L. Horlick

Absorption of dietary cholesterol in man

Abstract Cholesterol absorption was studied by balance methods in 10 hyperlipemic subjects. The cholesterol (401–1214 mg/day) and β-sitosterol (171–454 mg/day) were integral parts of the diets. The absorption was 37% ± 5% and the amounts of cholesterol absorbed were 149–508 mg/day. The amounts absorbed had an excellent correlation with the dietary intake of cholesterol (r = 0.98), and there was no leveling off of the amounts absorbed even at an intake of 1214 mg/day. Assuming that the percentage absorption of dietary and endogenous cholesterol in the gastrointestinal tract is identical, the amounts of endogenous cholesterol secreted into the gastrointestinal tract were calculated from the amounts of fecal neutral steroids of endogenous origin. The relationship of total (dietary plus endogenous) cholesterol in the gastrointestinal tract and its absorption was also excellent (r = 0.97) and also without any evidence of leveling off of the slope up to 2300 mg. These data indicate that within the normal range for North America, the amounts of dietary cholesterol absorbed are proportional to its intake.

Effects of plant sterols oncholesterol metabolism in man

Abstract Plant sterols (Cytellin, 3 g t.i.d.) were administered to 3 normal and 3 hyperlipemic subjects for periods of 12 or 15 days. There was a moderate decrease (9 ± 4%) in plasma cholesterol; and in 5 of 6 subjects there was a modest increase (6 ± 1%) in plasma triglycerides, and in the sixth, the levels were decreased by 20%. On cessation of treatment, plasma lipids returned gradually to pretreatment levels. Treatment with plant sterols increased the fecal excretion of metabolites of endogenous cholesterol by 35–73% over the control values. The increment was generally limited to the neutral metabolites except in one normal subject who showed significant increases both in neutral sterols and bile acids. The increment in fecal excretion was considerably in excess of the decrement in plasma cholesterol pool, suggesting one or more of the following: (1) a reduction in absorption of cholesterol from the intestinal tract, (2) an increase in cholesterol synthesis, and (3) mobilization of cholesterol from tissues. In all subjects the decay in plasma cholesterol specific activity showed an increase after commencement of treatment. Since treatment with plant sterols decreases the absorption of dietary (unlabelled) as well as endogenous (labelled) cholesterol in the intestinal lumen, the increased slope of the plasma cholesterol specific activity during this period may be a reflection of the increase in synthesis to compensate for the decrease in absorption. Similarly, the decreased slope on stopping the treatment in all probability reflects decreased synthesis of cholesterol.

Effects of chlorophenoxyisobutyrate on the synthesis and metabolism of cholesterol in man

Abstract The effects of an oral administration of clofibrate 500 mg. four times a day on plasma cholesterol were studied in 16 subjects on constant dietary intake. The lipoprotein patterns were normal in four, Type II in five and Type IV in seven subjects. There was a rapid decline in the levels of plasma cholesterol and in 15 days these had stabilized at 78 ± 5 per cent of the pretreatment values. No differences between the Type II, Type IV or normals were seen in these short-term studies. Eight subjects (three normal, two Type II and three Type IV) were given a mixture containing sodium acetate-1-14C and DL mevalonate-2-3H lactone intravenously, once before and once after 10 days of treatment with clofibrate. The peak specific activity of 14C (from acetate-1-14C) in plasma cholesterol was decreased (28.5 ± 14.6%) but that of 3H (from mevalonate-2-3H) was not affected by the treatment. The 14C specific activity of biliary cholesterol was decreased even more than that of plasma cholesterol supporting the hypothesis that the site of action of clofibrate in the hepatic synthesis of cholesterol in man is between acetate and mevalonate. The drug did not have any effect on the fractional turnover rates of plasma cholesterol esters; but, their net turnover rates were reduced on account of the decrease in their pool size. The ratios of 3 H 14 C in cholesterol during the first few hours after the injection of the radioactive precursors were not constant from hour to hour. There was also a steady difference in the 3 H 14 C ratios between plasma and bile cholesterol at all times. This suggested to us that the relative incorporation of the two precursors in different cholesterol tissue pools in rapid equilibrium with plasma cholesterol was different.

