treatment of the milk to prevent dissolution of the extraneous matter. Although clarification removes somatic cells, the elevated levels of lipoprotein lipase activators and plasmin that may be associated with increased numbers of white blood cells in the milk are not eliminated. Therefore, increased lipolysis of milk fat by lipoprotein lipase and proteolysis of casein by plasmin may not be deterred. 

The small particles of plasma lipoprotein, which carry triacylglycerols, can be separated according to their buoyant densities by centrifugation. They have been classified into five groups of increasing density but smaller size as chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density (LDL), and high density lipoproteins (HDL) (Table 21-1 and Fig. 21-2). Each lipoprotein particle contains one or more apolipoproteins (Table 21-2), whose sizes vary from the enormous 4536-residue apoB-100 to apoC-II and apoC-III, each of which contains just 79 residues73 and the 57-residue apoC-I.7b... 

Some freeze-dried antisera are difficult to reconstitute, or occasionally may lose activity. Test a small sample before drying the whole batch. Any cloudiness after reconstitution is denatured lipoprotein, and can be clarified by centrifugation and does not affect antibody binding. 

Phase I enzymes are located primarily in the endoplasmic reticulum of cells. These enzymes are membrane bound within a lipoprotein matrix and are referred to as microsomal enzymes. This is in reference to the subcellular fraction isolated by differential centrifugation of a liver homoge-... 

To determine what percentage of the activity released from antibody-complement-treated cells is associated with protein-containing macromolecules (e.g., lipoprotein), the supernatant from the treated cells is collected as described above, 0.7 ml of dextran sulfate and 2.0 ml of the MgClj are added to the supernatant, and the mixture is centrifuged at 4° for 15 min at 5000 g. Depending upon the lipid content of the macromolecules released from the cells, the j8-lipoprotein-dextran sulfate insoluble complexes thus formed may either float or pellet. The results in Table II... 

Lipoproteins have densities that are lower than the densities of plasma proteins, which do not contain lipids. This characteristic is used to purify and fractionate lipoproteins by sequential flotation centrifugation to... 

The lipoprotein density that should be employed in Eq. (2) is the buoyant density, which is the density at which the sedimentation coefficient is equal to zero. The buoyant density was obtained experimentally by measuring the sedimentation coefficient at several solvent densities and extrapolating to zero. Kahlon et al. (1982) have shown that the buoyant densities, which they call the a densities, vary with the rotor speed of the centrifuge, reflecting the different compressibilities of water and lipid. In order to convert the lipoprotein density, as determined from its composition, to the buoyant density at a rotor speed of 52,640, the data of Kahlon et al. (1982) were used calculate a correction factor of 0.0016 g/ml, which was added to the compositional densities. The values of buoyant densities listed in Table 11 have been calculated by adding 0.0016 g/ml to the density values determined from their compositions. 

Lipoproteins transport lipids in blood an early classification system was established using centrifugation methods, which are based on their different densities. Lipoproteins are classified as chylomicrons, largest and lowest in density... 

Standard centrifugation procedures used for fractionation of lipoproteins in human blood plasma are made by sequential flotation using KBr, NaBr, NaCl, or a combination of these salts to produce a solution with determined density, in which a selected lipoprotein fraction moves upward. 

Using self-formed iodixanol gradients, a novel rapid method for the separation of plasma lipoprotein has recently been described.19 The centrifugation time is reduced to 4 h, but the gradient must be collected carefully, in 0.1-0.2-mL fractions. 

Lipoprotein-X (Lp-X), an abnormal lipoprotein, occurs in patients with obstructive liver disease or LCAT deficiency. Lp-X floats in the density range of LDL and has the same electrophoretic mobility as LDL. It can be separated from LDL, however, by hydroxyapatite chromatography or by zonal centrifugation. The composition of Lp-X differs from that of LDL, and it does not react with antisera to LDL. The major apoproteins of Lp-X isolated from patients with LCAT deficiency are albumin, apo C, and apo A. Lp-X also contains small amounts of apo D and apo E. Lp-X from patients with obstructive liver disease has been reported to lack apo A-I, a powerful activator of LCAT. The lipid constituents of Lp-X are cholesterol (almost entirely unesterified) and phospholipids. In electron microscopy, negatively stained Lp-X preparations appear as stacks of disk-like structures rouleaux). 

If IgG is not further purified, lipoproteins can be eliminated by two successive dialysis cycles against water (12 h) and 50 mM sodium acetate (pH 5.0 24 h), and centrifugation to remove the precipitate (Harboe and Ingild, 1983). 

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