Cholesterol labeling

The second line of evidence suggesting metabolic stability of brain cholesterol was published shortly after the classical experiments of Waelsch et al. (1940). Bloch et al. (1943) fed cholesterol labeled with deuterium to an adult dog for 3 days although at the end of 6 days the marked material could be recovered from the other organs examined, none was found in the brain or spinal cord. Bloch and his colleagues concluded that their observations illustrated the lack or paucity of metabolic interchange between the sterol of the central nervous system and that of the blood. If 4-C -cholesterol is injected into newly bom animals... 

When cholesterol labeled in the 26-position was injected into an intact rat, most of the released by the animal was found as expired i C02 (9). However, when the identical experiment was performed using i C-4 labeled cholesterol, none of the isotope was recovered as i C02. Subsequent experiments (10) established the liver as the site of the side-chain oxidation. In vitro experiments with subcellular fractions (11,12) revealed that during the... 

Rosenfeld and his co-workers (1954, 1956, 1964) have studied the mechanism of reduction of cholesterol to coprostanol using cholesterol labeled at the 3 a position with deuterium or tritium and in the 4 position with carbon-14. Their data indicate that the coprostanol formed exhibits up to 81 % retention of the isotope at the 3a position thus, there is no oxidation at that position. However, the variable incorporation of hydrogen isotope into the B ring leaves the complete course of the conversion in doubt. The possibility exists that the products are the results of the action of more than one organism. 

Nutritional Value of Milk Products. Milk is considered one of the principal sources of nutrition for humans. Some people are intolerant to one or more components of milk so must avoid the product or consume a treated product. One example is intolerance to lactose in milk. Fluid milk is available in which the lactose has been treated to make it more digestible. The consumption of milk fat, either in fluid milk or in products derived from milk, has decreased markedly in the 1990s. Whole milk sales decreased 12% between 1985 and 1988, whereas the sales of low fat milk increased 165%, and skimmed milk sales increased 48% (35). Nutritionists have recommended that fat consumed provide no more than 30 calories, and that consumption of calories be reduced. Generally, a daily diet of 2000—3000 cal/d is needed depending on many variables, such as gender, type of work, age, body responses, exercise, etc. Further, there is concern about cholesterol [57-88-5] and density of fat consumed. Complete information on the nutritive value of milk and milk products is provided on product labels (36) (see also Table 4). 

Synthetic chemical approaches to the preparation of carbon-14 labeled materials iavolve a number of basic building blocks prepared from barium [ CJ-carbonate (2). These are carbon [ C]-dioxide [ CJ-acetjlene [U— C]-ben2ene, where U = uniformly labeled [1- and 2- C]-sodium acetate, [ C]-methyl iodide, [ C]-methanol, sodium [ C]-cyanide, and [ CJ-urea. Many compHcated radiotracers are synthesized from these materials. Some examples are [l- C]-8,ll,14-eicosatrienoic acid [3435-80-1] inoxn. [ CJ-carbon dioxide, [ting-U— C]-phenyhsothiocyanate [77590-93-3] ftom [ " CJ-acetjlene, [7- " C]-norepinephrine [18155-53-8] from [l- " C]-acetic acid, [4- " C]-cholesterol [1976-77-8] from [ " CJ-methyl iodide, [l- " C]-glucose [4005-41-8] from sodium [ " C]-cyanide, and [2- " C]-uracil [626-07-3] [27017-27-2] from [ " C]-urea. All syntheses of the basic radioactive building blocks have been described (4). 

In 1952, Konrad Bloch and Robert Langdon showed conclusively that labeled squalene is synthesized rapidly from labeled acetate and also that cholesterol is derived from squalene. Langdon, a graduate student of Bloch s, performed the critical experiments in Bloch s laboratory at the University of Chicago, while Bloch spent the summer in Bermuda attempting to demonstrate that radioactively labeled squalene would be converted to cholesterol in shark livers. As Bloch himself admitted, All I was able to learn was that sharks of manageable length are very difficult to catch and their oily livers impossible to slice (Bloch, 1987). 

Figure 26-3. Biosynthesis of cholesterol. The numbered positions are those of the steroid nucleus and the open and solid circles indicate the fate of each of the carbons in the acetyl moiety of acetyl-CoA. Asterisks Refer to labeling of squalene in Figure 26-2.

The hypothesis that polar carotenoids regulate membrane fluidity of prokaryotes (performing a function similar to cholesterol in eukaryotes) was postulated by Rohmer et al. (1979). Thus, the effects of polar carotenoids on membrane properties should be similar in many ways to the effects caused by cholesterol. These similarities were demonstrated using different EPR spin-labeling approaches in which the effects of dipolar, terminally dihydroxylated carotenoids such as lutein,... 

FIGURE 10.11 (a) Cross-sectional drawing of the lipid bilayer including lutein, cholesterol, and spin labels. 

