Lipoproteins methods

Clevidence, B.A. and Bieri, J.G., Association of carotenoids with human plasma lipoproteins, Methods Enzymol, 214, 33, 1993. 

Dobiasova M, Frohlich J (1996) Measurement of fractional esterification rate of cholesterol in plasma depleted of apoprotein containing lipoprotein methods and normal values. Physiol... 

None of the lipoprotein methods described in this section has been used widely enough to have been validated in independent studies to the same extent as have been the Friedewald and beta-quantification methods. In most cases, the identities of the lipoprotein contributing to the LDL cholesterol measurement have not been adequately established. Further evaluations should better define the relationships between these new methods and current reference and routine methods. 

Bachorik PS, Albers JJ. Precipitation methods for quantification of lipoproteins. Methods Enzymol 1986 129 78-100. 

Jonas, A. 1986. Reconstitution of high-density lipoproteins. Methods Enzymol. 128 553-582. 

In order to measure susceptibility to oxidation without the need to isolate the lipoproteins, methods have been developed for oxidizing whole serum or plasma and measuring diene formation (R2, S4). Such approaches may be subject to error as a result of variation in other oxidizable plasma components such as bilirubin, albumin, fibrinogen, and uric acid. A method that uses heparin affinity chromatography to separate LDL and intermediate density lipoproteins (IDL) from other serum proteins was described by Vinson et al. (V3, V5) and was later better standardized (K3). This approach has been shown to reflect susceptibility to oxidation in animal and human plasma under a variety of conditions (K3, V5). The heparin separation procedure is detailed in the following text. 

DeLalla, 0., and Gofman, J.W., 1954, Ultracentrifugal analysis of serum lipoproteins. Methods Biochem. Anal., 1 459. 

The antioxidant activities of carotenoids and other phytochemicals in the human body can be measured, or at least estimated, by a variety of techniques, in vitro, in vivo or ex vivo (Krinsky, 2001). Many studies describe the use of ex vivo methods to measure the oxidisability of low-density lipoprotein (LDL) particles after dietary intervention with carotene-rich foods. However, the difficulty with this approach is that complex plant foods usually also contain other carotenoids, ascorbate, flavonoids, and other compounds that have antioxidant activity, and it is difficult to attribute the results to any particular class of compounds. One study, in which subjects were given additional fruits and vegetables, demonstrated an increase in the resistance of LDL to oxidation (Hininger et al., 1997), but two other showed no effect (Chopra et al, 1996 van het Hof et al., 1999). These differing outcomes may have been due to systematic differences in the experimental protocols or in the populations studied (Krinsky, 2001), but the results do indicate the complexity of the problem, and the hazards of generalising too readily about the putative benefits of dietary antioxidants. 

Experimental evidence in humans is based upon intervention studies with diets enriched in carotenoids or carotenoid-contaiifing foods. Oxidative stress biomarkers are measured in plasma or urine. The inhibition of low density lipoprotein (LDL) oxidation has been posmlated as one mechanism by which antioxidants may prevent the development of atherosclerosis. Since carotenoids are transported mainly via LDL in blood, testing the susceptibility of carotenoid-loaded LDL to oxidation is a common method of evaluating the antioxidant activities of carotenoids in vivo. This type of smdy is more precisely of the ex vivo type because LDLs are extracted from plasma in order to be tested in vitro for oxidative sensitivity after the subjects are given a special diet. 

Wade, D.P., Knight, B.F., and Soutar, A.K. (1985) Detection of the low-density lipoprotein receptor with biotin-low-density lipoprotein. A rapid new method for ligand blotting. Biochem. J. 229, 785-790. 

This method is also used to measure ex vivo low-density lipoprotein (LDL) oxidation. LDL is isolated fresh from blood samples, oxidation is initiated by Cu(II) or AAPH, and peroxidation of the lipid components is followed at 234 nm for conjugated dienes (Prior and others 2005). In this specific case the procedure can be used to assess the interaction of certain antioxidant compounds, such as vitamin E, carotenoids, and retinyl stearate, exerting a protective effect on LDL (Esterbauer and others 1989). Hence, Viana and others (1996) studied the in vitro antioxidative effects of an extract rich in flavonoids. Similarly, Pearson and others (1999) assessed the ability of compounds in apple juices and extracts from fresh apple to protect LDL. Wang and Goodman (1999) examined the antioxidant properties of 26 common dietary phenolic agents in an ex vivo LDL oxidation model. Salleh and others (2002) screened 12 edible plant extracts rich in polyphenols for their potential to inhibit oxidation of LDL in vitro. Gongalves and others (2004) observed that phenolic extracts from cherry inhibited LDL oxidation in vitro in a dose-dependent manner. Yildirin and others (2007) demonstrated that grapes inhibited oxidation of human LDL at a level comparable to wine. Coinu and others (2007) studied the antioxidant properties of extracts obtained from artichoke leaves and outer bracts measured on human oxidized LDL. Milde and others (2007) showed that many phenolics, as well as carotenoids, enhance resistance to LDL oxidation. 

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. 

So, according to Emancipator (E8), a meal has a clinically significant influence on the Lp(a) level, as measured by ELISA methods (the effect decreases 2 h after the meal). Whether this is caused by a different distribution of apo(a) and Lp(a) over other lipoproteins, in which antigenic determinants are covered, is not clear. 

A competitive ELISA assay for Lp(a) was recently described (Y4) in which the microtiter plate was coated with Lp(a) purified from a pool of donors. The method is simple and easy to perform, with satisfactory analytical parameters. A good stability and a reproducible coating of plates with the large Lp(a) lipoprotein is, however, critical in this type of assay. Wang et al. (W6) described an indirect sandwich assay for the measurement of Lp(a) in plasma and in dried blood spots, which can be applied to screening elevated Lp(a) levels in newborns (V3, V4). 

Although most assays perform well with regard to specificity and reproducibility, the major problem remains their standardization (A9, Dl, K30, L4). There is currently no internationally accepted standard, and the selection of a reference material raised many problems (A8, G5, K30, L4). A number of questions have not been solved Should the standard consist of several apo(a) isoforms Can the reference material be lyophilized Should results be expressed as mass or as moles of apoprotein or lipoprotein How should the protein mass of the primary standard be determined What are optimal storage conditions for the secondary standard Which method can be used as a reference method Can recombinant apo(a) represent an alternative for a primary standard These problems came to light in the course of the international surveys whose results were presented at the Lp(a) Workshop in New Orleans (1992) (L4). 

Bl. Baldo-Enzi, G., Baiocchi, M. R., and Crepaldi, G., Comparison of lipoprotein(a) assay methods in serum and in a plasminogen-free fraction. Clin. Chim. Acta 218, 83-95 (1993). 

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