Secretion of the arginine-rich and A-I apolipoproteins by the isolated perfused rat liver.

Rates of secretion of the arginine-rich and A-I apolipoproteins into perfusates of rat livers were measured by specific radioimmunoassays. Livers were perfused for 6 hr in a recirculating system in the presence or absence of 5,5'-dithionitrobenzoic acid, an inhibitor of lecithin-cholesterol acyltransferase. Arginine-rich apoprotein (ARP) was secreted at a constant or increasing hourly rate of about 40 micro g/g liver, whereas the rate of accumulation of apoprotein A-I decreased progressively from about 12 to less than 5 micro g/g liver. These rates were not affected by inhibition of lecithin-cholesterol acyltransferase. The distribution of these two apolipoproteins was also measured in ultracentrifugally separated lipoprotein fractions from perfusates and blood plasma. Apoprotein A-I was mainly in high density lipoproteins, with the remainder in proteins of density > 1.21 g/ml. The percent of apoprotein A-I in the latter fraction was lowest in plasma (5%); in perfusates it was greater when the enzyme inhibitor was present (33%) than in its absence (11%). By contrast much less ARP was in proteins of d > 1.21 g/ml in perfusates than in blood plasma. Discoidal high density lipoproteins, recovered from perfusates in which lecithin-cholesterol acyltransferase was inhibited, contained much more arginine-rich apoprotein than apoprotein A-I (ratio = 10:1). The ratio in spherical plasma HDL was 1:7 and that in perfusate high density lipoproteins obtained in the absence of enzyme inhibitor was intermediate (2:1). It is concluded that: 1) the arginine-rich apoprotein is a major apolipoprotein whereas apoprotein A-I is a minor apolipoprotein secreted by the perfused rat liver; 2) the properties of the high density lipoproteins produced in this system are remarkably similar to those found in humans with genetically determined deficiency of lecithin-cholesterol acyltransferase.     

Chylomicron apoprotein alteration after plasma exposure

Purified rat lymph chylomicrons were incubated with chylomicron-free rat plasma and examined for changes in lipid and apoprotein constituents. Upon incubation there was a five-fold increase in the arginine rich apoprotein and a concomitant reduction in chylomicron Apo A-I to less than one-sixth its preincubation mass. These apoprotein changes were most faithfully reproduced when chylomicrons were incubated with the rat HDL fraction, although incubations of chylomicrons with rat lipoprotein-free plasma showed that arginine-rich apoprotein could readily associate with chylomicrons without concomitant changes in chylomicron lipid constituents. The gain in chylomicron apoprotein paralleled an increased affinity of the incubated chylomicron for heparin, when examined by heparin affinity chromatography. The apoprotein alterations were consistent in incubations in which the triglyceride concentrations varied from 330 mg/dl to 4200 mg/dl, and were not affected by inhibition of the Lecithin:Cholesterol Acyl Transferase (LCAT) reaction in the incubation mixture. The demonstration that in vivo alimentary lipemia chylomicrons have an apoprotein pattern identical to that of chylomicrons following in vitro plasma incubation suggests that these apoprotein alterations occur physiologically in alimentary lipemia.     

Human chylomicron apolipoprotein metabolism.

Chylomicron apolipoprotein metabolism was studied utilizing chylomicrons isolated from the pleural fluid of a patient with a recurrent chylous pleural effusion. Chylomicrons contained apolipoproteins A-I, A-II, B, C-I, C-II, C-III, D, E, and albumin. Following intravenous injection of [¹²⁵I] chylomicrons, almost all of the A apolipoprotein radioactivity was recovered in high density lipoproteins, while only a small amount of the B apolipoprotein radioactivity was recovered in low density lipoproteins. These observations indicate that intestinal chylomicron A apolipoproteins serve as precursors for plasma high density lipoprotein A apolipoproteins and only a small fraction of chylomicron apolipoprotein B is metabolized to form low density lipoprotein apolipoprotein B.     

