Relative effects of feeding saturated fats and cholesterol on fluidity of rabbit lipoproteins.

1.The effects of saturated fat and cholesterol on lipoprotein fluidity were tested in New Zealand white rabbits fed diets containing corn oil (CO) or cocoa butter (CB) with and without added 0.2% cholesterol. 2. Saturated fats had little effect on fluidity in any lipoprotein fraction. 3. Cholesterol feeding dramatically reduced fluidity in VLDL and LDL, but minimal change was noted in HDL. 4. Cholesterol-fed rabbits were hypercholesteroloemic throughout the 10-month study. 5. The rabbits became adapted to cholesterol feeding as VLDL became more fluid with time.     

Influence of experimental diets on cholesterol and triglyceride levels of rabbit blood serum lipoproteins

Groups of rabbits were fed for six weeks various diets: standard died + ethanol, high-cholesterol diet and a high-cholesterol + ethanol one. During the next six weeks every diet was supplemented with a fresh vegetable (carrot). Cholesterol and triglycerides were determined in the whole serum and in lipoprotein fractions. In rabbits fed standard diet ethanol caused a moderate elevation of VLDL cholesterol and triglyceride and LDL cholesterol levels. In animals on high-cholesterol diet cholesterol and triglyceride concentrations in these fractions were very high. Simultaneous consumption of large amounts of cholesterol and of ethanol resulted in a greater rise of cholesterol concentration in the whole serum and in VLDL and LDL fraction than did high-cholesterol diet alone. Addition of carrot caused a pronounced reduction of serum cholesterol concentration in animals fed all kinds of diets. The reduction concerned mainly VLDL.     

Deuterated delta 7-cholestenol analogues as mechanistic probes for wild-type and mutated delta 7-sterol-C5(6)-desaturase

Deuterium-labeled 5alpha-cholest-7-en-3beta-ol (1) bearing one or two deuteriums at the C-5alpha and (or) C-6alpha positions was synthesized in high isotopic and chiral purity. These compounds were used as substrates with the microsomal wild-type Zea mays and recombinant Arabidopsis thaliana Delta(7)-sterol-C5(6)-desaturases (5-DES) to probe directly the stereochemistry and the mechanism of the enzymatic reaction. Clearly, in the conversion of 1 by both 5-DESs, the 6alpha-hydrogen is removed. [6alpha-(2)H]-5alpha-Cholest-7-en-3beta-ol shows an intermolecular deuterium kinetic isotope effect (DKIE) on V and V/K, (D6)V = 2.6+/-0.3, (D6)V/K = 2.4+/-0.1; and (D6)V = 2.3 +/-0.3, (D6)V/K = 2.3+/-0.2 for the Zea mays and A. thaliana wild-type 5-DES, respectively. In contrast, negligible or minor isotope effects, (D5)V = 0.99+/-0.04, (D5)V/K = 0.91+/-0.08; and (D5)V = 0.93 +/-0.06, (D5)V/K = 0.96+/-0.04, respectively, were observed with [5alpha-(2)H]-cholest-7-en-3beta-ol. The observed pattern of isotope effects strongly suggests that the plant 5-DES initiates oxidation by cleavage of the chemically activated C6alpha-H bond, a step which appears to be partially rate-limiting in the desaturation process. Cleavage of the C5-H bond has a negligible isotope effect, indicating that the desaturation involves asynchronous scission of the two C-H bonds at C5 and C6. We showed previously [Taton, M., et al. (2000) Biochemistry 39, 701] that threonine 114 was not essential to maintaining desaturase activity, although V/K values for mutant T114I and T114S were respectively 10-fold lower and 4-fold higher than that of the native 5-DES. In this study, we combined variation in enzyme structure and DKIE studies and showed that (D6)V and (D6)V/K increased respectively to 3.8+/-0.3 and 3.8+/-0.4 in mutant T114I and decreased respectively to 1.6+/-0.4 and 1.7+/- 0.1 in mutant T114S. The data suggest that the conserved hydroxyl function at position 114 in the ERG3 family makes the abstraction of the 6alpha-hydrogen atom substantially less rate-limiting during the 5-DES reaction. Based on the data, a tentative mechanism for the desaturation of cholest-7-en-3beta-ol is proposed.     

