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The aim of this work was to study the bioavailability of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), i.e. eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), carried by marine phospholipids (PL) and formulated in different supramolecular forms. Marine PL were administrated in rats either (1) in bulk form, or (2) as an oil-in-water emulsion, or (3) as liposomes. Each dietary formulation was characterized by a similar fatty acid (FA) profile and provided the same n-3 LC-PUFA amount. Intestinal bioavailability of n-3 LC-PUFA was monitored in the lymph compartment in a duct fistula model. On the one hand, the emulsification of plant oils with PL increased the overall intestinal absorption of dietary FA by 84% without affecting the lymph FA profile compared with the bulk form, suggesting that emulsification favoured the absorption of the total dietary FA derived from both triglycerides (TG) and PL. On the other hand, the liposome form did not modify the lymph lipid amount compared with the bulk form, but specifically increased the n-3 LC-PUFA levels. The dietary forms of PL influenced the position of some FA on the glycerol backbone of lymph TG and PL. In conclusion, using marine PL as an emulsifier promoted total FA absorption independently of the dietary lipid carrier (TG or PL) and the FA type. Structuring PL as liposomes specifically increased the intestinal bioavailability of FA esterifed in this lipid class, such as DHA, resulting in a higher incorporation into lymph lipids. Thus, using specific PL supramolecular forms would guide n-3 LC-PUFA towards total lipid absorption or specific FA absorption, according to the dietary needs.

Objective: Dietary triglycerides are partially retained in the intestine within intracellular or extracellular compartments, which can be rapidly mobilized in response to several stimuli, including glucose and GLP-2 (glucagon-like peptide-2). To elucidate the mechanism of intestinal lipid mobilization, this study examined the patterns and time course of lymph flow and triglycerides after glucose and GLP-2 treatment in rats. Approach and Results: Lymph flow, triglyceride concentration, and triglyceride output were assessed in mesenteric lymph duct-cannulated rats in response to an intraduodenal (i.d.) lipid bolus followed 5 hours later by either (1) i.d. saline+intraperitoneal (i.p.) saline (placebo), (2) i.d. glucose plus i.p. saline, (3) i.d. saline+i.p. GLP-2, or (4) i.d. glucose+i.p. GLP-2. GLP-2 and glucose administered alone or in combination stimulated total triglyceride output to a similar extent, but the timing and pattern of stimulation differed markedly. Whereas GLP-2 rapidly increased lymph flow with no effect on lymph triglyceride concentration or triglyceride:apoB48 (apolipoprotein B48) ratio (a surrogate marker of chylomicron size) compared with placebo, glucose transiently decreased lymph flow followed by delayed stimulation of lymph flow and increased lymph triglyceride concentration and triglyceride:apoB48 ratio., Conclusions: Glucose and GLP-2 robustly enhanced intestinal triglyceride output in rats but with different effects on lymph flow, lymph triglyceride concentration, and chylomicron size. GLP-2 stimulated triglyceride output primarily by enhancing lymph flow with no effect on chylomicron size, whereas glucose mobilized intestinal triglycerides, stimulating secretion of larger chylomicrons. This suggests that these 2 stimuli mobilize intestinal lipid by different mechanisms.

Introduction: GLP-1 is secreted from the gut upon nutrient intake and stimulates insulin secretion. The lymph draining the intestine may transport high levels of GLP-1 to the systemic circulation before it is metabolized by DPP-4. The aims of this study were to investigate to what extent the lymphatic system might contribute to the final level(s) of systemic circulating intact GLP-1 and, in addition, whether secretory profiles in intestinal lymph might reflect lamina propria levels of GLP-1 i.e. before capillary uptake and degradation by endothelial dipeptidyl peptidase-4 (DPP-4)., Method: 7 pigs of the YDL-strain were catheterized in the portal vein, carotid artery and cisterna chyli (lymph). Neuromedin C (NC) was infused through an ear vein catheter, before and after injection of a selective DPP-4 inhibitor (vildagliptin). Total and intact GLP-1 levels were measured throughout the 150min experiments using specific sandwich ELISAs. DPP-4 activity was measured spectrophotometrically., Results: Concentrations of both total and intact GLP-1 were markedly lower in lymph compared to plasma samples, and did not increase significantly in response to stimulation with NC in the absence/presence of vildagliptin. In contrast, total and intact GLP-1 levels increased significantly in the portal vein and carotid artery. DPP-4 activity was lower in lymph than plasma, and was reduced further by vildagliptin., Conclusion: Our observations indicate that the lymphatic system does not transport high levels of intact GLP-1 to the systemic circulation, and that GLP-1 levels in cisternal lymph do not reflect the hormone levels in the intestinal lamina propria., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: Intestinal lymphatic transport of specific lipophilic drugs offers therapeutic advantages and maximises oral bioavailability. The aims of this study were; to compare intestinal lymphatic transport of a range of drugs and to investigate the influence of cyclosporine A on the mechanism/extent of lymphatic transport., Methods: Caco2 cells and an anaesthetised mesenteric lymphatic cannulated rat model were used for in vitro and in vivo studies. Lymphatic transport of three lipophilic drugs was directly compared in a long chain fatty acid formulation. In addition, the impact of cyclosporine A on triglyceride turnover was evaluated in vivo and in vitro., Results: The extent of intestinal lymphatic transport in rats was positively correlated with drug solubility in triglyceride and negatively correlated with drug aqueous solubility. Cyclosporine A displayed non-linear lymphatic transport kinetics and reduced intestinal lymph triglyceride. In vitro experiments indicated that the cellular processes affected were intracellular lipid processing and/or lipid secretion., Conclusions: The linear correlations obtained using a range of lipophilic drugs confirm that the simplified approach of determining aqueous or triglyceride drug solubility is useful in predicting the extent of lymphatic transport. In vitro experiments correlated with in vivo observations, demonstrating the usefulness of the Caco-2 model for mechanistic investigations.

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Objective: To investigate the active factors and the intervention effect of ω-3 polyunsaturated fatty acids (PUFAs) during intestinal ischemia-reperfusion (I/R) injury, which causes the inflammation of monocytes-macrophages cultured in lymph fluid and stimulated with ω-3 PUFAs., Methods: Forty-eight Sprague-Dawley male rats were randomly divided into the following two groups: A. (N + D) group and B. (I/R + D) group. The rats in the (N + D) group were drained of lymph for 180 min; the rats in the (I/R + D) group were subjected to 60 min ischemia by clamping the superior mesenteric artery followed by 120 min reperfusion and 180 min of lymph draining. Lymph fluid from each group was further divided into 4 subgroups, respectively: lymph group (A1, B1); eicosopentaenoic acid (EPA)-treated group (A2, B2); EPA + docosahexaeonic acid (DHA)-treated group (A3, B3); and DHA-treated group (A4, B4), then cultured monocyte-macrophage cell line., Results: The levels of tumor necrosis factor-α, interleukin (IL)-1 β, IL-6, soluble cell adhesion molecule-1, chemotactic factors macrophage chemoattractant protein-1, macrophage inflammatory protein-2, and high mobility group box protein 1 in the B1 group were significantly higher than in the A1 group. Importantly, addition of EPA, EPA + DHA, and DHA to the culture media significantly reduced the levels of the above-mentioned factors. Cell stimulation with EPA, EPA + DHA, and DHA also significantly decreased the expression of Toll-like receptor 4, nuclear factor-κB p65, macrophage chemoattractant protein-1, and macrophage inflammatory protein-2 with the combined treatment of EPA and DHA showing the strongest effect., Conclusions: The factors induced in lymph during intestinal I/R injury can cause inflammation in vitro. These data provide in vitro evidence that ω-3 PUFAs provide a protective effect by reducing the inflammatory response caused by intestinal I/R lymph. Moreover, the synergism of EPA and DHA had the greatest effect, which is possibly mediated through Toll-like receptor 4 and nuclear factor-κB p65., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objectives: To assess the role of intestinal lymphatic transport in the oral bioavailability and brain deposition of a highly lipophilic, centrally acting drug candidate (Org 49209) in comparison to cholesterol, a close structural analogue., Methods: The intestinal lymphatic transport of Org 49209 and cholesterol was assessed in lymph-cannulated anaesthetised rats and total bioavailability evaluated in non-lymph-cannulated animals. Parallel groups were employed to examine the brain deposition of Org 49209 after intraduodenal and intraperitoneal administrations., Key Findings: The contribution of intestinal lymphatic transport to total bioavailability was similar for Org 49209 and cholesterol (approximately 40% of the absorbed dose). However, the oral bioavailability of Org 49209 was significantly (fourfold) lower than cholesterol. Brain deposition of Org 49209 was similar after intraduodenal and intraperitoneal administration. Systemic exposure, however, was higher after intraduodenal administration and brain-to-plasma ratios were therefore reduced., Conclusion: The oral bioavailability of Org 49209 was significantly lower than that of its structural analogue cholesterol; however, intestinal lymphatic transport played a similar role in oral bioavailability for both compounds. Brain to plasma ratios were lower after intraduodenal versus intraperitoneal administration, suggesting that drug association with intestinal lymph lipoproteins may limit central nervous system access for highly lipophilic drugs., (© 2014 Royal Pharmaceutical Society.)