Inhibitory effects of C apolipoproteins from rats and humans on the uptake of triglyceride-rich lipoproteins and their remnants by the perfused rat liver

Like rat C apolipoproteins, each of the C apolipoproteins from human blood plasma (C-I, C-II, C-III-1, and C-III-2) bound to small chylomicrons from mesenteric lymph of estradiol-treated rats and inhibited their uptake by the isolated perfused rat liver. This inhibitory effect of the C apolipoproteins was independent of apolipoprotein E, which is present only in trace amounts in these chylomicrons. Addition of rat apolipoprotein E to small chylomicrons from mesenteric lymph of normal rats did not displace C apolipoproteins and had no effect on the uptake of these particles by the perfused liver, indicating that an increased ratio of E apolipoproteins to C apolipoproteins on chylomicron particles, unaccompanied by depletion of the latter, may not promote recognition by the chylomicron remnant receptor. The hepatic uptake of remnants of rat hepatic very low density lipoproteins (VLDL) and small chylomicrons, which had been produced in functionally eviscerated rats, was also inhibited by addition of C apolipoproteins. These observations are consistent with the hypothesis that the addition of all of the C apolipoproteins to newly secreted chylomicrons and VLDL inhibits premature uptake of these particles by the liver and that depletion of all of these apolipoproteins from remnant particles facilitates their hepatic uptake. Remnants of chylomicrons and VLDL incubated with rat C apolipoproteins efficiently took up C-III apolipoproteins, but not apolipoprotein C-II (the activator protein for lipoprotein lipase). Preferential loss of apolipoprotein C-II during remnant formation may regulate the termination of triglyceride hydrolysis prior to complete removal of triglycerides from chylomicrons and VLDL.     

Intestinal lipoprotein synthesis. Comparison of nascent Golgi lipoproteins from chow-fed and hypercholesterolemic rats

Hypercholesterolemia, induced by a cholesterol-enriched diet, is associated with distinctive modifications in the serum lipoproteins of a variety of species. Present in the serum of these animals are several classes of lipoproteins enriched in cholesteryl esters and apolipoprotein E. To investigate the role of intestinal lipoprotein synthesis in diet-induced hypercholesterolemia, we characterized nascent lipoproteins retrieved from Golgi apparatus-rich fractions of intestinal epithelial cells from chow-fed control and hypercholesterolemic rats. To eliminate chylomicrons from the preparations, rats were fasted overnight prior to the experiments. Golgi very low density lipoproteins (d less than 1.006 g/ml) from control rats were triglyceride-rich lipoproteins that migrated slightly slower than pre-beta migrating serum very low density lipoproteins. These particles contained apoproteins B-240, A-IV, and A-I. Golgi very low density lipoproteins from hypercholesterolemic rats were likewise triglyceride-rich lipoproteins migrating electrophoretically like control Golgi very low density lipoproteins and they contained apoproteins B-240, A-IV, and A-I. However, these latter particles contained less triglyceride and more cholesterol compared to control Golgi very low density lipoproteins. In addition, by radioisotope incorporation studies, Golgi very low density lipoproteins from hypercholesterolemic rats contained relatively more apoprotein A-IV (21.6 vs. 11.0%) and less apoprotein B-240 (17.0 vs. 27.0%) than found in control Golgi very low density lipoproteins. Approximately 60% of the total apoprotein radioactivity was found in apoprotein A-I in both preparations. We conclude that intestinal lipoprotein synthesis is modified by diet-induced hypercholesterolemia. The significance of these modifications with respect to the marked hypercholesterolemia observed in these animals remains to be determined.     

Comparison of the metabolic behavior of rat apolipoproteins A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins

Rat apolipoprotein (apo) A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins (HDL) were analyzed by isoelectric focusing. Lymph chylomicron apo A-I consisted for 81 +/- 2% of the pro form and for 19 +/- 2% of the mature form, while apo A-I isolated from serum HDL was present for 36 +/- 4% in the pro form and for 64 +/- 4% in the mature form. Apo A-IV also showed two major protein bands after analysis by isoelectric focusing. The most prominent component is the more basic protein that amounts to 80 +/- 2% in apo A-IV isolated from lymph chylomicrons and to 60 +/- 3% in apo A-IV isolated from serum HDL. Apo A-I (or apo A-IV), isolated from both sources (lymph chylomicrons or serum HDL), was iodinated and the radioactive apolipoproteins were incorporated into rat serum lipoproteins. The resulting labeled HDL was isolated from serum by molecular sieve chromatography on 6% agarose columns and injected intravenously into rats. No difference in the fractional turnover rate or the tissue uptake of the two labeled HDL preparations was observed, neither for apo A-I nor for apo A-IV. It is concluded that the physiological significance of the extracellular pro apo A-I conversion or the post-translational modification of apo A-IV is not related to the fractional turnover rate in serum or to the rate of catabolism in liver and kidneys.     

