Lymph High Density Lipoproteins Research Articles

Organ-specific lymphatics play distinct roles in regulating HDL trafficking and composition.

Recently, peripheral lymphatic vessels were found to transport high-density lipoprotein (HDL) from interstitial tissues to the blood circulation during reverse cholesterol transport. This function is thought to be critical to the clearance of cholesterol from atherosclerotic plaques. The role of organ-specific lymphatics in modulating HDL transport and composition is, however, incompletely understood. This study aimed to 1) determine the contribution of the lymphatics draining the intestine and liver (which are major sites of HDL synthesis) to total (thoracic) lymph HDL transport and 2) verify whether the HDLs in lymph are derived from specific organs and are modified during trafficking in lymph. The mesenteric, hepatic, or thoracic lymph duct was cannulated in nonfasted Sprague-Dawley rats, and lymph was collected over 5 h under anesthesia. Whole lymph and specific lymph lipoproteins (isolated by ultracentrifugation) were analyzed for protein and lipid composition. The majority of thoracic lymph fluid, protein, and lipid mass was sourced from the mesenteric, and to a lesser extent, hepatic lymph. Mesenteric and thoracic lymph were both rich in chylomicrons and very low-density lipoprotein, whereas hepatic lymph and plasma were HDL-rich. The protein and lipid mass in thoracic lymph HDL was mostly sourced from mesenteric lymph, whereas the cholesterol mass was equally sourced from mesenteric and hepatic lymph. HDLs were compositionally distinct across the lymph sources and plasma. The composition of HDL also appeared to be modified during passage from the mesenteric and hepatic to the thoracic lymph duct. Overall, this study demonstrates that the lipoproteins in lymph are organ specific in composition, and the intestine and liver appear to be the main source of HDL in the lymph.NEW & NOTEWORTHY High-density lipoprotein in lymph are organ-specific in composition and derive mostly from the intestine and liver. High-density lipoprotein also appears to be remodeled during transport through the lymphatics. These findings have implications to cardiometabolic diseases that involve perturbations in lipoprotein distribution and metabolism.

Mechanism and Physiologic Significance of the Suppression of Cholesterol Esterification in Human Interstitial Fluid

Cholesterol esterification in high density lipoproteins (HDLs) by lecithin:cholesterol acyltransferase (LCAT) promotes unesterified cholesterol (UC) transfer from red cell membranes to plasma in vitro. However, it does not explain the transfer of UC from most peripheral cells to interstitial fluid in vivo, as HDLs in afferent peripheral lymph are enriched in UC. Having already reported that the endogenous cholesterol esterification rate (ECER) in lymph is only 5% of that in plasma, we have now explored the underlying mechanism. In peripheral lymph from 20 healthy men, LCAT concentration, LCAT activity (assayed using an optimized substrate), and LCAT specific activity averaged, respectively, 11.8, 10.3, and 84.9% of plasma values. When recombinant human LCAT was added to lymph, the increments in enzyme activity were similar to those when LCAT was added to plasma. Addition of apolipoprotein AI (apo AI), fatty acid-free albumin, Intralipid, or the d < 1.006 g/ml plasma fraction had no effect on ECER. During incubation of lymph plus plasma, the ECER was similar to that observed with buffer plus plasma. When lymph was added to heat-inactivated plasma, the ECER was 11-fold greater than with lymph plus buffer. Addition of discoidal proteoliposomes of apo AI and phosphatidycholine (PC) to lymph increased ECER 10-fold, while addition of apo AI/PC/UC disks did so by only six-fold. We conclude that the low ECER in lymph is due to a property of the HDLs, seemingly substrate inhibition of LCAT by excess cell-derived UC. This is reversed when lymph enters plasma, consequent upon redistribution of UC from lymph HDLs to plasma lipoproteins.

