Don S. Lin

The Intestinal Absorption of Dietary Cholesterol by Hypercholesterolemic (Type II) and Normocholesterolemic Humans

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.

Effects of dietary n-3 fatty acids on the phospholipid molecular species of monkey brain

Abstract: We examined the changes in the molecular species of brain ethanolamine glycerophospholipids of monkeys fed diets containing widely ranging amounts of n‐3 fatty acids. Two groups of rhesus monkeys were fed pre‐ and postnatally either a control diet (soy oil; containing 8% of fatty acids as 18:3n‐3) or a deficient diet (safflower oil; containing <0.3% 18:3n‐3). The brains of these animals were analyzed at 22 months of age. A third group of monkeys was fed the safflower oil diet to 22 months of age and then switched to a fish oil diet (28% long‐chain n‐3 fatty acids) for 1–2 years before autopsy. The molecular species of the diacyl, alkylacyl, and alkenylacyl ethanolamine glycerophospholipids from frontal cortex were separated by HPLC. A total of 24 molecular species were identified. Fatty acids in the sn‐2 position differed markedly among the diet groups, but the sn‐1 position always contained only 16:0, 18:0, or 18:1. In the diacyl subclass of the control brain, the n‐3 molecular species represented 41% of total and the n‐6 species 45%, whereas in the deficient brain the n‐3 molecular species decreased to 9% and n‐6 molecular species increased to 77%. The fatty acid 22:5n‐6 did not replace 22:6n‐3 in a symmetrical fashion in the molecular species of the deficient brain. In the brains of the fish oil‐fed monkeys, the n‐3 molecular species amounted to 61% and n‐6 molecular species were reduced to 25%. The species 18:1–22:6, 16:0–22:6, and 18:0–22:6 generally changed proportionally in response to diet. However, 18:1–20:4, 16:0–20: 4, and 18:0–20:4 responded differently. The fish oil diet led to an increase in the proportion of 18:1–20:4 in the alkenylacyl subclass, whereas 16:0–20:4 and 18:0–20:4 decreased. Thus, total species containing sn‐1 18:1 increased at the expense of sn‐1 16:0 in the fish oil animals. Regardless of diet, each subclass of ethanolamine glycerophospholipid showed a strikingly different ratio of sn‐1 16:0 to 18:0 to 18:1 for a given sn‐2 fatty acid. In conclusion, the different diets had profound qualitative and quantitative effects on the molecular species of brain phospholipids, and these changes have implications for possible functional changes.

Can prenatal N-3 fatty acid deficiency be completely reversed after birth? Effects on retinal and brain biochemistry and visual function in rhesus monkeys.

Our previous studies of rhesus monkeys showed that combined prenatal and postnatal n-3 fatty acid deficiency resulted in reduced visual acuity, abnormal retinal function, and low retina and brain docosahexaenoic acid content. We now report effects of n-3 fatty acid deficiency during intrauterine development only. Rhesus infants, born to mothers fed an n-3 fatty acid deficient diet throughout pregnancy, were repleted with a diet high in alpha-linolenic acid from birth to 3 y. Fatty acid composition was determined for plasma and erythrocytes at several time points, for prefrontal cerebral cortex biopsies at 15, 30, 45, and 60 wk, and for cerebral cortex and retina at 3 y. Visual acuity was determined behaviorally at 4, 8, and 12 postnatal weeks, and the electroretinogram was recorded at 3–4 mo. Total n-3 fatty acids were reduced by 70–90% in plasma, erythrocytes, and tissues at birth but recovered to control values within 4 wk in plasma, 8 wk in erythrocytes, and 15 wk in cerebral cortex. At 3 y, fatty acid composition was normal in brain phospholipids, but in the retina DHA recovery was incomplete (84% of controls). Visual acuity thresholds did not differ from those of control infants from mothers fed a high linolenic acid diet. However, the repleted group had lower amplitudes of cone and rod ERG a-waves. These data suggest that restriction of n-3 fatty acid intake during the prenatal period may have long-term effects on retinal fatty acid composition and function.

