Uptake of vitamin A in macrophages from physiologic transport proteins: role of retinol-binding protein and chylomicron remnants

Vitamin A plays an important role in reducing infectious disease morbidity and mortality by enhancing immunity, an effect that is partly mediated by macrophages. Thus, knowing how these cells take up vitamin A is important. The results in the present study demonstrate that J774 macrophages efficiently take up chylomicron remnant retinyl esters and retinol-binding protein (retinol-RBP) bound retinol by specific and saturable mechanisms. The binding of (125)I-RBP to plasma membrane vesicles demonstrated that the macrophage receptor had a similar binding affinity, as was discovered previously for other cells. The B(max) for the macrophages was smaller than the values reported for placenta, bone marrow, and kidney, but larger than that reported for liver. The J774 cells also bound and took up [(3)H]retinol-RBP. Approximately 50 to 60% of the uptake may compete with excess unlabeled retinol-RBP and approximately 30 to 40% with excess transtyrethin. Following the uptake of [(3)H]retinol-RBP, an extensive esterification occurred: After 5 hours of incubation, 77.8 +/- 3.9% (SD; n = 3) of the cellular radioactivity was recovered as retinyl esters. The J774 cells also demonstrated saturable binding of chylomicron remnant [(3)H]retinyl esters, and a continuous uptake at 37 degrees C followed by an extensive hydrolysis of the retinyl esters. Binding could be inhibited by approximately 50% by excess unlabeled low density lipoprotein (LDL). In addition, lipoprotein lipase increased the binding of chylomicron remnant [(3)H]retinyl esters by approximately 30% and the uptake of chylomicron remnant [(3)H]retinyl ester by more than 300%. Furthermore, because sodium chlorate reduced binding with 40% and uptake with 55%, the results suggest that proteoglycans are involved in the uptake. Thus, the results suggest that both LDL receptor and LDL-related protein are involved in the uptake of chylomicron remnant [(3)H]retinyl ester in macrophages.     

Chylomicron remnant-vitamin A metabolism by the human hepatoma cell line HepG2

The binding and metabolism of [3H]vitamin A-containing chylomicron (CM) remnants by the human hepatoma cell line HepG2 were studied. Mesenteric lymph chylomicrons were collected from [3H]retinol-fed rats and incubated with lipoprotein lipase to obtain CM remnants. At 4 degrees C, specific CM remnant binding was inhibited by an excess of unlabeled CM remnants. Specific binding predominated at low concentrations and approached saturation while total binding continued to increase over an extensive concentration range (0.45-32 microgram triglyceride/ml). CM remnant uptake at 37 degrees C was greater than that of CM and at least 70 times more efficient than the pinocytosis of sucrose. CM remnant binding increased with the extent of lipolysis. Addition of human apolipoprotein E enhanced both CM remnant and CM binding. After internalization, HepG2 cells hydrolyzed CM remnant-[3H]retinyl esters, and radiolabeled metabolites accumulated. As a function of the concentration of [3H]retinoid initially bound to cells, retinol and retinyl esters accumulated as the major cell-associated metabolites. In contrast, retinol was the major metabolite in the medium only at low retinoid concentrations; other more polar metabolites accumulated at higher concentrations (greater than 110 pmol retinoid/mg cell protein). The accumulation in the medium of labeled metabolites derived from CM remnant-retinoid was reduced when cells were preincubated in unlabeled retinol-supplemented media. The specific activity of retinol in the medium indicated that CM remnant-vitamin A had mixed with the cellular store prior to its secretion as retinol. These results indicate that HepG2 cells internalize CM remnants in part by specific binding sites, and that the metabolism of CM remnant-retinoids by the HepG2 cell involves retinyl ester hydrolysis and the secretion of retinol and other more polar metabolites. These processes were regulated in part by the concentration of retinoid delivered by the CM remnant and by the initial retinoid content of the cell.     

