The interaction of cholesterol absorption and cholesterol synthesis in man

The total miscible pool of cholesterol in the body is determined largely by the interaction of cholesterol absorption and synthesis. In the present study we have examined the net effects of this interplay in one normal and five hypercholesteremic subjects when various amounts of cholesterol were made available for absorption. Feeding large amounts of cholesterol to the normocholesteremic patient caused an expansion of body pools by as much as 20 g before the amount of cholesterol re-excreted as fecal neutral steroids each day came into balance with the cholesterol absorbed from the diet. There was no detectable decrease in total body synthesis of cholesterol nor any increase in conversion of cholesterol into bile acids. However, feedback control of cholesterol synthesis was demonstrable when large quantities of plant sterols were fed: in the hypercholesteremic patients thus studied, the absorption of both endogenous and exogenous cholesterol was then greatly reduced, and a compensatory increase in synthesis occurred. Thus, the plant sterol experiments, but not the cholesterol feeding experiment, demonstrated that feedback control by dietary cholesterol does occur in man. That feedback control by dietary cholesterol is relatively unimportant in man seems to be due to the fact that in the metabolic "steady state" the absorption mechanism is essentially saturated by the large amounts of endogenous cholesterol available for reabsorption. These findings demonstrate that there are important differences between man and various laboratory animals in regard to the interaction of absorption and synthesis as factors controlling the size of tissue pools of cholesterol.     

Comparison of cholesterol turnover by sterol balance and input-output analysis, and a shortened way to estimate total exchangeable mass of cholesterol by the combination of the two methods

Daily turnover of cholesterol obtained by the balance method was compared to daily input rates calculated by input-output analysis in 43 experiments. The mean value of input rates for kinetic data of 10.1-16.4 weeks' duration (14 experiments) was 1.05 g/day vs. the chemical turnover of 0.94 g/day (difference 10.9 percent). For decay curves of 4.8-9.9 weeks' duration (29 experiments) the mean results were 1.67 g/day vs. 1.31 g/day, respectively (difference 20.1 percent). A combination of the balance method with input-output analysis is proposed to estimate the size of M (minimum value of the total exchangeable mass of cholesterol) in short-term experiments. Using this method, the analysis of curves of 10-12 weeks' duration showed a mean difference of 7.5 percent with the analysis of curves of 50-66 weeks' duration in 17 patients. However, because of considerable variations that can occur in individual cases, it is urged that a standard correction factor not be used, either in estimating turnover data or M from 10-12 weeks' kinetic data; rather, the proposed combined method will alert the investigator to the occurrence of discrepant results.     

Evaluation of an isotope ratio method for measurement of cholesterol absorption in man

Recently an isotope ratio method (IRM) was developed for measuring cholesterol absorption in rats by analysis of radioactivity in peripheral blood (Zilversmit, D. B. 1972. Proc. Soc. Exp. Biol. Med. 140: 862-865). To validate it in man we have compared cholesterol absorption by a fecal radioactivity method (FRM) with that simultaneously measured by IRM in 14 patients (15 experiments) hospitalized on a metabolic ward. Cholesterol absorption by FRM was assayed as fecal recovery of orally administered [(14)C]cholesterol, after correction with markers for fecal flow (chromic oxide) and cholesterol degradation (beta-sitosterol). Simultaneously, [(3)H]cholesterol was administered intravenously, and the dose-normalized ratio of [(14)C]- to [(3)H]cholesterol was repeatedly assayed in plasma. After 72 hours the ratio became constant in each patient and remained so for as long as 63 weeks (five additional outpatient studies). In three patients the fecal data were unsatisfactory because of poor recoveries of chromic oxide and radioactive cholesterol. In the remaining 11 patients (12 experiments) the mean cholesterol absorption by IRM was 42.1% (range 15.7-62.9%) and by FRM 36.6% (range 13.8-58.8%). There was good to excellent agreement between the two methods in the same patient, except in one experiment. Statistical analysis of these 12 comparisons by estimating confidence intervals showed that we can be 95% confident that the two absorption methods will produce results within 5 percentage points, and 99% confident that the differences are less than 7 percentage points. Although we conclude that IRM affords results that are concordant with those obtainable by earlier validated methods, we urge that its suitability for outpatient studies be further examined in more extensive trials.     

