Search

The metabolism of cholesterol and its 5-dihydro derivative, cholestanol, was investigated by means of sterol balance and isotope kinetic techniques in 3 subjects with cerebrotendinous xanthomatosis (CTX) and 11 other individuals. All subjects were hospitalized on a metabolic ward and were fed diets practically free of cholesterol and cholestanol. After the intravenous administration of [1,2-(3)H]cholestanol, the radioactive sterol was transported and esterified in plasma lipoproteins in an identical manner to cholesterol. In these short-term experiments, the specific activity-time curves of plasma cholestanol conformed to two-pool models in both the CTX and control groups. However, cholestanol plasma concentrations, total body miscible pools, and daily synthesis rates were two to five times greater in the CTX than control individuals. The short-term specific activity decay curves of plasma [4-(14)C]cholesterol also conformed to two-pool models in both groups. However, in the CTX subjects the decay was more rapid, and daily cholesterol synthesis was nearly double that of the control subjects. Plasma concentrations and the sizes of the rapidly turning over pool of exchangeable cholesterol were apparently small in the CTX subjects, and these measurements did not correlate with the large cholesterol deposits found in tendon and tuberous xanthomas. Despite active cholesterol synthesis, bile acid formation was subnormal in the CTX subjects. However, bile acid sequestration was accompanied by a rise in plasma cholestanol levels and greatly augmented fecal cholestanol outputs. In contrast, the administration of clofibrate lowered plasma cholesterol levels 50% and presumably reduced synthesis in the CTX subjects. Plasma cholesterol concentrations and fecal steroid excretion did not change significantly during this therapy. These findings indicate that the excessive tissue deposits of cholesterol and cholestanol that characterize CTX were associated with hyperactive neutral sterol synthesis. The demonstration of subnormal bile acid formation suggests that defective bile acid synthesis may predispose to the neutral sterol abnormalities.

Cholesterol balance studies were carried out in 11 patients with various types of hyperlipoproteinemia to determine the mechanism by which unsaturated fats lower plasma cholesterol. Unsaturated fats produced no increase in fecal endogenous neutral steroids in 10 of 11 patients and no decrease in absorption of exogenous cholesterol in 5 patients who received cholesterol in the diet. In 8 of 11 patients no changes occurred in excretion of bile acids during the period on unsaturated fat when plasma cholesterol was declining. However, in 3 of 11 patients small but significant increases in bile acid excretion were found during this transitional period; in 2 others increases also occurred after plasma cholesterol had become constant at lower levels on unsaturated fat.Since the majority of patients showed no change in cholesterol or bile acid excretions during the transitional period, we propose that when excretion changes did occur they were probably not the cause of the plasma cholesterol change. Furthermore, turnover data and specific activity curves suggested that cholesterol synthesis was not influenced by exchange of dietary fats. Thus, excluding changes in excretion and synthesis, we conclude that it is most likely that unsaturated fats cause plasma cholesterol to be redistributed into tissue pools. We have also examined the possibility that cholesterol which is redistributed into tissues could be secondarily excreted as neutral steroids or bile acids. In at least 5 of 11 patients excretion patterns were consistent with this explanation. However, we cannot rule out that excretion changes may have been due to alterations in transit time, to changes in bacterial flora, or to transitory changes in absorption or synthesis of cholesterol or bile acids. Our conclusion that unsaturated fats cause a redistribution of cholesterol between plasma and tissue pools points to the necessity in future to explore where cholesterol is stored, to what extent stored cholesterol can be mobilized, and to define the factors governing these fluxes.

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 metabolism of beta-sitosterol was compared to that of cholesterol in 12 patients. Sterol balance methods were supplemented by radiosterol studies, with the following results. (a) Plasma concentrations of beta-sitosterol ranged from 0.30 to 1.02 mg/100 ml plasma in patients on intakes of beta-sitosterol typical of the American diet. Plasma levels were raised little when intakes were increased greatly, and on fixed intakes they were constant from week to week. On diets devoid of plant sterols, the plasma and feces rapidly became free of beta-sitosterol. (b) The percentage of esterified beta-sitosterol in the plasma was the same as for cholesterol. However, the rate of esterification of beta-sitosterol was slower than that for cholesterol. (c) Specific activity-time curves after simultaneous pulse labeling with beta-sitosterol-(3)H and cholesterol-(14)C conformed to two-pool models. The two exponential half-lives of beta-sitosterol were much shorter than for cholesterol, and pool sizes were much smaller. Values of turnover for beta-sitosterol obtained by the sterol balance method agreed closely with those derived by use of the two-pool model. There was no endogenous synthesis of beta-sitosterol in the patients studied; hence, daily turnover of beta-sitosterol equaled its daily absorption. Absorption of beta-sitosterol was 5% (or less) of daily intake, while cholesterol absorption ranged from 45 to 54% of intake. (d) About 20% of the absorbed beta-sitosterol was converted to cholic and chenodeoxycholic acids. The remainder was excreted in bile as free sterol; this excretion was more rapid than that of cholesterol. (e) The employment of beta-sitosterol as an internal standard to correct for losses of cholesterol in sterol balance studies is further validated by the results presented here.

