Discrimination between cholesterol and sitosterol for absorption in rats

The intestinal absorption of cholesterol and sitosterol was compared in rats. The intragastric administration of a single emulsified lipid meal containing either 50 mg of [4-14C]cholesterol or [4-14C]sitosterol resulted in the lymphatic absorption of 18.2% and 0.42% of each sterol, respectively, in 6 hr. This difference was unaltered when the mucosal sterol load was equalized by reducing the cholesterol to 1 mg in the emulsified lipid meal while maintaining the same sitosterol load or when the physical state in the lumen was equalized by infusion of a micellar solution containing both sterols into bile-diverted intestine. Lymphatic cholesterol was 90% esterified compared to 12% for sitosterol. Both sterols were associated predominantly (greater than 70%) with the chylomicron fraction. Eighty percent of the chylomicron cholesterol was recovered as ester with the core lipids, while 77% of the sitosterol was recovered as free sterol with the chylomicron coat. In mucosal homogenates at 6 hr, sitosterol recovery was one-eleventh that of cholesterol. When [3H]cholesterol (10 mg) and [14C]sitosterol (10 mg) were co-administered in an emulsified intragastric lipid meal, sitosterol associated with the brush border isolated 2 hr later was one-fifth that of cholesterol. Similar differences were seen when brush border membranes were incubated in vitro with micellar solutions containing either 50 microM [3H]cholesterol or [14C]sitosterol and the relative uptake of each sterol was unaffected by micellar phospholipid type (egg yolk phospholipids, phosphatidylcholine, or phosphatidylethanolamine).(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the sterols and sterol esters of the intracellular organelles of maize shoots

1. The composition of the esterified and unesterified sterols of the nuclear, chloroplastidic, mitochondrial and microsomal fractions of 21-day-old maize shoots was examined. 2. The microsomal and mitochondrial fractions contain the bulk of the sterols of the tissue. 3. Only 1% of the sterol isolated from all the organelles is esterified. 4. The nuclear fraction has the greatest proportion of esterified sterol and the microsomal fraction the least. 5. 4-Demethyl sterols constitute the bulk of both esterified and unesterified sterols in all organelle fractions. 6. Cholesterol is the major esterified 4-demethyl sterol of the nuclear and chloroplastidic fractions, but only the nuclear fraction has an appreciable proportion of unesterified cholesterol. 7. Sterol esters of linolenic acid are more abundant in the mitochondrial and microsomal fractions than in the other two fractions.     

Regulation of cholesterol esterification by micellar cholesterol in CaCo-2 cells

The regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) activity by cholesterol was studied in an established enterocyte cell line. CaCo-2 cells were grown in culture to confluency and dome formation. They were characterized morphologically by light and transmission electron microscopy. During the culture period, ACAT activity remained stable while the activities of the brush border enzymes sucrase and alkaline phosphatase progressively increased with time and plateaued 12 days after plating. As determined by the rate of incorporation of oleic acid into the individual lipid classes, the rate of triglyceride synthesis was twice that of phospholipid and 15 times that of cholesteryl ester synthesis in these cells. Incubating CaCo-2 cells with cholesterol solubilized in taurocholate micelles resulted in a significant increase in ACAT activity (149 +/- 5 pmol/dish per 2 hr vs. 366 +/- 5, (P less than 0.001) without changing the rates of triglyceride or phospholipid synthesis. The stimulation of ACAT activity by micellar cholesterol was rapid, occurring within 5 min and reaching a maximal effect by 2 hr. The regulation of ACAT activity by cholesterol was directly dependent upon the concentration of cholesterol solubilized in the micelle and was independent of protein synthesis. Incubating CaCo-2 cells with micellar cholesterol did not increase the esterification of, nor did the cholesterol enter the pool of, newly synthesized or performed cholesterol within 2 hr. The micellar cholesterol that was taken up by the cells was esterified within 5 min after starting the incubation. Progesterone, a known ACAT inhibitor, significantly decreased the rate of esterification of intracellular micellar cholesterol proving that the cholesterol taken up by CaCo-2 cells was indeed entering the ACAT pool. Despite increasing amounts of unesterified cholesterol entering the cells via micelles, the percent of cholesterol that was esterified at any one time remained constant at 1%. The results suggest that ACAT activity in CaCo-2 cells is stimulated by cholesterol delivered to the cells by way of taurocholate micelles. The rapid entry of this sterol into the ACAT substrate pool suggests that ACAT activity in CaCo-2 cells is regulated by the expansion of the cholesterol substrate pool that is being utilized by an unsaturated ACAT enzyme.     

