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Focuses on studies of lowering of serum cholesterol and triglyceride by para-aminosalicylic acid in hyperlipoproteinemia. Method of the studies; Results and discussion; Conclusions.

The frequency of coronary risk factors was documented in 4,829 school children in Muscatine, Iowa, over a 14-month period of time. Serum cholesterol levels were similar for children at all ages; the mean serum cholesterol level was 182 mg/dl (SD lus or minus 29). Twenty four percent had levels larger than or equal to 200 mg/dl, 9% were larger than or equal to 220 mg/dl, 3 % were larger than or equal to 240 mg/dl, and 1% were larger than or equal to 260 mg/dl. Casual levels of serum triglyceride increased with age: the mean level was 71 mg/dl (SD plus or minus 36) at age 6 years and 108 mg/dl (SD plus or minus 45) at age 18 years. Only 15% of the children had serum triglyceride levels of 140 mg/dl or more. Blood pressure increased strikingly with age. No child between 6 and 9 years of age had blood pressures larger than or equal to 140 mm Hg systolic or larger than or equal to 90 mm Hg diastolic. In the age group 14 to 18 years, 8.9% had systolic blood pressures larger than or equal to 140 mm Hg, 12.2% had diastolic blood pressures larger than or equal to 90 mm Hg, and in 4.4% both pressures were at or above these levels. Obesity also increased through the school years. At ages 6 to 9 years, 20% had weights relative to those of the group as a whole of larger than or equal to 110%, and 5% were larger than or equal to 130%; in the 14 to 18 years age group, 25% had relative weights of larger than or equal to 110%, and 8% were larger than or equal to 130%. These data indicate that a considerable number of school-age children have risk factors which in adults are predictive of coronary heart disease.

After the establishment of a relatively linear decay curve for plasma [4-14C]cholesterol, rabbits were starved for 26-32 days. The plasma cholesterol concentration increased 400% during starvation. Concurrently, the plasma triglyceride level declined by 50%. While the plasma cholesterol was rising, the cholesterol specific radioactivity of the plasma remained unchanged in starved animals, but in control animals the plasma cholesterol specific radioactivity declined substantially. The cholesterol content of the liver and adipose tissue increased with starvation. The cholesterol specific radioactivities relative to plasma for adipose tissue were lower in the starved animals versus controls. These results support the hypothesis that cholesterol stored in the lipid droplet of the adipose tissue cell is released into plasma and is the chief source of the hypercholesterolemia observed during complete caloric starvation. Cholesterol metabolism in the starved animal can be depicted as a virtually closed system in both the input from biosynthesis and diet being low or zero and the output likewise being close to zero.

Two diets, identical except for differences in trans and cis fatty acids, were fed to rabbits in order to evaluate the effects of dietary trans fatty acids on the fatty acid composition of serum lipids, lipoproteins and adipose tissue. The animals were first starved to deplete adipose tissue stores and then were red a diet containing 15% of the calories as C18:1 trans fatty acid (elaidic acid). A second group received a diet containing an equivalent amount of C18:1 cis fatty acid (oleic acid). A third group of animals, the control group, was fed only the baseline low fat rabbit chow. Dietary trans fatty acid was incorporated into all serum lipid and lipoprotein fractions and into the adipose tissue. In the serum the triglycerides contained the greatest amount of trans fatty acid followed by the phospholipids with cholesterol esters containing the least. The trans fatty acids of the phospholipids and cholesterol esters were distributed equally among the lipoprotein fractions, but the triglycerides of high density lipoproteins contained less trans fatty acids than did the triglycerides of other lipoproteins. Adipose tissue developed a high content of C18:1 trans fatty acid (21.7%), apparently at the expense of C18:1 cis fatty acid. In addition to the incorporation of C18:1 trans fatty acid, the content of other fatty acids, including C18:2 and the total of C18:1 cis and trans, was influenced by the substitution of dietary trans for cis fatty acids. The serum phospholipids of the trans-fed rabbits contained more C18:2 than the phospholipids of the cis-fed rabbits. The serum cholesterol esters of the trans-fed rabbits contained more C18:2 and a smaller total of C18:1 (cis puls trans) than the cholesterol esters of the cis-fed rabbits. These differences indicated that C18:1 trans fatty acid was not necessarily the metabolic equivalent of 18:1 cis fatty acid. These results suggested that dietary trans fatty acids which are contained in hydrogenated margarines, shortenings and oils of the human diet are readily incorporated into the lipids of the body including membranous structures such as lipoproteins.