The mechanisms underlying resistance to challenge by gastrointestinal nematode parasites in sheep are complex. Using DIGE, we profiled ovine lymph proteins in lambs with host resistance (R), resilience (Ri) or susceptibility (S) to a daily trickle challenge with the nematode Trichostrongylus colubriformis. Efferent intestinal lymph was collected prior to infection (day 1) and on days 5 and 10 post-infection. Eight proteins identified by LC-MS/MS, showed differences relating to host genotype. Of these, Serpin A3-3 and Serpin A3-7 have not been reported previously in the lymph proteome. Three acute phase proteins showed significant differences relating to interactions between breeding line and parasite challenge, including complement C3β, C3α and haptoglobin (Hp) β. In the R lambs C3α was significantly up regulated (P<0.05) on day 10, while in the Ri lambs Hp β was significantly down regulated (P<0.05). In the S lambs, levels of C3β were up regulated and levels of Hp β down regulated (both P<0.05) on day 10. Hence we demonstrate that acute phase inflammation proteins contribute to differences in the innate immune response of sheep to challenge by T. colubriformis. The findings may lead to the development of new approaches to combat nematode infestations in sheep production systems., Biological Significance: Breeding lines of sheep with resistance (R), resilience (Ri) or susceptibility (S) to nematode infections provide an experimental model to examine the biological mechanisms underlying the ability of some sheep to expel worms and remain healthy without the use of an anthelmintic. Using proteomics we identified differences in the expression of acute phase lymph proteins in the R, Ri and S lambs. The results will assist the development of alternative control strategies to manage nematode infections in livestock., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Prenyl flavonoids are widely distributed in plant foods and have attracted appreciable attention in relation to their potential benefits for human health. Prenylation may enhance the biological functions of flavonoids by introducing hydrophobic properties in their basic structures. Previously, we found that 8-prenyl naringenin exerted a greater preventive effect on muscle atrophy than nonprenylated naringenin in a mouse model. Here, we aimed to estimate the effect of prenylation on the bioavailability of dietary quercetin (Q). The cellular uptake of 8-prenyl quercetin (PQ) and Q in Caco-2 cells and C2C12 myotube cells was examined. Prenylation significantly enhanced the cellular uptake by increasing the lipophilicity in both cell types. In Caco-2 cells, efflux of PQ to the basolateral side was <15% of that of Q, suggesting that prenylation attenuates transport from the intestine to the circulation. After intragastric administration of PQ or Q to mice or rats, the area under the concentration-time curve for PQ in plasma and lymph was 52.5% and 37.5% lower than that of Q, respectively. PQ and its O-methylated form (MePQ) accumulated at much higher amounts than Q and O-methylated Q in the liver (Q: 3400%; MePQ: 7570%) and kidney (Q: 385%; MePQ: 736%) of mice after 18 d of feeding. These data suggest that prenylation enhances the accumulation of Q in tissues during long-term feeding, even though prenylation per se lowers its intestinal absorption from the diet.

Aim: To investigate the impact of intestinal ischemia/reperfusion (I/R) injury and lymph drainage on distant organs in rats., Methods: Thirty-two Sprague-Dawley male rats, weighing 280-320 g, were randomly divided into blank, sham, I/R, and ischemia/reperfusion and drainage (I/R + D) groups (n = 8). All rats were subjected to 60 min ischemia by clamping the superior mesenteric artery, followed by 120 min reperfusion. The rats in the I/R + D group received intestinal lymph drainage for 180 min. In the sham group, the abdominal cavity was opened for 180 min, but the rats received no treatment. The blank group served as a normal and untreated control. A chromogenic limulus assay kit was used for quantitative detection of serum endotoxin. The serum concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, soluble cell adhesion molecules (sICAM-1), and high mobility group protein box 1 (HMGB1) were determined with an enzyme-linked immunosorbent assay kit. Histological evaluations of the intestine, liver, kidney, and lung were performed by hematoxylin and eosin staining and immunohistochemistry. HMGB1 protein expression was assayed by western blot analysis., Results: The serum levels of endotoxin and HMGB1 in the I/R and I/R + D groups were significantly higher than those in the sham group (endotoxin, I/R and I/R + D vs sham: 0.033 ± 0.004 EU/mL, 0.024 ± 0.003 EU/mL vs 0.017 ± 0.009 EU/mL, respectively, P < 0.05; HMGB1, I/R and I/R + D vs sham: 5.473 ± 0.963 EU/mL, 4.906 ± 0.552 EU/mL vs 0.476 ± 0.406 EU/mL, respectively, P < 0.05). In addition, endotoxin and HMGB1 were significantly lower in the I/R + D group compared to the I/R group (P < 0.05). The serum inflammatory factors IL-6, IL-1β, and sICAM-1 in the I/R and I/R + D groups were significantly higher than those in the sham group (IL-6, I/R and I/R + D vs sham: 41.773 ± 9.753 pg/mL, 19.204 ± 4.136 pg/mL vs 11.566 ± 2.973 pg/mL, respectively, P < 0.05; IL-1β, I/R and I/R + D vs sham: 144.646 ± 29.378 pg/mL, 65.829 ± 10.888 pg/mL vs 38.178 ± 7.157 pg/mL, respectively, P < 0.05; sICAM-1, I/R and I/R + D vs sham: 97.360 ± 12.714 ng/mL, 48.401 ± 6.547 ng/mL vs 33.073 ± 5.957 ng/mL, respectively; P < 0.05). The serum TNF-α in the I/R group were significantly higher than in the sham group (45.863 ± 11.553 pg/mL vs 18.863 ± 6.679 pg/mL, respectively, P < 0.05). These factors were significantly lower in the I/R + D group compared to the I/R group (P < 0.05). The HMGB1 immunohistochemical staining results showed no staining or apparent injury in the blank group, and slight staining at the top of the microvillus was detected in the sham group. In the I/R group, both the top of villi and the basement membrane were stained for HMGB1 in most areas, and injury in the I/R + D group was less than that in the I/R group. HMGB1 expression in the liver, kidney, and lung of rats in the I/R + D group was significantly lower than the rats in the I/R group (P < 0.05)., Conclusion: Lymph drainage could block the "gut-lymph" pathway, improve intestinal barrier function, and attenuate distant organ injury incurred by intestinal I/R.