The characterization and distribution of hexahydropolyprenyl esters in cultures of Aspergillus fumigatus Fresenius

The total mycelial lipid of Aspergillus fumigatus was analysed and over half of its hexahydropolyprenol was shown to be esterified with fatty acids. Comparison of the fatty acid content of the prenyl esters with the sterol ester and the total lipid indicated a marked predominance of saturated fatty acids in the polyprenyl esters. The predominant acids esterified to the prenols were palmitic acid, linoleic acid, oleic acid, lignoceric acid, stearic acid and palmitoleic acid. Most of the unesterified polyprenol was found in the mitochondrial fraction, but the esterified prenol was equally distributed throughout the cell fractions. This distribution was unlike that found for ergosteryl ester in the same tissue.     

Sterol distribution in intracellular organelles isolated from tobacco leaves

All membrane-containing fractions isolated from tobacco leaves contained free sterols, sterol glycosides, and sterol esters. The three sterol forms increased, on a dry weight basis, with a decrease in particle size. The supernatant fraction contained only trace amounts of sterol. The major sterols in all cellular fractions, in the order of decreasing amounts, were: stigmasterol, β-sitosterol, campesterol, and cholesterol. The 500g pellet contained the largest percentage of free sterol, while the 46,000g pellet contained the largest percentage of esterified sterol. The individual sterol composition of the free sterol and sterol glycoside fraction was very similar; however, the composition of the sterol ester fraction varied widely among intracellular fraction. The intracellular distribution pattern of cholesterol-¹⁴C added to the isolation medium provided evidence that the intracellular sterol distribution pattern is not an artifact. These results support the suggestion that sterols in plant cells may have a physiological function associated with membranes.     

Isolation and characterization of a small intestinal surfactant-like particle containing alkaline phosphatase and other digestive enzymes

Digestive brush-border enzymes in particulate form have been reported in the intestinal lumen in vivo and in medium from organ explants in vitro. It has been suggested that these particles derive from membrane shedding of the apical brush border. This study describes the isolation and characterization of particles derived from the 105,000 x g supernatant fraction of intestinal luminal washings and from light scrapings of the mucosa itself after fat feeding of rats. These fractions were separated in a continuous NaBr gradient, producing a visible band of 1.07-1.08 g/liter density and resulting in a 15-fold enrichment of intestinal alkaline phosphatase in the band fraction. Other brush-border hydrolases were represented in the banded fraction, but at specific activities only 1/5th to 1/36th that of the brush border. The major phospholipid in the fraction was phosphatidylcholine (58 +/- 15%), containing 75% saturated fatty acids. In contrast, the major brush-border phospholipid was phosphatidyl-ethanolamine. These characteristics showed that the particles derived from the lumen and mucosal surface were not identical to fragments of the brush border. Electron microscopy of the banded fraction revealed partially coiled membrane fragments. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots, some proteins (e.g. surfactant protein B, collagenous protein 4) were found in common between the intestinal particles and rat pulmonary surfactant. These data suggest the production of a particle secreted by rat intestine that differs from brush-border membranes and that shares some morphological and biochemical similarities with pulmonary surfactant.     