Effects of cholesterol feeding on primate serum lipoproteins. III. The change in high density lipoprotein components

Sooty mangabey (Cercocebus atys) monkeys had a lower serum HDL cholesterol concentration than any other Old World monkey species reported. In addition, they had a higher serum Lp(a) concentration than other species. The mangabeys were fed a cholesterol-fat diet for 5 weeks. HDL2 and HDL3 amounts were determined from the two peaks apparent upon analytical ultracentrifugation. In the first 1-3 weeks, 13 of the 14 mangabeys increased 30% (mean) in total HDL, this increase occurring only in the HDL2 fraction. After 5 weeks, HDL and HDL2 decreased markedly. During the cholesterol feeding, HDL3 continually decreased in flotation rate, indicating it was either smaller and/or denser. HDL2 and HDL3 separated well on molecular sieving agarose columns during the diet period, whereas a single symmetrical elution peak was found for chow-fed HDL. Thus on a cholesterol-fat diet, HDL2 and HDL3 increased in difference in molecular size.     

The effect of serum lipoproteins on cholesterol content and sterol exchange in cultured human endothelial cells.

Cultured human endothelial cells preincubated with the infranatant of human serum increased their content of cholesterol when subsequently exposed to low density lipoproteins (LDL) as compared to control cultures further incubated in the presence of infranatant only. Replacing LDL with high density lipoproteins (HDL) resulted in no change in the cellular cholesterol content compared to the control. The addition of HDL did not influence the increase in cellular cholesterol content mediated by LDL. HDL stimulated the efflux of endogenously synthesized 14C-labelled sterols compared to the infranatant fraction, whereas LDL had only a slight effect. Cells preincubated with whole serum did not change their cholesterol content when subsequently exposed to LDL, compared to cultures further incubated in presence of whole serum. Replacing whole serum (during the final incubation) with infranatant, resulted in a decrease of the cellular cholesterol content, which was not influenced by further addition of HDL.     

Microdetermination of cholesterol in serum lipoproteins.

A two-step procedure for the microdetermination of cholesterol in serum lipoproteins is compared with cholesterol quantitation after density gradient ultracentrifugation. Serum lipoproteins from 10 mul of serum are separated by electrophoresis on agarose and visualized by precipitation with dextran sulfate--CaCl2. The lipoprotein bands are cut off from the plates, the agarose slices are hydrolyzed by gas-liquid chromatography. The comparison between the two procedures reveals satisfactory correlations for beta-and pre-beta-lipoproteins and total serum. There is excellent recovery of cholesterol in fractionated lipoproteins.     

The metabolism of esterified cholesterol in rabbit plasma low density lipoproteins

The metabolism of esterified cholesterol in plasma low density lipoproteins (LDL) has been studied in rabbits. LDL labelled with ³H in the esterified and free cholesterol moieties was isolated from the serum of donar rabbits which has been injected with [³H]mevalonic acid, and subsequently either incubated at 37°C in vitro with unlabelled rabbit serum or unlabelled rabbit lipoprotein fractions, or reinjected into other rabbits.In vitro there was found to be a transfer of 40–60% of the esterified [³H]-cholesterol out of LDL into both the very low density lipoprotein (VLDL) and high density lipoprotein (HDL) fractions which could not be explained in terms of net transfer of esterified cholesterol mass. In the incubations of labelled LDL with either of the other unlabelled lipoprotein fractions, transfers were apparent only if the dialysed 1.21 g/ml infranatant of rabbit serum was also present. The transfer of esterified [³H]cholesterol out of LDL was enhanced when lecithin:cholesterol acyltransferase was active.After reinjecting labelled LDL into other rabbits, it was found that more than half of the esterified [³H]cholesterol removed from the recipient LDL fraction during the first 30 min was not lost from the plasma compartment, but rather was recovered in HDL. There was only minimal in vivo transfer of LDL esterified [³H]cholesterol into VLDL.It has been concluded that in vitro the esterified cholesterol in LDL exchanges with that in both the VLDL and HDL, and that in vivo the esterified cholesterol pools in LDL and HDL may represent parts of a progressively equilibrating plasma pool.     

Stimulation of cholesterol esterification in hepatic microsomes by lipoproteins from normal and hypercholesterolemic rabbit serum.

Incubation of plasma lipoproteins with rabbit hepatic microsomes enriched the microsomes with free cholesterol and stimulated cholesterol esterification. The rate of cholesterol esterification correlated well (r = 0.96) with the concentration of microsomal free cholesterol. Lipoproteins from normal and hypercholesterolemic serum varied in their propensity to stimulate cholesterol esterification. Among the normal lipoproteins, low density lipoproteins was more stimulatory than either high density lipoproteins or intermediate density lipoproteins. However, the intermediate density lipoproteins fraction from hypercholesterolemic serum was consistently more stimulatory than any of the normal lipoproteins. The augmentation of cholesterol content, when microsomes were exposed to mixed hyperlipidemic lipoproteins, was proportionately much greater than augementation of phospholipid or protein concentration.