The poor orally available lopinavir was successfully encapsulated in glyceryl behenate based solid lipid nanoparticles (Lo-SLN) for its ultimate use to target intestinal lymphatic vessels in combined chemotherapy-the so-called Highly Active Anti-Retroviral Therapy (HAART). SLN with mean particle size of 230 nm (polydispersity index, PDI<0.27) and surface electrical charge of approx. -27mV, were produced by hot homogenization process followed by ultrasonication. Particles were characterized using differential scanning calorimetry (DSC), wide angle X-ray scattering (WAXS) and atomic force microscopy (AFM) to confirm their solid character and the homogeneous distribution of drug within the lipid matrix. In vitro release studies at pH 6.8 phosphate buffer (PBS) and at pH 1.2 HCl 0.1N showed a slow release in both media. From the intestinal lymphatic transport study it became evident that SLN increased the cumulative percentage dose of lopinavir secreted into the lymph, which was 4.91-fold higher when compared with a conventional drug solution in methyl cellulose 0.5% (w/v) as suspending agent (Lo-MC). The percentage bioavailability was significantly enhanced. The AUC for the Lo-SLN was 2.13-fold higher than that obtained for the Lo-MC of similar concentration. The accelerated stability studies showed that there was no significant change in the mean particle size and PDI after storage at 25±2°C/60±5% RH. The shelf life of optimized formulation was assessed based on the remained drug content in the stabilized formulation and was shown to be 21.46 months., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Sepsis is a common problem in intensive care patients leading to multi-organ failure and gastrointestinal paralysis., Aim: The aim of the study was to investigate whether the gastrointestinal tract is not only the target but also the source of inflammatory mediators inhibiting gastrointestinal motility., Methods: Mesenteric lymph was obtained from rats in which a sepsis was induced by lipopolysaccharide (LPS) intraperitoneally. Gastrointestinal motility was recorded following mesenteric lymph- or TNFα infusion into the jugular vein of separate healthy recipient rats using the strain gauge transducer technique., Results: Infusion of sepsis lymph significantly impairs gastric and colonic motility, decreasing the motility-index in the stomach and colon by about 57% and 21% respectively in comparison to baseline motility. Among other inflammatory mediators, TNFα plays an important role in mediating the inhibitory effect of mesenteric lymph on gastrointestinal motility during sepsis., Conclusions: The gastrointestinal tract is the source and the target of inflammatory mediators released during sepsis causing paralytic ileus., (Copyright © 2011 S. Karger AG, Basel.)

The orexigenic hormone ghrelin is secreted from the stomach and has been implicated in the regulation of energy and glucose homeostasis. We hypothesized that ghrelin, like other gastrointestinal (GI) hormones, is present in intestinal lymph, and sampling this compartment would provide advantages for studying ghrelin secretion in rodents. Blood and lymph were sampled from catheters in the jugular vein and mesenteric lymph duct before and after intraduodenal (ID) administration of isocaloric Ensure, dextrin, or Liposyn meals or an equal volume of saline in conscious Sprague-Dawley rats. Total ghrelin levels were measured using an established radioimmunoassay. Acyl and des-acyl ghrelin were measured using two-site ELISA. Fasting ghrelin levels in lymph were significantly higher than in plasma (means +/- SE: 3,307.9 +/- 272.9 vs. 2,127.1 +/- 115.0 pg/ml, P = 0.004). Postingestive acyl and des-acyl ghrelin levels were also significantly higher, whereas the ratio of acyl:des-acyl ghrelin was similar in lymph and plasma (0.91 +/- 0.28 vs. 1.20 +/- 0.36, P = 0.76). The principle enzymes responsible for deacylation of ghrelin were lower in lymph than in plasma. Following ID Ensure, maximum ghrelin suppression occurred at 2 h in lymph compared with at 1 h in plasma. The return of suppressed ghrelin levels to baseline was also delayed in lymph. Similarly, dextrin also induced significant suppression of ghrelin (two-way ANOVA: P = 0.02), whereas Liposyn did not (P = 0.32). On the basis of these findings, it appears that intestinal lymph, which includes drainage from the interstitium of the GI mucosa, is enriched in ghrelin. Despite reduced deacylating activity in lymph, there is not a disproportionate amount of acyl ghrelin in this pool. The postprandial dynamics of ghrelin are slower in lymph than plasma, but the magnitude of change is greater. Assessing ghrelin levels in the lymph may be advantageous for studying its secretion and concentrations in the gastric mucosa.

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.

Glucagon like peptide 1 (GLP-1) is an intestinal hormone that plays an important role in glucose metabolism. GLP-1 is released from mucosal L cells following nutrient ingestion and contributes to the incretin effect, with the enhancement of insulin secretion occurring with enteral compared with intravenous glucose administration. The mechanisms linking nutrient absorption and GLP-1 secretion are unknown, and studies addressing this topic, particularly in small animal models, have been hampered by the relatively low concentrations of GLP-1 in the circulation. We hypothesized that GLP-1 levels would be higher in samples of intestinal lymph compared with plasma and could provide a novel system in which to study meal-induced hormone secretion. We addressed this hypothesis in conscious rats with indwelling catheters in the portal vein and distal intestinal lymph duct. These animals had plasma and lymph sampled before and for 240 min after instillation of a liquid meal in the gastrointestinal tract. Lymph contained detectable concentrations of glucose, insulin, and GLP-1 that were reliably measured using our assays. Before and after the Ensure feeding, plasma insulin levels were approximately two times as high in portal plasma as intestinal lymph. In marked contrast, GLP-1 levels were five to six times higher in lymph relative to portal plasma following nutrient administration. This relative difference in GLP-1 levels was even greater when lymph was compared with peripheral plasma and dramatically exceeded the ratio of lymph to plasma peptide tyrosine-tyrosine concentrations. This is the first observation of a gastrointestinal hormone being disproportionately transported in lymph. The remarkable levels of GLP-1 in intestinal lymph demonstrate the potential for lymphatic sampling as a more sensitive means of studying the secretory physiology of this hormone in vivo. In addition, these data raise the possibility that intestinal lymph may serve as a specialized signaling conduit for regulatory peptides secreted by gastrointestinal endocrine cells.

Unlabelled: Sitosterolemia is caused by mutations in either ABCG5 or ABCG8, but simultaneous mutations of these genes have never been observed. To explore whether ABCG8, the sterol efflux (hemi-)transporter, plays a major role in determining intestinal absorption efficiency and hepatic secretion rates of cholesterol and sitostanol, we performed direct measurements of the absorption and lymphatic transport of these sterols in mice with chronic biliary and lymphatic fistulae, as well as the transport rates of radiolabeled cholesterol and sitostanol from plasma high-density lipoprotein (HDL) into bile in male Abcg8(-/-) and wild-type mice. We observed that the absorption and lymphatic transport rates of radiolabeled cholesterol and sitostanol were increased by approximately 40% and approximately 500%, respectively, in Abcg8(-/-) mice in the setting of constant intraduodenal infusion of micellar taurocholate and lecithin. Both strains displayed identical intestinal Npc1l1 expression levels and small intestinal transit rates. After 45 minutes of intraduodenal infusion, acute intestinal uptake rates of trace [(14)C]cholesterol and [(3)H]sitostanol were essentially similar in both groups of mice with intact biliary secretion. Furthermore, in wild-type mice, mass transport rate of [(3)H]sitostanol from plasma HDL into bile was significantly faster than that of [(14)C]cholesterol; however, no [(3)H]sitostanol and only traces of [(14)C]cholesterol were detected in bile of Abcg8(-/-) mice., Conclusion: Deletion of the Abcg8 gene alone significantly increases the mass of intestinal cholesterol and sitostanol absorption and reduces but does not eliminate hepatic secretion of cholesterol. Moreover, the mutation has no influence on acute uptake of cholesterol and sitostanol by the enterocyte nor small intestinal transit time.