Distribution and dynamic state of sterols and steroids in the tissues of an insect, the roach Eurycotis floridana

The total concentrations of sterols in the tissues of the roach, Eurycotis floridana, reared under aseptic conditions and on semisynthetic diets, are similar to, but somewhat lower than, those of tissues of vertebrates. Total concentrations of tissue sterols are relatively independent of dietary concentration of sterols whether the diet contains 0.1% cholesterol as the sole sterol, or a "minimal cholesterol" mixture (0.1% cholestanol together with 0.005% cholesterol). Under the latter conditions the cholesterol is incorporated preferentially into most tissues and remains almost exclusively unesterified, while the cholesterol-sparing sterol is esterified to varying degree, depending upon the tissue. The turnover of tissue sterols has been studied. Cholesterol of the tissues of adult insects grown on a diet containing this sterol alone may be displaced by cholestanol fed as 5% of the total diet, initially at an appreciable rate but later much less rapidly. In growing insects that have received a diet containing cholestanol together with minimal cholesterol, the unesterified cholesterol turns over slowly in all tissues and immeasurably slowly in some. The unesterified sparing sterol, on the other hand, turns over at a much greater rate. The turnover of sterols during growth is accompanied by a shift of sterols from the unesterified to the esterified pool in all tissues. The fat body of the growing insect stores sterols (apparently as their esters) that have been displaced from other tissues. The fat body of the adult does not show evidence of sterol storage. Polar derivatives of sterols are present in minor amount in all tissues of the insect, most abundantly in the mid-intestine and gastric caeca. These compounds seem likely to be C(27) steroids.     

24-methylenepollinastanol and 24-dehydropollinastanol—two sterols of pollen

The esterified and unesterified sterol fractions of bee-gathered mixed pollens were examined, and total sterol composition was determined. Two new sterols of pollens, 14α-methyl-9β,19-cyclo-5α-cholest-24-en-3β-ol (24-dehydropollinastanol) and 14α-methyl-5α-ergost-24(28)-en-3β-ol (24-methylenepollinastanol) were isolated and identified. Both sterols were found primarily in the esterified sterol fraction, and 24-methylenepollinastanol accounted for 43% of the sterols of this fraction. 24-Dehydropollinastanol and four other sterols which also contain a 9β,19-cyclopropane ring were found only in the esterified sterol fraction. 24-Methylenecholesterol was the major sterol of the unesterified sterol fraction.     

Bile acid synthesis and intracellular and extracellular cholesterol concentrations in isolated rat hepatocytes: the effect of dietary cholesterol

Bile acid synthesis in isolated hepatocytes prepared from rats given 1% cholesterol in the diet and incubated for 1 h in suspension was not increased compared to that in cells from control rats. When the hepatocytes were maintained in monolayer culture for 24 h, however, increased production of bile acid (X2.5) was observed in the cholesterol-fed group. The amount of bile acid synthesised during incubation in suspension was significantly correlated with intracellular unesterified cholesterol levels, but showed no correlation with intracellular esterified or medium cholesterol concentrations after 1 h. Bile acid production in hepatocytes maintained in monolayer culture was also significantly correlated with the intracellular unesterified, but not esterified, cholesterol content. In addition, in this case, there was a significant correlation with the levels of both unesterified and esterified cholesterol found in the medium after 24 h. These results suggest that the amount of cholesterol available to liver cells from extracellular sources has a role in the regulation of bile acid synthesis in cholesterol-fed rats, while the concentrations of esterified cholesterol stored within the cells are not important in this process.     

Subcellular localization of enterokinase in human small intestine

Enterokinase (enteropeptidase, EC 3.4.4.8) was found to be purified to the same extent as sucrase and alkaline phophatase when human intestinal brush border membrane was isolated. It is concluded that, in man as in other mammals, enterokinase activity occurs in close association with the brush border membrane.However, a second localization was also found. A fraction of the mucosal homogenate containing only small amounts of brush border but large amounts of endoplasmic reticulum, basolateral membranes and mitochondria (Fraction P₁) contained a disproportionately high amount of enterokinase. The enzyme in this particulate fraction occurred in a not fully active form.     

