Apolipoprotein A-I stimulates the transport of intracellular cholesterol to cell-surface cholesterol-rich domains (caveolae).

We have studied the effect of lipid-free human plasma apolipoprotein A-I (apoA-I) on the transport of newly synthesized cholesterol to cell-surface cholesterol-rich domains, which in human skin fibroblasts are mainly represented by caveolae. Changes in transport of newly synthesized cholesterol were assessed after labelling cells with [(14)C]acetate at 15 degrees C and warming cells to permit the transfer of cholesterol, followed by the selective oxidation of cholesterol in cholesterol-rich domains (caveolae) in the plasma membrane before their partial purification. ApoA-I, but not BSA added in an equimolar concentration, enhanced the transport of cholesterol to the caveolae up to 5-fold in a dose- and time-dependent manner. The effect of apoA-I on cholesterol transport exceeded its effect on cholesterol efflux, resulting in an accumulation of intracellular cholesterol in caveolae. Methyl-beta-cyclodextrin, added at a concentration promoting cholesterol efflux to the same extent as apoA-I, also stimulated cholesterol trafficking, but was 3-fold less effective than apoA-I. Progesterone inhibited the transport of newly synthesized cholesterol to the caveolae. Treatment of cells with apoA-I stimulated the expression of caveolin, increasing the amount of caveolin protein and mRNA by approx. 2-fold. We conclude that apoA-I induces the transport of intracellular cholesterol to cell-surface caveolae, possibly in part through the stimulation of caveolin expression.

Dietary cholesterol is secreted in intestinally derived chylomicrons during several subsequent postprandial phases in healthy humans.

BACKGROUND: The process of intestinal absorption and chylomicron resecretion of dietary cholesterol in humans is poorly understood. OBJECTIVE: The present study aimed to test the hypothesis that dietary cholesterol ingested during a given meal is resecreted into chylomicrons (and plasma) during several subsequent postprandial periods. DESIGN: Seven healthy subjects ingested 3 comparable mixed test meals (at 0, 8, and 24 h) containing a given amount of fat (49 g) and cholesterol (157 mg); blood samples were taken 3 and 6 h after each test meal and 48 and 72 h after the beginning of the experiment. Heptadeuterated dietary cholesterol was present in the first test meal only, enabling its specific determination with use of gas chromatography-mass spectrometry. Chylomicrons, LDL, and HDL were isolated and lipids were quantified. RESULTS: In apolipoprotein B-48-containing chylomicrons, deuterated cholesterol concentrations were moderate after the first meal (1.3 x 10(-4) mmol/L), reached a maximum after the second meal (2.4 x 10(-4) mmol/L), and were still elevated after the third meal (1.7 x 10(-4) mmol/L). In plasma, LDL and HDL cholesterol enrichment in deuterated cholesterol was lower than in chylomicrons and plateaued after 24--48 h. Estimates of newly secreted exogenous deuterated cholesterol in chylomicrons indicate that 30.7%, 55.2%, and 14.1% of the total was secreted after the first, second, and third meals, respectively. CONCLUSION: Ingested dietary cholesterol is secreted by the small intestine in chylomicrons into the circulation during > or =3 subsequent postprandial periods in healthy humans. This likely results from a complex multistep intestinal processing of cholesterol with dietary fat as a driving force.

Method for simultaneous measurements of traces of heptadeuterated cholesterol and cholesterol by gas chromatography-mass spectrometry: application in humans.

An assay was developed to quantify deuterated cholesterol (used as a tracer) and cholesterol using gas chromatography-mass spectrometry. Ergosterol and epicoprostanol were used as internal standards. Deuterated cholesterol was quantified by comparing its peak area to that of epicoprostanol and cholesterol to ergosterol. The mean absolute recovery in spiked serum was 99.96%; the precision was in the range 0.16-10.9% and accuracy 90.4-100%; the limit of detection in plasma was 3x10(-5) mmol l(-1). Using two internal standards, the method described herein seems particularly suitable for application in humans i.e., measuring traces of deuterated cholesterol (range: 0-6.26 x 10(-4) mmol l(-1)) along with natural cholesterol (range: 0.065-4.42 mmol l(-1)) in human plasma and lipid fractions postprandially.