Saquinavir is a lipophilic, poorly water-soluble HIV protease inhibitor that undergoes extensive first-pass metabolism and exhibits poor oral bioavailability. Redirection of the absorption pathway of anti-HIV compounds from the portal blood to the HIV-rich intestinal lymphatics may enhance therapeutic efficacy and reduce the extent of the first-pass effect. This study investigates the potential of targeted intestinal lymphatic transport of saquinavir via a lipid formulation approach. Three formulations containing oleic acid were examined: cremophor-oleic acid mixed micelles, D-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS)-oleic acid mixed micelles and an oleic acid microemulsion. The mesenteric lymph duct cannulated anaesthetised rat model was employed. Plasma and lymph samples were analysed by HPLC. Lymph triglyceride was measured using an enzymatic colorimetric technique. The extent of lymphatic transport from the lipid vehicles was 0.025-0.05% of the dose administered. The microemulsion produced higher and more prolonged mesenteric lymph concentrations than the micellar formulations. A strong correlation existed between the concentration of saquinavir in intestinal lymph and lymph triglyceride levels. The systemic bioavailability was estimated to be 8.5% and 4.8% for the cremophor mixed micelle and the microemulsion, respectively. The cremophor mixed micelles produced higher bioavailability than TPGS mixed micelles, implying that the nature of the surfactant can influence the distribution of drug between lymph and plasma.

Plasma HDL cholesterol levels are inversely related to risk for atherosclerosis. The ATP-binding cassette, subfamily A, member 1 (ABCA1) mediates the rate-controlling step in HDL particle formation, the assembly of free cholesterol and phospholipids with apoA-I. ABCA1 is expressed in many tissues; however, the physiological functions of ABCA1 in specific tissues and organs are still elusive. The liver is known to be the major source of plasma HDL, but it is likely that there are other important sites of HDL biogenesis. To assess the contribution of intestinal ABCA1 to plasma HDL levels in vivo, we generated mice that specifically lack ABCA1 in the intestine. Our results indicate that approximately 30% of the steady-state plasma HDL pool is contributed by intestinal ABCA1 in mice. In addition, our data suggest that HDL derived from intestinal ABCA1 is secreted directly into the circulation and that HDL in lymph is predominantly derived from the plasma compartment. These data establish a critical role for intestinal ABCA1 in plasma HDL biogenesis in vivo.

The influence of the size and turnover kinetics of the enterocyte-based lymph lipid precursor pool (LLPP) on intestinal lymphatic drug transport has been examined. Mesenteric lymph duct-cannulated rats were infused intraduodenally with low (2-5 mg/h) or high (20 mg/h) lipid-dose formulations containing 100 microg/h halofantrine (Hf, a model drug) and 1 microCi/h (14)C-oleic acid (OA) (as a marker for lipid transport) until steady-state rates of lipid(dX(L)/dt)(ss) and drug (dD(L)/dt)(ss) transport in lymph were obtained. After 5 h, the infusion was changed to formulations of the same composition but excluding (14)C-OA and Hf, allowing calculation of the first order rate constants describing turnover of lipid (K(X)) and drug (K(D)) from the LLPP into the lymph from the washout kinetics. The mass of lipid (X(LP)) and drug (D(LP)) in the LLPP was also determined. Biliary-lipid output was determined in a separate group of rats that had been infused with the same formulations. The results indicate that after administration of high lipid doses, lymphatic drug transport is dependent on the mass of exogenous lipid available in the LLPP and the rate of lipid pool turnover into the lymph. In contrast, after administration of low lipid doses, biliary-derived endogenous lipids are most likely to be the primary drivers of drug incorporation into the LLPP and lymph. Therefore, the LLPP size and composition seem to be major determinants of lymphatic drug transport, and formulation components, which increase lipid pool size, may therefore enhance lymphatic drug transport.

Purpose: This study was conducted to determine the influence of (1) the source of recruited endogenous fatty acid (FA), and (2) bile on intestinal lymphatic transport of halofantrine (Hf) in fasted rats., Methods: Endogenous FA output in bile, and exogenous ((14)C radiolabeled) FA, endogenous FA, and Hf transport in mesenteric lymph were determined following administration of low dose lipid formulations containing either 4 or 40 mg of exogenous FA [oleic acid (OA)] and different amounts of bile salt (BS) and lysophosphatidylcholine (LPC) to fasted rats., Results: Administration of 40 mg of OA recruited endogenous FA and Hf transport into intestinal lymph, whereas 4 mg OA did not. However, addition of BS to the 4-mg OA dose led to stimulation of endogenous FA recruitment into lymph and an increase in lymphatic transport of Hf and endogenous FA output in bile. Addition of LPC to the 4-mg OA dose (dispersed in BS) caused a substantial increase in endogenous FA transport in lymph; however, no coincident increase in either lymphatic transport of Hf or endogenous FA output in bile was observed., Conclusion: Biliary-derived endogenous FA has a higher propensity to support lymphatic transport of Hf compared to other sources of endogenous FA. The results suggest that this is related to the disparate trafficking of these alternate sources of endogenous FA within the enterocyte.

Using adult rats, the characteristic transporting system for lactoferrin (LF) from intestinal lumen into the blood circulation was investigated. The rats were randomly divided into two groups, a non-collected thoracic lymph (NC) group and a collected thoracic lymph (LC) group. Peripheral blood and thoracic lymph were collected from a jugular vein and a thoracic lymph duct, respectively, under anaesthesia. Bovine LF (bLF) was infused into the duodenal lumen by needle over a 1-min period at a dose of 1 g kg(-1). The transported bLF in the plasma and lymph was assayed quantitatively by double-antibody enzyme-linked immunosorbent assay (ELISA). Morphological investigation was also carried out in the intestine, lymph node, and liver. Following intraduodenal administration of bLF, the transported bLF in the NC group was detected in the plasma, and reached a peak value at 2 h. Furthermore, the bLF concentration in the thoracic duct lymph fluid in the LC group increased significantly, and peaked 2 h after the administration. In addition, bLF was not detected in the plasma of the LC group. Immunohistochemical analysis clearly showed anti-bLF positive particles in the epithelial cells of the apical villi. The striated border and baso-lateral membrane were also bLF positive. These results suggest that intraduodenally infused bLF is transported into the blood circulation via the lymphatic pathway, not via portal circulation in adult rats.

Compelling evidence indicates that the small intestine is the primary source of factors inducing lung injury after major surgery and that the lymphatic system is the major route by which these gut-derived factors reach the pulmonary circulation. This study investigated the mechanism of lung edema induced by surgical stress. After subjecting male, fasted, pathogen-free Sprague-Dawley rats to surgical stress (laparotomy and intestinal handling for 5 min), followed by ventilation for 5 h, we measured H2O2 production in the mucosa of small intestine and in the lung using 2',7'-dichlorofluorescein and intravital fluorescence microscopy. In addition, H2O2 in mesenteric lymph was measured using a quantitative assay; lung permeability was assessed as a function of extravasation of Evans blue dye; neutrophil accumulation was visualized by intravital fluorescence microscopy and assessed as a function of myeloperoxidase activity; and TNF-alpha levels were measured using a specific ELISA. The intensity of 2',7'-dichlorofluorescein fluorescence in the mucosa of small intestine, H2O2 levels of mesenteric lymph, and lung permeability were all significantly higher in rats subjected to surgical stress than in control animals. Moreover, all of these effects were blocked by pretreatment with a specific xanthine oxidase inhibitor. Surgical stress did not increase neutrophil accumulation or TNF-alpha production in the lung. In conclusion, surgical stress induces xanthine oxidase-dependent H2O2 production in the small intestine. The H2O2 then enters the mesenteric lymph and travels to the lung, where it increases capillary permeability and thus induces edema.