Method for detection and isolation of cholesteryl ester-containing "foam" cells using flow cytometry

Accumulation of cholesteryl ester within vascular cells is a defining characteristic of atherosclerotic lesions. Therefore, it is of interest to be able to monitor this critical event in the development of atherosclerosis. With this objective in mind, we have developed a method for the detection of cholesteryl ester-containing cells (i.e., foam cells) in cell suspensions prepared from enzymatically dissociated aortas. Cholesteryl ester in aortic cells was selectively stained with the fluorescent dye filipin. Because filipin binds to unesterified cholesterol but not to esterified cholesterol, it was necessary first to remove unesterified cholesterol from cells by ethanol extraction so that its presence would not interfere with the specific detection of cholesteryl ester. Then unesterified cholesterol made available by enzymatic hydrolysis of cellular cholesteryl ester could be specifically stained with filipin. The filipin-stained cell suspensions were analyzed using flow cytometry. With a flow cytometer it was possible to detect and sort cholesteryl ester-containing cells onto glass slides for microscopic analysis. Cell suspensions prepared from either grossly normal or atherosclerotic swine aortas contained cells with cholesteryl ester inclusions. As expected, these cells were more numerous in the atherosclerotic aortas. Cells with higher levels of fluorescence contained more numerous cholesteryl ester inclusions. Flow cytometric detection of cholesteryl ester-containing cells should be generally useful in studies of cellular cholesterol metabolism as well as in specific studies of cellular cholesterol accumulation in atherosclerotic vessels.     

The origin of cholesterol in the mesenteric lymph of the rat

These studies were performed to quantitate the amounts of newly synthesized cholesterol secreted in the mesenteric lymph of the rat and to define the origin of this cholesterol. In control animals receiving no dietary fat, the amount of newly synthesized sterol entering the lymph increased linearly with respect to time over 24 hr. When a continuous intravenous infusion of chylomicrons was given or when the animals were prefed a diet containing 2.0% cholesterol to inhibit hepatic, but not intestinal or peripheral, cholesterol synthesis, the secretion of newly synthesized sterol in lymph was markedly suppressed, suggesting that the liver was its ultimate site of origin. When the animals were subjected to either blockade of intestinal cholesterol absorption or biliary diversion, there was a decrease in both the newly synthesized and total mass of cholesterol in lymph by approximately 60%, indicating that the majority was normally derived from the absorption of luminal (primarily biliary) sterol. In the absence of dietary cholesterol, the remainder was probably derived from plasma lipoproteins that were filtered through the intestinal capillaries into the lymph. In contrast, when lymph was collected during active fat absorption, the intestine was found to secrete sterol newly synthesized by the epithelium. Such newly synthesized cholesterol was found predominantly in the unesterified fraction and accounted for approximately 27% of the total sterol found in lymph at the end of the experiment. From these studies it was concluded that in the absence of fat absorption, sterol synthesized in the intestinal mucosa was incorporated predominantly into cell membranes and did not enter intestinal lymph to any significant degree. However, during fat absorption, a fraction of this newly synthesized sterol pool was incorporated into lipoproteins and so was delivered through the intestinal lymph to the body pools of cholesterol.     