An evaluation of four methods for measuring cholesterol absorption by the intestine in man

Critical comparisons have been made in 12 patients of four methods for measuring cholesterol absorption from the intestine. Methods I-III depend on the use of labeled cholesterol (intravenously or continuous labeling orally) in conjunction with sterol balance measurements; Method IV can be carried out with only a single test dose containing labeled cholesterol plus labeled beta-sitosterol. In the latter technique absorption is calculated as the loss of cholesterol relative to beta-sitosterol during intestinal transit. Method III (isotopic steady-state method) proved to be undependable because of uncertainties in determining the existence of an isotopic steady state. However, Method IV gave good agreement with Methods I and II, and it appears to have certain practical as well as theoretical advantages. Although Method IV requires collections of stools for up to 8 days, it is nevertheless the most rapid and the simplest of all the methods for estimating absorption. It can also be used in certain situations, such as in fur-licking animals, when Methods I and II are inadequate. Therefore, this method would seem to be a valuable addition to other isotopic techniques for estimating cholesterol absorption in man.     

The cholesterol turnover, synthesis, and absorption in two sisters with familial hypercholesterolemia (type IIa).

To explore the mechanisms of the profound plasma cholesterol elevations in familial homozygous hypercholesterolemia (type IIa), cholesterol turnover, sterol balance, cholesterol absorption, and low density lipoprotein studies were carried out under controlled dietary conditions in two sisters (aged 13 and 16). Cholesterol turnover was prolonged. The half-life of the first exponential of the plasma cholesterol specific activity decay curve was double that of normal adults. The rate constants for the removal of cholesterol from pool A (KAA = 0.0652) and for the excretion of cholesterol from the system (Kaa = 0.0197) were less than half of normal. The production rates of cholesterol were low, only 6.30 and 6.86 mg/kg per day as measured by cholesterol turnover and sterol balance techniques, respectively. Fecal neutral steroid and bile acid excretion were 5.22 and 1.64 mg/kg per day, which is remarkably low in comparison to those of normal and heterozygous children. Cholesterol absorption was within the upper limit of the values reported for normal adults. THE HDL cholesterol values were extremely low (27 mg/dl) in contrast to profoundly elevated LDL levels. The fractional catabolic rate of LDL (0.127 per day) and the rate of synthesis and catabolism of apo-LDL (15 mg/kg per day) were low in comparison to previously reported values in homozygotes. These composite data indicated that the definable metabolic defects of these two sisters with homozygous familial hypercholesterolemia were the sluggish clearance of cholesterol from the body coupled with low total body synthesis of cholesterol.     

Dietary cholesterol absorption, and sterol and bile acid excretion in hypercholesterolemia-resistant white rabbits

The New Zealand white (NZW) rabbit fed a 0.1% cholesterol-enriched diet (CD) typically responds (normoresponsive, NR) by quickly developing hypercholesterolemia. To study the underlying mechanisms responsible for the widespread phenomenon of inter-individual variability of response to dietary cholesterol, a unique hypercholesterolemia-resistant (RT) rabbit model was developed. These animals were utilized to investigate selected potential mechanisms that might enable the RT animal to compensate for dietary cholesterol overload. When rabbits were fed the low-cholesterol stock diet, there was no significant difference in the plasma cholesterol concentrations of the NR and the RT animals. However, a significant rise was observed in the NR rabbits within 1 month of their being placed on the cholesterol-enriched diet; the plasma cholesterol concentration of the RT animals was not affected. During consumption of the cholesterol diet the cholesterol absorption rate was somewhat greater in the NR rabbits (P less than 0.05), whereas intestinal transit times and the fecal excretion of neutral steroids were substantially the same in both groups. In contrast, the fecal bile acid excretion of the RT animals was more than twice as great (P less than 0.0001) as that of the NR animals. We conclude that the response to dietary cholesterol is a heritable trait in these rabbits and that, although less dietary cholesterol was absorbed by the RT animals, it appears that a major mechanism controlling plasma cholesterol levels involves the rate of conversion of cholesterol to bile acids and their subsequent excretion.     