Mode of Action of Chlorophenoxyisobutyric Acid on Cholesterol Metabolism in Man

In short-term trials chlorophenoxyisobutyric acid (CPIB) (Atromid-S) reduced the plasma cholesterol and triglyceride levels in eight subjects with type II and IV hyperlipidemias to an equal extent. In these subjects, who were maintained on constant solid food diets, CPIB administration resulted in increased excretion of fecal neutral and acidic sterols in the type II subjects only. There was an immediate increase in specific activity of plasma cholesterol in seven of the eight subjects, and a reduced rate of fall of specific activity in many of the subjects. It is suggested that CPIB inhibits the synthesis of cholesterol in vivo, and that the subsequent fall in plasma cholesterol is responsible for the release of cholesterol with higher specific activity from tissues into the plasma pool.

Hypocholesterolemic agents and mobilization of tissue cholesterol in man

Abstract Plasma cholesterol was pulse-labelled with [4- 14 C]cholesterol in 11 hyperlipemic subjects on cholesterol balance studies. When the decline in the specific activity of plasma cholesterol became exponential, various parameters of cholesterol metabolism were measured during steady state conditions of the control period. The subjects were then given clofibrate, nicotinic acid, or plant sterols and the changes in these parameters were noted. There was a prompt decline in the plasma cholesterol concentration and a marked upswing in the specific activity slope; the latter could only occur by the entry of cholesterol from the tissues. The amounts of tissue cholesterol entering plasma were estimated (a) from the increases in the fecal excretion of endogenous cholesterol and its metabolites, and (b) from the increases in the secretion of endogenous cholesterol into the lumen of the gastrointestinal tract. Both estimates were in good agreement. The increases in plasma cholesterol specific activity had excellent correlations with the amounts of cholesterol mobilized from the tissues. These parameters in turn correlated well with fall in plasma cholesterol concentration suggesting that the mobilization was secondary to the acute reduction in the plasma cholesterol cencentrations and was not caused directly by the drugs.

Mechanisms of Action of Cholestyramine in the Treatment of Hypercholesterolemia

Cholestyramine (12 g/day) was administered to four subjects with familial hyperchloesterolemia (type II) for 12 to 15 days. Plasma cholesterol values fell by 24 to 28% in all subjects. Total endogenous fecal steroids increased from 2.0 to 2.5 times over the control values. This increment was mainly in the acidic fraction which increased from 1.4 to 6.5 times during treatment. The neutral steroid fraction showed a slight increase (nonsignificant) in two subjects and a significant increase (P < 0.05) in one subject. The total fecal steroid increment was considerably in excess of the decrement in plasma cholesterol, thus indicating either (a) a substantial increase in cholesterol synthesis, (b) a transfer of cholesterol from depots, or (c) both. Plasma cholesterol specific activity time curves showed a sharp increase in the slope immediately after the commencement of treatment, reflecting an increase in the rate of entry of unlabeled cholesterol into the readily miscible pool. Since cholestyramine did not change the absorption of the dietary cholesterol, the increase in the contribution of unlabeled cholesterol could only be from an increase in endogenous synthesis. In accordance with previous findings from this laboratory, no evidence of degradation of the steroid nucleus was detected during its passage through the gastrointestinal tract.

Effect of chlorophenoxyisobutyrate on the metabolism of endogenous glycerides in man

Abstract The effects of chlorophenoxyisobutyrate (clofibrate) on the incorporation of acetate-1-C 14 and glycerol-2-H 3 into plasma triglycerides and phospholipids were investigated in nine subjects. After injection of the isotopes, the peak specific activities (SA) of plasma triglycerides for both isotopes were obtained simultaneously between 1 to 3 hr and the subsequent slopes of their SA were exponential and parallel for a period of up to 24 hr. The SA slopes after treatment were steeper than those obtained before treatment. If the rate limiting factor is the clearance of triglycerides from plasma as has been shown, these observations reflect increases in the fractional turnover rates (FTR) of plasma triglycerides. Another group of 10 subjects consuming diets of constant composition were given clofibrate and their plasma triglyceride concentrations were determined at intervals. Within 2 days the triglyceride concentrations began to drop and they stabilized at around 62 per cent of the pretreatment levels by the 15th day. There was an excellent correlation between the decrease and the pretreatment levels (r = 0.92). The relationship between the changes in FTR and concentrations of plasma triglycerides indicated that for the majority (six out of nine) of subjects there was no correlation between these two parameters. The remainder showed that reduction in plasma triglycerides was associated with increase in FTR. These results suggest that clofibrate may increase the FTR independent of decreased triglyceride concentrations. The drug appeared to increase the incorporation of acetate-1-C 14 into fatty acids and of glycerol-2-H 3 into triglycerides and phospholipids synthesized in the liver. The incorporation of glycerol-2-H 3 into plasma phospholipids occurred in two peaks suggesting that there were either marked differences in the synthetic rates of different phospholipids or the mono and/or diglycerides produced from the catabolism of plasma triglycerides were incorporated into plasma phospholipids.