Cholesterol synthesis and esterification in cultured intestinal mucosa. Evidence for compartmentation

The current studies were undertaken to define the optimal conditions for measuring the absolute rates of cholesterol synthesis in cultured rabbit intestine and to assess whether the rate of sterol synthesis affects the esterification of locally formed or absorbed cholesterol. Using both [3H]water or [14C]octanoate (3 mM) as a precursor, sterol formation was linear during the 24 h culture, resulting in comparable estimates of the rate of synthesis equivalent to 129.5 and 118.7 nmol acetyl CoA incorporated per g per h, respectively. The presence of liposomal cholesterol or the hydroxymethylglutaryl-CoA reductase inhibitor mevinolin suppressed the rates of cholesterol synthesis by 24 and 92% of controls, respectively. Only 12% of total newly synthesized cholesterol was recovered in the medium and more than 97% was in the unesterified form, in both medium and biopsy. Even when the rate of sterol synthesis was stimulated over 90-fold by increasing concentrations of [14C]mevalonolactone, less than 8% of the label in total cholesterol was found in the sterol nucleus of the esterified cholesterol. Rather, the majority of the cholesterol ester-bound radioactivity was incorporated into the fatty acid moiety. On the other hand, there was only a limited decrease in the esterification of absorbed [3H]cholesterol both when the rate of sterol synthesis was increased with 10 mM mevalonolactone and when it was inhibited with mevinolin. The data suggest that locally synthesized and absorbed cholesterol is organized in distinct functional pools with different degrees of esterification in the mucosal epithelial cell.     

Expression of scavenger receptor BI facilitates sterol movement between the plasma membrane and the endoplasmic reticulum in macrophages

Scavenger receptor BI influences multiple aspects of cellular sterol metabolism. In this series of studies, we evaluated the effect of scavenger receptor BI expression on the distribution and movement of sterol between the plasma membrane and the endoplasmic reticulum in macrophages, by comparing control J774 cells to J774 cells in which SR-BI expression was constitutively increased 3-fold. J774 cells with increased expression of SR-BI (J774-SRBI cells) esterified plasma membrane cholesterol more rapidly as compared to control cells. The esterification of endogenously synthesized cholesterol was also more rapid in cells with increased SR-BI expression; this could be partially suppressed by removing cholesterol from the plasma membrane. The increased plasma membrane sterol esterification in J774-SRBI cells was not due to increased acyl-coA:cholesterol acyltransferase activity and was observed even though J774-SRBI cells manifested a smaller free cholesterol pool in the endoplasmic reticulum. Cholesterol ester hydrolysis was also more rapid in J774-SRBI cells. Increased expression of SR-BI also facilitated the clearance of cellular cholesterol ester to HDL(3). This latter observation, combined with the measurement of the smaller ER free cholesterol pool in J774-SRBI cells, suggests that the free cholesterol derived from the hydrolysis of cholesterol ester was rapidly transported back to the plasma membrane. It is concluded that expression of SR-BI in macrophages increases the rate of free cholesterol transport, and modulates free cholesterol distribution between the plasma membrane and the internal membrane compartments in macrophages.     

Dietary cholesterol induces trafficking of intestinal Niemann-Pick Type C1 Like 1 from the brush border to endosomes

The transmembrane protein Niemann-Pick C1 Like 1 (NPC1L1) belongs to the Niemann-Pick C1 (NPC1) family of cholesterol transporters and is mainly expressed in the liver and the small intestine. NPC1L1 is believed to be the main transporter responsible for the absorption of dietary cholesterol. Like NPC1, NPC1L1 contains a sterol sensing domain, suggesting that it might be sensitive to dietary cholesterol. To test this hypothesis, mucosal explants were cultured in the presence or absence of cholesterol. In the absence of cholesterol NPC1L1 was localized mainly in the brush border of the enterocyte, colocalizing with the brush border enzyme aminopeptidase N (APN), and only a minor part was present in intracellular compartments. In contrast, following culture in the presence of cholesterol a major part of NPC1L1 was found in intracellular compartments positive for the early endosomal marker early endosome antigen 1, whereas only a minor fraction was left in the brush border. Neither APN, lactase, nor sucrase-isomaltase was endocytosed in parallel, demonstrating that this is a selective cholesterol-induced endocytosis of NPC1L1. Conceivably either the induced internalization could be due to NPC1L1 acting as an endocytic cholesterol receptor or it could be a mechanism to reduce the cholesterol uptake. The fluorescent cholesterol analog NBD-cholesterol readily labeled the cytoplasm also under conditions nonpermissible for endocytosis, arguing against a receptor-mediated uptake. We therefore propose that cholesterol is absorbed by NPC1L1 acting as a membrane transporter and that NPC1L1 is internalized to an endosomal compartment to reduce the absorption of cholesterol.     

