The Use of Kits in the Analysis of Tissue Lipids Requires Validation.

The ready availability and ease of use of kits for the measurement of serum lipids has greatly facilitated these measurements. In many cases it would be convenient to use these kits in the determination of lipid concentrations in tissues. The successful application of serum kits in tissue analysis requires that two important issues be considered. First, the solvent system for the extraction of the lipids and the solvent used for analysis by the kit must be compatible with the reactions in the kit. Second, the concentration range in the analyzed solution must be within the range for which the kit is used. We report here that lipids in liver and adipose tissues may be significantly underestimated by the use of some kits. We recommend that the use of kits for tissue analysis of lipids be validated for the specific analysis.

The effect of olestra on the absorption, excretion and storage of 2,2',5,5' tetrachlorobiphenyl; 3,3',4,4' tetrachlorobiphenyl; and perfluorooctanoic acid.

Mice were gavaged with either (14)C-labeled 2,2'5,5' tetrachlorobiphenyl; 3,3',4,4' tetrachlorobiphenyl; or perfluorooctanoic acid. Absorption of these compounds was determined by assay of feces collected for 48 h after the gavage. Part of the animals received test diets containing olestra during this 48-hour period to determine its effect on absorption of the compounds. Mice that received the diet without olestra during this period were divided into groups that either continued the diet without olestra or changed to a diet containing olestra. These diets were continued for 7 days, and a second 48-hour fecal collection was made to measure the effect of olestra on enterohepatic circulation of the compounds and their metabolites. The animals were sacrificed, and blood, fat, and liver concentrations of (14)C were measured. Olestra decreased the absorption of 2,2',5,5' tetrachlorobiphenyl. It also reduced tissue and blood concentrations of this compound. Olestra also decreased the absorption of 3,3',4,4' tetrachlorobiphenyl, but it did not alter enterohepatic circulation or tissue concentrations. Olestra significantly increased the excretion of perfluorooctanoic acid in the second 48-hour collection, suggesting an effect on enterohepatic circulation. It did not, however, alter tissue concentrations of perfluorooctanoic acid. These data are consistent with previously observed effects of olestra on the absorption and storage of lipophilic compounds.

Factors affecting the storage and excretion of toxic lipophilic xenobiotics.

Lipophilic toxins have been introduced into the environment both as functional compounds, such as pesticides, and as industrial waste from incineration or the manufacture of electrical transformer components. Among these substances are compounds that are carcinogenic and that affect the endocrine system. Accidental high exposures of humans to some lipophilic toxins have produced overt disease symptoms including chloracne and altered liver function. These toxic materials have been the recent focus of international effort to reduce or eliminate classes of halogenated hydrocarbons from the environment. Evidence of the widespread distribution of lipophilic toxins in the biosphere has been obtained by analyses of human tissues and human milk. The principal route of entry of lipophilic toxins into humans is through the food chain, and most of them are stored in adipose tissue. A common route of excretion is in bile, but there is also evidence of nonbiliary excretion into the intestine. Enterohepatic circulation of many of these compounds slows their removal from the body. Substances that interrupt the enterohepatic circulation of compounds that enter the intestine by the biliary and nonbiliary routes increase the rate of their removal from the body and reduce their storage half-lives. Reduction in body fat, along with these dietary substances that interrupt enterohepatic circulation, further enhances the excretion rate. Areas for further research include optimizing regimens for body burden reductions, understanding the nature of nonbiliary excretion, and following the effects of tissue redistribution during loss of body fat.

Intestinal absorption of beta-carotene ingested with a meal rich in sunflower oil or beef tallow: postprandial appearance in triacylglycerol-rich lipoproteins in women.

BACKGROUND: Evidence indicates that different types of fat have different effects on the postprandial plasma triacylglycerol response. Therefore, the type of fat may influence the appearance of beta-carotene in postprandial triacylglycerol-rich lipoproteins, which is used as an indicator of intestinal beta-carotene absorption. OBJECTIVE: We compared in female subjects the appearance of beta-carotene in plasma triacylglycerol-rich lipoproteins after beta-carotene was ingested with a meal containing sunflower oil or beef tallow. DESIGN: Women (n = 11) each ingested 2 different vitamin A-free, fat-rich meals that were supplemented with beta-carotene (47 micromol) and contained equivalent amounts (60 g) of sunflower oil or beef tallow. Blood samples were collected hourly from 0 to 10 h; additional samples were collected at selected intervals until 528 h. In a subgroup of the women (n = 7), plasma chylomicrons and 3 subfractions of VLDLs were separated by cumulative rate ultracentrifugation. RESULTS: The appearance of beta-carotene in chylomicrons and in each VLDL subfraction was lower after ingestion with the meal containing sunflower oil than after ingestion with the meal containing beef tallow (P < 0.03). In chylomicrons, the area under the concentration-versus-time curve (AUC) for beta-carotene was 38.1 +/- 13.6% lower (P < 0.03); in contrast, the AUC for triacylglycerol was higher (P < 0.05) after the sunflower-oil-rich meal than after the beef-tallow-rich meal. CONCLUSIONS: Ingestion of beta-carotene with a meal rich in sunflower oil as compared with a meal rich in beef tallow results in lower appearance of beta-carotene and greater appearance of triacylglycerol in triacylglycerol-rich lipoproteins.

