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SummaryThe cholesterol ester, triglyceride and phospholipid fatty acid composition (gas-liquid chromatography) of aortic media, thickened intima and plaque material were determined in 6 human subjects. For comparison, the fatty acid composition of those fractions in serum was determined in 6 subjects (aged 55-70 years) with occlusive atherosclerosis in a good nutritional status. The triglyceride fraction of those tissues and serum were similar in their fatty acid composition. Some slight differences were noted in the fatty acid composition of the phospholipid fractions, most notably in the arachidonic and long chain fatty acids. The cholesterol ester fraction of the tissues studied showed the greatest differences in fatty acid composition. Both early and advanced plaques had significantly less linoleic and more oleic acid in that fraction than serum or media. Media and serum CEFA were similar in their fatty acid composition. The significance of these findings in relation to atherogenesis is discussed.

SummaryThe fatty acids esterified with cholesterol have been determined by gasliquid chromatography in the media, free-lying lipid containing material of far-advanced plaques, and livers obtained at autopsy from 6 atherosclerotic subjects. For comparison, serum CEFA in 6 well-nourished subjects with occlusive atherosclerosis was also determined. Of tissues studied, composition of CEFA of the aortic media was very similar to that of serum. Plaque material contained a greater proportion of saturated and monoenoic fatty acids than either media or serum. Liver CEFA contained considerably less polyunsaturated fatty acids and more saturated and monoenoic acids than did serum or media. The major CEFA of media and serum was linoleic acid and major fatty acid of plaque material and liver was oleic acid. The significance of these findings in relation to the role of unsaturated fatty acids in atherogenesis is discussed.

SummarySoybean sterols inhibit the increase in blood cholesterol of rats receiving a diet containing 2% cholesterol and 1% sodium taurocholate. The degree of inhibition increased as the soybean sterol concentration of the diet was increased. It was also observed that pancreatic cholesterol esterase catalyzes the esterification of soybean sterols albeit at a slower rate than with cholesterol. Bile salts and a fatty acid source are required for their esterification. The mechanism of action of soybean sterols in inhibiting cholesterol absorption is discussed.

SummaryEntrance of cholesterol-4-C14 into the free cholesterol pool of mucosa and its subsequent transfer to the lymph requires the presence of a specific type of bile salt. This process does not require the presence of dietary fatty acid. It is suggested that free cholesterol in the lumen of the intestine complexes with bile salt which then enters the free cholesterol pool of mucosa.

The in vivo conversion of several 5 beta-cholestane intermediates to primary bile acids was investigated in three patients with total biliary diversion. The following compounds were administered intravenously: 5 beta-[G-3H]-cholestane-3 alpha, 7 alpha-diol, 5 beta-[G-3H]cholestane-3 alpha, 7alpha, 26-triol, and 5 beta-[24-14C]cholestane-3 alpha, 7 alpha-25-triol. Bile was then collected quantitatively at frequent intervals for the next 21 to 28 h. The administered 5 beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol was found to be efficiently converted to cholic and chenodeoxycholic acids in two patients; 61 and 75% of the administered label was found in primary bile acids. The proportion of labeled cholic to chenodeoxycholic acid was 1.20 and 1.02 in the bile of these patients, indicating that the C-26 triol was efficiently converted to cholic acid. The ratio of cholic to chenodeoxycholic acid (mass) in the bile of these patients was 1.23 and 2.32. The 5 beta-cholestane-3alpha, 7alpha-diol intermediate was also efficiently converted (71%) to both primary bile acids. The cholic to chenodeoxycholic acid ratios by mass and label were similar (2.97 versus 2.23). By contrast, the 5beta-cholestane-3alpha, 7alpha, 25-triol was poorly converted to bile acids in three patients. Following the administration of this compound almost all of the administered radioactivity found in the bile acid fraction was in cholic acid (5 to 19%) and very little (less than 5%) was found in chenodeoxycholic acid. These findings indicate that ring hydroxylation at position 12 is not materially hindered by the presence of a hydroxyl group on the side chain at C-26 in patients with biliary diversion. The labeled C-26-triol which was efficiently converted to both primary bile acids in a proportion similar to that which was observed for the bile acids synthesized by the liver suggests that this 5beta-cholestane derivative may be a major intermediate in the synthesis of both cholic and chenodeoxycholic acids.