Fate of intravenously administered particulate and lipoprotein cholesterol in the rat

Unesterified radioactive cholesterol, both bound to serum lipoproteins and dispersed in ethanol-saline, was injected into bile fistula and intact rats. Due to phagocytosis, mainly by the liver macrophages, intravenously injected cholesterol in ethanol-saline disappears from the bloodstream significantly faster than lipoprotein-bound cholesterol. Soon after the initial phagocytosis, the particulate isotopic cholesterol started to reappear in blood, reaching a maximal radioactivity in blood 10-24 hr after injection. Although the radioactive cholesterol reappears in serum in both esterified and unesterified form, it is likely that cholesterol is released from the phagocytic cells as unesterified cholesterol which is then esterified intravascularly or at other sites. In the bile fistula rats, somewhat more of the lipoprotein cholesterol than of the particulate cholesterol appeared in bile early after injection. However, cholesterol turnover calculated from a twopool model was the same for rats injected with lipoproteinbound or particulate cholesterol.     

Cholesterol ester concentrations and the ester hydrolases in cultured glioblastoma cells: Effect of lipoprotein depleted serum

We have investigated the effects of substituting lipoprotein depleted serum (LPDS) for normal fetal calf serum (FCS) in culture media on cholesterol ester concentrations and the activity of the ester hydrolases in cultured glioblastoma (C-6 glial) cells. Glial cells grown in media supplemented with 10% FCS contained 16–23% of total cholesterol as esterified sterol. Total sterol content of the cells cultured in media supplemented with LPDS was reduced by 55–75% as compared to cells cultured in FCS media and none of this sterol was in esterified form. Cholesterol ester hydrolase activity was maximal at pH values of 4.5 and 6.5 and required Triton X-100 for optimal activity. Cholesterol ester hydrolase activity at pH 4.5 was significantly higher in cells grown in FCS media than in cells cultured in LPDS media, but the activity at pH 6.5 was not significantly different. The protein: DNA ratio of cells cultured in FCS was higher than in cells cultured in LPDS. These findings indicate that the increase in cholesterol ester concentrations in cells is accompanied by increased activity of lysosomal cholesterol ester hydrolase; and suggest that, in cells cultured in FCS, the availability of free cholesterol for incorporation into cellular membranes is regulated by cholesterol ester hydrolase. The findings also indicate that changes in growth and differentiation of cells cultured in LPDS may be related to reduced availability of exogenous cholesterol.     

Variations of hepatic cholesterol precursors during altered flows of endogenous and exogenous squalene in the rat

Hepatic and serum levels of cholesterol precursors were analyzed in rats under basal (control) conditions and when cholesterol synthesis was activated by feeding 1% squalene or 5% cholestyramine. Exogenous squalene stimulated the activity of acyl-coenzyme A:cholesterol acyltransferase (ACAT) but strongly inhibited the activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase; cholestyramine did not affect ACAT but increased HMG-CoA reductase several-fold, indicating enhanced production of endogenous squalene. Activation of cholesterol synthesis by the two methods markedly increased the hepatic and serum contents of cholesterol precursor sterols. However, the sterol profiles were clearly different. Thus, exogenous squalene raised most significantly (up to 109-fold) free and esterified methyl sterols, and less so (up to 2-fold) demethylated C27 sterols (desmosterol and cholestenols) and also esterified cholesterol. Activation of endogenous squalene production by cholestyramine was associated with a depletion of esterified cholesterol and by a marked, up to 8-fold, increase of the free demethylated sterol precursor levels, whereas the increase of methyl sterols, up to 5-fold, was less conspicuous than during the squalene feeding. The changes were mostly insignificant for esterified sterols. The altered serum sterol profiles were quite similar to those in liver. Serum cholestenols and especially their portion of total serum precursor sterols were closely correlated with the hepatic activity of HMG-CoA reductase.     