Vitamin A metabolism in rats chronically treated with 3,3',4,4',5,5'-hexabromobiphenyl

Chronic dietary administration of 3,3',4,4',5,5'-hexabromobiphenyl (HBB), 1 mg/kg diet, caused a decrease in retinol (20-fold) and retinyl esters (23-fold) in the livers of female rats, but resulted in a 6.4-fold increase in retinol and 7.4-fold increase in retinyl esters in the kidneys. Liver acyl-CoA:retinol acyltransferase and retinyl palmitate hydrolase activities were reduced while serum concentration of retinol was unaffected by HBB feeding. Metabolism of a physiological dose of [11-3H]retinyl acetate (10 micrograms), was examined in rats fed either vitamin A-adequate diet, or marginal amounts of vitamin A, or vitamin A-adequate diet containing HBB. A 13-fold greater amount of the administered vitamin A was found in kidneys of HBB-treated rats. In rats fed adequate or low amounts of vitamin A, kidney radioactivity was primarily in the retinol fraction, while in HBB-fed rats the radioactivity was associated mostly with retinyl esters. Fecal and urinary excretion of radioactivity was greatly increased in HBB-treated rats. Chronic HBB feeding results in a loss of ability of liver to store vitamin A, and severely alters the uptake and metabolism of vitamin A in the kidneys. We conclude that HBB causes major disturbances in the regulation of vitamin A metabolism.     

Vitamin A metabolism in rats chronically treated with 3,3′,4,4′,5,′-hexabromobiphenyl

Chronic dietary administration of 3,3′,4,4′,5,5′-hexabromobiphenyl (HBB), 1 mg/kg diet, caused a decrease in retinol (20-fold) and retinyl esters (23-fold) in the livers of female rats, but resulted in a 6.4-fold increase in retinol and 7.4-fold increase in retinyl esters in the kidneys. Liver acyl-CoA: retinol acyltransferase and retinyl palmitate hydrolase activities were reduced while serum concentration of retinol was unaffected by HBB feeding. Metabolism of a physiological dose of [11-³H]retinyl acetate (10 μg), was examined in rats fed either vitamin A-adequate diet, or marginal amounts of vitamin A, or vitamin A-adequate diet containing HBB. A 13-fold greater amount of the administered vitamin A was found in kidneys of HBB-treated rats. In rats fed adequate or low amounts of vitamin A, kidney radioactivity was primarily in the retinol fraction, while in HBB-fed rats the radioactivity was associated mostly with retinyl esters. Fecal and urinary excretion of radioactivity was greatly increased in HBB-treated rats. Chronic HBB feeding results in a loss of ability of liver to store vitamin A, and severely alters the uptake and metabolism of vitamin A in the kidneys. We conclude that HBB causes major disturbances in the regulation of vitamin A metabolism.     

Hepatic uptake of [3H]retinol bound to the serum retinol binding protein involves both parenchymal and perisinusoidal stellate cells

We have studied the hepatic uptake of retinol bound to the circulating retinol binding protein-transthyretin complex. Labeled complex was obtained from the plasma of donor rats that were fed radioactive retinol. When labeled retinol-retinol binding protein-transthyretin complex was injected intravenously into control rats, about 45% of the administered dose was recovered in liver after 56 h. Parenchymal liver cells were responsible for an initial rapid uptake. Perisinusoidal stellate cells initially accumulated radioactivity more slowly than did the parenchymal cells, but after 16 h, these cells contained more radioactivity than the parenchymal cells. After 56 h, about 70% of the radioactivity recovered in liver was present in stellate cells. For the first 2 h after injection, most of the radioactivity in parenchymal cells was recovered as unesterified retinol. The radioactivity in the retinyl ester fraction increased after a lag period of about 2 h, and after 5 h more than 60% of the radioactivity was recovered as retinyl esters. In stellate cells, radioactivity was mostly present as retinyl esters at all time points examined. Uptake of retinol in both parenchymal cells and stellate cells was reduced considerably in vitamin A-deficient rats. Less than 5% of the injected dose of radioactivity was found in liver after 5-6 h (as compared to 25% in control rats), and the radioactivity recovered in liver from these animals was mostly in the unesterified retinol fraction. Studies with separated cells in vitro suggested that both parenchymal and stellate cells isolated from control rats were able to take up retinol from the retinol-retinol binding protein-transthyretin complex. This uptake was temperature dependent.     

In vitro transfer of chylomicron retinol and retinyl esters

The redistribution of rat chylomicron retinoids following incubation with fasting- or postheparin human plasma was investigated. With fasting plasma, chylomicron retinol appeared among higher density lipoprotein acceptors and density greater than 1.21 gm/ml plasma proteins; only small amounts of retinyl ester were found therein. With postheparin plasma, retinyl ester-containing chylomicron remnants with densities spanning the low- and high density lipoprotein distributions were generated; appreciable quantities of retinyl esters appeared among rho greater than 1.019 lipoproteins only in the presence of postheparin plasma. These observations are consistent with the conservation of retinyl esters, but not retinol, among chylomicrons and their catabolic products.     

Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT)

Lecithin:retinol acyltransferase (LRAT) is believed to be the predominant if not the sole enzyme in the body responsible for the physiologic esterification of retinol. We have studied Lrat-deficient (Lrat-/-) mice to gain a better understanding of how these mice take up and store dietary retinoids and to determine whether other enzymes may be responsible for retinol esterification in the body. Although the Lrat-/- mice possess only trace amounts of retinyl esters in liver, lung, and kidney, they possess elevated (by 2-3-fold) concentrations of retinyl esters in adipose tissue compared with wild type mice. These adipose retinyl ester depots are mobilized in times of dietary retinoid insufficiency. We further observed an up-regulation (3-4-fold) in the level of cytosolic retinol-binding protein type III (CRBPIII) in adipose tissue of Lrat-/- mice. Examination by electron microscopy reveals a striking total absence of large lipid-containing droplets that normally store hepatic retinoid within the hepatic stellate cells of Lrat-/- mice. Despite the absence of significant retinyl ester stores and stellate cell lipid droplets, the livers of Lrat-/- mice upon histologic analysis appear normal and show no histological signs of liver fibrosis. Lrat-/- mice absorb dietary retinol primarily as free retinol in chylomicrons; however, retinyl esters are also present within the chylomicron fraction obtained from Lrat-/- mice. The fatty acyl composition of these (chylomicron) retinyl esters suggests that they are synthesized via an acyl-CoA-dependent process suggesting the existence of a physiologically significant acyl-CoA:retinol acyltransferase.     

Effect of hexachlorobiphenyl on vitamin A homeostasis in the rat

Vitamin A status and turnover were examined in rats that had been exposed to chronic dietary treatment of 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), 1 mg/kg diet. HCB caused hepatic depletion and renal accumulation of vitamin A, and a 1.7-fold increase in the serum retinol concentration. Intravenously administered [3H]retinol bound to retinol binding protein-transthyretin complex (RBP-TTR complex) was used to study the dynamics of circulatory retinol in these rats. In HCB-treated rats, the plasma turnover rate of retinol was increased compared to vitamin A-adequate untreated controls. HCB caused a 50% reduction of total radioactivity in liver, and, except for 0.5 h after the [3H]retinol-RBP-TTR dose, the specific activity of the hepatic retinyl ester pool was greater compared to control rats. The kidneys of HCB-treated rats accumulated radioactivity in the retinyl ester fraction. HCB also caused a 50% reduction in adrenal radioactivity compared with control rats. Urinary and fecal excretion of radioactivity was 3-fold higher in HCB-treated rats as compared to controls. Our findings demonstrate that chronic HCB feeding results in expansion of plasma vitamin A mass, in changes of liver and kidney retinol and retinyl ester pool dynamics and in an increased metabolism of vitamin A.     

Metabolism of [11-3H]retinyl acetate in liver tissues of vitamin A-sufficient, -deficient and retinoic acid-supplemented rats

A study was conducted on the incorporation of [11-3H]retinyl acetate into various retinyl esters in liver tissues of rats either vitamin A-sufficient, vitamin A-deficient or vitamin A-deficient and maintained on retinoic acid. Further, the metabolism of [11-3H]retinyl acetate to polar metabolites in liver tissues of these three groups of animals was investigated. Retinol metabolites were analyzed by high-performance liquid chromatography. In vitamin A-sufficient rat liver, the incorporation of radioactivity into retinyl palmitate and stearate was observed at 0.25 h after the injection of the label. The label was further detected in retinyl laurate, myristate, palmitoleate, linoleate, pentadecanoate and heptadecanoate 3 h after the injection. The specific radioactivities (dpm/nmol) of all retinyl esters increased with time. However, the rate of increase in the specific radioactivity of retinyl laurate was found to be significantly higher (66-fold) than that of retinyl palmitate 24 h after the injection of the label. 7 days after the injection of the label, the specific radioactivity between different retinyl esters were found to be similar, indicating that newly dosed labelled vitamin A had now mixed uniformly with the endogenous pool of vitamin A in the liver. The esterification of labelled retinol was not detected in liver tissues of vitamin A-deficient or retinoic acid-supplemented rats at any of the time point studied. Among the polar metabolites analyzed, the formation of [3H]retinoic acid from [3H]retinyl acetate was found only in vitamin A-deficient rat liver 24 h after the injection of the label. A new polar metabolite of retinol (RM) was detected in liver of the three groups of animals. The formation of 3H-labelled metabolite RM from [3H]retinyl acetate was not detected until 7 days after the injection of the label in the vitamin A-sufficient rat liver, suggesting that metabolite RM could be derived from a more stable pool of vitamin A.     

