Lipoproteins lyophilization

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). 

B20. Borque, L., Maside, C., Rus, A., and del Cura, J., Addition of sucrose avoids effect of lyophilization on determinations of lipoprotein (a) in serum. Clin. Chem. (Winston-Salem. NC) 39, 553-554 (1993). 

Amphotericin B is available in the form of sterile lyophilized powder. Because it is insoluble in water, it is marketed with sodium deoxycholate for dispersal in sterile water and 5% dextrose. The polyene antibiotics — amphotericin B, nystatin, and candicidin — are all poorly absorbed from the gastrointestinal tract. In the plasma, amphotericin B binds to lipoproteins including cholesterol. It is extensively metabolized and the inactive metabolite, or metabolites, are slowly excreted in the urine. 

A powder of lipoprotein lipase (LPL) esterified an organic substrate in toluene at a rather poor reaction rate (Table 4), which was to some extent explained by adhesion of the sticky enzyme powder to the surface of the reaction vessel [7]. When polyethylene glycol (PEG) was bound covalently to LPL and this modified enzyme was dissolved in toluene, approximately 3.5 U mg 1 of enzyme protein were assayed. After simple addition of PEG to the reaction mixture together with LPL powder, the same poor reaction rate of the enzyme powder alone was observed. On the other hand, when LPL powder was lyophilized together with PEG the resultant preparation had an activity of 1.8 U mg. In this case, the enzyme... 

Gustafson (1964) has proposed a new procedure by which partial de-Iipidation of the various plasma lipoprotein classes is obtained by lyophilization in the presence of starch powder followed by extraction wiA n-heptane at —12°C. High recoveries of protein (93-100%) were obtained in the soluble protein-phospholipid residues from lipoproteins of Sf > 400 and HDL. This was contrasted by the low yields in de-lipi-dated protein of the LDL class. The technique would appear particularly applicable for the study of the triglyceride-rich very low-density lipoproteins, easily denatured by the action of diethyl ether. 

Attempts have been made in the author s laboratory to obtain a lipid-free water-soluble product from the d < 1.019 and d 1.019-1.063 low-density human serum lipoproteins by using the ethanol-ether procedure successfully applied to the high-density class (Scanu et al., 1958a). The precipitate obtained proved insoluble in the various buffered media alone or in the presence of 1-4 M urea. Banaszak and McDonald (1962) attempted separation of the protein moiety of d 1.019-1.063 serum lipoproteins by a pentane extraction of a lyophilized preparation. The product obtained, insoluble in common buffer solutions, was solubilized in carbonate buffer of pH 10.2 in the presence of 4 M urea, leading to prompt formation of a gel, which persisted after removal of urea by dialysis. 

Lyophilized, anatomically Intact, whole pituitary glands from hogs were kindly supplied by Canada Packers Anterior and posterior were carefully dissected from these when necessary. Isolation of Fractions H and L was exactly as described by Rudman et al. as was the preparation of the rabbits used in the bioassay. The extracts were dissolved in 0.9% NaCI and injected into the marginal ear vein. Blood samples were drawn from the marginal ear vein (except in 10 rabbits by cardiac puncture) and analyzed for FFA (Dole), triglycerides (Van Handel), cholesterol (Abell etal Levine and Zak), lipoproteins (Shandon), glucose, urea and uric acid (E.Seifter et al.). 

Detailed studies of the apolipoproteins require the removal of the lipid from the lipoproteins. This can be achieved by extraction of the lipoproteins either direct from plasma or in the precipitated form with ethanol-diethyl ether mixtures (3 1, v/v). The more powerful lipid solvent, chloroform-methanol (2 1, v/v) results in a poorly soluble apolipoprotein. With ethanol-ether, however the advantage of having a soluble apoprotein is partly offset by the loss of 20% of VLDL protein. Lyophilization before... 

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