Lipoprotein isolation

For the convenience of the reader, we have outlined the method of sequential flotation employed in our laboratory for separating chylomicrons VLDL, LDL, HDLa, HDLs, VHDL, and d> 1.25 bottom (Table 1). This method, the result of years of experience, has been highly reproducible in terms of the normal human population examined in this laboratory. Such a method may not necessarily apply to dyslipoproteinemic states, where modifications may be necessary, depending on the type of abnormality under consideration. It should also be stressed that any lipoprotein isolated is in need of purification this may be achieved by ultracentrifugation based on the assumption that contaminants are in loose association with the main complex. Whenever this purification is not achieved, other methods may be used as outlined below. For a discussion of the application of density gradient ultracentrifugation to the study of plasma lipoproteins, the reader is referred to a recent review (L3). 

Hazen SL, Heinecke JW (1997) 3-Chlorotyrosine, a Specific Marker of Myeloperoxidase-Catalyzed Oxidation, Is Markedly Elevated in Low Density Lipoprotein Isolated from Human Atherosclerotic Intima. J Clin Invest 99 2075... 

K16. Klein, R. L., and Rudel, L. L., Effect of dietary cholesterol level on the composition of thoracic duct lymph lipoproteins isolated from nonhuman primates. ]. Lipid Res. 24, 357-367 (1983). 

K18. Kostner, G., Studies of the composition and structure of human serum lipoproteins Isolation and partial characterization of apolipoprotein A-UI. Biochim. Biophys. Acta 336, 383-395 (1974). 

L7. Lee, D. M., and Alaupovic, P., Studies of the composition and structure of plasma lipoproteins. Isolation, composition and immunochemical characterization of low density lipoprotein subtractions of human plasma. Biochemistry 9, 2244-2252 (1970). 

Suenram, A., McConathy, W. J., and Alaupovic, P., Evidence for the lipoprotein heterogeneity of human plasma high density lipoproteins isolated by three different procedures. Lipids 14, 505-510 (1979). 

H13. Hazen, S. L., and Heinecke, J. W., 3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J. Clin. Invest. 99, 2075—2081 (1997). 

Traditionally, human LDLs have been defined as those lipoproteins isolated in the density interval between 1.063 g/ml > LDL > 1.019 g/ml. This density interval may be too broad, however, because a substantial contamination with apolipoproteins other than apoB may be present on those lipoproteins at the two extremes of this density range the contaminating apolipoproteins are principally apoE (present on IDEs and HDLs at the low- and high-density extremes of this density interval, respectively) and apo(a) [present on Lp(a) at the high-density extreme]. This contamination was almost absent in the more tightly defined LDL fraction lying between 1.024 and 1.050 g/ml (Chapman et al., 1988). However, it is possible that loosely associated apolipoproteins are normally bound to LDLs in plasma and are subsequently lost during the multiple flotation steps involved in lipoprotein isolation (Mahley and Holcombe, 1977). 

The scope of assays using microchips has also encompassed hpoproteins. Low-density lipoprotein analyses have been performed in an uncoated glass microchannel capillary electrophoresis chip. Analyses using mixtures of lipoproteins isolated from blood have been completed in under 25 seconds. However, no convenient method is yet available for the clinical laboratory,... 

TABLE 21—1. Composition of Lipoprotein Isolated from Normal Subjects... 

The only study located that specifically addressed a possible immunological effect of carbon disulfide exposure in humans reported data that indicated that the -lipoprotein isolated from carbon disulfide-exposed workers (presumably exposed via inhalation) is antigenically identical to lipoproteins isolated from healthy nonexposed controls (Bobnis et al. 1976). The authors concluded that these findings... 

II. Some Unique Problems of Ultracentrifugal Lipoprotein Isolation and Flotation Analysis... 

Leeuwenburgh C, Rasmussen JE, Hsu FF, Mueller DM, Pennathur S, Heinecke JW. Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques. J Biol Chem 1997 272 3520-3526. 

Table 2. Lipid composition of lipoproteins isolated from hemolymph or eggs of marine invertebrates...

Table 7. Properties of lipoproteins isolated from sea urchin eggs, polychaete eggs, and cuttlefish A Properties of lipoproteins isolated from sea urchin eggs...

B Properties of lipoproteins isolated from polychaete eggs... 

Fig. 5A -D. Spectra of lipoproteins from hemolymph and ovary of crabs. A Lipoprotein from ovary of crab Cancer pagurus) in 0.2 M phosphate buffer, pH 7. (Zagalsky et al. 1967) B Lipoprotein II (female-specific lipoprotein) isolated from hemolymph of vitellogenic blue crab Callinectes sapidus). Lipoprotein was in 0.05 M TRIS buffer (pH 8.0). (Lee and Puppione 1988). C Ovorverdin from Homarus gammarus in 0.5 M NaCl — 5 mm EDTA (pH 5). (Zagalsky 1985). D Lipoproteins from ovary of scallop, Pecten maximus, in 0.2 M phosphate buffer (pH 7). Free carotenoids were released from the lipoproteins by extraction with acetone. (Zagalsky et al. 1967)...

Sea urchin eggs are quite different from those of other marine invertebrates with respect to their lipoproteins. The lipoproteins isolated from sea urchins are in the low density lipoprotein class and contain a very high percent of lipid (75 to 63%) with triacylglycerol dominant. The vitellogenin of sea urchin eggs appears to be a glycoprotein. 

Figure 13.15. Headspace gas chromatograms of copper-oxidized low density lipoprotein isolated from a, unsupplemented subject, and b, fish oil supplemented subject. From Frankel et al (1994), with permission from the AOCS Press.

Studies of the composition and structure of serum lipoproteins isolation, purification, and characterization of very low density lipoproteins of human serum. Biochemistry 4, 596—605 (1965). 

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