Suppression of food intake, body weight, and body fat by jejunal fatty acid infusions.

Three experiments investigated effects of jejunal lipid infusions given on 4 or 21 consecutive days in adult, male Sprague-Dawley rats. In experiment 1, 7-h infusions of linoleic or oleic acid (0.2 ml/h for 7 h; total load = 11.5 kcal) on 4 consecutive days reduced total intake (ad libitum consumption of the liquid diet Boost, Mead Johnson, plus load) by approximately 15% and decreased weight gain compared with 4-day tests with saline administration. In experiment 2, linoleic acid at 0.1 ml/h for 7 h (5.7 kcal) was ineffective, whereas the same load delivered in 3.5 h produced effects similar in magnitude to those in the first experiment. In experiment 3, jejunal infusions of linoleic acid (0.2 ml/h for 7 h) on 21 consecutive days reduced mean total intake by 16%, body weight by 10%, and carcass fat by 48% compared with controls receiving saline. The net decrease in caloric intake may reflect the combined activation of pre- and postabsorptive mechanisms, and it suggests a possible treatment for obesity.

Olestra formulation and the gastrointestinal tract.

Olestra is a mixture of compounds comprising sucrose esterified with 6-8 long-chain fatty acids. It is not hydrolyzed by pancreatic lipase and as a result is not absorbed from the small intestine. Olestra in general has physical properties similar to those of a triacylglycerol with the same fatty acid composition. Foods made with olestra are virtually identical in taste and texture to those made with typical triacylglycerols. Olestra consumption does not generate hydrolytic products in the small intestine and, therefore, does not generate some of the signals that alter motility in the gastrointestinal tract. A reduction in gastroesophageal reflux with olestra, in contrast to triacylglycerols, is consistent with a lack of effect on stomach emptying. Unlike triacylglycerols that are absorbed in the proximal small intestine, olestra is distributed throughout the small intestine during transit and passes into the colon. In the colon, olestra's effects depend on its physical properties. Liquid nondigestible lipids result in separation of oil from the fecal matrix. Olestra formulations made with specific fatty acid compositions, particularly those containing a solid sucrose polyester component including behenic acid, possess appropriate rheology to hinder separation of oil from the rest of the fecal matrix, thereby reducing gastrointestinal symptoms.

Chylomicron triacylglycerol fatty acids in suckling northern elephant seals (Mirounga angustirostris) resemble the composition and the distribution of fatty acids in milk fat.

Following birth, the northern elephant seal (Mirounga angustirostris) pups rapidly gain weight by ingesting milk with a high fat content, as much as 50%. To better understand the metabolism of the pups during the suckling period, the positional distributions of triacylglycerol fatty acids in both the milk and chylomicra were determined. Extracts of enzymatically digested lipids were separated by thin layer chromatography and the constituent fatty acids were separated and quantified by gas liquid chromatography. Over 84% of the fatty acids were either monoenoic or saturated, with the ratio of monoenoic to saturated fatty acids ranging between 2.9-4.0. Positional distributional analyses revealed that the very long chain monoenoics (20:1 and 22:1) were located primarily at the sn-1,3 positions of milk triacylglycerols. In the interval between the onset of lactation to the time of weaning, the content of these very long chain monoenoic fatty acids at the sn-1,3 positions increased from 13-37%. At the sn-2 position, the percentage of 18:1 was 3-5-fold higher than 16:1. Analyses indicated that the triacylglycerols in both milk and suckling pup chylomicra were similar. This particularly was true for the distributions at the sn-2 position, indicating that milk fats are being absorbed primarily via the 2-monoacylglycerol pathway.

Relative effects on serum lipids and apolipoproteins of a caprenin-rich diet compared with diets rich in palm oil/palm-kernel oil or butter.