Purpose: To compare the influence of triglyceride vehicle intramolecular structure on the intestinal lymphatic transport and systemic absorption of halofantrine in conscious rats., Methods: Conscious, lymph cannulated and nonlymph cannulated rats were dosed orally with three structurally different triglycerides; sunflower oil, and two structured triglycerides containing different proportion and position of medium-(M) and long-chain (L) fatty acids on the glycerol backbone. The two structured triglycerides were abbreviated MLM and LML to reflect the structural position on the glycerol. The concentration of halofantrine in blood and lymph samples was analyzed by HPLC., Results: Both the lymphatic transport and the total absorption of halofantrine were enhanced by the use the MLM triglyceride. The estimated total absorption of halofantrine in the lymph cannulated animals was higher than in the nonlymph cannulated animals, and this was most pronounced for the animals dosed with the structured triglycerides., Conclusions: Using MLM as vehicle increases the portal absorption of halofantrine and results in similar lymphatic transport levels when compared to sunflower oil. Total absorption when assessed as absorption in the blood plus lymphatic transport for halofantrine after administration in the MLM triglyceride was higher than after administration in sunflower oil.

Postprandial dyslipidemia may be a major cause of atherosclerosis in diabetes. Microsomal triglyceride transfer protein (MTP) is essential for the synthesis of the chylomicron particle in the intestine and very low-density lipoprotein (VLDL) in the liver. The purpose of the present study was to examine the effect of diabetes on MTP mRNA expression in a rabbit model of diabetes, which develops atherosclerosis. Male New Zealand white rabbits were fed a 0.5% cholesterol diet. Diabetes was induced with alloxan monohydrate. The lymphatic duct was cannulated and lymph collected for isolation of chylomicrons by ultracentrifugation. Apolipoprotein B48 (apo B48) and apo B100 were separated by polyacrylamide gradient gel electrophoresis and quantified by densitometry. MTP mRNA was determined in liver and intestine by RNase protection analysis, and MTP activity was measured. Diabetic animals had significantly increased plasma triglyceride and decreased high-density lipoprotein (HDL) cholesterol (P <.05). They also secreted more lymph chylomicron apo B48 and apo B100 (P <.05) and more lymph chylomicron total and esterified cholesterol/h (P <.05). Lymph chylomicron particles in the diabetic animals contained significantly less lipid/apo B (P <.05). Intestinal MTP activity and mRNA were significantly higher in diabetic compared with control rabbits (0.07 +/- 0.01 v 0.04 +/- 0.015 fluorescent units/microg microsomal protein and 66 +/- 21 v 37 +/- 11 amol MTP mRNA/microg total RNA (P <.005). There was no difference in MTP activity or mRNA expression in the liver. This study suggests that MTP may play an important role in the postprandial dyslipidemia of diabetes., (Copyright 2002, Elsevier Science (USA). All rights reserved.)

The contribution of lymphatic transport and absorption directly into the portal blood to the overall oral bioavailability of a model lipophilic drug, halofantrine (Hf), was determined in lymph-cannulated, conscious, unrestrained rats after administration in lipidic vehicles with different fatty acid chain lengths. Both lymphatic transport (C(18)-based vehicle, 15.8% of dose > C(8-10), 5. 5% > C(4), 2.22% > C(0), 0.34%) and total systemic exposure (C(18), 22.7% of dose > C(8-10), 19.2% > C(4), 15.2% > C(0), 6.4%) of Hf were enhanced by the presence of lipids in the formulation and specifically by an increase in the fatty acid chain length of the coadministered lipid. Increases in lymphatic drug transport appeared to correlate with increases in lymphatic lipid transport. Surprisingly, where lymphatic transport was the primary mechanism of drug transport to the systemic circulation (i.e., after administration in a C(18)-based lipid vehicle), Hf bioavailability assessed in nonlymph-cannulated animals was lower than the extent of total availability measured in lymph-cannulated animals (estimated as percent appearing in the intestinal lymph plus percent transported directly into the blood), suggesting either presystemic drug clearance within the lymphatics or an altered systemic clearance pattern for lymphatically transported drug., (Copyright 2000 Wiley-Liss, Inc.)

Lymphatic lipid transport in the intestine of adult and ageing rats was compared. Adult (8-10 months old) and old (24-26 months old) male Wistar rats were cannulated into the mesenteric lymph under ethrane anesthesia. On the following day, lipid emulsion containing 35.4 mg/h of olive oil was infused intraduodenally for 7 h and lymph collected hourly was assayed for triglyceride and apolipoprotein A-IV (apo A-IV). The results showed there was no difference in lymphatic lipid and apo A-IV transport between adult and old rats. Since apo A-IV synthesis in the enterocytes is linked to the intracellular assembly of lipoprotein, it is likely that in addition to lymphatic transport, production of chylomicrons is not impaired in ageing rats.

A severe nutritional deficiency status is described in a 22-year-old patient with Crohn's disease. The clinical picture on admission was dominated by an episode of gastrointestinal bleeding secondary to clotting disturbances (vitamin K deficiency) and severe cachexia due to a protein energy malnutrition. The mechanisms of severe nutritional disturbances in Crohn's disease are multifactorial. In this patient, lymphatic leakage into the intestinal lumen was a major contributing factor in the pathogenesis of protein-losing enteropathy, fat malabsorption, and lymphocytopenia. The authors were able to demonstrate this intestinal lymph loss by nuclear imaging.

Aims/hypothesis: The aim of this study was to examine factors involved in chylomicron production in the streptozotocin diabetic rat, our hypothesis being that the synthesis of the chylomicron is abnormal in diabetes., Methods: Diabetic rats (n = 20) were paired with control rats (n = 20). Cholesterol emulsion was given by gavage and the lymph duct was cannulated. Lymph was collected for 4 h. Chylomicrons were prepared from the lymph by ultracentrifugation. Lymph apolipoprotein B48 was isolated by gradient gel electrophoresis and quantified by densitometric scanning. Intestinal microsomal triglycerol transfer protein mRNA was measured by solution hybridisation nuclease protection, using a rat specific [32P]-labelled cRNA probe., Results: Serum triglyceride and cholesterol were greatly increased in diabetic compared with control animals (258 +/- 77 and 8.9 +/- 6.4 mg/ml vs 1.04 +/- 0.37 and 0.54 +/- 0.03 mg/ml, p < 0.0001). Lymph chylomicron triglyceride and cholesterol were also higher in diabetic rats (29.4 +/- 27.3 and 0.28 +/- 0.3 mg/h vs 16.8 +/- 10.6 and 0.18 +/- 0.09 mg/h, p < 0.05). Lymph chylomicron apo B48 was similar in the two groups. Intestinal microsomal triglycerol transfer protein mRNA was higher in the diabetic rats (12.6 +/- 3.2 vs 3.8 +/- 3.0 amol/microgram RNA, p < 0.0001) and there was a positive correlation between lymph triglyceride and microsomal triglycerol transfer protein mRNA in the whole group (r = 0.65, p < 0.01)., Conclusion/interpretation: The study shows that microsomal triglycerol transfer protein mRNA is raised in diabetes without an increase in apolipoprotein B48 in the lymph suggesting that microsomal triglycerol transfer protein regulates chylomicron triglyceride content but not particle number.

We tested whether secretion of apolipoprotein (apo) A-IV depends upon intestinal triglyceride (TG) transport by comparing output kinetics of TG and apo A-IV during and after duodenal lipid infusion in lymph-fistula rats. Lipid infusion (triolein, 40 mumol/h, 8 h) produced increases in lymphatic TG and apo A-IV output. After 8 h, triolein infusate was replaced with glucose-saline; TG output returned to basal levels 4-5 h later. However, apo A-IV output continued at significantly elevated levels until 20 h after the start of the experiment. Bile diversion blocked this continued output of A-IV during the post-lipid period, and resulted in basal TG output that was 75% lower than in bile-intact rats. Return of bile or low-dose triolein infusion (5 mumol/h) into the intestine reversed these effects. There were no differences in hepatic synthesis or filtration of plasma A-IV into lymph between bile-intact and bile-diverted groups. Intestinal A-IV synthesis was elevated in both groups even during the post-lipid period. The results support the hypothesis that intestinal triglyceride transport drives apo A-IV secretion, and suggest the existence of a bile-dependent, post-translational mechanism for the control of lymphatic apo A-IV output.