Radioimmunoassay of apolipoprotein A-I of rat serum.

A double antibody radioimmunoassay technique was developed for quantification of apolipoprotein A-I, the major apoprotein of rat high density lipoprotein. Apo A-I was labeled with 125I by the chloramine-T method. 125I-labeled apo A-I had the same electrophoretic mobility as unlabeled apo A-I and more than 80% of the 125I was precipitated by rabbit anti apo A-I antibodies. The assay is sensitive at the level of 0.5-5 ng, and has intraassay and interassay coefficients of variation of 4.5 and 6.5% respectively. The specificity of the assay was established by competitive displacement of 125I-labeled apo A-I from its antibody by apo A-I and lipoproteins containing apo A-I, but not by rat albumin and other apoproteins. Immunoreactivity of high density lipoprotein and serum was only about 35% of that of their delipidated forms when Veronal buffer was used as a diluent. Inclusion of 5 mM sodium decyl sulfate in the incubation mixture brought out reactivity equivalent to that found after delipidation. Completeness of the reaction was verified by comparison with the amount of apo A-I in chromatographic fractions of the total apoprotein of high density lipoprotein. Content (weight %, mean values +/- S.D.) of immunoassayable apo A-I was: 62.3 +/- 5.9 in high density lipoprotein; 1.7 +/- 0.3 in low density lipoprotein; 0.09 +/- 0.03 in very low density lipoprotein and 25.0 +/- 5.0 in lymp chylomicrons. Concentration in whole serum was 51.4 +/- 8.9 mg/dl and 33.6 +/- 4.1 mg/dl for female and male rats, respectively (p less than 0.002), equivalent to the sex difference in concentration of high density lipoprotein. 95% of the apo A-I in serum was in high density lipoprotein, 5% in proteins of d greater than 1.21 g/ml and less than 1% in lipoproteins of d less than 1.063 g/ml.     

Composition, removal and metabolic fate of chylomicrons derived from diabetic rats

Tri[14C]acylglycerol-labelled chylomicrons, obtained from cannulated mesenteric lymph of streptozotocin-diabetic donor rats, when intravenously injected into non-diabetic recipient rats, disappeared from the circulation at a significantly slower rate than similarly prepared tri[14C]acylglycerol chylomicrons from non-diabetic donor rats (t1/2, 5.6 +/- 0.7 vs. 3.2 +/- 0.5 min-1, P less than 0.02). The appearance of labelled lipolysis products among plasma lipids (free fatty acid, cholesterol ester and phospholipid fractions) was delayed, indicating decreased availability for lipolysis of the chylomicron-borne triacylglycerol of diabetic origin. Tissue distribution of triacylglycerol, 15 min after the injection of chylomicrons to recipient rats, disclosed a 4-5-fold increase in uptake by muscles (heart and diaphragm) in relation to adipose tissues (epididymal and perirenal sites), in the case of chylomicrons of diabetic derivation. Since a large share of the chylomicron triacylglycerol was taken up by the liver, this tissue was perfused with chylomicron 'remnants' prepared by partial in vitro lipolysis with purified lipoprotein lipase. The 'remnants' of diabetic derivation were taken up by the liver at a 2-3-fold slower rate than those of non-diabetic origin. Chylomicrons derived from diabetic rats were found to be similar in size but markedly depleted of E apolipoproteins as determined by SDS-polyacrylamide gel electrophoresis, isoelectric focussing and a specific immunoassay. Decreases were also seen in A-I apolipoproteins by immunoassay and isoelectric focussing. Chylomicron 'remnants' were also markedly apolipoprotein E-deficient. In vitro incubation of the 'diabetic remnants' with high-density lipoproteins raised their apolipoprotein E content approx. 3-fold and considerably increased their hepatic uptake. Injection of intact chylomicrons preincubated with high-density lipoproteins likewise increased their in vivo removal rate toward the range of that of 'non-diabetic' chylomicrons. We conclude that diabetes-induced changes in the apolipoprotein composition of the chylomicrons and chylomicron remnants play an important role in their removal from the circulation. It appears that their recognition pattern is altered, reducing their ability to interact with receptor sites in the peripheral tissues and the liver, respectively.     