Selective evaluation of high density lipoprotein from mouse small intestine by an in situ perfusion technique

The small intestine (SI) is the second-greatest source of HDL in mice. However, the selective evaluation of SI-derived HDL (SI-HDL) has been difficult because even the origin of HDL obtained in vivo from the intestinal lymph duct of anesthetized rodents is doubtful. To shed light on this question, we have developed a novel in situ perfusion technique using surgically isolated mouse SI, with which the possible filtration of plasma HDL into the SI lymph duct can be prevented. With the developed method, we studied the characteristics of and mechanism for the production and regulation of SI-HDL. Nascent HDL particles were detected in SI lymph perfusates in WT mice, but not in ABCA1 KO mice. SI-HDL had a high protein content and was smaller than plasma HDL. SI-HDL was rich in TG and apo AIV compared with HDL in liver perfusates. SI-HDL was increased by high-fat diets and reduced in apo E KO mice. In conclusion, with our in situ perfusion model that enables the selective evaluation of SI-HDL, we demonstrated that ABCA1 plays an important role in intestinal HDL production, and SI-HDL is small, dense, rich in apo AIV, and regulated by nutritional and genetic factors.

Composition and ultrastructure of size subclasses of normal human peripheral lymph lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids

Peripheral lymph lipoproteins have been characterized in animals, but there is little information about their composition, and none about their ultrastructure, in normal humans. Therefore, we collected afferent leg lymph from 16 healthy males and quantified lipids and apolipoproteins in fractions separated by high performance-size exclusion chromatography. Apolipoprotein B (apoB) was found almost exclusively in low density lipoproteins. The distribution of apoA-I, particularly in lipoprotein A-I (LpA-I) without A-II particles, was shifted toward larger particles relative to plasma. The fractions containing these particles were also enriched in apoA-II, apoE, total cholesterol, and phospholipids and had greater unesterified cholesterol-to-cholesteryl ester ratios than their counterparts in plasma. Fractions containing smaller apoA-I particles were enriched in phospholipid. Most apoA-IV was lipid poor or lipid free. Most apoC-III coeluted with large apoA-I-containing particles. Electron microscopy showed that lymph contained discoidal particles not seen in plasma. These findings support other evidence that high density lipoproteins (HDL) undergo extensive remodeling in human tissue fluid. Total cholesterol concentration in lymph HDL was 30% greater (P < 0.05) than could be explained by the transendothelial transfer of HDL from plasma, providing direct confirmation that HDL acquire cholesterol in the extravascular compartment. Net transport rates of new HDL cholesterol in the cannulated vessels corresponded to a mean whole body reverse cholesterol transport rate via lymph of 0.89 mmol (344 mg)/day.

Lipid and apolipoprotein concentrations in prenodal leg lymph of fasted humans: associations with plasma concentrations in normal subjects, lipoprotein lipase deficiency, and LCAT deficiency

The extent to which lipid and apolipoprotein (apo) concentrations in tissue fluids are determined by those in plasma in normal humans is not known, as all studies to date have been performed on small numbers of subjects, often with dyslipidemia or lymphedema. Therefore, we quantified lipids, apolipoproteins, high density lipoprotein (HDL) lipids, and non-HDL lipids in prenodal leg lymph from 37 fasted ambulant healthy men. Lymph contained almost no triglycerides, but had higher concentrations of free glycerol than plasma. Unesterified cholesterol (UC), cholesteryl ester (CE), phosphatidylcholine (PC), and sphingomyelin (SPM) concentrations in whole lymph were not significantly correlated with those in plasma. HDL lipids, but not non-HDL lipids, were directly related to those in plasma. Lymph HDLs were enriched in UC. However, as the HDL cholesterol/non-HDL cholesterol ratio in lymph exceeded that in plasma, whole lymph nevertheless had a lower UC/CE ratio than plasma. Lymph also had a significantly higher SPM/PC ratio. The lymph/plasma (L/P) ratios of apolipoproteins were as follows: A-IV > A-I and A-II > C-III and E > B. Comparison with the L/P ratios of seven nonlipoprotein proteins suggested that apoA-IV was predominantly lipid free. Concentrations of apolipoproteins A-II, A-IV, C-III, and E in lymph, but not of apolipoproteins A-I or B, were positively correlated with those in plasma. The L/P ratios of apolipoproteins B, C-III, and E in two subjects with lipoprotein lipase (LPL) deficiency, and of apolipoproteins A-I and A-IV in a subject with lecithin:cholesterol acyltransferase (LCAT) deficiency, were low relative to those in normal subjects. Thus, the concentrations of lipids, apolipoproteins, and lipoproteins in human tissue fluid are determined only in part by their concentrations in plasma. Other factors, including the actions of LPL and LCAT, are at least as important.

