Cholesterol derivatives, labelled

Ftgttre 13.20 Bloch s etpertment Incorpomtion the carton atoms acetate Into cholestervL (A) Carbon atoms of cholesterol derived Jrcm carboxyl carbon of acetate. (B) Carbon atoms of cholesterol derived from methyl carbon of acetate. The shaded boxes denote isotopic label both C and C were used as labels. Bloch and Langdon went on to show that the route to cholesterol is through the intermedtate squalene. 

The efificiency of various hydroxylated cholesterol derivatives to form pregnenolone is summarized in Table III. At the substrate concentrations studied, first-order reaction kinetics were realized the fractions formed, therefore, represent fractional rates. As may be seen, the (22R)-22-hydroxycholesterol served as a much better precursor of pregnenolone than the 20a-hydroxycholesterol. (It is noteworthy that in Digitalis labeled 22 -hydroxycholesterol or 22-oxocho-lesterol do not appear to be converted to tigogenin or gitogenin... 

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). 

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). 

Isotopic labeling experiments show that cholesterol is derived from ethanoate by way of squalene and lanosterol. The evidence for this is that homogenized liver tissue is able to convert labeled squalene to labeled lanosterol and thence to labeled cholesterol. The conversion of squalene to lanosterol is particularly interesting because, although squalene is divisible into isoprene units, lanosterol is not—a methyl being required at C8 and not C13 ... 

The carbon skeleton of isoprenoids is derived from the branched C5 skeleton of isoprene. Isopentenyl diphosphate and dimethylallyl diphosphate represent the biologic equivalents of isoprene. From research on cholesterol biosynthesis in liver tissues and on ergosterol in yeast, mevalonate was accepted as the universal precursor of isoprenoids. However this assertion is inaccurate. Incorporation of labeled acetate and glucose isotopomers into bacterial isoprenoids and into diterpenes of ginkgo embryos indicated fortuitously the existence of an alternative mevalonate-independent route. Its full elucidation required experiments using and H-labeled precursors followed by extensive nuclear magnetic resonance analyses as well as a combination of biochemical and molecular biology methods. These additional studies revealed a complete set of novel unsuspected enzymes. 

Other lipid compositions with synthetic lipids, hydrogenated SPC (HSPC) and PEG-modified phospholipids are often used, especially for liposome formulations intended for parenteral applications use (long circulating or stealth liposomes) (41). Several analytical methods to follow loss of lipids during the preparation steps are available. Radioactively labeled lipids ( H-DPPC, C-DPPC) or cholesterol ( H-cholesterol) or H-cholesteryl hexadecyl ether (NEN Life Science Products, Boston, MA, USA) or lipophilic fluorescence dyes (e.g. lipophilic BODIPy derivatives. Molecular Probes) are added at appropriate amounts to the initial lipid mixtures. 

Reduction of the A " -Double Bond.— The reduction of the A -double bond is again a trans-process. Caspi et have shown that in cholesterol the (24R)-hydrogen was labelled by [2- C,3R,4R- H]mevalonic acid, and that on hydroxy-lation to (25R)-cholest-5-ene-3/S,26-diol the hydroxymethyl group was labelled by [2- C]mevalonic acid [see (74)]. The latter result is the opposite of that found for tigogenin (88) in higher plants. NADPH provides the hydrogen at C-25 while the hydrogen at C-24 is derived from the medium. 

Very early in the investigation of sterol biosynthesis it was estabUshed that acetate was the primary precursor. In 1942 Bloch and Rittenberg found that deutero-acetate could be converted to cholesterol in the intact animal in high yields [7]. This was in accord with the earlier observation of Sonderhoff and Thomas that the nonsaponifi-able lipids from yeast (primarily sterol) were heavily labeled by the same substrate [8]. Degradation of the sterol molecule in the laboratories of both Bloch and Popjak showed that all of the carbon atoms of cholesterol were derived from acetate and that the labeling pattern of methyl and carboxyl carbons originating from acetate indicated that the molecule was isoprenoid in nature [9]. It was apparent then that sterols have as their fundamental building block, acetate, a molecule that resides at the center of intermediary metabolism. 

Spin labels are stable, paramagnetic molecules that, by their structme, easily attach themselves to various biological macromolecular systems such as proteins or cell membranes. Examples of spin labels that can be covalently bonded to specific sites of biological systems include nitroxide derivatives of A-ethylmaleimide, which bind specifically to -SH groups, and nitroxide derivatives of iodoacetamide, which bind specifically to methionine, lysine, and arginine residues of amino acids. Nonco-valently bonded spin-labels that can be incorporated into biological systems include nitroxide derivatives of stearic acid, of phospholipids, and of cholesterol. 

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