Distribution of apolipoproteins A-I and B among intestinal lipoproteins

Chylomicrons and very low density lipoproteins (VLDL) are produced by the intestine and these nascent particles are thought to be similar to their counterparts in intestinal lymph. To study the relationship between these lipoproteins within the cell and those secreted into the lamina propria and lymph, we have isolated enterocytes, lamina propria, and mesenteric lymph from rats while fasted and after corn oil feeding. Apolipoprotein A-I and B content were measured by radioimmunoassay in cell, lamina propria, and lymph fractions separated by Sepharose 6B and 10% agarose chromatography, and by KBr isopycnic density centrifugation. ApoA-I in the cell and the underlying lamina propria was found partly in those fractions in which chylomicron and very low density lipoproteins (chylo-VLDL) and high density lipoproteins (HDL) elute, but more abundantly where unassociated 125I-labeled apoA-I was eluted. In the lymph, however, 74% of apoA-I eluted in the HDL region and no peak of free apoA-I was found. ApoB and apoC-III within the enterocyte were found distributed in the position of particles eluting not only with chylomicrons and VLDL, but also in the regions corresponding to LDL and HDL. In the lamina propria and lymph, on the other hand, most of the apoB was found in the region of VLDL and chylomicrons. These results indicate that the patterns in lymph lipoproteins and the lamina propria do not exactly mirror the distribution of apoA-I and B among lipoproteins inside the cell. This may be because intracellular apoproteins may be unassociated with lipoproteins, or they could be associated with lipoproteins in various stages of assembly of protein with lipids. Furthermore, the apoprotein composition of intestinal lipoproteins is altered after secretion from the enterocyte. Finally, not all apoproteins seem to be secreted in association with identifiable lipoprotein particles from the enterocyte.     

Radioimmunoassay of arginine-rich apolipoprotein of rat serum.

A double-antibody radioimmunoassay was developed for quantification of rat arginine-rich apolipoprotein in sodium decyl sulfate. Arginine-rich protein, labeled with 125I by the chloramine-T method, had the same chromatographic characteristics on Sephadex G-200 as unlabeled arginine-rich protein and up to 70% of 125I-labeled arginine-rich protein was precipitated by antisera to arginine-rich protein in rabbits. The assay is sensitive at the level of 1-10 ng and has intraassay and interassay coefficients of variation of 5.4 and 6.8%, respectively. The specificity of the assay was established by competitive displacement of 125I-labeled arginine-rich protein from its antiserum by arginine-rich protein and lipoproteins containing this protein, but not by rat albumin or other purified apolipoproteins. Immunoreactivity of rat serum and lipoproteins was complete as demonstrated by comparison with their delipidated form. The accuracy of the immunoassay was further substantiated by comparison with the amount of arginine-rich protein in chromatographic fractions of total apoprotein of very low and high density lipoproteins, and by recovery experiments in ultracentrifugally separated fractions of serum. In contrast to an immunoassay reported previously for rat apo A-I, sodium decyl sulfate was not required for complete immunoreactivity of serum and lipoproteins. The inclusion of sodium decyl sulfate (9 mM final concentration) was necessary, however, for stability of labeled and unlabeled preparations of arginine-rich protein. Content (weight %, means values +/- S.D.), of immunoassayable arginine-rich protein in isolated lipoproteins was 15 +/- 1.5% in very density lipoproteins; 6.8% in low density lipoproteins (1.02 less than d less than 1.04 g/m); 7.1 +/- 0.3% in high density lipoproteins; and 4.8 +/- 0.5% in lymph chylomicrons. Concentration in whole serum was 18.1 +/- 1.4 and 20.4 +/- 2.3 mg/dl for male and female rats, respectively. Only about 55% of arginine-rich protein was recovered in the major lipoprotein classes and about 40% was in "lipoprotein-free" serum (d greater than 1.25 g/ml). Among the lipoproteins, the high density lipoprotein fraction contained twice the amount of arginine-rich protein recovered in very low or low density lipoproteins (26.6 vs. 13.5 and 13.4%, respectively). The significance of the large amount of arginine-rich protein in the 1.25 g/ml infranatant fraction is not apparent. Although repetitive centrifugation did not alter the amount recovered in this fraction, the possibility of an artifact induced by centrifugation and high salt concentration cannot be excluded.     

The lecithin-cholesterol acyl transferase activity of rat intestinal lymph.

