Lymph HDL Research Articles

Intestinal lymphatic HDL miR-223 and ApoA-I are reduced during insulin resistance and restored with niacin.

The intestine is involved in whole-body lipid and cholesterol homeostasis and secretes lipoproteins containing apolipoprotein (Apo)B48 and discrete ApoA-I into the mesenteric lymph. The lymphatic system has been proposed to have a significant role in the reverse cholesterol transport pathway associated with HDL-ApoA-I. In conditions of insulin resistance (IR), there is intestinal overproduction of chylomicrons containing ApoB48; however, there is limited data on the intestinal synthesis and secretion of HDL-ApoA-I. microRNA (miR)-223 has been shown to regulate peripheral HDL metabolism and may impact intestinal-derived HDL. Niacin (nicotinic acid; vitamin B3) is known to regulate lipid metabolism, but the role of niacin in modulating intestinal lipid and lipoprotein (ApoB48 and ApoA-I) metabolism is unknown. The aim of this study was to determine the secretion of intestinal lymphatic HDL-ApoA-I and the effect of dietary intervention with niacin on these pathways in a rodent model of IR. HDL was isolated from intestinal mesenteric lymph by density ultracentrifugation, and subsequent HDL miR analysis was developed in collaboration with Exiqon Services. Insulin-resistant rodents were fed chow or chow with niacin (1% w/w) for 6 wk. Intestinal lymph HDL-ApoA-I and miR-223 expression were lower by at least 45 and 60%, respectively, and lymph HDL was associated with 85% higher triglyceride (TG) content in IR compared to non-IR control group. Niacin was found to increase secretion of lymph HDL and miR-223 by at least 50-60% and to deplete the TGs associated with HDL compared with the nontreated IR group. Niacin significantly increased peroxisome proliferator-activating nuclear receptor α and carnitine palmitoyltransferase I α mRNA and annulled Tnf-α mRNA expression in intestinal (jejunal) explants. Altered intestinal lymphatic HDL-ApoA-I and miR-223 metabolism in IR and modulation by niacin may provide insight into the intestinal-mediated regulation of the reverse cholesterol transport pathway.-Mangat, R., Borthwick, F., Haase, T., Jacome, M., Nelson, R., Kontush, A., Vine, D. F., Proctor, S. D. Intestinal lymphatic HDL miR-223 and ApoA-I are reduced during insulin resistance and restored with niacin.

Enhanced incorporation of dietary DHA into lymph phospholipids by altering its molecular carrier

Several previous studies indicated that for optimal uptake by the brain, docosahexaenoic acid (DHA) should be present as phospholipid in the plasma. However most of dietary DHA is absorbed as triacylglycerol (TAG) because it is released as free fatty acid during digestion of either TAG-DHA (fish oil) or sn-2-DHA phospholipid (krill oil), and subsequently incorporated into TAG of chylomicrons. We tested the hypothesis that the absorption of DHA as phospholipid can be increased if it is present in the sn-1 position of dietary phospholipid or in lysophosphatidylcholine (LPC), because it would escape the hydrolysis by pancreatic phospholipase A2. We infused micelle containing the DHA either as LPC or as free acid, into the duodenum of lymph cannulated rats, and analyzed the chylomicrons and HDL of the lymph for the DHA-containing lipids. The results show that while the total amount of DHA absorbed was comparable from the two types of micelle, the percentage of DHA recovered in lymph phospholipids was 5 times greater with LPC-DHA, compared to free DHA. Furthermore, the amount of DHA recovered in lymph HDL was increased by 2-fold when LPC-DHA micelle was infused. These results could potentially lead to a novel strategy to increase brain DHA levels through the diet.