The present report has presented the first clear evidence in man for the existence of specific hepatic cholesterol precursor sites associated with the formation and secretion of bile acids and biliary cholesterol. These hepatic compartments derive virtually all their cholesterol from newly synthesized and lipoprotein free cholesterol. The model which is presented was formulated on current concepts of cholesterol metabolism in man and is concerned, at this initial stage, with the elucidation of the bile acid and biliary cholesterol compartments. The complexity of cholesterol metabolism in man necessitated an initial approach that would minimize the number of inputs of cholesterol into the system, allow for the sampling of several cholesterol compartments, and permit the simultaneous labeling of newly synthesized cholesterol and preformed cholesterol. To achieve these objectives, we studied the patient with a total bile fistula. Six patients were administered simultaneously pulse injections of labeled mevalonic acid and [(14)C]cholesterol. The qualitative features of the specific activity time course curves after labeled mevalonic acid revealed no precursor-product relationship between bile acid, biliary cholesterol, and plasma free cholesterol. The peak specific activity of the bile acids was reached in approximately 100 min and was higher than the biliary cholesterol, which was higher than the plasma free cholesterol. The plasma free cholesterol specific activity became higher than the other lipids after 12 h and remained higher throughout the period of study. Similar related observations were made with [(14)C]cholesterol. The data were then subjected to simulation analysis and modeling using the SAAM-27 computer program. Computer least-square fits of the data were obtained after the model was evolved. During the model development, the least number of compartments and transport pathways were introduced consistent with a good fit of the data. Of particular importance was the constraint that the model fit the data obtained from both [(14)C]cholesterol and labeled mevalonic acid. The same parameter values were used to fit the data from both tracers. The fluxes arrived at in the model indicate that 31% and 20%, respectively, of the cholesterol input into the bile acid and biliary cholesterol precursor sites were derived directly from the newly synthesized hepatic cholesterol. The remainder had its origin predominantly from lipoprotein free cholesterol. Plasma esterified cholesterol (as free) made a small contribution (11%) to the bile acid compartment. Similarly, 10% of the biliary cholesterol arose from an unknown hepatic site. The present report has provided the basis for a new procedure for studying in vivo cholesterol metabolism in man. Examination of the derived cholesterol flux rates between the compartments suggests the presence of an important mechanism regulating the partitioning of lipoprotein free cholesterol between the bile acid and biliary cholesterol precursor sites. Aberrations in the proportioning of precursor cholesterol between these sites could be a causative factor precipitating the excessive secretion of biliary cholesterol and the production of lithogenic bile.

The purpose of this study is to delineate the immediate sources and fractional turnover of high density lipoprotein (HDL) esterified cholesterol in man. Various labeled preparations were administered in 11 experiments to six subjects who had either a complete bile fistula (maximally stimulated cholesterol metabolism) or an intact enterohepatic circulation. The administered tracers included [(3)H]mevalonic acid; [(14)C]cholesterol bound to albumin; low density lipoprotein (LDL) free [(3)H] or [(14)C]cholesterol; HDL free [(3)H] or [(14)C]cholesterol; HDL esterified [(3)H]cholesterol; and LDL esterified [(3)H]cholesterol. Blood samples were obtained at frequent intervals for up to 5 d after the administration of tracers. The mass and radioactivity in individual plasma lipoprotein (very low density lipoprotein [VLDL], HDL, and LDL) free and esterified cholesterol were determined. The data were subjected to multicompartmental analysis using the SAAM-27 computer program. The analysis revealed that plasma free cholesterol was not the only immediate source of either a single- or two-compartment HDL ester system. When LDL esters and plasma (HDL) free cholesterol were tested together as sources of one HDL ester compartment, data from all the experiments were readily fit. The fluxes arrived at with the final model indicated that only approximately 20% of the esterified cholesterol in HDL was newly synthesized from plasma (HDL) free cholesterol (2.36 mumol/min); the remaining 80% was from LDL ester (8.92 mumol/min). The presence of a bile fistula had no obvious effect on HDL esterified cholesterol metabolism. The rate of HDL cholesterol ester turnover was 3-12 times/d, indicating that the ester component of the HDL particle is in a very dynamic state.