Bile acid synthesis in man: assay of hepatic microsomal cholesterol 7 alpha-hydroxylase activity by isotope dilution-mass spectrometry

The present work describes an accurate assay of the rate-limiting enzyme in bile acid synthesis, the cholesterol 7 alpha-hydroxylase, in human liver. The assay is based on isotope dilution-mass spectrometry, and endogenous microsomal cholesterol is used as the only substrate for the enzyme. Operative liver biopsies were obtained from patients undergoing elective cholecystectomy under highly standardized conditions. In ten gallstone patients, the enzyme activity of the microsomal fraction averaged 9.6 +/- 1.4 (mean +/- SEM) pmol X min-1 X mg protein-1 corresponding to a daily synthesis of about 0.5 mmol of bile acids. Three cholestyramine-treated patients displayed a four-fold higher enzyme activity. No evidence was obtained supporting the concept that the cholesterol 7 alpha-hydroxylase is modulated by phosphorylation-dephosphorylation.     

Inhibition of cholesterol absorption and synthesis in rats by sesamin

The effects of sesamin, a lignan from sesame oil, on various aspects of cholesterol metabolism were examined in rats maintained on various dietary regimens. When given at a dietary level of 0.5% for 4 weeks, sesamin reduced the concentration of serum and liver cholesterol significantly irrespective of the presence or absence of cholesterol in the diet, except for one experiment in which the purified diet free of cholesterol was given. On feeding sesamin, there was a decrease in lymphatic absorption of cholesterol accompanying an increase in fecal excretion of neutral, but not acidic, steroids, particularly when the cholesterol-enriched diet was given. Sesamin inhibited micellar solubility of cholesterol, but not bile acids, whereas it neither bound taurocholate nor affected the absorption of fatty acids. Only a marginal proportion (ca. 0.15%) of sesamin administered intragastrically was recovered in the lymph. There was a significant reduction in the activity of liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase after feeding sesamin, although the activity of hepatic cholesterol 7 alpha-hydroxylase, drug metabolizing enzymes, and alcohol dehydrogenase remained uninfluenced. Although the weight and phospholipid concentration of the liver increased unequivocally on feeding sesamin, the histological examination by microscopy showed no abnormality, and the activity of serum GOT and GPT remained unchanged. Since sesamin lowered both serum and liver cholesterol levels by inhibiting absorption and synthesis of cholesterol simultaneously, it deserves further study as a possible hypocholesterolemic agent of natural origin.     

Sterol balance studies in the rat. Effects of dietary cholesterol and beta-sitosterol on sterol balance and rate-limiting enzymes of sterol metabolism.

Sterol balance measurements using isotopic and chromatographic techniques were carried out in rats fed diets containing beta-sitosterol (0.8%) and cholesterol (1.2%). The activities of the rate-limiting enzymes of cholesterol synthesis (beta-hydroxy-beta-methylglutaryl-CoA reductase, EC 1.1.1.34) and bile acid synthesis (cholesterol 7 alpha-hydroxylase) were determined in the same animals. Cholesterol feeding increased cholesterol absorption from 1.2 to 70 mg/day. The increased absorption was compensated for by inhibition of hepatic cholesterol synthesis, enhanced conversion of cholesterol to bile acids (from 13.7 to 27.3 mg/day) and a slight increase in the excretion of endogenous neutral steroids (from 7.7 to 11.2 mg/day). Despite the adaptation there was accumulation of cholesterol in the liver (from 2.2 to 9.2 mg/g). Beta-Sitosterol feeding inhibited cholesterol absorption (calculated absorption was zero). In these rats there was enhanced cholesterol synthesis (from 20.0 to 28.8 mg/day, but no change in the rates of bile acid formation. Measurements of the activities of the rate-limiting enzymes showed fair correlation with cholesterol-bile acid balance. In cholesterol fed animals, beta-hydroxy-beta-methylglutaryl-CoA reductase was inhibited 80% and cholesterol 7 alpha-hydroxylase was enhanced 61%. In beta-sitosterol-fed animals, the reductase was increased 2-fold and cholesterol 7 alpha-hydroxylase was not significantly different from controls.     

