Cholesterol labeling pattern

The tritium labelling pattern of lanosterol has not been studied in great detail. However, it may be deduced to a large extent from the results with cholesterol, which are in agreement with the incorporation of and... 

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. 

FIGURE 21.25 The labeling pattern of cholesterol. Each letter m indicates a methyl carbon and each letter c indicates a carbonyl carbon, all of which come from acetyl-CoA. 

The search for precursors and pathways was brought into sharper focus through the observation by Little and Bloch (1950) that the labeling pattern of the cholesterol side chain suggested the recurrence of a five carbon atom unit (Fig. 2). 

Whereas squalene has 30 carbons, cholesterol has only 27 carbons, and other steroids have even fewer carbons. Therefore degradation must accompany (or follow) cyclization. Robinson was the first to propose a manner in which squalene might cyclize to provide the tetracyclic ring system of the steroids. This was followed by an alternate hypothesis by Bloch and Woodward. The two proposals suggest different labelling patterns in tetracyclic products. For example, if one thinks about cholesterol as the product, the two proposals suggest that the black methyl groups would have to be lost. These are different for the two proposals. Experiments eventually disproved the Robinson hypothesis and supported the Bloch-Woodward hypothesis. For an eariy experiment see Woodward,... 

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. 

Cholesterol, like long-chain fatty acids, is made from acetyl-CoA, but the assembly plan is quite different. In early experiments, animals were fed acetate labeled with 14C in either the methyl carbon or the carboxyl carbon. The pattern of labeling in the cholesterol isolated from the two groups of animals (Fig. 21-32) provided the blueprint for working out the enzymatic steps in cholesterol biosynthesis. 

More than 100 years later, Comforth, Hunter, and Popjak made use of this reaction in determining the pattern of isotope distribution in cholesterol produced by biosynthesis from labeled acetic acid. One degradation liberated ring A in the form of 2-methylcyclohexanone (1), which was converted by the Schmidt reaction into the lactam (2), which in turn was converted by hydrolysis and methylation into the... 

In one step in the degradation of biosynthetic cholesterol from labeled acetate to establish the pattern of distribution in ring A, Comforth, Hunter, and Popjak succeeded in converting A -cholestene into the ozonide in 80% yield by passing ozone into a solution of the hydrocarbon in dry n-hexane until a dilute solution of... 

The general pathway of isoprenoid biosynthesis was elucidated largely through studies of the biosynthesis of sterols in yeast and higher animals. It was shown that specifically labeled [ CJacetate was incorporated in specific patterns into cholesterol. A variety of branched C5 compounds, notably j8,jS-dimethylacrylate, were also tested as possible isoprenoid precursors. Incorporation of label was observed, but the results were not definitive. The key... 

The rate of equilibration between liver and serum-free cholesterol is rapid, being attained within a few hours in dogs (Eckles et al., 1955). In humans, the peak specific activity of the serum-free cholesterol is attained within 2—4 hours after administration of labeled acetate. Serum ester cholesterol and serum-free cholesterol equilibrate after 4—7 days (Hellman et al., 1954 Gould et al., 1955). The same pattern of equilibration is observed after administration of labeled cholesterol (Biggs et al., 1952). 

Patterns of cholesterol biosynthesis and transport in the baboon parallel those observed in man (Kritchevsky et al., 1965). Peak specific activity of serum-free cholesterol synthesized from mevalonic acid-2-is reached within 4—10 hours and the free and esterified forms equilibrate by 72 hours. Peak specific activities of exogenously labeled serum-free and ester cholesterol are observed at three days. The specific activity of the total cholesterol of the serum a and lipoproteins is equal over a 10-day period, but there is the possibility that the specific activities of the free and ester cholesterol moieties of the serum oc and jS lipoproteins differ. The lipoprotein cholesterol findings are similar to results reported from similar human studies (Gidez and Eder, 1963). The equilibration of serum and tissue cholesterol is reached at about two weeks in the dog (Gould, 1952) and rat (Chevallier, 1953) and one month in man (Chobanian and Hollander, 1962). 

When trihydroxycoprostanic acid labeled in the 4 position (prepared in the same manner as the 26-labeled acid) is incubated with rat liver mitochondria, labeled cholic acid is obtained. Danielsson had been carrying out parallel experiments in mouse liver mitochondria, and his work yielded valuable information concerning the pattern of hydroxylation of cholesterol. Cholesterol can be converted to 26 hydroxycholesterol and also to the 3/5, 7a, 26-triol. Danielsson also showed that liver mitochondrial preparations could convert 3 a, 7 a, 12 a trihydroxy coprostane to 3a, 7 a, 12 a, 26-tetrahydroxycoprostane, 3 a, 7 a, 12a-tri-... 

The conversion of cholesterol-4-to corticosterone and cortisol has been achieved in perfused cow adrenal (Hechter et al., 1953) and a cell-free homogenate of cow adrenal cortex (Saba and Hechter, 1955). In man, administration of labeled cholesterol yields the characteristic urinary metabolites of cortisol (Wer-BiN and LeRoy, 1954, 1955). The conversion by human ovarian tissue of labeled acetate to progesterone (Sweat et al., 1960) and other hormones (Ryan and Smith, 1961) has also been reported. If cholesterol is indeed the precursor of the adrenocortical steroids, a predictable pattern of labeling should appear in hormones synthesized from labeled acetate. Caspi et al. (1956, 1957, 1962) have shown this to be the case. 

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