Fifteen patients with liver disease were found to have marked reductions in both plasma lecithin-cholesterol acyltransferase activity (62%) and cholesterol ester concentration (46%) when compared with 11 normal subjects. The changes in cholesterol ester concentration were confined to high-density lipoprotein and very-low-density lipoprotein; the low-density lipoprotein fraction was not significantly altered. Examination of the plasma lecithin and lipoprotein cholesterol esters revealed that patients with liver disease had significantly greater amounts of saturated and monounsaturated fatty acids and less linoleic acid than normal subjects. Lipoprotein cholesterol ester kinetic studies were carried out in 2 normal subjects and 2 severely cirrhotic patients who simultaneously received [14C]mevalonic acid and high-density lipoprotein labeled with free [3H]cholesterol. A lipoprotein cholesterol ester model was developed that fit both the 3H and 14C data. The precursors of high-density lipoprotein cholesterol esters were free cholesterol and low-density lipoprotein cholesterol esters; the distribution pattern was in favor of low-density lipoprotein cholesterol esters in both groups of subjects (71% for normals and 88% for cirrhotics). The turnover of high-density lipoprotein cholesterol esters was similar in both groups of subjects, but the flux of free cholesterol into high-density lipoprotein cholesterol esters was greatly reduced in the cirrhotic patients. In cirrhotic patients, this represented only 9% and 12% of the total lipoprotein cholesterol ester synthesized; in the normal subjects, the values were 68% and 74%. The turnover of very-low-density lipoprotein cholesterol esters was greatly suppressed in the cirrhotic patients, and their precursor sources were high-density lipoprotein cholesterol esters and tissue free cholesterol. The low-density lipoprotein cholesterol ester precursors were very-low-density lipoprotein, low-density lipoprotein, and tissue free cholesterol. In the cirrhotic patients, much greater proportions (78% and 88%) of low-density lipoprotein cholesterol ester were derived from free cholesterol. Also, the turnover rates of these esters were reduced in the cirrhotic patients. The present report has provided evidence that cirrhotic patients derive most of their plasma lipoprotein cholesterol esters from an acyl-CoA-cholesterol acyltransferase free-cholesterol tissue source--most likely the intestine. By contrast, in normal subjects, the major contribution to plasma esters is via the introduction of newly formed cholesterol esters into the high-density lipoprotein fraction.

A bile fistula patient was administered intravenously a constant infusion of [3H]mevalonic acid for 4 hrs, and then after 2 weeks he was given a pulse of [3H]mevalonic acid. Bile and blood were collected at frequent intervals. The specific activity-time course curves did not show a precursor-product relationship between biliary cholesterol, plasma free cholesterol and bile acids. Both the constant infusion and pulse labeling data indicated that the bile acid precursor had a more rapid rate of turnover than plasma or biliary cholesterol; the biliary cholesterol precursor turned over more rapidly than plasma cholesterol. The data suggest the presence of multiple hepatic cholesterol precursor compartments. Bile acids may be derived predominantly from newly synthesized cholesterol in man.

Metabolic pathways involved in the conversion of cholesterol to cholic and chenodeoxycholic acids have been investigated in bile fistula patients treated with a number of labeled potential bile acid intermediates. The findings of the present report indicate that the human liver cell has the capacity to synthesize both primary bile acids via multiple routes from cholesterol and 7 alpha-hydroxycholesterol. Evidence has been obtained for the existence of a major pathway to chenodeoxycholic acid via the 26-hydroxylation of 7 alpha-hydroxycholest-4-en-3-one. Cholic acid is synthesized preferentially via pathways from 5 beta-cholestane 3 alpha, 7 alpha-diol and a pathway from cholesterol not involving an initial 7 alpha-hydroxylation.

Several recent studies have demonstrated that patients with cirrhosis frequently lack deoxycholic acid in bile and plasma. In order to explain this observation, comparative experiments on the colonic absorption of deoxycholic acid and on the colonic conversion of cholic to deoxycholic acid were carried out in the cirrhotic patients with normal and very low percentages of deoxycholic acid. Deoxycholic or cholic acid (100 mg) plus 5 muc of each [14C] bile acid were administered by enema to 8 patients with and 5 without liver disease. Deoxycholic acid produced a significant increase in the percentage of biliary deoxycholic acid in patients with cirrhosis. However, the rate of appearance of 14C-deoxycholic acid in patients with cirrhosis was slower than in normal control subjects. Distribution of the 14C activity among the bile acids indicated that rehydroxylation of deoxycholic to cholic acid did not occur. The distribution of 14C activity in biliary bile acids after the rectal administration of [14C]cholic acid showed that patients with severe cirrhosis converted [14C]cholic to [14C]deoxycholic acid at a much slower rate than did cirrhotic patients with normal percentages of biliary deoxycholic acid. Feeding of cholic acid (450 mg per day) for 3 days to 4 cirrhotic patients resulted in a 2-fold increase in the percentage of biliary cholic acid, but only a small increase in the percentage of deoxycholic acid. In a separate group of 9 cirrhotic patients, fecal bile acid analysis indicated that cirrhotic patients had a significantly lower percentage of deoxycholic acid than 12 patients without liver disease; there was no significant difference in fecal lithocholic acid. The data suggest that alteration of bacterial flora and/or altered conditions for bacterial 7alpha-dehydroxylase enzyme activity in the colon could account for the virtual absence of biliary deoxycholic acid in severely cirrhotic patients.