We have estimated the daily synthesis of cholesterol in man by measuring the excretion of cholesterol and its conversion products during periods of controlled sterol intake (sterol balance method), using isotopic or chromatographic procedures (or a combination of the two). Estimates of daily synthesis by this method are based on the premise that the subject is in the metabolic steady state; i.e., the synthesis of cholesterol is equal to the balance (or difference) between the intake of cholesterol and the excretion of cholesterol and its products. To test this premise, we carried out sterol balances in 11 patients; simultaneously, after administration of isotopic cholesterol, turnover was calculated according to previously described models (one-pool, two-pool, or isotopic steady state models for the distribution of radioactive cholesterol within various pools of the body). With calculations based on the one-pool model, turnover rates were considerably higher than estimates based on all other models, and reasons are given for considering these to be overestimates. Good agreement was obtained between results calculated from the two-pool model and those based on sterol balance data; neither method is theoretically preferable to the other. However, with the sterol balance method supplemented by isotopic techniques, valid measurements of cholesterol absorption can be obtained; this in turn permits the essential distinction to be made between daily synthesis and daily turnover of cholesterol when the diet contains cholesterol. In addition, the use of chromatographic isolation procedures provides an accurate measurement of the balance of -sitosterol. This in turn permits valid corrections to be made for losses (which may be large) of neutral steroids during intestinal transit; this is a unique advantage of the chromatographic method.

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.

In the course of carrying out sterol balance studies in 19 patients, we gathered the following evidence that, in some but not all patients, considerable amounts of neutral sterols are "lost" during their passage through the intestinal tract. (a) Since plant sterols are largely nonabsorbable in man, they should be totally recovered in the feces; yet in many patients significantly less plant sterol than expected was recovered, the loss amounting to as much as 56% of daily intake. (b) In two patients in whom cholesterol-(14)C and beta-sitosterol-(3)H were instilled into the terminal ileum, from which neither sterol is absorbed, the feces contained 25% less of each isotope than was instilled. (c) In four patients fed radioactive cholesterol daily until the isotopic steady state was closely approximated, 28-50% of the isotope could not be accounted for. On the other hand, in five patients fed radioactive bile acids until the isotopic steady state was approximated, input equalled output as predicted. Since the amount of -sitosterol absorbed in man is limited (5% or less), this sterol can be used as an internal standard for upward correction of the figure obtained for the amount of neutral steroids excreted. The use of beta-sitosterol for this purpose is based on three considerations: (a) it passes through the intestine in the same physicochemical state as cholesterol; (b) it accompanies cholesterol at every step of its isolation and chromatographic measurement; and (c) it is lost to the same extent as cholesterol. Excretion data for fecal neutral steroids can therefore be corrected for irregular fecal flow as well as for the "unexpected loss" referred to. This loss seems to be due not to errors in stool collection or to technical errors, but to intestinal bacterial degradation of neutral 3beta-OH,Delta(5)-sterols to products not recognized as steroids in the analytical methods used.

Lacking reliable data on cholesterol concentrations in muscle, adipose tissue, skin, and connective tissues (i.e., the "bulk tissues") in "normal" man, we have completed these analyses in 21 men and 8 women who died suddenly and unexpectedly; their ages ranged from 23 to 78 yr. In 11 of these subjects aged 20-40 yr, the mean cholesterol concentrations ranged from 180-440 mg/100 g dry tissue. In contrast, in 13 subjects aged 60-80 yr the values were 23-28% higher in muscle, adipose, and skin, while in dura mater, biceps, and psoas tendons the concentrations were 130, 260, and 460% higher (respectively). Esterified cholesterol in these connective tissues was found to be the major contributor, increasing 5- to 10-fold in the older group as opposed to a less than 5-fold rise in free cholesterol. In view of the large proportion of total body weight represented by these tissues, it is clear that large amounts of cholesterol can be accumulated there over a lifetime; indeed, the dense connective tissues appear to act uniquely as a trap for cholesterol, especially in the esterified form. Whether analyses of tendinous tissues in man, accessible during life, mirror the pattern of cholesterol deposition in arterial connective tissue remains to be determined.