Two randomized, blind studies measured changes in serum cholesterol, other serum lipids, and apolipoprotein (apo) concentrations in hypercholesterolemic men consuming caprenin (Cap)-rich diets after either baseline diets enriched in palm oil/palm-kernel oil (PO/PKO) or butter. The triglyceride Cap contains 45% 22:0 and 50% 8:0-10:0. Compared with baseline values established at 3 wk on the PO/PKO diet, the 17 subjects on the Cap diet showed significant reductions after 6 wk in HDL cholesterol (HDL-C), HDL2-C, and HDL3-C and a significant increase in the ratio of total cholesterol, LDL-C, triglycerides, apo B-100, or apo A-I were seen. Compared with baseline values established at 3 wk on the butter diet, after 6 wk the seven subjects receiving the Cap diet showed no significant changes in the lipid and apolipoprotein indexes analyzed. These data show that one or more of 8:0, 10:0, and 22:0 fatty acids can contribute to hypercholesterolemia in men.

Positional analyses of triacylglycerol fatty acids in the milk fat of the antarctic fur seal (Arctocephalus gazella).

The positional distribution of fatty acids has been determined for the milk triacylglycerols of the Antarctic fur seal, Arctocephalus gazella. Of particular interest was the positional distribution of the polyunsaturated n-3 fatty acids in milk triacylglycerols (TG). In adipocytes of pinnipeds, TG are synthesized with the n-3 fatty acids primarily in the sn-1,3 positions. To determine the positional distribution, extracts of enzymatically digested lipids were separated by thin-layer chromatography, and the constituent fatty acids were separated and quantified by gas-liquid chromatography. Monoenoic and saturated fatty acids comprised over 75% of the total, the ratio of monoenoic to saturated fatty acids being 2:1. The percent content of the long-chain n-3 fatty acids, 20:5, 22:5 and 22:6, ranged between 15-20%. The positional analyses revealed that at the sn-2 position of milk TG, saturated fatty acids were in excess (57%), and the content of n-3 fatty acids was less than 5%. More than 80% of the n-3 fatty acids in milk were located in the sn-1,3 positions. The data indicate that in pinnipeds TG are synthesized in the mammary gland and adipose tissue with fatty acids having similar positional distributions.

Distribution among tissues of intravenously administered sucrose octaoleate.

The distribution of [14C]oleate label in rat tissues in the 6 hr after intravenous administration of sucrose octa[14C]oleate (7.5 mg; SuO8) was compared with that observed after administration of [14C]triolein. The [14C]oleate label, whether injected as triolein emulsion, or as chylomicrons obtained from donor animals, rapidly cleared from the serum; only 10% or less remained in the serum 15 min after injection. Labeled SuO8 disappeared less rapidly from the serum; about one-third of the dose was present after 15 min, and after 120 min 14% remained. In the liver, there was an initial greater accumulation of fatty acid label when an emulsion of either triolein or SuO8 was given rather than the chylomicrons. The octaester continued to accumulate in the liver throughout the 6 hr of study, and 78% of the initial dose was present at that time. By contrast, although one-third of the triolein, as of SuO8, was found in the liver shortly after injection, levels subsequently decreased; at 6 hr, 12% of the label remained associated with that organ. A small portion, up to 8% of the acid label, whether administered as chylomicrons or as a triolein emulsion, was found in the epididymal fat pads. Smaller amounts, usually 1% or less, of the [14C]oleate label were found in fat pads following the injection of labeled SuO8. In a separate study, the levels of acid label in the liver and spleen were monitored for 21 days following the intravenous administration of [14C]SuO8. There was an initial accumulation of approximately half of the injected lipid label in the liver and one-quarter in the spleen. By day 21, the level in the liver had decreased to one-third of that administered, while the level in spleen remained at one-quarter.

The excretion and characterization of intravenously administered olestra.

Although sucrose octaoleate that is consumed is neither digested or absorbed, following intravenous injection it is found mainly in the liver. Olestra is a mixture of the hexa-, hepta-, and octaesters of sucrose. To follow the metabolic fate of intravenously administered [14C]sucrose-labeled olestra, we measured its urinary elimination, and the rate of excretion of 14C in the feces, and characterized the 14C-labeled material that appeared in bile. The fecal excretion for days 4-14 after dosing was found to be first order with the half-life of the injected olestra being 5.0 +/- 0.5 days. The 14C recovered in the bile was soluble in chloroform. Two-dimensional thin-layer chromatography autoradiograms of the biliary lipid showed the pattern of the biliary 14C to be essentially the same as that of the dosed olestra. Biliary excretion and subsequent fecal egestion of essentially unhydrolyzed sucrose esters is the principal route for the removal of intravenously administered olestra. Only traces of 14C were found in the urine.