Abstract 321: Lymphatic derived HDL Is an Effective Donor for the Trans-Intestinal Cholesterol Efflux Pathway That Is Impaired During Insulin Resistance

Introduction: Emerging evidence shows that the proximal small intestine secretes cholesterol into the intestinal lumen via the trans-intestinal cholesterol excretion (TICE) pathway. It is thought TICE can contribute up to 30-40% of fecal neutral sterol excretion. Plasma apoB containing lipoproteins are known to donate cholesterol to the TICE pathway whilst conflicting evidence exists on the role for plasma HDL. Due to the anatomical proximity of lymphatic vessels to the basolateral membrane of enterocytes, it may serve as a candidate donor to TICE, but to date this has not been tested. Objective: To determine if HDL derived from mesenteric lymph can act as cholesterol donor for TICE in JCR:LA cp rats as a model of insulin resistance (IR). Methods: Mesenteric lymph was collected following intralipid infusion via lymphatic cannulation from control rats. Lymph HDL was isolated using ultra-density centrifugation and labeled with H3 cholesterol. Jejunal explants were obtained from control and IR rats (as a model of reduced TICE) fed chow or a high fat/cholesterol diet. TICE was measured with Ussing Chambers as appearance of H3-cholesterol labeled lymph HDL using micelles as acceptors. Results: Relative to free cholesterol [FC; used as marker of non-specific lipid permeability], lymph derived HDL TICE was 77% higher in control tissue (n=5, p<0.05) under chow fed conditions, suggestive of an effective donor for TICE. Lymph HDL TICE was also reduced (89%, p<0.05) in IR rats compared to control. Furthermore SR-B1 mRNA was reduced (-65%) in enterocytes from IR rats compared to control, and may explain reduced TICE by lymph HDL in IR. Under conditions of high fat/cholesterol fed diet, TICE from FC [and mannitol as a marker of paracellular transport] was increased in both control and IR rats, suggesting an elevated non-specific permeability of lipids by the basolateral membrane. Conclusions: Consistent with evidence that the lymphatics have a role in reverse cholesterol transport, this data supports that lymph HDL is an effective donor for TICE possibly by the SR-B1 pathway. While we have shown that the lymph HDL TICE pathway may be impaired during insulin resistance, a high fat/cholesterol diet may exacerbate lipid permeability via non-specific efflux pathways.

Absorption of Molecular Forms of Dietary Docosahexaenoic acid (DHA) and Their Potential to Provide DHA to the Brain

In this study, we tested the hypothesis that the presence of dietary DHA in the sn‐1 position of phosphatidylcholine (PC) results in greater incorporation of absorbed DHA into the phospholipids of plasma lipoproteins, compared to its presence in triacylglycerol (TG) or in the sn‐2 position of PC, because of the positional specificities of the pancreatic enzymes. Micelle containing the DHA either in lyso PC (LPC) form (surrogate for the dietary sn‐1 DHA PC) or as free fatty acid (surrogate for dietary DHA TG and sn‐2 DHA PC) were infused intraduodenally into lymph fistula rats, and the concentration of DHA in PC and TG of lymph chylomicrons and HDL was determined by GC and LC/MS. The total amount of DHA absorbed was comparable with the two preparations. However the ratio of DHA in PC/TG in lymph was much higher (0.4) after LPC‐DHA infusion, compared to free DHA infusion (0.1). Also, more DHA appeared in HDL fraction of lymph after LPC‐DHA infusion, and more of the HDL DHA was in PC than in TG. Similar results were obtained with CACO‐2 cells grown on micropore membranes in transwell plates. The two types of DHA micelle were added to the apical medium and the lipoproteins in the basolateral medium were analyzed after 24 h. The results showed a 4‐fold higher incorporation of DHA into PC in presence of LPC DHA compared to free DHA. These data indicate that the dietary DHA in the sn‐1 position of PC is potentially more available than the DHA in sn‐2 position of PC (krill oil) or in TG (fish oil) for uptake by the brain since the phospholipid form of DHA, especially from HDL, is transported more efficiently into the brain. Supported by NIH R21 AT008457.

NIACIN ALTERS THE SECRETION OF INTESTINAL LYMPHATIC HDL AND ASSOCIATED MIRNA PROFILE IN A RAT MODEL OF INSULIN RESISTANCE