Intestinal apolipoprotein synthesis and secretion in the suckling pig.

The present studies report characterization of intestinal apolipoprotein (apoLp) synthesis and secretion in the suckling pig. Lipoproteins (d less than 1.006 g/ml) from mesenteric lymph were found to contain both apoB-100 and B-48, in addition to apoA-IV, E, A-I, and Cs. Lymph low density lipoproteins (LDL) and high density lipoproteins (HDL) contained mainly apoB-100 and apoA-I, respectively. Analysis of core cholesteryl ester fatty acid composition suggested filtration from plasma as the major source of lymph LDL and HDL. Dual radioisotope labeling of intestinal and hepatic apoLps in lymph, as well as immunoprecipitation of radiolabeled intestinal mucosa, demonstrated intestinal synthesis of apoB-48, A-IV, and A-I. There was no evidence for apoB-100 synthesis by intestinal mucosa. By contrast, piglet liver synthesized apoB-100, E, A-I, and Cs, but not apoB-48. Newly synthesized intracellular intestinal apoA-I was mainly (basic) isoform 1 (pI 5.58), while lymph and plasma HDL apoA-I were predominantly isoform 3 (pI 5.33), mature apoA-I. Lymph apoB (P less than 0.001) and apoA-I (P less than 0.04) mass output increased significantly during lipid absorption. Studies were subsequently conducted in fasting, fat-fed, bile-diverted, and sham-operated animals to determine the role of both dietary and biliary lipid in regulating intestinal apoLp biosynthesis. Proximal and distal small intestinal loops were pulse-radiolabeled with [3H]leucine, and apoB-48 and A-I were immunoprecipitated from cytosolic supernatants. Although a proximal to distal gradient in intestinal synthesis rates for both apoB and A-I was noted in all groups, the acute absorption of dietary lipid did not significantly increase apoB or A-I synthesis in either location. Complete removal of biliary lipid for 48 hr did not alter synthesis rates in jejunum or ileum. These studies suggest that mesenteric lymph apoLps in the suckling pig are derived both by filtration from plasma and by direct secretion from the intestine. Physiologic regulation of intestinal apoA-I and B-48 synthesis rates appears to be independent of luminal lipid availability.

Isoforms of apolipoprotein A-II in human plasma and thoracic duct lymph. Identification of proapolipoprotein A-II and sialic acid-containing isoforms.

Proapolipoprotein (apo-) A-II and several isoforms of apo-A-II including sialylated isoforms were identified in human plasma and thoracic duct lymph. Proapo-A-II secreted by HepG2 cells was identified by a combination of immunoblots and [14C]arginine incorporation. Proapo-A-II which contains 2 arginine residues could be readily differentiated from mature apo-A-II which contains no arginine. The pI of proapo-A-II is 6.79, whereas the pI of the major apo-A-II isoform in plasma and lymph is 4.90. Minor apo-A-II isoforms have pI values of 5.17, 4.68, 4.42, and 4.20, respectively. Sialoisoforms of apo-A-II were identified, which had a higher apparent molecular weight on sodium dodecyl sulfate-gel electrophoresis than the major isoform and disappeared following neuraminidase treatment. The relative quantity of proapo-A-II was relatively constant in lymph very low density lipoproteins, lymph high density lipoproteins, and plasma high density lipoproteins, whereas the sialoforms and the other minor isoforms of apo-A-II were greater in lymph very low density lipoproteins and the lowest in plasma high density lipoproteins.

Utilization of individual lecithins in intestinal lipoprotein formation in the rat.