The role of the kinin system (KS) in pathogenesis of endotoxin shock was investigated. In the lymph and blood flowing out of the small intestine and in the liver the activity of KS was measured 30, 60, 90 and 120 min after injection of the sublethel dose of endotoxin E coli 0 111 B4. The results suggest that in the lymph and blood flowing from the intestine KS activity occurs during the first minutes of endotoxin shock. In the liver KS activity was observed during the second hour of shock development followed by enhancing synthesis of KS components in the presence of KS higher activity and kinin developing function exhaustion to the end of the third hour of shock duration.

Hydrophobic surfactants such as Poloxalene inhibit triglyceride secretion into lymph by enterocytes. The inhibitory effect of these agents on triglyceride secretion is reversed when lipid presented for absorption is exclusively in the form of phosphatidylcholine (PC) and not triglyceride. The present investigation performed in conscious mesenteric lymph fistula rats was designed to determine whether various mixtures of triglyceride and PC given intraduodenally with Poloxalene would also reverse the inhibitory effect of Poloxalene on triglyceride secretion into lymph. A 50-50 mixture of triolein (TO) and PC resulted in normal triglyceride secretion into lymph. However, when the mixture of lipids was 75-25, TO to PC, results for triglyceride recovery in lymph were considerably reduced. The transport rate for triglyceride into lymph was not as depressed, however, as observed for Poloxalene treated rats given lipid for absorption basically in the triglyceride form. Substitution of phosphatidylethanolamine for PC had no beneficial effect on triglyceride secretion in Poloxalene treated rats. It is concluded that PC can reverse the inhibitory effect of Poloxalene on triglyceride secretion into lymph even when considerable amounts of triglyceride along with PC are presented for absorption.

The adhesion molecules integrin alpha 4 beta 7 and L-selectin have been hypothesized to help direct selective migration (homing) of lymphocytes to the gut and peripheral lymph nodes, respectively. An important prediction of current models is that lymphocytes selectively recirculating through an organ will preferentially express adhesion receptors for organ-specific endothelial ligands. To test this prediction, we directly examined the expression of cell adhesion molecules on lymphocytes recirculating through the gut, the periphery, and the lung. Integrin beta 7 was highly expressed on CD4+CD45R- "memory/effector" T cells recirculating through the gut (mesenteric efferent and lower thoracic duct lymph). In contrast, cells recirculating through the periphery (prescapular efferent lymph) and the lung (caudal mediastinal efferent lymph) had much less beta 7 expression. A similar pattern of organ-specific beta 7 expression was seen on B cells. Beta 7 expression corresponded with adhesion to the gut mucosal addressin, MAdCAM-1, in vitro. The peripheral lymph node homing receptor, L-selectin, was expressed at higher levels on CD4+CD45R- T cells from peripheral lymph than on cells from gut or lung lymph. These results provide additional strong support for alpha 4 beta 7 and L-selectin involvement in lymphocyte homing to the gut and to peripheral lymph nodes, respectively. Lymphocytes emigrating from the lung expressed low levels of both homing receptors and likely utilize molecules other than alpha 4 beta 7 and L-selectin for migration to the lung and associated lymphoid tissue.

The aim of the present paper was to summarize histamine-mediated repair of rat intestinal mucosa. To evaluate intestinal repair, we examined lipid transport (an index of intestinal mucosal function) after 15 minutes occlusion of the superior mesenteric artery. Rats were pretreated with alpha-fluoromethylhistidine (a suicide inhibitor of histidine decarboxylase, a synthesizing enzyme of histamine), H1-receptor antagonist (chlorpheniramine maleate), H2-antagonist (cimetidine), or H3-antagonist (thioperamide) before ischemia-reperfusion (I/R). Lipid transport to rat mesenteric lymph decreased significantly 24 hours after I/R in all groups tested compared to sham-treated rats. Lipid transport was restored 48 hours after I/R in the vehicle-pretreated control group. Lipid transport was not restored to the control level 48 hours after I/R in rats pretreated with H1-antagonist and a suicide inhibitor of histidine decarboxylase. In contrast, intestinal function was restored to the control level 48 hours after I/R in rats pretreated with H2- and H3-antagonists. These results support our previous findings that newly formed histamine after I/R plays an important role in mucosal recovery through H1-receptors.

This study evaluated 1) the effect of increased submucosal lymph osmolarity on the regulation of first-order (1A) and second-order (2A) intestinal arterioles and 2) the role of endothelium-derived relaxing factor (EDRF) in hypertonic-induced vasodilation. Increasing the submucosal lymph osmolarity from 280 to 400 mosM, in increments of 30 mosM, resulted in a dose-dependent dilation of 1A and 2A. A submucosal lymph tonicity of 340 mosM, as occurs during glucose and oleic acid absorption, caused dilation of 1A (118%) and 2A (124%) equivalent to that during absorptive hyperemia. The dilation caused by 400 mosM mannitol (137%) was similar to that with 340 mosM NaCl (131%) and approximately 70% of that with 400 mosM NaCl (152%). After EDRF blockade, the responses to sodium hypertonicity decreased by about one-half; blockade reduced mannitol-induced dilation by 22%. These results indicate that sodium hypertonicity, as occurs during absorption, can play a major role in absorptive hyperemia, and about one-half of the dilation is related to a sodium-coupled release of EDRF.

The fate of a small oral dose of protein given to overnight-starved rats was studied. After 3 h, 62% of the protein amino acids had been absorbed. Most of the absorbed N went into the bloodstream through the portal in the form of amino acids, but urea and ammonia were also present. About one-quarter of all absorbed N was carried as lymph amino acids. The liver was able to take all portal free ammonia and a large proportion of portal amino acids, releasing urea. The hepatic N balance was negative, indicating active proteolysis and net loss of liver protein.

The output of hyaluronan in mesenteric lymph was studied in anaesthetized rats to allow estimation of the turnover rate. The duodenum/jejunum contained 45 micrograms of this glycosaminoglycan per g wet tissue weight. In fasted rats the concentration of hyaluronan in postnodal lymph averaged 15 micrograms ml-1 and the mean efflux was 2.1 micrograms h-1, corresponding to a daily removal of 10% of the intestinal hyaluronan content. An intravenously injected bolus of 0.9% saline 4 ml 100 g-1 followed by an infusion of the same amount per hour increased the hyaluronan concentration in lymph transiently to 22 micrograms ml-1. During the 8-h i.v. infusion the hyaluronan output remained five times above control due to the high lymph flow. Water and hyaluronan content of the small intestine remained unaltered despite the saline load, the maintained tissue level of hyaluronan suggests an increased rate of synthesis.

The present study was designed to investigate the uptake and metabolism of circulating fatty acids by the intestinal mucosa in rats actively absorbing glyceryl trioleate given intraduodenally to determine the plasma fatty acid contribution to mucosal triacylglycerol. Rats with duodenal, femoral vein, carotid artery, and mesenteric lymph duct cannulas were used. [3H]oleate was constantly infused into the femoral vein while glyceryl trioleate was infused into the duodenum (135 mumol/h). After 5 h of infusion, a mass and radioactive steady state existed in the plasma and mucosa. At 6 h of infusion, the plasma oleate specific activity was sixfold greater than mucosal oleate and 50 times greater than mucosal triacylglycerol oleate; 86% of the mucosal oleate disintegrations/minute were in triacylglycerol. Chylomicron triacylglycerol oleate specific activity was less than that of the mucosa. Furthermore, the percentage of mucosal triacylglycerol acyl groups composed of oleate was greater than the percentage of oleate in mucosal free fatty acids. The data indicate that fatty acids are taken up by the mucosa during active fat absorption and metabolized primarily to triacylglycerols by the mucosa. The triacylglycerols in the mucosa synthesized from circulating fatty acids are selected against as a precursor of chylomicron triacylglycerol. The results support our previous hypothesis suggesting that the mucosa has at least two pools of neutral lipid (J. Lipid Res. 23: 1009-1019, 1982) and that steady-state conditions as performed here yield different results from previous work using bolus tracer injection techniques.