A simple method for calculating absorption of dietary cholesterol in man.

Summary The currently available methods for estimating the absorption of dietary cholesterol are so difficult that only a few studies under rather restricted experimental conditions have been conducted. A very simple approach circumventing these difficulties is based on the determination of two isotopes (3H and 14C) in a single unmeasured “test” sample of feces after feeding a mixture of cholesterol-1-2-3H (5 μCi), β-sitosterol-4-14C (1 μCi) and carmine red (300 mg). The two sterols undergo similar conversions and degradations in the gastrointestinal tract, except for the differences in their absorption. Thus, the fraction of dietary cholesterol which is absorbed in excess of the absorption of β-sitosterol can be calculated merely from the difference in the 3H/14C ratios between the mixture given by mouth and that obtained from the feces. It is important to examine the first sample of feces containing the red color of the carmine red since this “test” sample contains the unabsorbed sterols uncontaminated by radioactivity from the biliary cholesterol and bile acids.

Effect of Cholestyramine on Tissue Pools of Cholesterol a Preliminary Report

Summary Four Type II Hyperlipopro-teinemic subjects were investigated before and after treatment with cholestyramine. Plasma cholesterol was significantly reduced (365 ± 23 vs 273 ± 34 mg/100 ml) and tri-glycerides significantly increased (149 ± 55 vs 181 ± 59 mg/100 ml) on cholestyramine treatment. The daily turnover of cholesterol, as determined by the method of Goodman and Noble, was nearly doubled by the treatment (0.813 ± 0.11 vs 1.595 ± 0.176 g). Although previous workers have already suggested that cholestyramine does not decrease tissue cholesterol pools, we observed a significant increase in tissue pools in each of the three subjects given cholestyramine alone (10.1 ± 1.4 vs 16.2 ± 6.9 g) for Pool A—excluding plasma; and 27.2 ± 4.6 vs 43.7 ± 6.4 g for Pool B). Treatment for the fourth subject consisted of a combination of cholestyramine and clofibrate. This combination appeared to prevent increases in the size of Pool B and in the size and production rate of Pool A. These preliminary observations suggest that the hypocholesterolemic effect of cholestyramine may be enhanced and its effects on tissue cholesterol prevented by giving it in combination with other agents such as clofibrate.

Lack of Degradation of Dietary and Endogenous Sterols in Gastrointestinal Tract of Man

Abstract Nine hyperlipidemic subjects consuming their habitual solid diets were investigated by sterol balance techniques to see if there was any degradation of the steroid nucleus by the intestinal microflora. After the subjects had been equilibrated on their controlled habitual diets, 3-day fecal pools were collected first in a steady state for 9–12 days and then for another 12–15 days during the treatment with clofibrate and nicotinic acid. The amounts of cholesterol, β-sitosterol, and their microbial metabolites were estimated from each fecal pool. They were corrected for fecal flow and methodological losses. There was a modest variation in the recovery of the dietary β-sitosterol from pool to pool within a given subject. The mean variation was 9 ± 7%. The mean recovery of dietary β-sitosterol in nine subjects was 94 ± 5% with a range between 89 and 104%. If the absorption of β-sitosterol is assumed to be 5%, these results rule out significant degradation of the steroid nucleus in the gastrointestinal tract. This was true even when the subjects were given clofibrate or nicotinic acid. The mean recovery of β-sitosterol during this period was 96 ± 6%. The proportions of microbial conversion products (stanone and stanol) of cholesterol and β-sitosterol were similar. Clofibrate treatment did not significantly influence these proportions. However, in response to the treatment with nicotinic acid, significant increases in the stanol derivatives of both cholesterol and β-sitosterol were seen, but the changes were identical for the two sterols.