To determine the molecular species composition of lecithins of different nascent lipoproteins, high density lipoproteins (HDL), very low density lipoproteins (VLDL), and chylomicrons (CM) were isolated from the mesenteric lymph of rats. Lymph was collected at 0 degrees C with 5,5'-dithiobis-2-dinitrobenzoic acid added to inhibit lecithin-cholesterol acyl transferase. CM were separated by ultracentrifugation and HDL from VLDL by dextran SO4-MG+2 precipitation. Molecular species of lecithin were directly isolated by reverse phase high performance liquid chromatography. In fasted animals, the lecithin compositions of lymph HDL and VLDL were virtually the same and closely resembled the lecithin composition of intestinal mucosa. When bile lecithin was eliminated (by bile diversion), there was a marked change in lecithin composition of all lipoprotein and mucosal samples, which was most notable for a reduction in 16:0-species (which are predominant in bile) and a relative increase in the corresponding 18:0-species. Feeding unsaturated triglycerides (triolein, trilinolein, or a combination of triolein and trilinolein) also resulted in a change in HDL and VLDL lecithin composition. The effect was similar whether bile lecithin was present or eliminated and was notable for a reduction in 16:0-species, an increase in 18:0-species, and the emergence of large amounts of diunsaturated lecithins that corresponded to the fatty acid composition of the triglycerides fed (i.e., 18:1-18:1, 18:2-18:2, and 18:1-18:2 lecithins). When bile-diverted rats were infused via the duodenum with a mix of [14C]choline-labeled lecithins (isolated from the bile of other rats), the incorporation of infused lecithins into different lymph lipoproteins was distinctly different. Individual lecithins were incorporated to a variable extent into each lipoprotein. In fasted rats the specific activities of all major molecular species of lecithin were relatively greater in VLDL than HDL, indicating that HDL derived proportionately more of its lecithins from an endogenous pool than did VLDL. Feeding triolein changed the specific activities of more of the lecithin species of VLDL than of HDL. The specific activities of lecithins in CM were more similar to VLDL than to HDL after triolein feeding. Results thus indicate that, although the lecithins of different mesenteric lymph lipoproteins are similar and may be derived from membrane sites with the same lecithin composition, lecithins incorporated into different lipoproteins originate from different metabolic pools and/or by different mechanisms.

Characterization of dog peripheral lymph lipoproteins: the presence of a disc-shaped "nascent" high density lipoprotein.

The distribution, chemical, and apoprotein composition of plasma and peripheral lymph lipoproteins were compared in control and cholesterol-fed dogs. In both groups of animals, the agarose electrophoretic patterns of plasma and lymph lipoproteins were similar. In hypercholesterolemic dogs, beta-very low density lipoprotein, beta-migrating intermediate density lipoprotein, and HDLc were major components both in plasma and lymph, providing evidence for a potential interaction of these atherogenic particles with macrophages and other peripheral cells. The chemical composition and physical appearance of peripheral lymph HDL was markedly different from that of plasma HDL (high density lipoprotein), especially in the cholesterol-fed animals. Lymph HDL had a higher cholesterol to protein ratio and a markedly increased free cholesterol content (free cholesterol to cholesteryl ester ratio of 1.7 as opposed to 0.2 in plasma HDL in cholesterol-fed animals). The phospholipid content of lymph HDL was higher than that of plasma HDL, while the protein content was lower. A significant proportion of lymph HDL obtained from cholesterol-fed dogs was in the form of disc-shaped particles stacked in rouleau structures. Changes in plasma apolipoprotein concentrations due to cholesterol feeding were reflected in peripheral lymph to different degrees, depending largely on the relative size of the lipoproteins containing the individual lipoproteins. A considerable enrichment of lymph HDL with apoE and apoA-IV was observed by both immunochemical and electrophoretic methods. In lymph HDL from control and cholesterol-fed dogs, the apoE/apoA-I and apoA-IV/apoA-I ratios were several-fold elevated, compared to those of plasma HDL. It is concluded, therefore, that during cholesterol feeding a substantial portion of interstitial HDL is assembled de novo in the periphery as a crucial stage of reverse cholesterol transport to the liver. It is likely that further modification occurs upon entry to plasma and exposure to lecithin:cholesterol acyltransferase, possibly leading to generation of HDLc. Alternatively, these particles may be directly and rapidly removed by the liver.

Intestinal lipoproteins in the rat with D-(+)-galactosamine hepatitis.