The metabolism of the small pool of triglyceride in human high-density lipoproteins (HDL) has been studied. In 28 postabsorptive and unmedicated subjects the previously reported reciprocal relationships between the concentration of very low-density lipoprotein (VLDL) lipids and HDL cholesterol was confirmed. It was also found that the HDL triglyceride correlated positively and significantly with the concentration of VLDL triglyceride, and there was a significant negative correlation between the concentrations of triglyceride and cholesterol in HDL. Five normal and 3 hypertriglyceridemic subjects were given 5,000 units of heparin intravenously. Not only was there a pronounced reduction in VLDL triglyceride, but there was a 60 to 80 per cent reduction in HDL triglyceride; the low-density lipoprotein (LDL) triglyceride was unchanged. To test a possible precursor-product relationship between the triglyceride pools in VLDL and HDL, 5 subjects were given an intravenous pulse injection of (14-C)-palmitic acid. The specific activity-time curves of triglyceride in VLDL and HDL did not conform to a typical precursor-product relationship; rather the HDL curve reached its peak well before it crossed the VLDL curve, quite compatible with an origin of HDL triglyceride which was independent of VLDL. However, when the lipoproteins of density smaller than 1.006 (VLDL and chylomicrons) were labeled in the intestine after an oral tracer dose of (3-H)-palmitic acid in 2 subjects, the relationship of the triglyceride labeling in HDL to that in d smaller than 1.006 lipoproteins was very similar to the relationship observed after the intravenous dose. Although an independent intestinal origin of HDL triglyceride could not be excluded, this result did suggest that the labeling of triglyceride in d smaller than 1.006 lipoproteins and in HDL was related and was consistent with a transfer from the larger lipoproteins to the HDL. In three subjects given a constant intravenous infusion of (3-H)-palmitic acid, the specific activity of triglyceride in VLDL became constant after 3 to 4 hours; it also became constant in the HDL after about 4 hours but at a level of only 40 to 70 per cent of the VLDL level. It appeared that 30 to 60 per cent of the HDL triglyceride was either derived from an unlabeled source or else turned over too slowly to become labeled to any extent during the infusions. Whatever the explanation, the same phenomenon would be likely to influence the level of the peak specific activity of HDL triglyceride after the pulse intravenous injections of label. It is possible that the specific activity of the "labeled" fraction of HDL triglyceride may have reached its peak at the point at which it crossed the VLDL curve, which would be consistent with a precursor-product relationship between VLDL and one fraction of the HDL triglyceride. It has been concluded that HDL triglyceride is heterogeneous and includes at least one pool, possibly transferred from VLDL, which is relatively rapidly turning over.

Although the usual diet may contain 150-250 mg of plant sterols, chiefly beta-sitosterol, only trace amounts of these sterols have heretofore been found in human or animal blood and tissues. We now report elevated plant sterol levels in the blood and tissues of two sisters with extensive tendon xanthomas but normal plasma cholesterol levels. Besides beta-sitosterolemia and xanthomatosis, no other physical, mental, or biochemical abnormalities were detected.Repeatedly, the plasmas of the two sisters have contained 27.1 and 17.7 mg/100 ml of beta-sitosterol, 9.7 and 8.2 mg/100 ml of campesterol, and 0.5 and 0.5 mg/100 ml of stigmasterol, respectively. These plant sterols constituted 15.6 and 11.3% of the total plasma sterols. Some 60% of the plasma beta-sitosterol and campesterol was esterified; the measurable stigmasterol was entirely unesterified. The transport of the plasma beta-sitosterol and campesterol was largely in low density lipoproteins (76 and 83%, respectively). High density lipoproteins carried the remainder. Plant sterols were barely detectable in the very low density lipoprotein fraction. Only trace amounts of stigmasterol could be detected in the low density and high density lipoprotein fractions. The plant sterol content of the red blood cells averaged 12-13 mg/100 ml packed cells or about 13% of the total sterols. Two tendon xanthoma biopsies with the usual high concentration of cholesterol had 36.7 and 4.0 mg of plant sterols/g dry wt, of which 25.7 and 2.9 mg were beta-sitosterol, entirely in the free form. Plant sterols were also found in adipose tissue (0.2 mg/g wet wt) and in skin surface lipids (3.2 mg/g of lipid). The intestinal absorption of beta-sitosterol in both the patients, measured by two techniques, indicated greatly increased absorption of this sterol (about 24 and 28% in the patients L. H. and R. H., respectively, normal absorption being <5%). We suggest that increased absorption of beta-sitosterol must be considered as one cause of this disease. The reason for the extensive xanthomatosis in these two patients remains unknown. Perhaps in some way plant sterols initiated the development of xanthomas with otherwise normal plasma cholesterol levels. Clinical atherosclerosis has not yet occurred. The occurrence of beta-sitosterolemia in these two sisters with un-affected parents suggests an inherited recessive trait.