Essential fatty acid deficiency and adrenal cortical function in vitro.

Adrenocortical cells were prepared from rats maintained on essential fatty acid-deficient diets and control litter mates. Cells from control rats had high concentrations of essential fatty acids in the cholesteryl ester fraction of which approximately 22% was arachidonate. In contrast, cells from EFA-deficient rats had only 2.5% arachidonate in the cholesteryl esters, even though the total esterified cholesterol level was comparable to that of controls. In place of the essential fatty acids, the cholesteryl esters of these cells were rich in 20:3(n--9) and 22:3(n--9). When cells from EFA-deficient rats were incubated with ACTH or dibutyryl cyclic AMP, the output of corticosterone was the same as in controls. Also sterol esters were hydrolyzed to the same extent as in controls despite the unusual composition of the fatty acid esters. The phospholipids in both control and EFA-deficient cells contained high levels of arachidonate but were not hydrolyzed in either type of cell during incubation with ACTH or dibutyryl cyclic AMP. The results indicate that high levels of the prostaglandin precursors, namely linoleate and arachidonate, are not a sine qua non for the steroidogenic action of ACTH or cyclic AMP.     

Cholesterol exchange and synthesis in the live rat

The turnover of plasma cholesterol and de novo cholesterol synthesis were measured simultaneously in the live rat, immediately after administration of [3H]water together with a large volume exchange transfusion of whole blood prelabeled with [14C]cholesterol. It was possible to separate the exchange of unesterified cholesterol from the uptake and secretion of lipoprotein cholesteryl ester, and also to assess the impact of plasma cholesterol exchange on the measurement of in vivo rates of cholesterolgenesis by individual tissues. Cholesterol was measured by an HPLC procedure that effectively separated cholesterol from other structurally similar sterols, and synthesis was determined by the incorporation of [3H]water into cholesterol. Plasma unesterified cholesterol turnover was multiphasic and exceedingly rapid (initial T1/2, 4.1 min) in contrast to the near linear and much slower turnover of plasma cholesteryl ester (initial T1/2, 59.4 min). Plasma unesterified cholesterol equilibrated with different tissues at different rates, with the liver and adrenal equilibrating most rapidly. Full equilibration of plasma unesterified cholesterol was not achieved with any tissue during the course of this study. For rapidly exchanging tissues like the liver, which was responsible for about 60% of plasma unesterified cholesterol exchange, unesterified cholesterol appeared to be kinetically compartmentalized into rapidly, and much less rapidly, exchangeable pools. After [3H]water administration, the content of newly synthesized cholesterol was greatest in the liver, adrenal, and intestine, and appreciably lower in all other tissues studied. Hepatectomy and intestinal resection resulted in a profound reduction of newly synthesized cholesterol in the plasma and adrenal, but no certain change in the already low amounts at other sites. Thus, while it is clear that appreciable amounts of newly synthesized cholesterol in the adrenal were derived from the plasma by exchange, it was not possible to make this assessment for other selected individual tissues. When, however, newly synthesized cholesterol was determined in the total mass of all extrahepatic and extraintestinal tissues together, exchange could be calculated to account for close to 50% of the new cholesterol recovered in the carcass (in studies of 60 min duration). After correcting for exchange, the liver accounted for 82% of all newly synthesized cholesterol, the intestine for about 10%, and the remaining tissues of the body for just 9%. These results are in marked contrast to recent findings of others and demonstrate that in the live rat cholesterol synthesis is principally confined to the liver.