Recently, due to increased interest in membrane raft domains, extensive attention has been paid to the cholesterol-dependent liquid-ordered phase in the membrane (Subczynski and Kusumi 2003). The pulse EPR spin-labeling DOT method detected two coexisting phases in the DMPC/cholesterol membranes the liquid-ordered and the liquid-disordered domains above the phase-transition temperature (Subczynski et al. 2007b). However, using the same method for DMPC/lutein (zeaxanthin) membranes, only the liquid-ordered-like phase was detected above the phase-transition temperature (Widomska, Wisniewska, and Subczynski, unpublished data). No significant differences were found in the effects of lutein and zeaxanthin on the lateral organization of lipid bilayer membranes. We can conclude that lutein and zeaxanthin—macular xanthophylls that parallel cholesterol in its function as a regulator of both membrane fluidity and hydrophobicity—cannot parallel the ability of cholesterol to induce liquid-ordered-disordered phase separation. 

Kusumi, A., W. K. Subczynski, M. Pasenkiewicz-Gierula, J. S. Hyde, and H. Merkle. 1986. Spin-label studies on phosphatidylcholine-cholesterol membranes Effects of alkyl chain length and unsaturation in the fluid phase. Biochim. Biophys. Acta 854 307-317. 

Subczynski, W. K., J. S.Hyde, and A. Kusumi. 1991. Effect of alkyl chain unsaturation and cholesterol intercalation on oxygen transport in membranes A pulse ESR spin labeling study. Biochemistry 30 8578-8590. 

Yin, J.-J. and W. K. Subczynski. 1996. Effect of lutein and cholesterol on alkyl chain bending in lipid bilayers A pulse electron paramagnetic resonance spin labeling study. Biophys. J. 71 832-839. 

In the experiments with acetic acid labelled radioisotopically and fed to ani-mals, it has been established that the cholesterol carbon framework is made up entirely of the acetic acid carbon. 

Another example comes from the work of Johnson, et a/.18 These workers studied spin labels dissolved in lipid bilayer dispersions of dipalmitoylphos-phatidylcholine and cholesterol (9 1 by weight) in the hope that anisotropic rotational diffusion of the spin label would mimic the motion of the bilayer components. In addition to 5-DS, which is sensitive to rotational motion about the NO bond, they used the steroidal nitroxide 8, which tends to rotate about an axis perpendicular to the N-O bond. ESR measurements were carried out at both 9 and 35 GHz and at temperatures ranging from 30 to 30 °C. Rather different results were obtained with the two spin labels, largely as a result of the different axes of rotation. Because the rotation rates were very slow, ESR spectra appeared as powder patterns rather than isotropic spectra and special methods were needed to extract the motional data. 

Recently, Voogt et al. [91] have reported on the d5-pathway in steroid metabolism of Asterias rubens. These workers established the existence of the d5-pathway (Scheme 20), analogous to the pathway found in mammals this conclusion was based on the observation that radiolabeled cholesterol (1) was converted to pregnenolone (112), 17a-hydroxypregnenolone (141), and androstenediol (142). Labeled pregnenolone was converted additionally to progesterone (129). Androstenediol (142) was the main metabolite of de-hydroepiandrosterone (143), a reaction catalyzed by 17/i-hydroxysteroid dehydrogenase (17/1-HSD). The metabolic conversion of androstenedione (131) to testosterone (132) is also mediated by 17/J-HSD and is related to... 

In more recent studies the use of HPLC allowed isolation and counting of individual sterols after administration of labelled precursors. The sterols isolated from mantles and viscera of the nudibranch Doris verrucosa were identified as cholestanol, cholesterol, 24-dehydrocholesterol and 7-dehydrocholesterol [103]. After injection of dl-[2-14C]-mevalonic acid DBED salt, cholesterol (57) and 7-dehydrocholesterol (58) were isolated as the acetates by reversed phase HPLC. Both sterols were found significantly labelled specific radioactivity associated with 7-dehydrocholesterol was higher by one order of magnitude than that associated with cholesterol. This fact would indicate either that the reduction of the A1 double bond of 7-dehydrocholesterol to afford cholesterol occurs at a low rate, or that the cholesterol found in D. verrucosa comes partly from a dietary source. 

The ability of bivalve molluscs to synthesize sterols is questioned [106]. Approximately forty sterols have been identified from the oyster Crassostrea virginica and, since it appeared that many of the sterols identified must be of dietary origin, the ability of the oyster to incorporate injected radioactive acetate was studied [110]. Of the forty sterols naturally occurring in the oyster, only four were labelled by injection of labelled acetate cholesterol, desmosterol, 24-methylenecholesterol and fucosterol. However, when an oyster hearth tissue culture was grown aseptically with addition of labelled acetate, the sterols were found to be non-radioactive [111], This finding does not rule out the possibility... 

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