Effects of partial replacement of dietary fat by olestra on dietary cholesterol absorption in man.

Olestra, a nonabsorbable fat substitute comprising long-chain fatty acid esters of sucrose, had been previously shown to reduce cholesterol absorption in humans when ingested at a level of 50 g/d. To determine whether or not a lower level of dietary olestra would also reduce cholesterol absorption, we studied the effect of 7 g of olestra twice a day in 20 normocholesterolemic male inpatients in a double-blind, crossover trial. Two 6-day diet treatment and stool collection periods were separated by a 14-day washout period. Half of the subjects received butter, and half, a butter-olestra blend during each treatment period according to a crossover design. All subjects ingested trace amounts of 3H-cholesterol and 14C-beta-sitosterol with the butter or the butter-olestra blend. Cholesterol absorption was determined from the 3H/14C ratios in the diet and in saponified and extracted stools according to previously validated methodology. Cholesterol absorption during the butter regimen was significantly greater than that during the olestra regimen (56.1% +/- 1.6% v 46.7% +/- 1.1%, P less than .01).

Reduction in the body content of DDE in the Mongolian gerbil treated with sucrose polyester and caloric restriction.

It has previously been shown that oral administration to rats of sucrose polyester (SPE4), a nonabsorbable lipophilic binding agent, greatly stimulates the fecal excretion of coorally administered DDT5 (R.J. Jandacek, 1982, Drug Metab. Rev., 13, 695-714). To determine whether this agent would stimulate the excretion of persistent metabolites of DDT stored in body tissues, we treated a group of gerbils with [14C]-DDT and monitored the fecal excretion of radioactivity for several months until a terminal, log-linear phase of excretion was observed. At this point, when greater than 75% of the fecal radioactivity was identified as [14C]DDE, we fed the animals diets containing up to 10% sucrose polyester and found that the rate of excretion of radioactivity in the stool promptly increased two to three times as compared to the rate in the preceding control period. Some rats were subjected to a 25-50% restriction in total food allotment, but this produced no significant change in fecal excretion of total radioactivity. However, when food restriction was combined with administration of sucrose polyester, there was a dramatic, eightfold average increase in excretion of fecal radioactivity. This synergistic effect was reversed (within 24 hr) when the animals were transferred to a normal diet. Measurement of total body radioactivity confirmed that food restriction plus sucrose polyester treatment reduced the body content of the pesticide. We conclude that stimulation of intestinal excretion may offer a new approach to treatment of patients exposed to lipophilic environmental contaminants.

The rapid hydrolysis and efficient absorption of triglycerides with octanoic acid in the 1 and 3 positions and long-chain fatty acid in the 2 position.

We describe rapid hydrolysis of triglycerides with medium-chain fatty acids in the 1 and 3 positions and a long-chain fatty acid in the 2 position. The triglycerides, 2-linoleoyl-1,3-dioctanoyl glycerol (8L8) and 2-oleoyl-1,3-dioctanoyl glycerol, hydrolyzed more rapidly than triglycerides comprising all long-chain fatty acids. The in vitro hydrolysis rate of 8L8 was similar to that of a medium-chain triglyceride of octanoic and decanoic acids in random positions. From intestinal recovery of 14C 45 min after injection into the isolated, irrigated loop of the small intestine of an anesthetized rat, the amount of 2-[1-14C]linoleoyl-1,3-dioctanoyl glycerol absorbed was greater than 2 1/2 times that of its long-chain analog, 2-[1-14C]linoleoyl-1,3-dioleoyl glycerol. These data support the ease of hydrolysis and absorption of 1,3-dioctanoyl triglycerides with long-chain fatty acids in the 2 position.

The effect of a non-absorbable fat on the turnover of plasma cholesterol in the rat.

Rats were injected with [4-14C]-cholesterol and then fed diets that contained sucrose polyester (SPE) at levels of 0 and 8% of the diet. 14C was measured in neutral and acidic steroid fractions of the feces collected during days 35-39 post i.v. injection. Periodic blood samples were used to measure the specific activity of the plasma cholesterol. The plasma data were consistent with a two-pool model for the decay of the plasma specific activity. The slow component of the decay curve decreased more rapidly in animals that received SPE. The half-life corresponding to this component was approximately 20% shorter in the SPE-fed animals compared to the control group. The mass of cholesterol calculated for the first pool was similar for all groups of animals. The 14C found in the feces was consistent with the more rapid removal of cholesterol from the body in the SPE-fed animals. The mass of excreted steroid was equal to the calculated rate of cholesterol production in each group of animals.