The Interaction of Dietary Fibers and Cholesterol upon the Plasma Lipids and Lipoproteins, Sterol Balance, and Bowel Function in Human Subjects

To identify any metabolic effects of dietary fiber upon cholesterol metabolism in man, six adult volunteer subjects were fed eucaloric cholesterol-free formula diets, with and without added dietary fiber for two 4-wk periods. A large quantity of dietary fiber was fed, some 60 g of plant cell wall material (or 16 g of crude fiber) derived from corn, beans, bran, pectin, and purified cellulose. This provided about five times the fiber intake of the typical American diet. The addition of fiber to the cholesterol-free diet did not change either the plasma cholesterol level (171+/-21 mg/dl, SEM, to 167+/-18) or the triglyceride (103+/-39 to 93+/-27 mg/dl). The excretion of both endogenous neutral steroids and bile acids were unchanged with fiber (505+/-41 to 636+/-75 mg/day and 194+/-23 to 266+/-47 mg/day, respectively.) However, total fecal steroid excretion was increased 699+/-29 to 902+/-64 mg/day, P < 0.025). With fiber, intestinal transit time was decreased (59+/-9 to 35+/-8 h, P < 0.005), and both the wet and dry stool weights were greatly increased.A second group of six subjects was fed similar diets containing 1,000 mg cholesterol derived from egg yolk. The addition of fiber to the 1,000-mg cholesterol diet did not alter either plasma cholesterol level (233+/-26 to 223+/-36 mg/dl) or triglyceride (102+/-19 to 83+/-11 mg/dl). The excretion of endogenous neutral steroids (618+/-84 to 571+/-59 mg/day), of bile acids (423+/-122 to 401+/-89 mg/day), and of total fecal steroids (1,041+/-175 to 972+/-111 mg/day) were unchanged by fiber. The absorption of dietary cholesterol was not altered when fiber was added to the 1,000-mg cholesterol diet (44.0+/-3.3 to 42.9+/-2.5%). A two-way analysis of variance utilizing both groups of subjects indicated a significant (P < 0.001) effect of dietary cholesterol upon the plasma cholesterol concentration. We concluded that a large quantity of dietary fiber from diverse sources had little or no effect upon the plasma lipids and sterol balance in man in spite of the fact that intestinal transit time and stool bulk changed greatly.

Dietary eicosapentaenoic acid and docosahexaenoic acid from fish oil. Their incorporation into advanced human atherosclerotic plaques.

The incorporation of fatty acids from dietary fish oil was measured in obstructive atherosclerotic plaques removed from 11 patients fed fish oil, rich in omega-3 fatty acids, for 6-120 days before a planned arterial endarterectomy. The fatty acids of plasma and atheroma were analyzed with special reference to docosahexaenoic acid (DHA, 22:6) and eicosapentaenoic acid (EPA, 20:5), the principal omega-3 fatty acids of fish oil. The omega-3 fatty acid content increased greatly in plasma from 0.9% of fatty acids to 14.8% in cholesteryl esters, from 3.8% to 22.1% in phospholipids, and from 1.3% to 21.9% in triglycerides. The omega-3 fatty acid content of the atherosclerotic plaques was also greater when compared with that of plaques removed from 18 non-fish oil-fed controls. The omega-3 fatty acid in cholesteryl esters of the plaques was 4.9% in the experimental group versus 1.4% in control plaque, in phospholipids it was 8.8% versus 1.8%, and in triglycerides it was 4.7% versus 0.7% (p less than 0.001 for each lipid class). The two major omega-3 fatty acids (DHA and EPA) behaved differently. Compared with their respective plasma levels, relatively more DHA than EPA was deposited into the plaques. Whereas the increase of omega-3 fatty acids in plasma reached a plateau 3 weeks after initiation of fish oil feeding, a linear increase in plaque omega-3 fatty acids continued with time. As a result of the changes in fatty acid composition, the lipid classes of both plasma and plaque had a higher unsaturation index in the fish oil-fed group.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol Metabolism and Placental Transfer in the Pregnant Rhesus Monkey