To determine the appropriateness of the ferret as a model for human beta-carotene (beta-C) metabolism, we have perfused both 15,15'-beta-[14C]C and unlabeled beta-C through the upper 30-cm portion of the small intestine of ferrets in vivo. The effluents of a mesenteric lymph duct cannulation and a common bile duct cannulation, as well as portal vein blood periodically sampled via an indwelling catheter, were collected. Ten percent (9.5 +/- 0.06%) of the total administered beta-C was taken up by the intestine after a 4-h perfusion. Of the radioactivity taken up, 68.6 +/- 6.5% remained in the intestinal mucosa, 3.2 +/- 0.2% was recovered in the lymph, and 28.2 +/- 6.5% (calculated) was absorbed via the portal system. The total uptake/absorption of beta-C was 12.9 +/- 6.8 nmol.h-1.30 cm intestine-1. Large amounts of unchanged beta-C and relatively small amounts of both beta-apo-12'-carotenal and beta-apo-10'-carotenal were isolated in the intestinal mucosa after a 4-h perfusion with beta-C. Considerable amounts of metabolites more polar than retinol were formed and comprised 35% of the total radioactivity recovered in the intestinal mucosa. Polar metabolites were absorbed mostly into the portal venous system, whereas retinol and retinyl esters were absorbed mainly into the mesenteric lymph. Of the total absorbed radioactivity in lymph, 10 +/- 1.0% appeared as unchanged beta-C, with peak absorption occurring at 3 h after beginning the perfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to quantify free radical-mediated lipid, protein, and sulfhydryl oxidation in intestinal interstitial fluid (lymph) and plasma of fasted rats. Free radicals and oxidants were generated either by thermal decomposition of 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH), which yields peroxyl radicals, or by activated polymorphonuclear neutrophils (PMNs). Incubation of intestinal lymph with AAPH resulted in a time-dependent increase in the formation of thiobarbituric acid-reactive substances (TBARS; lipid peroxidation) and carbonyl content (protein oxidation). TBARS formation was completely inhibited by removal of the apo B-containing lipoproteins in lymph suggesting that very low-density lipoprotein is the major substrate for lipid peroxidation in fasted interstitial fluid. The sulfhydryl content of lymph was reduced significantly on exposure to the peroxyl radical generator. Incubation with activated PMNs revealed qualitatively similar changes in protein and sulfhydryl oxidation; however, there was no detectable TBARS formation. Exposure of plasma to AAPH produced similar increases in protein and sulfhydryl oxidation when plasma protein concentration was adjusted to that of lymph; however, TBARS formation was significantly lower compared with lymph. Incubation of dialyzed plasma with AAPH produced significantly greater amounts of TBARS. Taken together, our data suggest that plasma is more resistant to AAPH-induced lipid peroxidation than interstitial fluid and the substrate for TBARS formation in intestinal interstitial fluid is different from that of plasma.

Intestinal lymph flow in rats receiving an oral load of 3 mmoles of glucose was determined by measuring the amount of tritiated gavage water entering via the portal route [computed differential arterio-venous tritium water concentrations multiplied by portal blood flow) and measuring the total amount of tritium transferred from the intestinal lumen to the rest of the rat in a given time [from the blood tritium specific activity and the water space (795 ml.kg-1) of the rat]. The approximate figure obtained (79.8 ml.kg-1.h) was used for the evaluation of the amount of glucose (from 14C-labelled glucose in the gavage) transfer via intestinal lymph from the label present in the lymph and its flow. Lymph glucose concentration (c. 11 mM) and specific radioactivity were higher than those of blood. The amount of glucose actually taken up from the intestinal lumen was determined measuring the remaining label in the alimentary canal. The portal contribution to label distribution was measured as for tritium water transfer. Most glucose label passed through in the form of glucose, with much smaller proportions as CO2 and even smaller as lactate and other labelled compounds. The results suggest that the contribution of lymph circulation to dietary glucose incorporation into the bloodstream may be significant, amounting to values higher than 10% of the glucose actually incorporated.

Mesenteric ischemia stimulates both A delta- and C-fiber afferents to reflexly activate the cardiovascular system. Leukotriene B4 (LTB4) concentration is increased in intestinal mucosa following prolonged ischemia (3 h) followed by reperfusion. Because LTB4 sensitizes afferent nerve endings in the skin, we determined whether LTB4 is produced during brief mesenteric ischemia and thus would be present to sensitize afferent nerve endings in the abdominal visceral region. Cannulas were placed in the portal vein and in a mesenteric lymphatic vessel distal to the lymph node. Mesenteric lymph and portal venous immunoreactive LTB4 (iLTB4) and immunoreactive thromboxane B2 (iTxB2) concentrations were measured before, during, and after 5-7 min of ischemia induced by occlusion of the descending thoracic aorta in cats. Simultaneously, lymph and plasma lactate concentrations were measured. During arterial occlusion, femoral arterial pressure dropped to less than 30 mmHg, and portal venous and mesenteric lymph lactate concentrations were increased significantly (3.3 +/- 0.6 to 6.3 +/- 1.0 mM and 5.2 +/- 0.9 to 7.2 +/- 1.1 mM, respectively, P less than 0.05). During ischemia, iLTB4 concentration increased in lymph from 261 +/- 70 to 424 +/- 102 pg/0.1 ml (P less than 0.05) but did not increase in portal venous blood (135 +/- 26 vs. 168 +/- 44 pg/0.1 ml, control vs. ischemia). iTxB2 concentration was not increased during ischemia in either portal venous blood or lymph (12 +/- 4 to 24 +/- 9 pg/0.1 ml and 19 +/- 7 to 24 +/- 11 pg/0.1 ml, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

The major objective of this study was to estimate how microvascular changes occur in the intestinal segment in relation to fat absorption and also to assess the role played by cholecystokinin (CCK) in fat-induced intestinal hyperemia. A 12-cm loop of rat middle small intestine was exposed to a 120-min infusion of oleic acid micelle solution containing [14C]oleic acid. Time-course changes of vascular diameter and red blood cell (RBC) velocity of submucosal arterioles, venules, and periglandular and muscular capillaries were determined simultaneously in their different order of branching by using intravital microscopy equipped with a high-speed video camera system. Lymphatic transport of oleic acid was also monitored by collecting lymph from intestinal lymphatics. The instillation of micelle produced significant dilatation of submucosal arterioles and venules and significant increase in RBC velocity in submucosal microvessels and periglandular capillaries at 15 to 30 min after the fat instillation. This corresponds well to the appearance of [14C]oleic acid in intestinal lymph. This microvascular dilatation and RBC velocity increase continued throughout the exposure to fat and started to attenuate 30 min after restoration to saline infusion. There was also heterogeneity in microvascular responses among different orders of branches to fat administration, and RBC velocity increase was not observed in muscular capillaries. Local intra-arterial infusion of CCK inhibitor, loxiglumide (CR 1505, 10 mg.kg-1.h-1), significantly attenuated both fat-induced microvascular dilatation and RBC velocity increase but not the fat absorptive function. Our results indicate that CCK plays a significant role in intestinal hyperemia induced by fat absorption. However, CCK-mediated intestinal microvascular change was not a prerequisite for fat absorption.

