Robert M. Glickman

Apolipoprotein Synthesis in Normal and Abetalipoproteinemic Intestinal Mucosa

The genetic disease abetalipoproteinemia is characterized by a total absence of apolipoprotein B-containing lipoproteins from plasma. A presumed synthetic defect in apolipoprotein B synthesis was thought to be responsible for this disorder. The present study quantitates apoprotein B synthesis and apolipoprotein B messenger RNA levels in duodenal mucosa from normal patients and four patients with abetalipoproteinemia. After in vitro [3H]leucine incorporation, small intestinal biopsy specimens from three of four patients with abetalipoproteinemia synthesized immunoprecipitable apolipoprotein B of identical mobility (on sodium dodecyl sulfate gel electrophoresis) to normal apolipoprotein B. In abetalipoproteinemia, the apolipoprotein B content of intestinal mucosa by radioimmunoassay was 15% of normal mucosal values, whereas apolipoprotein B messenger RNA quantitation showed 3-20-fold increased levels compared with normal mucosa. In one patient, smaller-molecular-weight fragments of apolipoprotein B were immunoprecipitated from duodenal biopsy specimens. The synthesis rates and messenger RNA levels of two other chylomicron apoproteins (apolipoprotein A-I and apolipoprotein A-IV) were found to be reduced by 50%. These results show the synthesis of immunologically recognizable apolipoprotein B48 in abetalipoproteinemia. The significance of mucosal apolipoprotein B content in abetalipoproteinemia is discussed in terms of factors controlling apolipoprotein B synthesis in normal mucosa and in abetalipoproteinemia.

A method to screen apolipoprotein polymorphisms in whole plasma: description of apolipoprotein A-IV variants in dyslipidemias and a reassessment of apolipoprotein A-I in Tangier disease

A sensitive and rapid immunological detection method was used to screen for apolipoprotein A-IV variants. Antibodies to human lymph chylomicron or plasma apolipoprotein A-IV, and plasma apolipoprotein A-I were raised in rabbits. Antibodies to apolipoprotein A-I or apolipoprotein A-IV were shown to be monospecific to their respective antigens by reactivity against human chylomicron apolipoproteins by immunoblot analysis. Plasma samples were obtained from dyslipidemic subjects from the Lipid Research Clinic of Columbia University. The plasma samples were isoelectrically focused (pH 4-6) on slab gels. Plasma proteins were then transferred to nitrocellulose paper for immunoblotting. Apolipoprotein A-IV polymorphism was determined by specific immunological detection of apolipoprotein A-IV. Identical apolipoprotein A-IV isoprotein patterns were observed when either antibodies to lymph or plasma apolipoprotein A-IV were used for immunoblotting. All the dyslipidemic plasma samples screened contained the two major and one or two minor isoproteins of normal plasma. In two instances, new apolipoprotein A-IV variants having an additional isoform were detected. One subject was hypertriglyceridemic (triacylglycerols = 342 mg/dl, cholesterol = 251 mg/dl) and had an additional major acidic apolipoprotein A-IV isoform. Another subject with mild hypocholesterolemia (triacylglycerols = 209 mg/dl, cholesterol = 120 mg/dl) was found to have additional major and minor basic apolipoprotein A-IV isoforms. The specificity of this technique allows detection of polymorphism of apolipoproteins of similar isoelectric points by use of a single dimension isoelectric focusing gel. This technique also demonstrated the presence of altered apolipoprotein A-I isoforms in the plasma of a patient with Tangier disease. These isoforms were previously identified as isoforms 2 and 4 of normal plasma by use of two-dimensional gel electrophoresis. However, by use of this new technique and careful evaluation of previously published two-dimensional gels, we now identify these apolipoprotein A-I isoforms as being more acidic than those of normal plasma.

Intestinal Lipoprotein Formation

The average western diet contains approximately 40% of total calories as dietary fat or approximately 100 g of fat. The efficiency of the entire process of fat absorption can be judged by the fact that under normal conditions less than 5% of ingested fat is recovered in the stool. In the past several years, new concepts have greatly added to our understanding of the process by which dietary fat is digested, absorbed and processed in the intestinal epithelial cell for delivery to the body via the intestinal lymph and the portal venous system. These newer concepts include an understanding of the physical chemistry of lipids, the physiology of bile salts and the formation and metabolisms of lipoprotein all directly influencing the process of fat absorption. The present discussion will emphasize the formation of lipoproteins within the intestinal mucosa. New information suggests that the small intestinal mucosa is a quantitatively important source of lipoprotein constituents for systemic lipoproteins. This is hardly surprising when one considers the large quantities of lipid transversing the intestinal mucosa each day which must exit in the form of lipoproteins.

Apoprotein Secretion from the Human Intestine

There is mounting evidence that the small intestine is a quantitatively important source of lipoprotein constituents (apoproteins and phospho- lipids) which eventuate in circulating lipoproteins. This is especially true of apoproteins which are directly synthesized by the intestine and secreted as lipoprotein constituents. Recent work in our laboratory suggests that for certain of these apoproteins the intestine synthesizes and secretes from 30–50% of the total daily synthesis of apoproteins such as apoA-I and apoA-II. We will summarize the evidence for these conclusions.

Products of delipidation of intestinal chylomicrons with diethyl ether

Abstract To investigate potential modes of dissociation of their surface constituents, chylomicrons, obtained from human subjects with chyluria or from rat mesenteric lymph, were delipidated with diethyl ether. The ether dissolved most of the chylomicron apolar lipids. The polar surface constituents remaining in the acqueous phase were incorporated into discrete “low” and “high density” lipoproteins, as judged by preparative ultracentrifugation or agarose gel chromatography. The density 1.006–1.063 particles consisted primarily of phospholipid, apoA-I, apoA-IV and C-apoproteins and the density 1.063–1.21 particles of phospholipid, ApoA-I and apoA-IV. The density 1.006–1.063 particles appeared as phospholipid vesicles when examined by negative stain electron microscopy. When chylomicrons were delipidated in the presence of [ 3 H]glucose, radioactivity was associated with the vesicles, consistent with trapping of [ 3 H]glucose in their internal aqueous space. In further experiments human plasma high density lipoproteins (HDL) were added to the aqueous phase after delipidation of chylomicrons with diethyl ether. Following incubation for 1h, vesicle phospholipids, apoA-I and apoA-IV were largely incorporated into the column HDL region, with release of vesicle-associated [ 3 H]glucose. Following incubation for 16 h, part of this HDL fraction was transformed into a larger lipoprotein, enriched in apoE and apoA-II. Solvent delipidation of the chylomicron core leads to dissociation of the surface polar lipids primarily as lipid bilayer vesicles containing adsorbed water-soluble apoproteins. Upon interaction with plasma HDL, there is disruption of the vesicle structure and incorporation of phospholipid and probably apoA-I and apoA-IV into HDL-like particles.