The substitution of sucrose polyester for dietary fat in obese, hypercholesterolemic outpatients.

Our aim was to determine the effects of the substitution of sucrose polyester (SPE) for dietary fat in a 16-week outpatient study in 36 obese subjects with primary hypercholesterolemia. The subjects were randomized into three groups who followed a 16-week treatment period where all subjects received hypocaloric diets which provided approximately 7 kcal/lb body weight, a polyunsaturated/saturated (P/S) fat ratio of 0.9, and 180 mg cholesterol/day. The percentages of calories as fat in the 3 groups were as follows: a low fat diet group (n = 12) received 27% of dietary calories as fat, a low fat plus SPE group (n = 13) received 25% of calories as fat plus 27 g SPE/day as a bread spread and salad dressing, and a third group (placebo, n = 11) received 37% of calories as fat with a 27 g/day conventional fat placebo (bread spread and salad dressing). Mean weight loss from baseline in the 16 week treatment period was 2.6, 3.9, and 3.4% respectively in the placebo, diet, and SPE groups, p less than .05 for each group, without significant differences between the groups. There was a mean reduction of low density lipoprotein cholesterol (LDL-C) of 16% in the SPE group (p less than .05), more than twice the reductions in the placebo and diet groups, 5% and 6%, respectively. There was a mean 20% reduction in the SPE group in triglyceride and very low density lipoprotein cholesterol (p less than .05), compared to 7 and 10% reductions in the placebo and diet groups respectively. The degree of weight loss was correlated with the degree of reduction in LDL-C in the low fat diet group, and in the low fat diet group plus SPE (r = 0.59 for both groups). Without confounding by different levels of dietary cholesterol or P/S, SPE induced significant reductions in LDL-C in hypercholesterolemic obese subjects beyond the effects of weight loss alone. The effects of SPE were significantly greater than those achieved by the use of a diet which severely limited conventional dietary fat intake (to 40 g/day). SPE in the form of a bread spread and a salad dressing is a practical formulation for outpatient hypocholesterolemic low fat diets and provides the lubricity and organoleptic benefits of authentic foods without the dense caloric content of digestible fats.

Studies with sucrose polyester.

Sucrose polyester (SPE) is a lipid synthesized from sucrose and fatty acid methyl esters. SPE has physical and organoleptic properties that closely resemble triglycerides, but it is not hydrolysed in the intestine and therefore not absorbed. By providing a persistent lipophilic phase in the intestine, SPE reduces the absorption of lipophilic substances such as cholesterol. SPE leaves the stomach more rapidly than triglyceride oils, presumably because of the absence of an effect on duodenal receptors. In studies with obese subjects, dietary SPE reduced total and LDL cholesterol by reducing the absorption of cholesterol from the intestine. In a study, in which SPE was covertly substituted for dietary fat, ten obese subjects in a weight-loss regimen did not increase dietary intake to compensate for the energy removed from the diet by SPE substitution. In a similar study with five naive, obese subjects, the investigators concluded that the mean energy intake decrease during SPE treatment [184 (769 kJ) kcal/d] was not significant. SPE continues to be studied as a means of reducing energy intake.

A double-blind, placebo-controlled study of sucrose polyester in hypercholesterolemic outpatients.

Sucrose polyester (SPE) was studied in a double-blind, placebo-controlled trial in 91 outpatients with primary hypercholesterolemia. All patients maintained an isocaloric diet with cholesterol intake of 400 mg/day and a polyunsaturated to saturated fat ratio of 0.8 to 1.2 for the duration of the study. The study sequence consisted of a diet lead-in period, a first 8-wk treatment period, a 4-wk washout period, and a second 8-wk treatment period. Subjects were randomly assigned to six groups that differed by SPE dose (8, 16, and 32 g/day) and by the treatment period in which either SPE or an olive oil placebo was given in a bread spread formulation. Compared to placebo, the 8, 16, and 32 g/day doses of SPE decreased low-density lipoprotein cholesterol by 2%, 4% (p less than 0.05), and 5% (p less than 0.05) respectively, without changing high-density lipoprotein cholesterol. On SPE, 14/91 (15%) of the subjects experienced a decrease in low-density lipoprotein cholesterol greater than or equal to 10%, while only 2/91 (2%) showed this decrease with placebo.