Uptake and metabolism of retinol in cultured Sertoli cells: evidence for a kinetic model

When cultured Sertoli cells derived from 20-day-old weanling rats were supplied [3H]retinol bound to serum retinol binding protein-transthyretin complex, [3H]retinol was rapidly incorporated and [3H]retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied [3H]retinol in culture under identical conditions likewise incorporated [3H]retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular [3H]retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of [3H]retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of [3H]retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of [3H]retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of [3H]retinol. During the time course the specific activity of [3H]retinyl palmitate eventually exceeded that of intracellular [3H]retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells.     

Fatty acid, carotenoid and vitamin A composition of tissues of free living gulls

The aim of this study was to investigate fatty acid and carotenoid profile as well as vitamin A (retinol and retinol esters) content in gull (Larus fucus) tissues. Palmitic (16:0) and stearic (18:0) fatty acids were major saturates in all the tissues studied. Oleic acid (18:1n-9) was the major monounsaturate in the tissue phospholipids varying from 11.9% (liver) up to 18.2% (lung). Arachidonic acid (20:4n-6) was the major unsaturate in the phospholipid fraction in all the tissues. Liver contained the highest total carotenoid concentration which was 5 and 7 fold higher compared to kidney and pancreas. In the liver beta-carotene was major carotenoid. In contrast, in all other tissues beta-carotene was minor fraction with lutein being major carotenoid. Zeaxanthin, canthaxanthin, beta-cryptoxanthin and echinenone were also identified in the gull tissues. Liver and kidney were characterised by the highest vitamin A concentrations (1067.5 and 867.5 microg/g, respectively). Retinol comprised from 55.3% (pancreas) down to 8% (kidney) of the total vitamin A but was not detected in the abdominal fat. Retinyl palmitate was the major retinyl ester in the liver, kidney and heart (44.2; 38.1 and 46.0% of total retinyl esters). In muscles and abdominal fat retinyl stearate was the major retinyl ester fraction. Therefore high proportions of beta-carotene were found in gull liver and peripheral tissues were enriched by lutein and zeaxanthin compared to the liver, a very high concentration of retinyl esters in the kidney was observed and tissue-specificity in retinyl ester proportions in peripheral tissues was found.     

Novel aspects of vitamin A metabolism in the dog: distribution of lipoprotein retinyl esters in vitamin A-deprived and cholesterol-fed animals

Retinyl ester concentrations in plasma from fasting humans, rabbits and rats are usually negligible. In contrast, plasma from fasting dogs contains appreciable amounts of retinyl esters, associated almost entirely with the low-density lipoproteins. This study was undertaken to gather additional information about the nature and origin of canine retinyl ester-containing lipoproteins. We examined the metabolism of endogenous lipoprotein retinyl esters in adult mongrel dogs with moderate vitamin A deficiency. Four animals were fed a diet of oatmeal and tuna fish that provided only 4% of the vitamin A contained in their control rations (15 vs. 367% of the canine recommended daily intake). There was an initial rapid decline in plasma retinyl esters. However, measurable concentrations persisted in plasma for up to 1 year of restricted vitamin A intake. Total plasma retinyl ester concentrations after 6 months of vitamin A deprivation, extrapolated from best-fit monoexponential decay curves for each animal, ranged from 11 to 89% of control, suggesting that there was sustained secretion of retinyl esters from endogenous stores. Density gradient ultracentrifugation of plasma from fasting vitamin A-deprived dogs showed retinyl esters in the very-low- and low-density lipoproteins. After fat and vitamin A feeding retinyl esters appeared among the very-low-, intermediate- and low-density lipoproteins, consistent with the suggestion that chylomicron retinyl esters are first taken up by the liver, and then resecreted as density less than 1.006-1.063 g/ml lipoproteins. Maximal incorporation of dietary retinyl esters into low-density lipoproteins was not reached until 24-48 h. Intermediate-density and beta-migrating low-density lipoprotein retinyl esters were increased markedly in fasting animals maintained on cholesterol- and saturated fat-enriched diets. These observations provide further evidence for the proposal that the canine liver secretes retinyl ester-containing particles, in amounts governed by dietary composition and vitamin A content. What selective advantage this unusual transport pathway might provide is not apparent.     