Hypolipidemic effects of niacin (nicotinic acid) include lowering plasma triglycerides (TG) whilst increasing HDL-C concentration. However the mechanism of action of niacin still remains elusive. Emerging evidence shows that in addition to the liver, the intestine also contributes to whole body HDL metabolism. The effect of insulin resistance (IR) and niacin on intestinal HDL secretion is not known. Therefore the objective of this study was to determine the effect of niacin on the secretion, composition and miRNA profile of intestinal lymphatic HDL in a model of insulin resistance (IR) (JCR:LA-cp rat). IR rats were fed control chow or chow supplemented with niacin (1% w/w) for 6 weeks. The mesenteric lymph duct was cannulated and lymph sampled following an intra-gastric intralipid (fed) infusion for 6 hrs. The lymphatic HDL (1.063-1.21g/ml) fraction was separated by density ultracentrifugation; associated lipid, protein and miRNA composition was analyzed. For ex-vivo experiments, Jejunal explants from IR rats were treated with 5mg/ml niacin or nicotinamide and mRNA expression was assessed. In IR rats, apo-AI lymphatic HDL secretion was reduced (-47%) and associated with an enriched (86%) TG content, compared to non-IR rats. Interestingly, niacin was found to stimulate the secretion (number) of lymph HDL (>60%), as well as depleting the TG content compared to non-treated IR rats. Niacin (but not nicotinamide) significantly increased PPAR alpha and cpt1a mRNA, and annulled TNF alpha mRNA in jejunal explants. In lymph associated HDL from IR rats, miR223 and miR29c were both decreased compared to control. Interestingly niacin treatment increased both miR223 and miR29c in lymph associated HDL, suggesting a benefit to restore anti-inflammatory action of miR223 as well as the collagen/fibrotic repair functions of miR29c. IR may reduce the secretion of apoAI HDL into mesenteric lymph, potentially contributing to lipid enrichment and particle dysfunction. Niacin may contribute to improving CVD risk by increasing the number of lipid depleted intestinal apoAI particles. The ability of niacin to modulate miRs in the lymphatic HDL fraction may provide novel insight into hypolipidemic mechanisms of action of niacin.

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.

Abstract 629: Niacin Beneficially Alters the Secretion of Intestinal HDL in a Rat Model of Insulin Resistance by Opposing Effects on PPAR Alpha and TNF Alpha

INTRODUCTION: Niacin is the oldest hypolipidemic drug that effectively lowers plasma triglycerides whilst increasing HDL-C concentration. However the mechanism of action of niacin still remains elusive. Emerging evidence shows that in addition to the liver, the intestine also contributes to whole body HDL metabolism. The effect of insulin resistance and niacin on intestinal HDL secretion is unclear. OBJECTIVE: To determine the effect of niacin on the secretion and composition of intestinal lymphatic HDL in a model of insulin resistance (JCR:LA-cp rat). METHODS: Insulin resistant (IR) rats were fed control chow or chow supplemented with niacin (1% w/w) for 6 weeks. The mesenteric lymph duct was cannulated and lymph sampled following an intra-gastric intralipid (fed) infusion for 6 hrs. The lymphatic HDL (1.063-1.21g/ml) fraction was separated by density ultracentrifugation; associated lipid and protein composition was analyzed. Primary enterocytes were isolated by an adaptation of the Wieser method. Jejunal explants from IR rats were treated with 5mg/ml niacin or nicotinamide and mRNA expression was assessed. RESULTS: In IR rats, apo-AI lymphatic HDL secretion was reduced (-47%), but associated TG content enriched (86%), compared to non-IR rats. Interestingly, niacin was found to stimulate the secretion of lymph HDL (>60%) compared to non-treated IR rats. Niacin treatment also normalized the TG content of lymphatic HDL particles. In parallel experiments, niacin was also shown to beneficially reduce lymphatic apoB secretion, suggesting concomitant pathways. Primary enterocytes isolated from niacin treated animals had increased ABCA1 and PPAR alpha mRNA compared to untreated control. Further, niacin (but not nicotinamide) significantly increased PPAR alpha and CPT1a mRNA, while annulling TNF alpha mRNA in treated jejunal explants. Conversely, there was no effect of niacin or nicotinamide treatment on ATGL, PLIN2 or ATG5. CONCLUSION: Insulin resistance may reduce the secretion of apoAI HDL into mesenteric lymph, potentially contributing to particle dysfunction. Niacin may contribute to improving CVD risk by restoring the number and TG enrichment of intestinal apoAI particles.

