Cholesterol esters labeled

A change in the opposite direction occurred in the distribution of the label between esterified and free cholesterol when the uptake of cholesterol ester-labeled chylomicrons by the liver was studied (O. Stein et al., 1969). Since both the lability toward dehydrating agents and the ultrastructural localization of free and esterified cholesterol may differ considerably, it is important to determine chemically and define the nature of the compound at the time of its visualization. 

Figure 11. Microscopic autoradiography of liver of a mouse treated with tritium labelled CPIA-cholesterol (X540) 5 weeks after single intravenous injection of ca. 30 mg/kg of 3-H-[2R]-CPIA-cholesterol ester. 3-H label is localized in Kupffer cells (arrow and giant cells (long arrows).

Gavey et al. [22] studied the participation of SCP2 in the conversion of cholesterol to cholesterol ester (acyl-CoA cholesterol acyl transferase, ACAT) by rat liver microsomes. The effect of rat liver cytosol on the conversion of [4- Cjcholesterol to cholesterol ester was examined as a function of cytosol protein added. In the absence of cytosol, microsomes formed 1.0 nmole of labeled cholesterol ester/mg microsomal protein/2 h. The addition of rat liver cytosol to the microsomes produced a marked, concentration-dependent, increase in the amount of exogenously added [4- C]cholesterol that was converted to cholesterol ester. Specifically, with the addition of 37.6 mg of cytosol to the microsomes, 12.9 nmoles of labeled cholesterol ester/mg microsomal protein/2 h were formed. When cytosol was incubated with [4- C]cholesteroI in the absence of microsomes, no cholesterol esters were formed. 

Dietary pectin affects lipid metabolism, especially that of cholesterol. One of the explanations proposed to explain an action of pectin on cholesterol metabolism is through its ability to bind bile acids and bile salts. However, pectin also has the property of forming a gel in water. This gel lowers the intestinal absorption of cholesterol and thereby decreases liver cholesterol. Recently, evidence has been obtained that the presence of pectin in a cholesterol diet increases the excretion of cholesterol esters. Results from the administration of cholesterol-4-l C in the diet showed that the presence of pectin slows gastric emptying and results in more labeled cholesterol as well as cholesterol esters in all segments of the gut. 

TLC is ideally suited for analysing the products of radiochemical syntheses and isolation of labelled compounds from complex reaction mixtures. The method has proved its value, for example, in the preparation of H- or C-tagged fatty acids [628], cholesterol esters [416], glyceryl ethers [425], phospholipids [569] and steroids [328]. 

The use of labeled cholesterol or its precursor, mevalonate, has the appeal that a limited number of products are presumably formed and that the lipid is believed to turn over very slowly within the nervous system. It should be noted, however, that the observed slow cholesterol turnover reflects primarily the major brain pool of this lipid, myelin. Axonal flow studies are however directed at neurons, not at the glial cells that synthesize myelin. MacGregor et al., (1973) noted that following injection of cholesterol into the lumbar region of the chick, aproximodistal gradient of cholesterol was found in the sciatic nerve. The rate was thought to be about that observed for protein. Both cholesterol and cholesterol ester were detected, but the relative proportions were variable. A slow and fast rate of axonal flow were... 

One of the first attempts to improve retention of cholesterol ester was that of Idelman (1964, 1965), who studied the lipid droplets in the adrenal gland and was able to reduce their extraction with the use of partial dehydration, in which the highest ethanol concentration was 70%, and dehydration was completed by a mixture of 70% ethanol and Epon containing the accelerators. That study was corroborated and further extended by Friihling et al. (1969), who determined the extent of extraction by radiochemical determination after injection of cholesterol- to intact rats. During the first hour after injection most of the labeled cholesterol recovered in the adrenal was in the free form, but later on it became esterified therefore, this system provided a good model for the study of the compound in its free and esterified form. It became apparent that it was possible to reduce the loss of labeled cholesterol ester to 11-13%, when dehydration was carried through 70% or even 80% ethanol, but that abso-... 

A far better preservation of cholesterol, whether free or esterified, was obtained in the liver after intravenous injection of labeled chylomicrons, by the use of Aquon (O. Stein et al., 1969). The latter is a highly water-miscible derivative of Epon, and its preparation has been described by Gibbons (1959). This resin was used both for dehydration and embedding and allowed retention of 67-73% of the label in the tissue, irrespective whether the cholesterol ester amounted to 68 or 15%. 

In order to ascertain whether the cholesterol ester taken up by the liver can be removed readily, livers of rats injected with chylomicrons containing labeled cholesterol ester were subjected to perfusion with plasma or Krebs-Ringer bicarbonate for 30 minutes, a procedure which removed no more than 9% of the label (O. Stein et al., 1969). These results indicated that the chylomicron-derived cholesterol ester is not easily removable by perfusion, and its more accurate localization was studied with the help of radioautography. Thirty minutes after injection of chylomicrons labeled with cholesterol (70% in the form of cholesterol ester) and in situ perfusion of the liver with buffer the radioautographic reaction was found predominantly over the sinusoidal cell boundary (which included the plasma membrane, the microvilli, and the intervillar space), but was not associated with any particulate lipid in the form of chylomicrons (Fig. 16). At that time 68% of the label in the liver were in the form of cholesterol ester. Some label was already intracellular in the paren-... 

Labeled precursor Cholesterol ester- H Free cholesterol-H ... 

Labeled product Cholesterol ester Free cholesterol Cholesterol esteF ... 

Contrary findings have been reported in which the percentage as well as the total content of cholesterol in the liver of rats were appreciably less for starch- than for sucrose-fed animals (Adams et at, 1959). No alteration in the amount of cholesterol esters was found in the livers of rats during 12 weeks on high-sucrose, fructose, and dextrose diets (Macdonald and Roberts, 1965) and, though the dietary carbohydrate was uniformly labeled with very little incorporation was found in the sterol esters in the liver. Thus there seems to be uncertainty about the effect of dietary carbohydrate on the amount of cholesterol, free or combined, in the liver and the confused results probably reflect the influence of the other components of die diet. 

Most of the mention of cholesterol in the popular press positions this molecule as a threat to human health. Many foods are proudly labeled cholesterol-free. People are properly warned to pay attention to their plasma cholesterol level, particnlarly that carried in the low-density lipoproteins, LDLs, commonly known, with pretty good reason, as bad cholesterol. LDLs are lipoprotein particles containing a large protein known as B-100 associated with cholesterol, cholesteryl esters, phospholipids, and some triglycerides. 

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