D-(+)-galactosamine (GalN) induces severe reversible hepatocellular injury in the rat accompanied by lecithin: cholesterol acyltransferase (LCAT) deficiency, defective chylomicron (CM) catabolism, and accumulation of abnormal plasma lipoproteins (Lps), including discoidal high density lipoproteins (HDL). These abnormalities are presumed to result from hepatic injury alone, but the effect of GalN on intestinal Lps has not been studied. To assess possible effects on intestinal Lp formation and secretion, mesenteric lymph fistula rats were injected with GalN or saline. Twenty-four hours later a 2-hr fasting lymph sample was collected; this was followed by an 8-hr duodenal infusion of a lipid emulsion containing 17.7 mM [3H]triolein at 3 ml/hr. Fasting lymph and fat-infused lymph flow rates, 3H, triglyceride, and cholesterol output, residual 3H in intestinal lumen and mucosa, total 3H recovery, and d less than 1.006 g/ml Lp size and lipid composition were unchanged by GalN treatment, but d less than 1.006 g/ml Lps were depleted of apoE and C. Fat-infused lymph phospholipid (PL) output was higher in GalN rats due to PL-enriched d greater than 1.006 g/ml Lps. Electron microscopy of lymph and plasma LDL and HDL revealed spherical Lps in all samples. GalN plasma, fasting lymph, and fat-infused lymph also contained large abnormal LDL and discoidal HDL. Control lymph LDL and HDL did not differ in size from control plasma LDL and HDL. Control lymph LDL contained both apoB240K and B335K. However, spherical LDL and discoidal HDL in fasting lymph from GalN rats differed significantly in size from the corresponding plasma particles and became closer in size to the plasma particles with fat infusion. GalN lymph LDL contained only apoB240K and had a lower PL/CE than GalN plasma LDL. GalN fasting lymph HDL, depleted of apoC and having a PL/CE of 5, became enriched in apoE and the PL/CE increased to 10 with fat infusion to closely resemble GalN plasma HDL. GalN reduces apoE and C (mainly of hepatic origin) in d less than 1.006 g/ml gut Lps, which may contribute to the CM catabolic defect in GalN rats. Lymph LDL and HDL, especially in fasting lymph, may be partially gut-derived with increased filtration of plasma Lps into lymph with fat infusion. GalN fat-infused lymph HDL is enriched in apoE, but unable to transfer apoE to d less than 1.006 g/ml intestinal Lps. We conclude that GalN hepatitis is a model that allows study of intestinal Lps with normal lipid digestion and absorption in the face of severe hepatic injury and LCAT deficiency.

Lecithin:cholesterol acyltransferase-mediated modification of discoidal peripheral lymph high density lipoproteins: possible mechanism of formation of cholesterol-induced high density lipoproteins (HDLc) in cholesterol-fed dogs.

Peripheral lymph high density lipoproteins (HDL) of the cholesterol-fed dog differ in a number of characteristics from plasma HDL of the same animal. Their high content of free cholesterol, phospholipid, apoprotein E, and apoprotein A-IV, their greater heterogeneity in size, and the presence of many discoidal particles suggest that a portion of lymph HDL is assembled within the interstitial fluid. The present experiments demonstrate that the endogenous lecithin:cholesterol acyltransferase (LCAT) activity of whole peripheral lymph of the cholesterol-fed dog is far less (less than 1%) than that found in the plasma of the same animal (0.3 nmol/hr per ml versus 40.6 nmol/hr per ml). Addition of partially purified LCAT to whole lymph induced many changes in the chemical composition of peripheral lymph lipoproteins. After incubation with LCAT, the free cholesterol and phospholipid contents of lymph HDL decreased, from 17% to 12% and from 46% to 33%, respectively, whereas cholesteryl ester content increased from 7% to 13%. These changes were accompanied by a mass transfer of apoprotein E and cholesterol to the p less than 1.05 g/ml fraction, the complete disappearance of the discoidal particles, and a decrease in size heterogeneity of lymph HDL. These results suggest that, in the cholesterol-fed dog, cholesterol efflux into the interstitial spaces may occur in the absence of significant LCAT activity. Furthermore, our studies suggest that the subsequent reaction between lymph HDL and LCAT within the vascular compartment leads to the generation of apoprotein E and cholesteryl ester-rich cholesterol-induced HDL.

Effect of biliary diversion on rat mesenteric lymph apolipoprotein-I and high density lipoprotein.