SummaryThe fatty acid composition of adrenal cholesterol ester fraction has been determined by gas-liquid chromatography in 7 species. Characteristic CEFA patterns for each type of animal were apparent. Rat and dog had the highest content of polyunsaturated fatty acids in the adrenal cholesterol ester fraction. The remaining species had considerably less of those acids and the major CEFA of dog, man, goose, chicken and rabbit was oleic acid. In the dog, rat and human, an acid tentatively identified as a C22 tetraenoic acid was found in the adrenal CEFA. The significance of these results in relation to hormone synthesis and polyunsaturated fatty acid metabolism is discussed.

SummaryThe serum CEFA and cholesterol level of children (6-10 years old) and older subjects (60-87 years old) of both negro and white races have been compared. Children of both races had significantly less oleic acid and significantly more linoleic acid in their serum CEFA fraction than older subjects. Arachidonic acid did not show significant changes with increasing age. Negro and white subjects of the same age group did not show significant differences in CEFA spectrum. Also, children had a significantly lower total blood cholesterol level than older individuals. The significance of these findings as related to atherosclerosis is discussed.

SummarySerum CEFA composition of 8 different species has been determined by gas-liquid chromatography. Different CEFA spectrums were found for each species. The outstanding variation was the proportions of arachidonic acid in CEFA of the serums. That of the rat had 50% of arachidonic acid and that of the dog had 17%. All species also had a large proportion of linoleic acid in the CEFA of their serums. The rat and the dog, with high arachidonic acid levels in CEFA of their serums, are known to be highly resistant to development of atherosclerosis. Those species having small proportions of arachidonic acid in the CEFA of their serums are susceptible to atherosclerosis and develop the disease spontaneously. This study provides evidence, by association, that level of arachidonic acid in the CEFA may be related to susceptibility to atherosclerosis. It remains open to investigation whether the proportion of arachidonic acid in the CEFA is a characteristic of the species or whether it, and the susceptibility to ...

ConclusionsThe data on the different bile salts suggest that several aspects of the structure of the bile salt molecule are involved in their effects on cholesterol esterase activity. Firstly, taurocholic acid has a distinctly greater effect than either glycocholic or the unconjugated cholic acid (the latter in the esterifying system only). Secondly, conjugation with glycine inhibits the effect of cholic acid. Thirdly, free taurine and glycine have no influence on the effect of cholic acid, i.e. the effects of taurocholic and glycocholic acids are due to the intact molecules. Fourthly, in the series of unconjugated acids the degree of effect was greatest with cholic acid which has 3 hydroxyl groups in the molecule and the effect decreased with the decreasing number of hydroxyl groups until with dehydrocholic, which has no hydroxyl groups, there was no effect. The results clearly demonstrate that both the hydroxyl groups and the carboxyl group, free or conjugated, are involved in the effect of bile salts o...

SummaryAdministration of 44 mg of H3-β-sitosterol to lymph fistula rats and analysis of feces, intestinal contents, intestine, and lymph gave the following results: (1) balance data indicated approximately 40% absorption in 48 hours, (2) there was increased fecal excretion of cholesterol and related sterols, (3) there was considerable uptake of H3-β-sitosterol by the intestinal wall, but essentially no transfer into the lymph. Explanations for the apparent disappearance of H3-β-sitosterol are discussed.

SummaryCholesterol-4-C14 oleate and cholesterol-4-C14 were fed in a test meal to lymph fistula rats. The data indicate that free cholesterol was absorbed to a greater extent than esterified cholesterol. This study also provides evidence to support the concept that preliminary hydrolysis is obligatory for absorption of cholesterol esters and that only free cholesterol can enter the intestinal wall. The significance of these findings is discussed.

SummaryA decreased capacity of liver homogenate to metabolize cortisol in sodium deficient rats has been demonstrated. The rate of Ring A reduction was diminished by about 25% below that of control animals. The possibility that this effect may result in a decreased adrenal corticosteroid secretion via the negative-feedback mechanism by which plasma corticoid levels are stabilized is discussed.

SummarySmall doses of fed cholesterol-4-C14 lead to labeling of cholesterol fractions of mucosa and lymph without increase in level or turnover rate of free cholesterol pool of the mucosa or in amount of cholesterol in lymph. Feeding tracer dose with sodium taurocholate and oleic acid increases the turnover rate of pool which leads to an increased amount of labeled and unlabeled cholesterol in lymph.