The lecithin-cholesterol acyl transferase (LCAT) activity in rat mesenteric lymph was examined as a possible source of chylomicron cholesteryl ester. Lymph activity was only 2-3% of rat serum activity. Removal of d less than 1.006 lipoproteins increased lymph LCAT activity, but only to 6-8% of that of serum. Relative to total cholesterol in the d greater than 1.08 g/ml fractions, lymph LCAT activity in lymph from fasting rats was less than serum, but in lymph from nonfasting rats the ratio LCAT/HDL-cholesterol reached levels greater than serum, suggesting a contribution of enzyme from the gut. Both LCAT activity and HDL concentration in mesenteric lymph increased during feeding. Subfractions of lymph that inhibited serum LCAT were: chylomicrons, VLDL, chylomicron lipid, VLDL apoprotein, and HDL apoprotein. In the rat, the low LCAT activity of mesenteric lymph was in part due to the low enzyme concentration present, and the activity was apparently lowered further by lipid-rich lipoproteins that inhibited the reaction. Enzyme inhibition due to the apoprotein fractions of lipoproteins is probably minor in the rat in vivo.     

Intracellular apoA-I and apoB distribution in rat intestine is altered by lipid feeding

Intracellular forms of chylomicrons, very low density lipoprotein (VLDL) and high density lipoprotein (HDL) have previously been isolated from the rat intestine. These intracellular particles are likely to be nascent precursors of secreted lipoproteins. To study the distribution of intracellular apolipoprotein among nascent lipoproteins, a method to isolate intracellular lipoproteins was developed and validated. The method consists of suspending isolated enterocytes in hypotonic buffer containing a lipase inhibitor, rupturing cell membranes by nitrogen cavitation, and isolating lipoproteins by sequential ultracentrifugation. ApoB and apoA-I mass are determined by radioimmunoassay and newly synthesized apolipoprotein characterized following [3H]leucine intraduodenal infusion. Intracellular chylomicron, VLDL, low density lipoprotein (LDL), and HDL fractions were isolated and found to contain apoB, and apoA-IV, and apoA-I. In the fasted animal, less than 10% of total intracellular apoB and apoA-I was bound to lipoproteins and 7% of apoB and 35% of apoA-I was contained in the d 1.21 g/ml infranatant. The remainder of intracellular apolipoprotein was in the pellets of centrifugation. Lipid feeding doubled the percentage of intracellular apoA-I bound to lipoproteins and increased the percentage of intracellular apoB bound to lipoproteins by 65%. Following lipid feeding, the most significant increase was in the chylomicron apoB and HDL apoA-I fractions. These data suggest that in the fasting state, 90% of intracellular apoB and apoA-I is not bound to lipoproteins. Lipid feeding shifts intracellular apolipoprotein onto lipoproteins, but most intracellular apolipoprotein remains non-lipoprotein bound. The constant presence of a large non-lipoprotein-bound pool suggests that apolipoprotein synthesis is not the rate limiting step in lipoprotein assembly or secretion.     

Interaction of plasma apolipoproteins with lipid monolayers.

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidy[14C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipids by apolipoprotein C-III. The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface. Although apolipoprotein A-I and arginine-rich protein gave surface pressure increases, phospholipid was only slightly removed fromthe interface by the addition of liposomes. Based on these findings, we conclude that the apolipoproteins C interact specifically with phosphatidylcholine at the interface. This interaction is important as it relates to the transfer of the apolipoproteins C and phospholipids from very low density lipoproteins to other plasma lipoproteins. The addition of human plasma high density lipoproteins or very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of phosphatidyl[14C]choline from the interface 3--4 fold. Low density lipoproteins did not affect the rate of decrease. During lipolysis of very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of with the lipid monolayer. Lipolysis experiments were performed in a monolayer trough containing a surface film of egg phosphatidyl[14C]choline and a subphase of very low density lipoproteins and bovine serum albumin. Lipolysis was initiated by the addition of purified milk lipoprotein lipase to the subphase. As a result of lipolysis, there was a decrease in surface radioactivity of phosphatidylcholine. The pre-addition of high density lipoproteins decreased the rate of decrease in surface radioactivity...     

Interaction of plasma apolipoproteins with lipid monolayers

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidyl[¹⁴C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipid by apolipoprotein C-III.The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface.     