The effect of biliary diversion on intestinal apolipoprotein (apoA)-I and high density lipoprotein formation was studied in mesenteric lymph fistula rats. Bile diversion was produced by an exteriorized catheter that allowed interruption and reconstitution of the enterohepatic circulation. Bile diversion reduced lymph cholesterol output from 0.47+/-0.05 mumol/h to 0.17+/-0.03 mumol/h (P < 0.025), and lymph triglyceride output from 3.6+/-0.3mumol/h to 0.6+/-0.05 mumol/h (P < 0.025) after 24 h. This was due to depletion of lymph chylomicrons and very low density lipoprotein (VLDL). Despite the reduced lipid outputs, lymph apoA-I output was maintained during biliary diversion (basal: 119+/-15 mug/h; diverted 140+/-20 mug/h, n = 12). During biliary diversion, high density lipoprotein (HDL) were maintained in mesenteric lymph as shown by lipoprotein and immunoelectrophoresis. Bile diversion altered the lipid composition of lymph HDL. Bile-diverted lymph HDL was depleted in total cholesterol and has a greater phospholipid/cholesterol ester ratio than basal lymph HDL. Lymph HDL contained discoidal particles when examined by negative stain electron microscopy. Bile diversion was associated with a reduction in the size of discoidal HDL particles (basal, nondiverted, 165+/-7A (n = 112) compared with diverted 126+/-5A (n = 98, P < 0.025). Experiments were then carried out to determine the source of the apoA-I and HDL found in lymph from bile-diverted animals. The transfer of HDL from plasma into lymph was determined by the intravenous infusion of (125)I-apoA-I labeled HDL into lymph fistula rats. In both nonbile-diverted and diverted rats, the specific activity of apoA-I in the HDL fraction of lymph was 23% of the specific activity of apoA-I in plasma HDL, indicating that the major portion (75%) of mesenteric lymph apoA-I did not come from plasma filtration. In other experiments the intraduodenal infusion of [(3)H]leucine to bile fistula, lymph fistula rats resulted in relative fivefold increase in the specific activity in apoA-I in lymph HDL when compared with the specific activity of apoA-I in plasma HDL from the same animal. We conclude that intestinal apoA-I secretion is maintained during biliary diversion and that synthesis of this apoprotein occurs in the absence of chylomicron formation. We also conclude that discoidal HDL are present in mesenteric lymph despite reduced triglyceride absorption and secretion into lymph.

Transport of apolipoproteins A-I and A-II by human thoracic duct lymph.

The daily transport of human plasma apolipoproteins A-I and A-II, triglyceride, and total cholesterol from the thoracic duct lymph into plasma was measured in two subjects before and three subjects after renal transplantation. Lymph triglyceride transport was approximately 83% of the daily ingested fat loads, whereas lymph cholesterol transport was consistently greater than the amount of daily ingested cholesterol. Lymph apolipoprotein transport significantly (P < 0.05) exceeded the predicted apolipoprotein synthesis rate by an average of 659+/-578 mg/d for apolipoprotein A-I and 109+/-59 mg/d for apolipoprotein A-II among the five subjects. It is estimated that 22-77% (apolipoprotein A-I) and 28-82% (apolipoprotein A-II) of daily total body apolipoprotein synthesis takes place in the intestine. Lymph high density lipoprotein particles are mostly high density lipoprotein(2b) and high density lipoprotein(2a) and have a greater overall relative triglyceride content and a smaller relative cholesteryl ester content when compared with homologous plasma high density lipoproteins. The major quantity of both lymph apolipoprotein A-I (81+/-8%) and apolipoprotein A-II (90+/-11%) was found within high density lipoproteins with almost all of the remainder found in chylomicrons and very low density lipoproteins. The combined results are consistent with a major contribution of the intestine to total body synthesis of apolipoprotein A-I and apolipoprotein A-II. An important role of lymph in returning filtered apolipoprotein to plasma in association with high density lipoproteins is proposed. Accompanying the return of filtered apolipoprotein to the plasma is a probable transformation, both in size and composition, of at least some of the lymph high density lipoprotein(2b) and high density lipoprotein(2a) particles into high density lipoprotein(3).

