Cholesterol radioactive

P. Samuel, W. Perl, C. M. Holtzman, N. D. Rochman and S. Lieberman, Long-term kinetics of serum and xanthoma cholesterol radioactivity in patients with hypercholesterolemia. J Clin Invest 51 266 (1972). 

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). 

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... 

C-labelled cholesterol was used to test the recovery of 5-100 pg of faecal sterols from seawater (labelled coprostanol not being available). The radioactivity of the samples and eluates was measured by a two-channel liquid scintillation counter. Percentage recovery was calculated on the basis of the amount of labeled material recovered in the acetone eluant. The results indicate that column extraction efficiency is not adversely affected by the salinity of the water samples, i.e., in the range 95-97%. 

Myelin components exhibit great heterogeneity of metabolic turnover. One of the novel characteristics of myelin demonstrated in early biochemical studies was that its overall rate of metabolic turnover is substantially slower than that of other neural membranes [1]. A standard type of experiment was to evaluate lipid or protein turnover by injecting rat brains with a radioactive metabolic precursor and then follow loss of radioactivity from individual components as a function of time. Structural lipid components of myelin, notably cholesterol, cerebro-side and sulfatide, as well as proteins of compact myelin, are relatively stable, with half-lives of the order of many months. One complication in interpreting these studies is that the metabolic turnover of individual myelin components is multiphasic - consisting of an initial rapid loss of radioactivity followed by a much longer slower loss. 

Wolfe has presented an excellent description of the systematic application of stable and radioactive isotope tracers in determining the kinetics of intestinal fat absorption, hepatic triglyceride synthesis, lipid mobilization, triglyceride-fatty acid recycling, and cholesterol turnover. 

Human skin fibroblasts are cultured from skin biopsy samples. The dermis is cut into small pieces (0.5 mm on each side) and placed into a dish in DMEM containing 10% (v/v) FCS and 1% (v/v) antibiotic-antimycotic solution. When these primary cultures are confluent they are split and cells between passage three and six are used for experiments. For the cholesterol efflux assay, cells are grown in 24-well plates to 60-80% confluence and are labeled with [1,2-3H]-cholesterol (1 pCi/well) for 24 h. Cells are then washed with DMEM and incubated for 4 h at 37°C with DMEM containing BSA (0.2%, v/v) and either 0 (negative control) or 5-30 pg/ml apoA-I. The efflux medium is collected and centrifuged to remove cell debris. Cells are solubilized in 0.1 mol/1 NaOH and the radioactivity in the efflux media and in the cell lysates is determined by scintillation counting [11, 30, 75]. 

Fractional cholesterol efflux is calculated as radioactivity in the medium/(radioactivity in the medium + cellular radioactivity). 

LCAT acts preferentially on lipids transported by HDL (so-called a-LCAT activity), but also on lipids transported by apoB-containing lipoproteins (so-called jS-LCAT activity) [58, 85]. In practice, LCAT activity is measured either as the activity required to esterify radioactive cholesterol that has been exogenously incorporated into native HDL or into artificial HDL-like particles (a-LCAT activity) or which has been equilibrated with endogenous lipoproteins of the plasma sample (cholesterol esterification rate, CER) [21, 58, 85]. Several variations of these assays have been reported, some of which are available as commercial test kits (e.g., Roar Biomedical, New York, USA). In addition, LCAT concentration can be determined by either laboratory-made tests or by a commercial ELISA kits [57]. However, the decrease in LCAT concentration is difficult to judge since it also decreases secondary to HDL deficiency due to causes other than LCAT deficiency. Plasma from patients with LCAT deficiency fails to esterify radioactive cholesterol provided by any substrate. By contrast, plasmas of patients with fish-eye disease show a near-normal cholesterol ester-fication rate but have a selective inability to esterify radioactive cholesterol provided to plasma with native HDL or reconstituted HDL (a-LCAT activity) [58, 85]. 

A study carried out in the U.S. showed a 56% decrease in serum total lipid radioactivity in rats fed the equivalent of 5 g of fresh garlic bulbs/d for 7 d. Rats fed an experimental diet containing cholesterol were treated either with intraperitoneal injections of C-14 labeled acetate or a diet containing C-14 sucrose. 

Various modifications are reported with respect to the experimental setup (single pass or recirculated intestinal perfusion) as well as the site of blood collection, e.g. mesenteric vessels for estimation of the intestinal absorption rate (DeGraw RT, Anderson BD 2004). vs. peripheral veins for estimation of systemic availability of the candidate compound. This method is widely used for investigation of intestinal absorption of nutrients by using radioactive tracers (e.g. cholesterol, glucose) and their interference with the candidate compound (Arts et al. 2004). In addition the secretion of the candidate compound into the intestine can be studied by peripheral administration of the compound into a peripheral vein and subsequent determination of the appearance of the candidate compound in the intestinal perfusate (Merino et al. 2003 Berggren et al. 2004). Also variations are reported using chronically isolated intestinal loops in rats (Poelma et al. 1992). 

Mevalonic acid, radioactively labeled at the a-carbon atom, was fed to an organism that synthesizes cholesterol. Which atoms in the cholesterol will be labeled ... 

Radioactive carbon dioxide was detected in the breath of rats and men almost immediately after the administration, either orally or by injection, of (RS)-[5-14C]MVA,36 and up to 6.5% of the administered dose was exhaled within 100 minutes. Since the carbon dioxide was not derived from the unnatural S-enantiometer of MVA, or from degradation of cholesterol biosynthesized from the additive MVA, the observations support the hypothesis that there exists a metabolic shunt of intermediates of sterol biosynthesis which, although derived from MVA, do not lead to sterol formation. The significance of this shunt is that its occurrence could explain some of the human hypercholesterolaemias. The authors claim that MVA has no metabolic fate (hitherto known) except the biosynthesis of terpenoids is quite... 

In contrast to dendrobatid alkaloids, which are not present in captive-raised dendrobatid frogs, lire salamanders produce samandarine alkaloids when reared in captivity (G. Habermehl, personal communication, 1989). No apparent differences in alkaloid content occurred for at least three generations. Incubation of secretions from salamander parotoid glands with radiolabeled cholesterol in buffer for 3 days at room temperature led to some apparent incorporation of radioactivity into samandarine alkaloids (53). 

Metabolism of the Steroid Nucleus.—Two routes may be considered for the reduction of the A -double bond, either directly or via a A -3-one. Use of [3a- H,4- C]cholesterol gave coprostanol retaining about half of the tritium. However, with a mixture of [3a- H]sitosterol and [4- C]cholesterol the coprostanol again contains some tritium which must have been transferred from the j -sitosterol. Partial retention of tritium from [4/ - H,4- C]cholesterol was also shown to be misleading since most of the tritium was at C-6. Reduction in the presence of D2O gave deuterium mainly at C-5 and C-3. The two-step reduction of a A" -3-one system was examinedfor nicotinamide specificity. More radioactivity was incorporated from [4R- H]NADPH than from the 4S-isomer into C-3 and C-5. 

The measurements were performed on a cholesterol monolayer spread at an air/water interface and at a temperature of 22 + 0.5 °C. The mass concentration lor the radioactive tracer molecule in the monolayer is given by Fick s second law lor surface diffusion [1.6.5.46)... 

In the integral method, the amount of radioactive cholesterol, M(t), that has dil-... 

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