Abstract 445: Niacin Beneficially Alters the Secretion of Intestinal-derived TRL and HDL in a Rat Model of Insulin Resistance

Introduction The intestine secretes discrete fractions of triglyceride (TG) rich apoB-chylomicrons (CM) and HDL particles into mesenteric lymph. Insulin resistance (IR) is associated with overproduction of intestinal CM however, the effect of IR on intestinal HDL secretion remains unknown. Niacin has been shown to reduce plasma TG while increasing HDL-C, however it is unknown if niacin modulates intestinal lymphatic CM and HDL secretion. Objective To determine the effect of niacin on the secretion and composition of intestinal lymphatic CM and HDL in a model of IR (JCR:LA- cp rat). Methods IR rats were fed control chow or chow supplemented with niacin (0.5% or 1% w/w) for 6 weeks. The mesenteric lymph duct was cannulated and lymph sampled following an intra-gastric intralipid ( fed ) infusion for 6hrs. The lymphatic CM (<1.006g/ml) and HDL (1.063-1.21g/ml) fractions were separated by density ultracentrifugation. Results IR rats over-secreted (2-fold) lymph apoB-CMs, while simultaneously reducing lymphatic apoA1-HDL (-47%) secretion compared to non-IR rats. In addition, CM and HDL particles from IR rats were found to be enriched in TG (64 and 86% respectively). Interestingly, niacin stimulated the secretion of lymph HDL (>60%), as well as attenuated lymph CM (-53%) secretion compared to control. Niacin treatment was also found to normalize the TG content of both lymph-CM and lymph-HDL particles. Conclusion IR appears to stimulate the intestine to over-secrete apoB-CM as well as reduce apoAI-HDL secretion into lymph, potentially contributing to particle dysfunction. Niacin may contribute to improving CVD risk by reducing the over-secretion of intestinal pro-atherogenic apoB-particles, whilst restoring the number and TG enrichment of intestinal anti-atherogenic apoAI-particles.

LXR Agonist Increases the Lymph HDL Transport in Rats by Promoting Reciprocally Intestinal ABCA1 and apo A‐I mRNA Levels

Liver and intestine are major sites of apo A-I synthesis in mammals. ABCAI is reported to be involved in the secretion of nascent HDL from cultured intestinal cells. However, whether ABCA1 participates in the secretion of nascent HDL from the intestine has not been assessed directly in vivo. This study examined the effect of a synthetic LXR-agonist "TO" on the lymphatic transport of HDL in thoracic duct-cannulated rats. The feeding of a TO-containing diet resulted in an increased transport of cholesterol and apo A-I in the lymph d > 1.063 g/ml lipoprotein fraction than did the feeding of a control diet without TO. The transport of cholesterol in whole lymph was lower, whereas the transport of apo A-I was higher, in the TO group. The abundance of mRNAs for ABCAI and apo A-I in the intestine was increased in the TO group. Furthermore, although the TO-containing diet reportedly increased the serum HDL concentration in intact mice and rats, no such effect was observed in the cannulated rats. The LXR agonist stimulated in vivo the synthesis of nascent HDL by increasing reciprocally the mRNA for ABCAI and apo A-I in the intestine, thereby contributing to an increase in the circulating HDL.

Intravenous apoA-I/lecithin discs increase pre-β-HDL concentration in tissue fluid and stimulate reverse cholesterol transport in humans

The extent to which plasma HDL concentration regulates reverse cholesterol transport (RCT) is not known. The principal acceptors of unesterified cholesterol (UC) from cultured cells are small pre-β-HDL, which we have shown increase in plasma during intravenous infusion of apolipoprotein A-I/phosphatidylcholine (apoA-I/PC) discs in humans. We have now examined the effects on tissue fluid HDL and RCT. ApoA-I/PC or proapoA-I/PC discs were infused into 16 healthy males. Eleven had been given intravenous radiocholesterol to label tissue pools; in 12 prenodal leg lymph was collected throughout; and in 8 all feces were collected. The rise in small pre-β-HDL in plasma was associated with increases in 1) pre-β-HDL concentration in lymph (all subjects), 2) the size of other lymph HDL (four of four subjects), 3) the cholesterol content of lymph lipoproteins relative to plasma lipoproteins (P < 0.01, n = 4), 4) cholesterol-specific radioactivity in lymph (five of nine subjects), 5) plasma lathosterol (P < 0.004, n = 4), 6) plasma cholesterol esterification rate (P < 0.001, n = 4), and 7) fecal bile acid excretion (P < 0.001, n = 8).These results support the hypothesis that small pre-β-HDL generated in plasma readily cross endothelium into tissue fluid, and thereby promote efflux of UC from peripheral cells. After delivery to the liver, peripheral cholesterol appears to be utilized more for bile acid synthesis than for biliary cholesterol secretion in humans.