Rapid and sensitive liquid chromatographic method using a conductivity detector for the determination of phytic acid in food.

An LC method was developed for the determination of phytic acid in food. The separation was carried out by gradient elution on an anion-exchange column using a conductivity detector. Earlier reversed-phase LC procedures for the quantitation of phytic acid usually required a prepurification step. The prepurification can be avoided by the separation method described in this paper. The method is sensitive and selective, and can be rapidly and easily performed. It is therefore suitable for routine determination.

Lipid content and fatty acid composition in foods commonly consumed by nursing Congolese women: incidences on their essential fatty acid intakes and breast milk fatty acids.

The fat content and fatty acid (FA) composition of nearly 40 foods, currently consumed by 102 nursing Congolese mothers living in Brazzaville, were determined to assess their impact on mothers' essential fatty acid (EFA) intakes and breast milk FA. Data on mothers' milk FA and dietary habits which allowed food selection were recently published (Rocquelin et al., 1998). Most foods were locally produced. Food samples were collected at local markets, bleached if necessary to avoid microbial degradation, and stored at +4 degrees C or -20 degrees C. They were lyophilized upon their arrival in the laboratory before lipid analyses. FA composition of food lipids was determined by capillary gas chromatography. Staple diets included low-fat, high-carbohydrate foods (processed cassava roots, wheat bread) and high-polyunsaturated fatty acid (PUFA) foods: soybean oil (high in 18 : 2 n-6 and alpha-18 : 3 n-3), bushbutter (dacryodes edulis), peanuts, avocado (high in fat and 18 : 2 n-6), freshwater and salt-water fish (high in LC n-3 and/or n-6 PUFA), and leafy green vegetables (low in fat but very high in alpha-18 : 3 n-3). Their frequent consumption by nursing mothers provided enough EFA to meet requirements due to lactation. It also explains why mothers' breast milk was rich in C8-C14 saturated FA (26% of total FA) and in n-6, n-3 PUFA (respectively 15.0% and 2.4% of total FA) highly profitable for breastfed infants' development. From this point of view, dietary habits of Congolese mothers have to be sustained for they are more adequate than most Western-type diets.

Postprandial studies on dietary cholesterol in human subjects using stable isotopes and gas chromatography-mass spectrometry analysis.

We hypothesized that intestinal absorption and postprandial re-secretion of dietary cholesterol may be a particularly complex process in humans. To test this hypothesis, we used deuterium-enriched cholesterol to specifically label meal cholesterol and developed an improved method for quantitative measurement of traces of deuterated cholesterol as well as cholesterol with reference to two different internal standards by gas chromatography-mass spectrometry (GC-MS) measurement. In the first study, a group of healthy subjects ingested a single test meal containing deuterated cholesterol with a 7 h postprandial follow-up. In the second one, a group of healthy subjects ingested a first test meal containing deuterated cholesterol and a follow-up was performed during three consecutive test meals and later until 72 h. The most striking observations were that the occurrence of dietary cholesterol in chylomicrons is not concomitant to triglycerides and is very low after a single meal while most dietary cholesterol is re-secreted in chylomicrons after a second, and even a third, fat test meal. The data obtained show that the re-secretion of dietary cholesterol from the small intestine is a slow and complex process in humans.

Total dietary restriction and thymus, spleen, and phenotype and function of splenocytes in growing mice.

To study the immunological effect of total dietary restriction, BALB/c mice were limited to reduced body growth or maintained at practically constant body weight between 21 and 61 days of age by giving them 70% (R70%) or 55% (R55%) of the average daily food intake of the control group fed ad libitum. Thymus and spleen weight were decreased, but thymus size was maintained in proportion to body weight in R70% mice, whereas the ratio of thymus weight to body weight was significantly decreased in R55% mice. In restricted mice, splenocytes showed a lower percentage of B lymphocytes and a higher percentage of T lymphocytes. Results of stimulation showed that proliferation capacity was increased for B lymphocytes and decreased for T lymphocytes in restricted mice. Our data show the importance of studying the thymus over a period, because normal thymus growth was first greatly impaired by dietary restriction, and subsequent thymus involution was delayed, showing that an isolated comparison between the thymus of growing mice is not enough. Our data show the interest of determining the percentage of lymphocyte populations parallel to their response to mitogen stimulation.