Tritiated Cholesterol Research Articles

Steroid production from plasma cholesterol. II. In vivo conversion of plasma cholesterol to ovarian progesterone and adrenal C19 and C21 steroids in humans.

ABSTRACT Tritiated cholesterol was injected intravenously into 3 ovulating women, 10, 14 and 23 days prior to tubal ligation. The specific activity of urinary pregnanediol (SA in dpm/μmole) was consistently lower than the SA of plasma free cholesterol of the corresponding day. This was observed in the luteal phase of 2 normal cycles and in 1 normal cycle prolonged with hCG from day 22 to day 31. This finding would suggest that the corpus luteum of a normal cycle as well as in early pregnancy produces progesterone from at least two sources, plasma cholesterol and probably newly formed cholesterol within the gland. The steroid production in the adrenal gland from plasma cholesterol was studied in a similar manner, by the determination of the SA of 2 C21 urinary metabolites, tetrahydrocortisone and tetrahydrocortisol and 3 C19 compounds, dehydroepiandrosterone, androsterone and etiocholanolone. The SA of all 5 compounds were similar to the SA of the corresponding plasma free cholesterol in the control period...

The role of dietary triglycerides in cholesterol metabolism

Ratios of free cholesterol to cholesteryl esters in thoracic duct lymph from rabbits fed varying amounts of cholesterol and triglycerides were estimated to study the influence of dietary triglycerides on cholesterol metabolism. Lymph was collected from groups of animals 6 hours after feeding a marker of tritiated cholesterol in food containing by weight 0, 0.8 or 2 % cholesterol and 0, 15 or 30% of various triglycerides. At constant levels of dietary cholesterol, the proportion of free cholesterol increased as dietary triglyceride increased. At constant levels of a triglyceride, the proportion of cholesteryl esters increased as dietary cholesterol and total cholesterol in lymph increased. These findings may have metabolic significance since the surface of chylomicrons carries diffusible free cholesterol while their core contains the cholesteryl esters which are metabolized by the liver and perhaps inhibit synthesis of cholesterol by that organ.

Transport of cholesterol in thoracic duct lymph and serum of rhesus monkeys fed cholesterol with various food fats

Rhesus monkeys were fed cholesterol with several food fats for 3, 10 and 30 days. After cannulation of the thoracic duct, lymph and serum were collected and fractionated into various lipoprotein classes in the preparative ultracentrifuge. It was found that in serum with high cholesterol levels the major increases in cholesterol were carried by low density lipoproteins. The amount of cholesterol in high density lipoproteins on the other hand decreased in the hypercholesterolemic animals. In lymph the chylomicron fraction transported most of the cholesterol. Slight variations in cholesterol content of different lipoprotein fractions in both lymph and serum were noted depending on the type of food fat fed. Recently absorbed cholesterol, as determined by radioactive methods after feeding tritiated cholesterol, was present in thoracic duct lymph more as cholesterol ester after the ingestion of corn oil, but more as free cholesterol after the ingestion of coconut oil. A hypothesis is proposed which suggests that the form in which absorbed cholesterol reaches the blood circulation from thoracic duct lymph may to some extent alter its subsequent metabolism.

Arterial repair and atherosclerosis after mechanical injury: Part 4. Uptake and composition of cholesteryl ester in morphologically defined regions of atherosclerotic lesions

Atherosclerotic lesions with defined morphological properties were induced by mechanical trauma in the aorta of normo-lipidemic rabbits. In all parts of the lesion and regardless of the morphological distribution of the lipid, cholesteryl esters with mono-unsaturated fatty acids dominated, whereas cholesteryl esters with di-unsaturated fatty acids dominated in plasma. The similarity between the cholesteryl ester composition in non-endothelialized regions of the lesion and that in endothelialized regions suggests that the cholesteryl ester composition is primarily determined by local, cellular activity, independently of plasma influx. Twenty-four hours after the injection of tritiated cholesterol, no consistent differences between the specific activities in the three more saturated cholesteryl ester fractions were encountered, suggesting that both differential hydrolysis and differential esterification might contribute to the characteristic cholesteryl ester pattern of the lesions. The specific activity of cholesteryl esters with fatty acyl groups containing three or more double bonds was lower than that of other cholesteryl ester fractions, and the proportion of poly-unsaturated cholesteryl esters increased during restoration of the arterial wall structure, suggesting a slow removal of such esters. These results suggest that hydrolysis of cholesteryl esters may be a prerequisite for the elimination of cholesterol deposits from atherosclerotic lesions.