Rat Intestine Secretes Discoid High Density Lipoprotein

High density lipoprotein was isolated from pooled rat serum and mesenteric lymph of lymph fistula rats. In most experiments, 5,5'-dithionitrobenzoic acid, an inhibitor of the enzyme lecithin:cholesterol acyltransferase, was added during the collection of lymph to prevent modification of the lipid composition of newly secreted lipoproteins. Negative staining electron microscopy of lymph high density lipoprotein revealed discoidal particles (190+/-3 x 55+/-1 A) which tended to form rouleaux, smaller spherical particles were also present. Serum high density lipoprotein contained only spherical particles (diameter 93+/-4 A). Lipid analysis showed that lymph high density lipoprotein was enriched in phospholipid and deficient in cholesterol esters when compared to serum high density lipoprotein. The phospholipid to cholesterol esters ratio was greatest in basal lymph high density lipoprotein when compared to fatty lymph and serum high density lipoprotein. From analysis of the lipid compositional data and direct particle measurement by electron microscopy, it could be determined that congruent with50% of basal lymph high density lipoprotein and 30% of fatty lymph high density lipoprotein was discoid. Basal lymph high density lipoprotein was enriched in apoA-I and deficient in the arginine-rich peptide, and the apoprotein composition of fatty lymph high density lipoprotein more closely resembled serum. These observations demonstrate that intestinal lymph contains two types of high density lipoprotein particles, a discoid nascent particle deficient in cholesterol ester and rich in apoA-I, and spherical high density lipoprotein derived from plasma. A significant amount of lymph high density lipoprotein appears to be secreted by the intestine.

The intestine as a source of apolipoprotein A1.

The major apoprotein of rat mesenteric lymph chylomicrons has been isolated and characterized and shown to be identical to apoprotein A1 (apo A1) isolated from serum high density lipoprotein (HDL). During intestinal lipid absorption, active synthesis of apo A1 was demonstrated by radioactive amino acid incorporation into lymph chylomicron A1 as well as lymph HDL. Immunofluorescence studies of intestinal epithelium demonstrated a marked increase in apo A1 fluorescence, confirming an active synthesis of this apoprotein during lipid absorption. Quantitative immunoelectrophoretic methods were used to measure apo A1 in lymph and peripheral blood during various conditions designed to estimate the quantitative importance of intestinal apo A1 to the levels of circulating lipoproteins. During lipid feeding there was an increase in lymph apo A1 that was associated with lymph lipoproteins (50%) of density less than 1.006 g/ml whereas in basal lymph most apo A1 (85%) was in the lipoproteins of density greater than 1.006 g/ml. Lipid feeding in animals without lymph fistulas resulted in a significant increase in serum apo A1 levels; biliary diversion, designed to eliminate intestinal lipoproteins of density less than 1.006 g/ml, resulted in a significant decrease in serum apo A1 levels. These studies demonstrate that the intestine actively synthesizes apo A1 and is a significant source of this apoprotein for circulating lipoproteins.

Biosynthesis of lymph and plasma lipoprotein apoproteins by isolated perfused rat liver and intestine

The ability of rat intestine and liver to synthesize the various apoproteins of plasma lipoproteins was investigated. After the individual isolated organs were perfused with blood containing [(3)H]lysine, chylomicrons plus very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL) were isolated from the perfusates and the intestinal lymph. After lipoprotein delipidation, apoproteins were separated by polyacrylamide gel electrophoresis and the (3)H content was determined. Livers incorporated [(3)H]lysine into all apoprotein bands of VLDL and HDL. The (3)H content was greater in large proteins that remained in the stacking gel (group I, predominantly beta-apoprotein) than in proteins with apparent molecular weights near 50,000 (group II) or in the smaller peptides (molecular weights near 10,000, group III). In the intestine, (3)H was incorporated into group I and, in larger amounts, into group II apoproteins of lymph VLDL. No labeled VLDL appeared in the perfusate. (3)H was also incorporated into group II apoproteins of lymph and perfusate HDL. Significantly, no [(3)H]lysine was found in the group III peptides of any lymph or intestinal perfusate lipoproteins. Since these peptides were always present in VLDL from mesenteric lymph collected in vivo, the results suggest that nascent VLDL of gut origin acquires group III peptides from other lipoproteins that penetrate lymph from plasma.