Determination of cholesterol turnover rate by a one-and two-pool kinetic analysis.

The cholesterol turnover rate in rabbits with alimentary cholesterol atherosclerosis and in guinea-pigs with chronic vitamin C deficiency was studied by a one- and two-pool kinetic analysis. The one-pool analysis yielded exaggeraged values for the turnover rate, but the turnover rate differences between the control and experimental groups in the one- and the two-pool analysis were very similar. One-pool analysis can be used in initial studies to obtain preliminary data on the effect of nutritional, pharmacological and other factors on cholesterol turnover rate.     

Cholesterol absorption and turnover in rhesus monkeys as measured by two methods

We compared the absorption of cholesterol in seven rhesus monkeys (four high-responders and three low-responders) as measured by two methods: 1) the dual isotope plasma ratio method of Zilversmit (1972. Proc. Soc. Exp. Biol. Med. 140: 862) and 2) the single isotopic meal feeding technique of Borgstr?m (1969. J. Lipid Res. 10: 331). We also compared the cholesterol pool sizes calculated by kinetic analysis of the plasma cholesterol specific activity decay curves obtained after simultaneous administration of [(3)H]- and [(14)C]cholesterol, one given intravenously and the other orally. The ratio of orally to intravenously administered cholesterol radioactivity in plasma did not attain constancy until 6 weeks after isotope administration. Therefore, the percent absorption of cholesterol was calculated by the Zilversmit method 8 weeks after the administration of isotopes. The mean percent absorption of cholesterol by the Borgstr?m method was 66.3 +/- 5.1 (S.E.) and by the Zilversmit method was 70.3 +/- 7.4. The differences were not statistically significant. However, in two of seven monkeys the percent absorption of cholesterol calculated by the Zilversmit method was higher by 10.4 and 22.6 percentage points than the values obtained by the Borgstr?m method. Cholesterol absorption by either method was higher in the high-responding monkeys than in the low-responding group. The sizes of the rapidly exchangeable pool or the minimum estimate of the total body pool of cholesterol were similar for all monkeys or for either the low-responding or the high-responding animals and were also similar when calculated using the data from either the orally or the intravenously administered radioactive cholesterol. Cholesterol synthesis was significantly higher in the low-responding monkeys (115 mg/day) than in the high-responding (64 mg/day). The present study and our previous studies support the hypothesis that a major factor causing the difference in response of plasma cholesterol to dietary cholesterol between the high- and low-responding rhesus monkeys is a difference in the intestinal absorption of cholesterol.-Bhattacharyya, A. K., and D. A. Eggen. Cholesterol absorption and turnover in rhesus monkeys as measured by two methods.     