Vitesse des processus de renouvellement du cholestérol contenu dans son espace de transfert, chez le rat: III. Modifications et étude critique de la méthode d'équilibre isotopique

Since the isotopic equilibrium method as applied to rat cholesterol was described, some practical modifications have been made which are reported here. Sometimes the use of a subcutaneous implantation of capsules containing tritiated cholesterol leads to an over-estimate of the rate of fecal cholesterol excretion. For the questionable cases, it is necessary to use the initial method of daily subcutaneous injections of radioactive cholesterol. In another connection, 17 experiments using the isotopic equilibrium method have shown great variability of the weekly radioactive balance. The radioactivity of cholesterol and its transformation products released from the organism of the rat and that of absorbed cholesterol are rarely equal as would be expected. To define the origins of these losses an experiment using the isotopic equilibrium method was carried out on rats which were killed after 4, 8 or 12 weeks. Bacterial degradation cannot explain the deficit in the weekly radioactive balances, for the total radioactive balance which includes the radioactive cholesterol of the rat is always nil. In fact, these deficits can be explained on the basis that isotopic equilibrium is not really obtained or that the organism is not in a steady state but in an expansion state. In both cases the application of suitable relations permits the calculation of all the rates involved in the dynamics of cholesterol with certainty. But for an organism in an expansion state it is necessary to know the rate of cholesterol increase. After these corrections have been made, the percentage of error on each rate is defined. In the particular case of this critical experiment, animals were fed with a basal diet to which, 0.2% taurocholate was added. Although this results in an expansion state, the rates of synthesis, excretion and absorption are constant. The increase of mobile cholesterol in the organism is associated with a decrease of its transformation to bile acids.

Determination of tritium in microgram tissue samples: distribution of exogenous 3H-cholesterol in the rat adrenal cortex.

An internal sample metal cathode proportional counter for measurements of low tritium activity in biologic samples of microgram size has been designed. Using a guard detector operating in anticoincidence together with a thick steel castle, a background of 0.5 cpm and an efficiency of 54.6% for tritium were realized. Radioactivities down to 2 pC (4.4 dpm) can be measured with an accuracy of ±16%. Tritium activity was measured in single adrenal cortical sections of 7 µm in thickness and 0.4 mm in diameter having a mean dry weight of 1.2 µg. The cortical layers were identified histologically from alternate sections. Two days after the injection of tritiated cholesterol the highest radioactivity was encountered in the outer fascicular zone (39 dpm/µg). The distribution of the radioactivity within the cortex was equal in all animals, although the mean level varied individually. The glomerulosa and reticularis contained usually about one-third of the activity in the outer fasciculata. Ether anesthesia and unilateral adrenalectomy caused significant decrease in the incorporation of the cholesterol, which was not further reduced by an injection of adrenocorticotropin. Ninety-five per cent of the radioactivity in the adrenals was bound to cholesterol or its esters 2 days after the injection.

Penetration of exogenous testosterone, pregnenolone, progesterone and cholesterol into the seminiferous tubules of the rat.

Tritiated cholesterol, pregnenolone, progesterone and testosterone were injected intravenously into adult male rats. They were killed from 30 min to 10 days after the injection of cholesterol, and from 2 to 30 min after the other steroids. The interstitial tissue of the testis was separated by free hand dissection from the seminiferous tubules. The tissue samples were dried, weighed, combusted in closed glass ampules and the tritium gas was subsequently counted in a metal cathode internal gas counter. Pregnenolone, progesterone and testosterone behaved equally, producing maximal radioactivity in both tissue compartments between 2 and 5 min after the injection, but the maximal radioactivity appeared at 24 hr after the cholesterol injection. The radioactivity in the seminiferous tubules was always significantly lower than in the interstitial tissue, the difference being most marked in the case of cholesterol. Very low or negligible counts were obtained in the seminiferous tubules throughout the cholesterol ...

Free cholesterol exchange in vitro: a comparison of endogenous and exogenous cholesterol.

SummaryThe exchange of free cholesterol of endogenous or exogenous origin between plasma and red blood cells has been studied. Using tritiated cholesterol and 14C-mevalonic acid or sodium-1-14C-acetate, it has been shown that after incubation of labeled plasma with control RBC or of labeled RBC with control plasma, the free cholesterol 3H/14C ratio in plasma or RBC is unchanged. This is true for incubation of 6 or 24 hr. In a parallel series of experiments it was observed that the plasma 3H/14C free cholesterol ratio was unchanged after 24 hr of incubation in the presence of native lecithin cholesterol acyl transferase. The data confirm the findings in man and baboon which suggest that it is not possible to distinguish between plasma free cholesterol of endogenous or exogenous origin.

