Lipoproteins precipitation

Burstein M, Legmann P. Lipoprotein precipitation. Monogr Atheroscler 1982 11 1-131. 

It is commonly known that lipids, carbohydrates, and glycolipids are present in the Golgi apparatus (27). The determination of the components that react with the ZIO mixture was carried out by removing each component from tissues before incubation in the ZIO mixture. After lipid extraction by acetone (14), chloroform-methanol (15), or propylene oxide (27), no osmium-zinc precipitates could be detected in structures that normally reacted with ZIO. Blumcke et al. (15) summarized the nature of the lipids that react with the ZIO mixture as follows lipids and lipoproteins of cell membranes, neutral fat droplets (41), and lipid globules of type II pneumocytes and alveolar macrophages were, however, not as electron dense as the normally reactive lamellae containing highly unsaturated fatty acids. 

The discovery of Lp(a) by Berg in 1962 (B6) relied on the production of rabbit antisera against beta-lipoprotein and on the selective absorption of these antisera with individual human sera. When certain human sera were used for absorption, the antisera retained precipitation capacity in radial immunodiffusion with 30-35% of individual human sera, which obviously contained a previous unknown antigen. The particle carrying the new antigen shared antigenic properties with beta-lipoprotein, but had an additional antigenic structure. This was evidenced from the only partial fusion of the precipitin bands formed between a positive human serum, the antibeta lipoprotein antiserum and the new absorbed antiserum. 

Similar problems occur for the nephelometric and turbidimetric methods, where the sizes of the IgG-Lp(a) complexes depend upon that of apo(a) itself (L2, W4). Furthermore, problems due to interferences from elevated plasma triglyceride are commonly encountered in the precipitation techniques (C3). As Lp(a) can be redistributed among the Lp(a) fraction and the triglyceride-rich lipoproteins, especially in patients after a fatty meal (B11), these methods are not appropriate for monitoring Lp(a) levels and distribution in plasma. 

Primarily through the work of Oncley et al. [for reviews, see references (C4) and (Bll)], relatively simple procedures have been devised for the specific precipitation of the low- or the high-density lipoproteins by the use of polyanions in the presence of divalent cations. It is necessary, however, to purify the preparations thus obtained, but the procedures thus far described do not permit subfractionation of the major lipopro-... 

A5. Albers, J. J., and Aladjem, F., Precipitation of I-labeled lipoproteins with specific polypeptide antisera. Evidence for two populations with differing polypeptide compositions in human density lipoproteins. Biochemistry 10, 3436-3442 (1971). 

BH. Burstein, M., Scholnick, H. R., and Morhn, R., Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions. J. Lipid Res. 11, 583-595 (1970). 

IgM, an immunoglobulin in milk, forms a complex with lipoproteins. This complex, known as cryoglobulin, precipitates onto the fat globules and causes flocculation. The process is known as cold agglutination. As fat globules cluster, the speed of rising increases and sweeps up the smaller globules with them. The cream layer forms very rapidly, within 20 to 30 min, in cold milk. 

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. 

Two complementary experiments show that the orientation and hiding of one or the other face of the steroid ring of cholate can occur when mixtures of lecithin and bile salt are considered. One of these experiments was performed by Etienne (4), who observed the following facts incidentally while extracting lipids from the serum lipoproteins by Delsal s method. This method utilizes a mixture of methanol and methylal (1 to 4) in the cold. The proteins are precipitated, while the lipids are dissolved in the methanol-methylal solvent mixture. If this solution of the lipids is evaporated, the residue is soluble in nonpolar solvents, such as chloroform. However, if sodium cholate is added to the lipoproteins before their extraction, the residue obtained after the methylal-methanol solvent evaporates is insoluble in chloroform. More precisely, while cholesterol and the triglycerides of the lipidic residue are extracted by chloroform, all of the lecithin remains insoluble, associated to the bile salt. The explanation is probably as follows. During evaporation, methylal with its low boiling point (44°C.), evaporates first, and the solvent becomes more and more concentrated with methanol and the residual water from the lipoprotein aqueous solution. Therefore, in the lecithin plus... 

Cemm will be determined by performing the described cholesterol assay directly on serum. CHDL will be determined on a separated, soluble HDL fraction of serum. Very low-density lipoproteins and low-density lipoproteins are selectively removed from serum by precipitation with magnesium-phos-photungstate reagent. 

Polyanion precipitation has been used for separating apo-B-containing lipoproteins from other lipoproteins. Gidez et al. (G4) have used a fractional precipitation procedure with dextran sulfate and Mn2+ to precipitate apoB-containing lipoproteins first, then HDL2. 

G4. Gidez, L. I, Miller, G. J., Burstein, M., and Eder, H. A., Analyses of plasma high density lipoprotein subclasses by a precipitation procedure Correlation with preparative and analytical ultracentrifugation. In Report of the High Density Lipoprotein Methodology Workshop (K. Lippel, ed.), pp. 328-340. U.S. Department of Health, Education and Welfare. NIH publication No. 79-1661, Bethesda, Md., 1979. 

R15. Rooke, J. A., and Skinner, E. R., The dissociation of apolipoproteins from rat plasma lipoproteins during isolation by precipitation with polyanions. Int. J. Biochem. 10, 329-335 (1979). 

The interaction of partially purified, commercial preparations of P. vulgaris with a large number of serum glycoproteins was studied by Morse.105 The lectin precipitated a2-macroglobulin, /3-lipoproteins, and immunoglobulin M. Preparations ofarglycoprotein, orosomucoid, and several immunoglobulin A myeloma proteins also reacted.105 Chon-droitin 4-sulfate, dermatan sulfate, and heparin also precipitated a partially purified preparation of red kidney-bean the precipitation reaction was completely inhibited by 0.5 M sodium chloride solution.3663... 

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