The placental transfer of cholesterol (5-cholesten-3beta-ol) was investigated by giving pregnant rhesus monkeys cholesterol-1alpha-(3)H or cholesterol-4-(14)C and then determining the cholesterol specific activity (SA) in maternal serum and in fetal serum and tissues. An isotopic steady state was established in five pregnant animals by the daily feeding of a tracer dose of cholesterol-4-(14)C. Comparison of maternal and fetal serum cholesterol SA revealed that an average of 42.6% of the serum cholesterol in the term fetus originated by transfer from the maternal blood. The remainder presumably arose by fetal synthesis de novo. Fetal tissues had cholesterol SA equal to or slightly less than that of fetal serum, except for brain which had a SA only 5% that of fetal serum. In other studies a single intravenous dose of radioactive cholesterol was given to either mother or fetus in late pregnancy. The time for detectable passage across the placenta in either direction was between 4 and 24 hr. With maternal administration of the isotope, there was equilibration of maternal and fetal serum cholesterol SA after 10-12 days. With fetal injection of isotopic cholesterol, however, the maternal cholesterol SA never attained a level more than 5% of fetal SA. This indicated that the net cholesterol flux was strongly in the direction of mother to fetus. Serum cholesterol levels were significantly greater in maternal than in fetal serum (80.3+/-18.5 vs. 59.6+/-15.6 mg/100 ml). Maternal serum cholesterol concentration in the monkey was significantly lower in late pregnancy than during the puerperium. Studies of breast milk indicated that approximately two-thirds of milk cholesterol was transferred from the maternal blood.

Cholesterol supplementation with egg yolk increases plasma cholesterol and decreases plasma 7-dehydrocholesterol in Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive condition comprising multiple malformations, mental retardation, and growth failure, results from reduced activity of the final enzyme in cholesterol biosynthesis, 7-dehydrocholesterol Delta(7)-reductase (DHCR7). Reduced plasma and tissue cholesterol concentrations and accumulation of cholesterol precursors including 7-dehydrocholesterol (7-DHC) are characteristic biochemical abnormalities. While it is still unclear what role these potentially toxic precursors have in the pathogenesis of this disorder, the accumulation of 7-DHC in the brain has been associated with impaired learning in rats and oxidized 7-DHC has been shown to induce growth retardation in cultured rat embryos. We hypothesized that supplemental dietary cholesterol would increase plasma cholesterol levels and suppress synthesis of 7-DHC and other abnormal sterols in individuals with SLOS. After baseline sterol levels were obtained, patients were provided supplemental cholesterol as egg yolk. Plasma sterols were analyzed by capillary-column gas chromatography over time in four children with SLOS. When evaluated at 4-8 weeks after the initiation of cholesterol supplementation, there was a marked increase in mean plasma cholesterol, from 53 mg/dl to 82 mg/dl. While the percent of total sterols as 7-DHC decreased from 15% to 10%, there was no change in total plasma 7-DHC levels. However, when evaluated 35-90 weeks after the institution of cholesterol supplementation, mean plasma 7-DHC decreased, from 11.3 mg/dl to 3.5 mg/dl (-67%, P < 0.05), along with an increase in mean plasma cholesterol from 53 mg/dl to 114 mg/dl (+116%, P < 0.05). These results support the hypothesis that over time dietary cholesterol supplementation from egg yolk increases the plasma cholesterol levels and decreases levels of 7-DHC which may be toxic. These data have important therapeutic implications in the management of SLOS.

The incorporation of n-3 and n-6 essential fatty acids into the chick embryo from egg yolks having vastly different fatty acid compositions.