The concentration of apolipoprotein A-II in human peripheral lymph

The concentration of apolipoprotein A-I (apoA-I) and of apolipoprotein A-II (apoA-II) was determined immunoelectrophoretically in lymph and plasma of six subjects. The concentration in lymph of apoA-I was 20.3 +/- 3.1 mg/dl, that of apoA-II was 4.6 +/- 0.5 mg/dl. The ratio of the concentration in lymph over that in plasma (CL/CP) for apoA-I was 0.14 +/- 0.1, that for apoA-II 0.14 +/- 0.01. Lymph and plasma samples from two subjects were fractionated by exclusion chromatography and the concentration of both apolipoproteins in resulting fractions was determined immunoelectrophoretically. ApoA-I of lymph eluted with fractions spanning a wider range of particle sizes than plasma apoA-I, while lymph apoA-II eluted predominantly with fractions that contained particles corresponding in size to plasma apoA-II-containing particles. It appears that the largest and smallest lymph HDL represent subspecies of Lp(A-I without A-II). These findings are discussed in the context of their possible bearing on initial stages of reverse transport of cholesterol.

Composition, morphology and distribution of high-density lipoproteins in plasma and peripheral lymph: effect of feeding cholesterol and saturated fat

In euthyroid dogs fed a diet rich in cholesterol and saturated fat, the cholesterol concentration in both plasma and peripheral lymph increased progressively with the appearance of HDL C ( d 1.006–1.063). This HDL C fraction was heterogeneous and could be separated into ‘slow’ and ‘fast’ migrating fractions by Pevikon block electrophoresis. On SDS-polyacrylamide gel electrophoresis, plasma ‘slow’ HDL C was appreciably enriched in apolipoprotein (apo) E, while plasma and lymph ‘fast’ HDL C were apo E-poor. In contrast, no apo E was visible in lymph ‘slow’ HDL C in either plasma or lymph HDL 2 fractions ( d 1.087–1.21). The interstitial HDL fractions containing apo A-IV (‘fast’ HDL C and HDL 2) were also rich in free cholesterol, implying that apo A-IV-containing particles are involved in reverse cholesterol transport. Plasma and peripheral lymph HDL 2 and ‘fast’ HDL C cholesterol/protein ratios were not different, whereas lymph ‘slow’ HDL C was 24% that of plasma, indicating that interstitial ‘slow’ HDL C was poor in cholesterol compared to plasma. This marked reduction in lymph ‘slow’ HDL C cholesterol suggests that this particle was either selectively retarded from egress by the endothelial barrier, or that interstitial ‘slow’ HDL C represents a depleted particle involved in the delivery of cholesterol to peripheral tissues. These findings taken together support the hypothesis that interstitial ‘slow’ HDL C may represent a particle involved in cholesterol ester delivery, in contrast with HDL 2 and ‘fast’ HDL C, which could serve as an efflux acceptor of tissue free cholesterol. This study demonstrates significant heterogeneity of interstitial peripheral lymph lipoproteins compared to plasma lipoproteins, and indicates selective distribution of these particles in the extravascular space.

Interstitial fluid lipoproteins.