Isolated and autoperfused ileal segments from pentobarbital-anesthetized cats were used to study turnover of hyaluronan in the intestine. A postnodal lymphatic was cannulated, and transcapillary and interstitial fluid fluxes were increased by raising venous pressure. Lymph hyaluronan concentration in control averaged 20.2 +/- 18.8 (SD) micrograms/ml (range 4.6-50) and increased with increasing lymph flow in all experiments to peak at concentrations two to three times above control values (at 15-20 mmHg increase in venous pressure). At higher lymph flows, hyaluronan concentration fell to below 5 micrograms/ml to an average of 21.3 +/- 19.5% of control value at the highest venous pressures used (30-40 mmHg). Tissue hyaluronan content fell from 349 +/- 191 micrograms/g dry wt in control to 148 +/- 78 micrograms/g dry wt (P less than 0.05) at the end of the experiment. In a second group, vasodilators were administered before elevation of venous pressure to prevent redistribution of blood flow between mucosal and muscular layers. The results were similar to those obtained above. In a third experimental group, venous pressure was elevated in one step to 30 mmHg and maintained at this level. Again, hyaluronan concentration initially increased and later fell well below control values. We conclude that a major part of the intestinal hyaluronan is easily mobilized by increased interstitial fluid flux.

Addition of triglyceride and phospholipid to sodium taurocholate when chylomicron output was blocked by L81 did not increase lymphatic total cholesterol output or mucosal unesterified (UC) content more than with sodium taurocholate alone, but mucosal esterified cholesterol (CE) was increased slightly. In these animals with defective chylomicron formation, excess cholesterol accumulated in the intestinal mucosa mainly as CE. The mucosal cholesterol content of animals with normal chylomicron transport expanded during cholesterol and triglyceride absorption, and the expansion led to increased lymphatic secretion of CE. These animals accumulated significantly less CE in their mucosa than did rats treated with L81, but had about the same amount of mucosal UC. However, the overall uptake of cholesterol from the lumen, as determined by either radioactivity or mass of cholesterol in mucosa and lymph, was significantly less in the L81 rats. Also, more radioactive cholesterol remained in the lumen of the L81-treated rats. Finally, the data on specific activities of free and esterified cholesterol showed that the mucosal cholesterol derived from the lumen does not mix evenly with the free cholesterol pool in the enterocytes and is preferentially esterified for export in lymph as triglyceride-rich lipoprotein.

1. In the isolated perfused rat gut oestrone was not taken up from the perfusate but was rapidly taken up from the gut lumen when administered in dimethyl sulphoxide or in 10% acetone in water. 2. Uptake from corn oil was much slower and the rate was concentration-dependent. 3. When given in dimethyl sulphoxide or in aqueous acetone, the residual oestrone in the gut lumen was largely unchanged, with only about 10% being present as the glucuronide. Of the oestrone in the gut perfusate, some 40% was glucuronidated. 4. No evidence was obtained for any reduction or hydroxylation, or for any sulphation, during uptake of oestrone by the gut. 5. No secretion of oestrone or its metabolites into lymph could be detected.

We have recently evaluated the in vivo role of the liver in lipoprotein homeostasis in the preruminant calf (Bauchart, D., D. Durand, P. M. Laplaud, P. Forgez, S. Goulinet, and M. J. Chapman, 1989. J. Lipid Res. 30: 1499-1514). We now present the partial characterization of lipoprotein particles in postprandial intestinal lymph at peak lipid absorption (i.e., 10 h after a meal) in the preruminant calf fed a curdled milk replacer. Intestinal lymph from four male preruminant calves was analyzed for its content of lipids and fractionated by sequential and density gradient ultracentrifugation into chylomicrons (Sf greater than 400), very low density lipoproteins (VLDL) (Sf less than 400; d less than 1.006 g/ml), and a series of lipoprotein subfractions with d greater than 1.006 g/ml. Postprandial lymph contained predominantly triglycerides (1099 +/- 611 mg/100 ml), with lesser amounts of phospholipids (197 +/- 107 mg/100 ml) and cholesterol (52 +/- 30 mg/100 ml). The most abundant particles were triglyceride-rich chylomicrons and VLDL which accounted for approximately 76% and approximately 19%, respectively, of total d less than 1.21 g/ml lipoproteins. As judged by negative stain electron microscopy, chylomicron particle diameters ranged from 650 to 2400 A, while VLDL were smaller and distributed over a distinct size range (340-860 A). These two lipoprotein classes each presented protein components with Mr comparable to those of human apoB-48, apoA-I, and C apoproteins, together with an Mr 52,000 protein resembling human beta 2-glycoprotein-I. In addition, VLDL exhibited a polypeptide with Mr approximately 61,000. Lymph lipoproteins with d greater than 1.006 g/ml consisted primarily (approximately 81% of total) of particles distributed over the 1.053-1.119 g/ml density range. Electrophoretic analysis of the latter lipoprotein fraction showed it to be heterogeneous, including particles with the migration characteristics of low and of high density lipoproteins, respectively. Subfractions in the d 1.053-1.076 g/ml range were dominated by particles with Stokes diameters typical of high density lipoproteins (HDL), but also contained three different populations of low density lipoprotein-like particles. The high molecular weight apolipoproteins in these same cholesteryl ester-rich (greater than 30% of lipoprotein mass) subfractions comprised components with Mr resembling those of human apoB-100 and apoB-48, respectively, and with the latter protein predominating to a varying degree. A counterpart to human apoA-I was the major protein component over the entire density range from d 1.053 to 1.119 g/ml.(ABSTRACT TRUNCATED AT 400 WORDS)

Intestinal lipid absorption is associated with marked increases in the synthesis and secretion of apolipoprotein A-IV (apoA-IV) by the small intestine. Whether the increased intestinal apoA-IV synthesis and secretion results from increased fat uptake, increased cellular triglyceride (TG) content, or increased secretion of TG-rich lipoproteins by the enterocytes is unknown. Previous work from this laboratory has shown that a hydrophobic surfactant, Pluronic L-81 (L-81), is a potent inhibitor of intestinal formation of chylomicrons (CM), without reducing fat uptake or re-synthesis to TG. Furthermore, this inhibition can be reversed quickly by the cessation of L-81 infusion. Thus L-81 offers a unique opportunity to study the relationship between lymphatic TG, apoA-I and A-IV secretion. In this study, we studied the lymphatic transport of TG, apoA-I, and apoA-IV during both the inhibitory phase (L-81 infused together with lipid) and the subsequent unblocking phase (saline infusion). Two groups of lymph fistula rats were used, the control and the experimental rats. In the experimental rats, a phosphate-buffered taurocholate-stabilized emulsion containing 40 mumol [3H]triolein, 7.8 mumol of phosphatidylcholine, and 1 mg L-81 per 3 ml was infused at 3 ml/h for 8 h. This was then replaced by glucose-saline infusion for an additional 12 h. The control rats received the same lipid emulsion as the experimental rats, but without L-81 added, for 8 h. Lymph lipid was determined both by radioactivity and by glyceride-glycerol determination, and the apoA-I and apoA-IV concentrations were determined by rocket electroimmunophoresis assay. L-81 inhibited the rise in lymphatic lipid and apoA-IV output in the experimental rats after the beginning of lipid + L-81 infusion. Upon cessation of L-81 infusion, the mucosal lipid accumulated as a result of L-81 treatment was rapidly cleared into lymph as CM. This was associated with a marked increase in apoA-IV output; the maximal output was about 3 times that of the fasting level. There was a time lag of 4-5 h between the peak lymph lipid output and the peak lymph apoA-IV output during the unblocking phase in the experimental rats. There was also a comparable time lag between the maximal lipid and apoA-IV outputs in the control animals. Incorporation studies using [3H]leucine showed that apoA-IV synthesis was not stimulated during lipid + L-81 infusion, perhaps explaining the lack of increase in lymphatic A-IV secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

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

In a survey the present state of our knowledge about lipolysis, formation of micelles, lipid resorption, intracellular lipid transport, lipid resynthesis, lipoprotein synthesis, lipoprotein secretion and their regulation are summarized. Before their resorption the nutritive lipids are saponified by lipases. Fatty acids, monoglycerides, cholesterol and phosphatides are resorbed via a micellary phase. In the enterocytes the resorbed lipids are resynthetized together with endogenic constituents to triglycerides, phosphatides and cholesterol esters, which together with apoproteins as chylomicrons and VLDL reach the blood circulation via the lymphatic channels.