Adrenal cholesterol: localization by electron-microscope autoradiography.

As determined by electron-microscope autoradiography of adrenal glands containing tritiated cholesterol and by modified differential centrifugation techniques, 70 to 80 percent of adrenal cholesterol is contained within lipid droplets of rat adrenal cortical cells.

Autoradiographic study of the incorporation of tritiated cholesterol into the zona reticularis of the rat adrenal cortex.

In the zona reticularis of the rat adrenal cortex three types of cells are to be observed: light, dark, and very dark cells. A time study of the incorporation of cholesterol-3H into these elements has been made. The data obtained from autoradiographic study are discussed in relation to the hypothesis that the light cells are elements in active steroido-synthesis, the dark cells are resting elements, and the very dark cells are elements in degeneration.

Stimulation of aldosterone biosynthesis in vitro by serotonin.

ABSTRACT The dialysable fraction of rat serum, which was previously shown to stimulate aldosterone production in rat adrenal sections, was chromatographed on Sephadex G-25. Fluorescence spectra indicated that a 5-hydroxyindole derivative was present in the active eluate fraction. The aldosterone-stimulating activity of serum dialysate had a similar retention volume to serotonin and 5-hydroxytryptophan, but was separated from other indole derivatives. Addition of small amounts of pure serotonin to the incubation medium led to a significant and dose-dependent increase of aldosterone and deoxycorticosterone production but did not affect corticosterone production. At higher concentrations, 5-methoxytryptamine, bufotenine and 5-hydroxytryptophan also stimulated aldosterone biosynthesis, whereas a number of other substances chemically or pharmacologically related to serotonin were found to be inactive. Two different serotonin antagonists completely blocked the aldosterone-stimulating effects of serotonin and rat serum but did not influence aldosterone stimulation by ACTH. Serotonin stimulated the incorporation of tritiated cholesterol into aldosterone but not the incorporation of tritiated pregnenolone, indicating that it acts on the conversion of cholesterol to pregnenolone.

Variations in biosynthesis of progesterone and response to interstitial cell-stimulating hormone in vitro in bovine corpora lutea.

Eighteen corpora lutea were obtained from 10 lactating dairy cows of three breeds at three stages of the estrual cycle. Synthesis of progesterone in vitro in the presence of cholesterol-7 a -H3 and in the presence or absence of ICSH was studied in these glands. The response to ICSH in vitro was influenced by the age of the corpus luteum. Two criteria of ICSH activity, namely: (1) an increase in progesterone synthesis during incubation with ICSH, and (2) increased conversion of cholesterol-7 a -H3 to progesterone-H3 produced by ICSH, were greater at days 11 to 13 than with younger or older glands. Corpora lutea obtained at days 19 and 20 did not respond to ICSH. Breed of cow appeared to influence corpus luteum weight and concentration of progesterone following incubation and, perhaps, also the conversion of exogenous tritiated cholesterol to progesterone.

ALDOSTERONE STIMULATION IN VITRO

ABSTRACT Tritiated precursors of corticosteroids were incubated with rat adrenal sections. At the end of the incubation, tritium-labelled aldosterone and corticosterone were determined by double isotope dilution procedures. When corticosterone-[3H], deoxycorticosterone-[3H], progesterone-[3H] or pregnenolone-[3H] were used as substrates, 2–5% of the total radioactivity in the incubation medium could be recovered in the aldosterone fraction, while 20–25% of the latter three substrates were incorporated into corticosterone. Potassium ions, ammonium ions, angiotensin II and ACTH markedly stimulated the biosynthesis of unlabelled aldosterone from endogenous precursors. However, they did not influence the amounts of radioactive substrate converted to aldosterone or corticosterone. NADP and glucose 6-phosphate stimulated both the total production of aldosterone and corticosterone and the conversion of deoxycorticosterone-[3H] and of pregnenolone-[3H] to corticosterone. Only minute amounts of tritiated cholesterol were converted to corticosteroids. Angiotensin II and ammonium ions significantly stimulated the conversion of cholesterol-[3H] to aldosterone, while ACTH, potassium ions and particularly NADP and glucose 6-phosphate enhanced its incorporation into both aldosterone and corticosterone. These findings indicate that, like ACTH and NADPH2, monovalent cations and angiotensin act mainly on the conversion of cholesterol to pregnenolone and do not directly stimulate hydroxylation or dehydrogenation in position 18.