Radioimmunoassay of human high density lipoprotein apo-protein A-1.

A double antibody radioimmunoassay technique was developed for the measurement of apolipoprotein A-I, the major apoprotein of human high density lipoproteins. Apolipoprotein A-I was prepared from human delipidated high density lipoprotein (d equal to 1.085-1.210) by gel filtration and ion-exchange chromatography. Purified apolipoprotein A-I antibodies were obtained by means of apolipoprotein A-I immunoadsorbent. Apolipoprotein A-I was radiolabeled with 125-I by the iodine monochloride technique. 65-80% of 125 I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125-I-labeled apolipoprotein A-I was displaced by unlabeled apolipoprotein A-I. The immunoassay was found to be sensitive for the detection of about 10 ng of apolipoprotein A-I in the incubation mixture, and accurate with a variability of only 3-5% (S.E.M.). This technique enables the quantitation of apolipoprotein A-I in whole plasma or high density lipoprotein without the need of delipidation. The quantitation of apolipoprotein A-I in high density lipoprotein was found similar to that obtained by gel filtration technique. The displacement capacity of the different lipoproteins and apoproteins in comparison to unlabeled apolipoprotein A-I was: very low density lipoprotein, 1.8%; low density lipoprotein, 2.6%; high density lipoprotein, 68%; apolipoprotein B, non-detectable; apolipoprotein C, 0.5%; and apolipoprotein A-II, 4%. The distribution of immunoassayable apolipoprotein A-I among the different plasma lipoproteins was as follows: smaller than 1% in very low density lipoprotein and low density lipoprotein; 50% in high density lipoprotein, and 50% in lipoprotein fraction of density greater than 1.21 g/ml. The amount of apolipoprotein A-I in the latter fraction was found to be related to the number of centrifugations.     

Apolipoprotein A-IV: a protein occurring in human mesenteric lymph chylomicrons and free in plasma. Isolation and quantification.

1. Human mesenteric lymph chylomicrons were isolated from chylous ascites fluid by ultra-centrifugation and agarose/gel chromatography and their apoprotein composition was analysed by dodecylsulfate/polyacrylamide gel electrophoresis, analytical isoelectric focusing and immuno-chemically. Major components of mesenteric lymph chylomicrons were apoprotein A-I, proteins of Mr less than 15 000 including the C-group apoproteins and a protein of Mr 46 000. Minor components were apoprotein E and a protein of Mr approximately equal to 200 000 (B-like protein). This apoprotein composition was qualitatively identical with that of chylomicrons from intestinal lymph of the rat, but was distinctly different from plasma chylomicrons of humans with fasting chylomicronaemia. 2. The protein of Mr approximately equal to 46 000 has been isolated by preparative dodecylsulfate/polyacrylamide gel electrophoresis from human and rat lymph chylomicrons and was compared to a protein of identical Mr present in rat high-density lipoproteins (apoplipoprotein A-IV) and in the rho less than 1.006 g/ml serum lipoprotein fraction of individual humans with alimentary hypertriglyceridaemia. In both species the 46 000-Mr proteins isolated from lymph and serum were identical according to amino acid composition and isoelectric point in 6 M urea. The human proteins from both sources were also immunologically identical. The similarities in the molecular properties of the human apolipoprotein and rat apolipoprotein A-IV indicate that these proteins are homologous. 3. Plasma levels of human apolipoprotein A-IV determined by electroimmunodiffusion were 14.15 +/- 3.66 mg/100 ml (n = 59), but greater than 90% of the protein was unassociated with the major lipoprotein fractions. It is concluded, that apolipoprotein A-IV is a main protein component of human lymph chylomicrons, that is removed from the particles in the plasma compartment.     

Alterations of the plasma lipoproteins and apoproteins following cholesterol feeding in the rat.