ABSTRACT: The effect of egg yolk fatty acid composition on essential fatty acid utilization by the developing chick embryo was studied by feeding laying hens a fat-free diet supplemented with oils containing widely divergent contents of the essential n-6 and n-3 fatty acids. A control hen was fed a commercial feed for laying hens. The diets contained 20 to 4370 mg/100 g n-3 fatty acids and 360 to 8020 mg/100 g n-6 fatty acids. Fertile eggs were collected in pairs: one was incubated and the other served as an unincubated control. The fatty acid content of the unincubated egg and the newly hatched chick from each pair was compared. Some 50% of the total fatty acids in the egg yolk were incorporated into the tissues of the newly hatched chick. Regardless of diet, more yolk n-6 fatty acids were incorporated into the chick compared to saturated or monounsaturated fatty acids. The percentage of incorporation especially increased from the eggs containing relatively low amounts of n-6 fatty acids. The percentage of incorporation of n-3 fatty acids was similar to that of saturated and monounsaturated fatty acids when n-3 fatty acids were plentiful in the egg yolk, but increased significantly when n-3 fatty acids were low in the eggs. There was a generally linear relationship between essential fatty acids in the egg and in the chick, although levels of docosabexaenoic acid [DHA; 22:6(n-3)] in the brain did not respond proportionally. The developing chick preferentially removed DHA from the yolk, but did not synthesize more DHA when the amount of the DHA precursor, 18:3(n-3), in the yolk was increased. We concluded that the developing chick embryo requires 0.4–1.1% of egg energy as n-3 fatty acids and 4.8–6.2% as n-6 fatty acids, or a “dietary” ratio of n-6/n-3 of 5 to 14. This requirement may have relevance for humans as well.

THE LIPIDS OF SLUGS AND SNAILS : EVOLUTION, DIET AND BIOSYNTHESIS

There is a considerable gap in current knowledge of the lipid composition of snails and slugs, both of which belong to the phylum Mollusca. We have therefore analyzed the sterol and fatty acid compositions of three species of slugs and three species of snails. The sterols of slugs included eight different sterols: cholesterol contributed 76–85% of the total sterols, brassicasterol accounted for 4–13%; other sterols we identified were lathosterol, 24-methylene cholesterol, campesterol, stigmasterol, sitosterol and sitostanol. In contrast, snails contained two additional sterols, desmosterol and cholestanol. Of the polyunsaturated fatty acids in slugs, linoleic (18∶2n−6) and arachidonic acids (20∶4n−6) were the major n−6 fatty acids, while linolenic (18∶3n−3) and eicosapentaenoic acids (20∶5n−3) were the predominant n−3 fatty acids. Docosahexaenoic acid (22∶6n−3), the end product in the n−3 fatty acid synthetic pathway and an important membrane fatty acid of mammals, fish and birds, was absent in both slugs and snails. However, the analogous product of n−6 fatty acid synthesis, 22∶5n−6, was found in both snails and slugs. This raises speculation about preference for n−6 fatty acid synthesis in these species. Our data show the unique sterol and fatty acid compositions of slugs and snails, as well as similarities and differences in sterol composition between the two. The results between the two land mollusks are contrasted with those of marine mollusks, such as oysters, clams and scallops.

The effect of shellfish in the diet upon the plasma lipid levels in humans

In order to document any hypercholesterolemic effects from the ingestion of shellfish, 6 normal men were given two diets containing different shellfish, each preceded by a low cholesterol baseline diet. Diet I contained 449 mg cholesterol per day from lobster, crab, and shrimp. Diet II contained clam, oyster, and scallop and provided 447 mg of sterols of which cholesterol constituted only 40 percent. The other sterols are uniquely characteristic of these shellfish (i.e. brassicasterol, 24-methylene cholesterol, etc.). In a second study, 2 normal men and 1 type II hypercholesterolemic woman were fed the baseline diet and shellfish diet II to provide 623 mg of sterols per day. The plasma cholesterol of the 6 subjects averaged 184 +/- 35 mg/dl during baseline, 192 +/- 35 mg/dl in shellfish diet I (p less than 0.05) and 182 +/- 24 mg/dl during shellfish diet II. In the second study, the plasma cholesterol of the 2 normal men did not change. The cholesterol of the hypercholesterolemic woman increased from 311 mg/dl (baseline) to 352 during the shellfish diet (p less than 0.05). Plasma triglyceride levels remained unchanged. Our data indicated that large quantities of lobster, crab, and shrimp were only mildly hypercholesterolemic in normals, but less so than other cholesterol-containing foods. Clams, oysters, and scallops were not hypercholesterolemic in normal subjects but were in a hypercholesterolemic patient.