The influence of clofibrate on cholesterol metabolism in patients with hyperlipidemia was studied by means of sterol balance and isotope kinetic techniques and by measurements of flow rates of cholesterol through the biliary tract. Long-term balance studies were carried out on a metabolic ward in 24 patients with all currently recognized types of hyperlipidemia; in five other patients with hypercholesterolemia, pool sizes and turnover rates of cholesterol were defined by compartmental analysis before and after three years' daily administration of the drug. Except in fat-induced hypertriglyceridemia (two patients), clofibrate caused reduced plasma levels of triglycerides and cholesterol in all categories of hyperlipidemia. As a general rule, excretion of cholesterol into bile and feces was significantly increased and fecal bile acid excretion was decreased, regardless of the type of lipoprotein abnormality. Despite a net increase in steroid excretion in most patients with hyperlipidemia, cholesterol synthesis was not increased; indeed, in many patients synthesis appeared to be decreased. While the data obtained in 29 patients were not always consistent, the bulk of the evidence suggests that, in all forms of hyperlipidemia except fat-induced hyperglyceridemia, the drug causes an increased output of cholesterol while simultaneously inhibiting any compensatory increase in cholesterol synthesis. Therefore, it appeared that the increased excretion of steroids was most likely derived from cholesterol stored in tissues. This conclusion was strengthened by finding that long-term administration of the drug can cause marked reduction in body pools of cholesterol. These findings are reflected clinically by resolution of skin and tendon xanthomatosis. However, it is not yet known whether the accumulation of cholesterol in arterial walls that is part of the process of atherogenesis can be inhibited or reversed by the drug.

Studies on the interaction of cholesterol absorption, synthesis, and excretion were carried out in eight patients using sterol balance techniques. Absorption of dietary cholesterol was found to increase with intake; up to 1 g of cholesterol was absorbed in patients fed as much as 3 g per day. In most patients, increased absorption of cholesterol evoked two compensatory mechanisms: (a) increased reexcretion of cholesterol (but not of bile acids), and (b) decrease in total body synthesis. However, the amount of suppression in synthesis was extremely variable from one patient to another; one patient had no decrease in synthesis despite a large increment in absorption of dietary cholesterol, and two patients showed a complete suppression of synthesis. In the majority of cases the accumulation of cholesterol in body pools was small because of adequate compensation by reexcretion plus reduced synthesis, but in a few patients large accumulations occurred on high cholesterol diets when absorption exceeded the compensatory mechanisms. These accumulations were not necessarily reflected in plasma cholesterol levels; these increased only slightly or not at all.

Hepatic secretions of biliary lipids were estimated in 43 patients with and without cholesterol gallstones. Studies were carried out by a marker dilution technique employing duodenal intubation with a three-lumen tube. Hourly secretion rates of cholesterol, bile acids, and phospholipids were determined during constant infusion with liquid formula. In 17 American Indian women with gallstones, hourly outputs of biliary bile acids were significantly less than those in 7 Indian men and 12 Caucasian women without gallstones. These findings suggest that a decreased hepatic secretion of bile acids contributes significantly to the production of a lithogenic bile in Indian women. However, in Indian women with gallstones, secretion of biliary cholesterol was also significantly increased, as compared with Caucasian women without stones. Therefore, lithogenic bile in Indian women was, in most cases, due to a combined decrease in bile acid output and increase in cholesterol secretion. In an attempt to determine the mechanisms for these abnormalities, cholesterol balance studies were done in Indian women with gallstones and normal Indian men. Balance data were compared with results reported previously in non-Indian patients studied by the same techniques, and in general, Indian women showed a slight increase in fecal excretion of bile acids. Since bile acids in the enterohepatic circulation were relatively depleted in Indian women, these patients had a reduced fractional reabsorption. However, previous studies have shown that Caucasians can rapidly replenish bile acid pools in the presence of much greater intestinal losses, and it is suggested that among Indian women with gallstones, reduced secretion rates of bile acids are primarily the result of defective homeostatic regulation of bile acid synthesis. In Indian women with gallstones, at least two factors may have contributed to an increased availability of cholesterol in the liver for secretion into bile. First, cholesterol was inadequately converted into bile acids, and secondly, an increased amount of cholesterol was synthesized, as shown by the balance technique. This enhanced production of cholesterol can partially be explained by obesity, but other factors may also play a role.