The feeding of cholesterol to rats resulted in marked alterations in the type and distribution of the plasma lipoproteins and their apoproteins. The hyperlipoproteinemia was characterized by an increase in the d < 1.006 lipoproteins (B-VLDL and VLDL), an increase in the intermediate and low density lipoproteins (LDL), and the appearance of HDL(c). Associated with these lipoproteins was a prominence of the arginine-rich apoprotein. The high density lipoproteins (HDL) were decreased. A two-dimensional immunoelectrophoretic procedure was adapted to quantitate the changes in distribution of the arginine-rich apoprotein in the plasma and various ultracentrifugal fractions obtained from control and cholesterol-fed rats. In rats fed the cholesterol diet, the total plasma arginine-rich apoprotein increased from a control value of approximately 29 mg/dl to 47 mg/dl. The method of ultracentrifugation, however, was found to markedly alter the quantitative results. When the 60 Ti rotor was used at maximum speed to isolate the ultracentrifugal fractions, less than 50% of the total plasma arginine-rich apoprotein was associated with the lipoproteins in the d < 1.006 or the d 1.006-1.02, 1.02-1.063, or 1.063-1.21 ultracentrifugal fractions. By contrast, after limited ultracentrifugation with the 40 rotor, much less arginine-rich apoprotein was lost, with approximately 20% of the arginine-rich apoprotein in control rats and 10% in cholesterol-fed rats found in the d > 1.21 fraction. Significant alterations in the arginine-rich apoprotein quantitation notwithstanding, the observations of increased arginine-rich apoprotein in the B-VLDL, intermediate fraction, and HDL(c) following cholesterol feeding remained valid. However, precise quantitation awaits refinements in lipoprotein isolation techniques.     

Development and physiological regulation of intestinal lipid absorption. I. Development of intestinal lipid absorption: cellular events in chylomicron assembly and secretion

The newborn mammal must efficiently absorb dietary fat, predominantly as triacylglycerol, and produce chylomicrons to deliver this lipid to peripheral tissues. The cellular mechanisms involved in enterocyte chylomicron assembly have recently been elucidated, and data on their regulation in the immature gut are beginning to emerge. This review focuses on key proteins involved in chylomicron assembly: apolipoprotein B-48, microsomal triglyceride transfer protein, and apolipoprotein A-IV. Recent studies support a role for apolipoprotein A-IV in enhancing chylomicron secretion by promoting production of larger particles. These proteins are regulated in a manner to maximize the lipid absorptive capacity of the newborn intestine.     

Identification and partial characterization of discrete apolipoprotein A-containing lipoprotein particles secreted by human hepatoma cell line HepG2

The purpose of this study was to identify the apolipoprotein A-containing lipoprotein particles produced by HepG2 cells. The apolipoprotein A-containing lipoproteins separated from apolipoprotein B-containing lipoproteins by affinity chromatography of culture medium on concanavalin A were fractionated on an immunosorber with monoclonal antibodies to apolipoprotein A-II. The retained fraction contained apolipoproteins A-I, A-II and E, while the unretained fraction contained apolipoproteins A-I and E. Both fractions were characterized by free cholesterol as the major and triglycerides and cholesterol esters as the minor neutral lipids. Further chromatography of both fractions on an immunosorber with monoclonal antibodies to apolipoprotein A-I showed that 1) apolipoprotein A-II only occurs in association with apolipoprotein A-I, 2) apolipoprotein A-IV is only present as part of a separate lipoprotein family (lipoprotein A-IV), and 3) apolipoprotein E-enriched lipoprotein A-I:A-II and lipoprotein A-I are the main apolipoprotein A-containing lipoproteins secreted by HepG2 cells.     

Involvement of intestinal alkaline phosphatase in serum apolipoprotein B-48 level and its association with ABO and secretor blood group types

Serum levels of intestinal alkaline phosphatase (IAP), a protein implicated in transcellular transport of chylomicrons, vary among ABO blood groups. In rat enterocytes, IAP is associated with chylomicron secretion, but the rat expresses only blood group A. It is not known whether chylomicron secretion may be affected in humans who express multiple blood group types. Serum samples from 40 healthy subjects were obtained after overnight fast and 3h after a high-fat meal, and assayed for IAP and apolipoprotein B-48 (apoB-48), both proteins exclusive to intestine, although only apoB-48 is found in chylomicrons. The two proteins were greater in subjects without blood antigen A (B and O) than in those with this antigen (A and AB); 2.4- and 4.7-fold for IAP and 1.5- and 2.0-fold for apoB-48 before and after the meal, respectively. Moreover, IAP and apoB-48 levels were strongly correlated in the subjects with the secretor phenotype (r > 0.81). These results indicate that IAP is strongly involved in chylomicron formation and fatty acid metabolism might change among ABO blood type. In addition, ABO blood type classification in apoB-48 measurement would improve the diagnostic value in the evaluation of metabolic syndrome.