While a wide variety of techniques has been used to collect samples of interstitial fluid, most of our detailed knowledge about the composition of interstitial fluid lipoproteins has come from lymph collection studies. The considerable variability of lymph data probably reflects the effect of variable metabolic modification and different capillary permeabilities on the lipoprotein composition of interstitial fluid. All density classes of plasma lipoproteins are present in lymph. In peripheral lymph, the lymph/plasma concentration ratios of lipoproteins vary from 0.03 for VLDL-sized particles to 0.2 for HDL. Lymph from more permeable vascular beds, such as lung and myocardium, contains proportionately more lipoproteins. Their lymph/plasma concentration ratios vary from 0.1 to 0.6. In general, lymph lipoproteins are more heterogeneous in size than their plasma counterparts. Lymph HDL and LDL contain larger and smaller particles than their plasma equivalents. Lymph lipoproteins have unusual shapes (square packing and discoidal), chemical compositions, and molecular charge, which suggest de novo formation and/or extensive peripheral modification. Lymph HDL and LDL are enriched in free cholesterol. Lymph HDL also has increased cholesterol/protein and phospholipid/protein (especially sphingomyelin) ratios (Sloop, C.H., L. Dory, and P.S. Roheim, unpublished observations). Lymph HDL apoprotein composition differs from that of plasma, with an increase in apoE and apoA-IV content relative to apoA-I. These discoidal HDL particles may be products of an initial stage of reverse cholesterol transport. We believe further study of their metabolic fate would give important information concerning the later stages of reverse cholesterol transport.

Distribution of apolipoproteins A-I and A-IV among lipoprotein classes in rat mesenteric lymph, fractionated by molecular sieve chromatography

The distribution of apolipoproteins A-I and A-IV among lymph lipoprotein fractions was studied after separation by molecular sieve chromatography, avoiding any ultracentrifugation. Lymph was obtained from rats infused either with a glucose solution or with a triacylglycerol emulsion. Relative to glucose infusion, triacylglycerol infusion caused a 20-fold increase in the output of triacylglycerol, coupled with a 4-fold increase in output of apolipoprotein A-IV. The output of apolipoprotein A-I was only elevated 2-fold. Chromatography on 6% agarose showed that lymph apolipoproteins A-I and A-IV are present on triacylglycerol-rich particles and on particles of the size of HDL. In addition, apolipoprotein A-IV is also present as ‘free’ apolipoprotein A-IV. The increase in apolipoprotein A-I output is caused by a higher output of A-I associated with large chylomicrons only, while the increase in apolipoprotein A-IV output is reflected by an increased output in all lymph lipoprotein fractions, including lymph HDL and ‘free’ apolipoprotein A-IV. The increased level of ‘free’ A-IV, seen in fatty lymph, may contribute to, and at least partly explain, the high concentrations of ‘free’ apolipoprotein A-IV present in serum obtained from fed animals.

Characterization of sheep lung lymph lipoproteins: chemical and physical properties.

We have determined the composition and distribution of plasma and lung lymph lipoproteins from unanesthetized ewes. Cholesterol, triglyceride, and phospholipid levels in lung lymph were 45%, 50%, and 50%, respectively, of those in plasma. Lipoproteins from both lymph and plasma were separated into two major fractions: d less than 1.063 g/ml or "LDL", and d 1.063-1.21 g/ml or HDL. HDL was the major lipoprotein species in the plasma and lymph. Gradient gel electrophoresis of HDL on 4-30% gels showed that, in lymph, HDL particles were shifted to larger sizes; in addition to a peak at 8.5 nm, which was similar to plasma HDL, there were two additional components of larger size, one at 9.2 nm and the other at 12 nm. Electron microscopy revealed that lymph HDL contained two new particles not seen in plasma: large, round particles, 13.6 nm diameter, and discoidal particles, 18.7 by 4.9 nm, long and short axis, respectively. Compositional analysis of lymph HDL revealed a relative enrichment in free cholesterol as well as an enrichment in apolipoprotein E. Lymph "LDL" on gradient gel electrophoresis was extremely heterogeneous. Several peaks were evident in the 23-30 nm size range (similar to plasma "LDL"), but a supplementary component at approximately 15-16 nm was also present. Whereas plasma "LDL" on electron microscopy contained only round particles 26 nm in diameter, lymph contained an additional, unusual particle which was close-packed, with square geometry, and was 15 nm in diameter. Lymph apolipoprotein composition differed from that of plasma by the appearance of apoE and A-I as well as apoB. Particles containing apoE and A-I were separated from apoB-containing particles in a fraction of d 1.047-1.063 g/ml by density gradient centrifugation. On electron microscopy, this fraction revealed square-packing particles; the density and apolipoprotein composition suggest that these unusual particles are a continuum of HDL. Changes in the physical and chemical properties of lung lymph lipoproteins suggest that these particles are metabolically modified.