The incomplete absorption of dietary cholesterol may represent an adaptive intestinal barrier that prevents hypercholesterolemia. To explore this mechanism, we compared cholesterol absorption in 15 normocholesterolemic and 6 hypercholesterolemic (type II) subjects fed background cholesterol-free formula diets with 40% of calories as fat. Each test meal consisted of a breakfast into which was incorporated scrambled egg yolk containing 300-500 mg of cholesterol and [4-(14)C]cholesterol (3-22 muCi), either naturally incorporated into the yolk cholesterol by previous isotope injection into the laying hen or added in peanut oil to the yolk of the test breakfast. In some instances [1alpha-(3)H]cholesterol was the radioactive marker. The radioactivity of the fecal neutral sterol fraction was determined in daily stool samples for the next 7 days to provide an estimate of unabsorbed dietary cholesterol. The amount of absorbed and reexcreted labeled cholesterol proved negligible. Most unabsorbed dietary cholesterol appeared in the stool on the second or third day after the meal, and 95% or more was recovered in the stool by 6 days. Plasma specific activity curves were usually maximal at 48 h. Normal subjects absorbed 44.5+/-9.3 (SD) of the administered cholesterol (range 25.9-60.3). Hypercholesterolemics absorbed the same percentage of cholesterol as normals: 47.6+/-12.6% (range 29.3-67.3). Absorption was similar whether the radiolabeled cholesterol was added to egg yolk or naturally incorporated in it (42.1+/-9.3 vs. 48.9+/-9.8%). Six normal subjects were fed a cholesterol-free formula for 4 wk, and then different amounts of cholesterol (110-610 mg/day) were added for another 4 wk. At the end of each period, single test meals containing either 110, 310, or 610 mg of cholesterol and [1alpha-(3)H]cholesterol were administered. Cholesterol absorption was 42.3+/-6.0% and 45.4+/-8.3% for the two dietary periods, respectively. The absolute cholesterol absorption was linearly related to the amount of cholesterol in the test meal, and absorption was not affected by background diets high or low in cholesterol content.

In one normal subject, J.S., fed several formula diets in a sterol balance study, only 25-58% of the ingested plant sterols were recovered from the stool. The dietary plant sterols were completely recovered from the stools of five other men. Plant sterol recovery was complete in all men when a diet of mixed general foods was consumed. Since the chief differences in composition of the formula and the diet of mixed general foods were related to the different contents of cellulose and lactose, these components were added to the formula diet of J.S., and plant sterol balance studies were then carried out. The addition of fresh celery or pulverized cellulose to the formula diet partially corrected the usual fecal loss of plant sterols (80% being recovered). Lactose in the formula was only slightly corrective. However, the addition of both cellulose and lactose led to complete recovery of the ingested plant sterols in the feces. Bacterial cultures of stools were incubated with added cholesterol-4-(14)C, and a linear relationship between losses of sterol during balance studies and in vitro incubations was observed; that is, a considerable loss of the labeled cholesterol from cultures after the formula diet, but not after the diet of mixed general foods. This in vitro loss was also corrected by the addition of cellulose and lactose to the formula diet. The loss of the sterol nucleus in the intestinal tract may occur at times because of the lack of certain dietary constituents. It is hypothesized that the metabolism of intestinal tract bacteria is altered when certain constituents are not present in the diet, and that these bacteria may then degrade the sterol nucleus.

The effect of cholestane-3,5alpha,6-triol (CT) on the intestinal absorption of cholesterol and oleic acid, as well as the absorption of labeled CT, was studied in lymph ductcannulated rats. Intragastric administration of 50 mg of CT in an emulsion with cholesterol-7alpha-(3)H and oleic acid-1-(14)C resulted in 50% inhibition of sterol transfer into lymph but only 8% depression of fatty acid absorption over an 8 hr period. The absorption of labeled CT into lymph was only 2-3% compared with 50% absorption of cholesterol when each was fed alone. 10% of the fed CT was recovered in the intestinal mucosa, and of this, one-half was associated with the brush border fraction. In rats fed CT 6 days prior to cholesterol and fatty acid administration, there was no effect on fatty acid absorption, while cholesterol absorption was reduced by almost 30%. When the intestinal mucosa from these animals were investigated by electron microscopy, it appeared that CT feeding resulted in numerous enlarged mitochondria and a marked increase in length of the microvilli. If animals were allowed to recover for 6 days from the CT prefeeding regime, the intestinal mucosa appeared normal, and the absorption of cholesterol approached that in controls. A possible mechanism for CT inhibition of cholesterol absorption was shown to be competition for the enzyme cholesterol esterase which esterifies cholesterol prior to entrance into the lymphatic system. CT itself is poorly esterified and poorly absorbed, but it is effective in inhibiting esterification of cholesterol in vitro.