Changes in cholesterol absorption and cholesterol synthesis caused by ezetimibe and/or simvastatin in men

This study evaluates changes in cholesterol balance in hypercholesterolemic subjects following treatment with an inhibitor of cholesterol absorption or cholesterol synthesis or coadministration of both agents. This was a randomized, double blind, placebo-controlled, four-period crossover study to evaluate the effects of coadministering 10 mg ezetimibe with 20 mg simvastatin (ezetimibe/simvastatin) on cholesterol absorption and synthesis relative to either drug alone or placebo in 41 subjects. Each treatment period lasted 7 weeks. Ezetimibe and ezetimibe/simvastatin decreased fractional cholesterol absorption by 65% and 59%, respectively (P < 0.001 for both relative to placebo). Simvastatin did not significantly affect cholesterol absorption. Ezetimibe and ezetimibe/simvastatin increased fecal sterol excretion (corrected for dietary cholesterol), which also represents net steady state cholesterol synthesis, by 109% and 79%, respectively (P < 0.001). Ezetimibe, simvastatin, and ezetimibe/simvastatin decreased plasma LDL-cholesterol by 20, 38, and 55%, respectively. The coadministered therapy was well tolerated. The decreases in net cholesterol synthesis and increased fecal sterol excretion yielded nearly additive reductions in LDL-cholesterol for the coadministration of ezetimibe and simvastatin.     

Genetic regulation of cholesterol absorption and plasma plant sterol levels: commonalities and differences

The molecular basis of the processes that control two closely related traits, the absorption of cholesterol from the intestines and plasma plant sterol levels, are only partially understood. The discovery that mutations in two novel hemitransporters, ATP binding cassette transporter G5 (ABCG5) and ABCG8, underlie a rare inborn error in plant sterol metabolism, beta-sitosterolemia, represents a major breakthrough in this field. More recently, genetic studies in the mouse that mapped loci in linkage with cholesterol absorption and plasma plant sterol levels and studies in humans that examined the relationship of plasma plant sterol levels to sequence variation in the ABCG5/ABCG8 locus suggested the involvement of other genes. Moreover, studies in beta-sitosterolemic patients, in ABCG5/ABCG8-targeted animals, and on a newly developed cholesterol absorption inhibitor, ezetimibe, suggest commonalities and differences in the regulation of the two traits. This review summarizes the evidence for genetic control of cholesterol absorption and plasma plant sterol levels, presents the evidence for commonalities and differences between the two traits, and discusses recent developments and future perspectives in this field.     

Corn fiber oil and sitostanol decrease cholesterol absorption independently of intestinal sterol transporters in hamsters

OBJECTIVE: The aim of this study was to investigate the cholesterol-lowering mechanisms of corn fiber oil (CFO), ferulate phytostanyl esters (FPEs) and parent compounds of FPE, including sitostanol and ferulic acid, in hamsters. METHOD: Seventy male Golden Syrian hamsters were randomly assigned to six experimental diets for 4 weeks: (1) cornstarch-casein-sucrose-based control diet (control); and (2) control diet plus 0.1% (wt/wt) cholesterol (cholesterol-control). The remaining four groups were given cholesterol-control diet with: (3) 10% (wt/wt) CFO; (4) 0.5% (wt/wt) sitostanol; (5) 0.23% (wt/wt) ferulic acid; and (6) 0.73% (wt/wt) FPE. At the end of dietary intervention, total plasma cholesterol, high-density lipoprotein cholesterol and triglyceride concentrations were determined. Parameters of cholesterol kinetics, including cholesterol absorption and synthesis, as well as mRNA expression of sterol transporters such as Niemann-Pick C1 like 1 (NPC1L1), ATP-binding cassette G5 (ABCG5) and ABCG8, were assessed. RESULTS: Supplementation with CFO decreased (P<.0001) plasma total cholesterol levels by 29% as compared with the cholesterol-control group, while FPE and sitostanol reduced (P<.02) cholesterolemia by 15% and 14%, respectively. CFO and sitostanol decreased (P<.05) cholesterol absorption by 24% compared to the cholesterol-control group. Dietary intervention did not alter the intestinal gene expression of ABCG5, ABCG8 and NPC1L1. CONCLUSION: The present results show that the CFO-induced and sitostanol-induced decrease in cholesterol absorption is independent of intestinal enterocyte sterol transporters such as ABCG5, ABCG8 and NPC1L1 in hamsters.