Lipoproteins and apolipoproteins in peripheral lymph of normal and cholesterol-fed dogs

Prenodal peripheral lymph was used as a model of interstitial fluid to obtain information on the composition of lipoproteins and apolipoproteins which are in direct contact with peripheral cells. Lipoproteins resembling plasma lipoproteins in size and electrophoretic mobility were present in the prenodal peripheral lymph of control as well as cholesterol-fed dogs. Most of the lipoproteins in control dogs were high density lipoproteins, both in the plasma and in the lymph. Cholesterol feeding resulted in an increased concentration of lipoprotein particles with decreased electrophoretic mobility (β-VLDL) and, in plasma, HDL c) and decreased concentration of HDL, both in plasma and in lymph. Size distribution of lipoproteins was also markedly altered by cholesterol feeding; most of the lipoproteins were present as IDL and VLDL both in plasma and in lymph. Judged by agarose gel chromatography, the size of the lymph HDL as consistently larger than plasma HDL in both groups of dogs. Furthermore, it appears that cholesterol feeding increased the size of an HDL subfraction, partially resolved by agarose chromatography, both in lymph and plasma. All apolipoproteins present in plasma were also present in lymph. Cholesterol feeding resulted in 3–10-fold increases in plasma apo B, E, and A-IV while apo A-I was drastically decreased. These changes were reflected in lymph to

Effect of dietary cholesterol level on the composition of thoracic duct lymph lipoproteins isolated from nonhuman primates

The effect of two different levels of dietary cholesterol (0.16 mg/Kcal and 0.79 mg/cal) on the composition of thoracic lymph duct lipoproteins was studied in two species of nonhuman primates, Ceropithecus aethiops (African green monkey) and Macaca fascicularis (cynomolgus monkey). Diet was infused intraduodenally at a constant rate to facilitate comparisons among animals. The higher level of dietary cholesterol resulted in an increase in the amount of cholesteryl ester in lymph chylomicrons and VLDL. Cholesteryl oleate was the predominant cholesteryl ester present in lymph d less than 1.006 g/ml lipoproteins and it was the predominant cholesteryl ester formed from exogenous radiolabeled cholesterol. The percentage of saturated and monounsaturated cholesteryl esters in lymph chylomicrons and VLDL significantly increased with the higher dietary cholesterol level. The apoprotein distribution of chylomicrons and VLDL was qualitatively similar during infusions of both diets. The apoprotein B of intestinal chylomicrons and VLDL, termed apoprotein B2, was qualitatively similar during low and high cholesterol diet infusion and was significantly smaller than that of plasma LDL apoB, termed apoprotein B1, as indicated by its electrophoretic mobility in SDS-polyacrylamide gels. The major phospholipid present in lymph chylomicrons and VLDL was phosphatidylcholine and the phospholipid composition of the particles was not affected by diet. Lymph d greater than 1.006 g/ml lipoproteins were separated and the cholesterol mass distribution among lipoprotein fractions was found to be similar during both diet infusions. With an increase in the level of dietary cholesterol, the percentage esterification of cholesterol mass and of exogenous cholesterol radioactivity increased in LDL and HDL from lymph. Lymph LDL and HDL contained less free and esterified cholesterol when their composition was compared to that for these lipoproteins in plasma. We conclude that the primary effect of increased dietary cholesterol level was to increase the cholesteryl ester content of all lymph lipoproteins; cholesterol distribution among lymph lipoproteins was unaffected.

Evidence for the presence of tissue-free cholesterol in low density and high density lipoproteins of human peripheral lymph

The specific radioactivity of free and esterified cholesterol in the lipoproteins of human peripheral lymph was measured in 4 normal human subjects at various intervals after labelling the tissue cholesterol by a single intravenous injection of [ 14C] cholesterol. The free : esterified cholesterol specific-activity ratios in lymph LDL and HDL at short and long intervals after labelling in vivo suggest that both lipoproteins were capable of acting as acceptors for tissue-free cholesterol, but that in 3 of the 4 subjects the predominant acceptor was HDL.