The majority of vitamin A is transported as retinyl esters in the blood of most carnivores

1. In canines and mustelides total vitamin A was 10-50 times higher compared to other species due to a high amount of retinyl esters (40-99% of total vitamin A) in blood plasma. The dominant vitamin A ester was in most species retinyl stearate. 2. In Ursidae, Procyonidae, Viveridae and Felidae, total vitamin A was much lower. When present, however, retinyl esters also represented 10-65% of total vitamin A in plasma. 3. Only retinol was detected in plasma of the family, Hyaenidae, and the suborder, Pinnipedia. 4. In maned wolf cubs it was found that retinol, retinyl esters and alpha-tocopherol increased with the age of the animals, reaching values comparable to adult animals at the age of 5 months.     

Protocol for the study of the metabolism of retinyl esters in plasma lipoproteins during postprandial lipemia.

An efficient protocol is described for the study of the kinetics of retinyl esters in whole plasma and several lipoprotein fractions following the consumption of an oral fat load containing vitamin A (retinol). To allow for a more complete characterization of the kinetics of retinyl esters in different lipoprotein fractions, a simplified two-step ultracentrifugation procedure is reported for the efficient and reproducible isolation of triglyceride-rich chylomicrons from nonfasting subjects, VLDL-sized lipoprotein particles, and the triglyceride-poor lipoprotein fraction. The present method for the determination of retinyl esters is based on the direct application of the lipid fraction onto a normal phase HPLC column without requiring the lipid extract to be desiccated and resolubilized. All of the commonly occurring esters of retinol elute as a single peak with a retention time of 1.6-1.8 min followed by retinyl acetate (serving as the internal standard) and retinol with retention times of 2-2.5 min and 5-5.5 min, respectively. With this system, a new sample can be processed every 10 min and a complete set of 60 samples from a typical oral fat load can analyzed in one working day with minimal technical interaction. By normalizing to the area under the internal standard to correct for variability in the injected volume, the coefficient of variability for the concentration retinyl esters within a single run is less than 5% and less than 10% between runs.     

Perisinusoidal fat-storing cells are the main vitamin A storage sites in rat liver

Highly purified sinusoidal (fat-storing, Kupffer and endothelial cells) and parenchymal cells were isolated to assess the cellular distribution of vitamin A in liver of adult vitamin A-sufficient rats. A modified simple procedure was developed for the purification of fat-storing cells from rat liver. This was achieved by a single centrifugation step in a two-layer density Nycodenz gradient. Endothelial and Kupffer cells were obtained from the same gradient and further purified by centrifugal elutriation. Reverse-phase HPLC analysis showed that fat-storing cells contained about 300-fold the amount of retinyl esters present in parenchymal cells on a mg cell protein basis. In fat-storing cells, the same retinyl esters, viz. retinyl palmitate, retinyl stearate and retinyl oleate, were present as in whole liver. It was also observed that, within 12 h after intravenous injection of chylomicron [3H]retinyl ester, most of the radioactivity had accumulated in the fat-storing cells. It is concluded that fat-storing cells are the main storage sites for vitamin A in rat liver.     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

Compartmental modeling of whole-body vitamin A kinetics in unsupplemented and vitamin A-retinoic acid-supplemented neonatal rats

Little is known about the contribution of different tissues to whole-body vitamin A (VA) kinetics in neonates. Here, we have used model-based compartmental analysis of tissue tracer kinetic data from unsupplemented (control) and VA-retinoic acid (VARA)-supplemented neonatal rats to determine VA kinetics in specific tissues under control and supplemented conditions. First, compartmental models for retinol kinetics were developed for individual tissues, and then an integrated compartmental model incorporating all tissues was developed for both groups. The models predicted that 52% of chylomicron (CM) retinyl ester was cleared by liver in control pups versus 22% in VARA-treated pups, whereas about 51% of VA was predicted to be extrahepatic in 4- to 6-day-old unsupplemented neonatal rats. VARA increased CM retinyl ester uptake by lung, carcass, and intestine; decreased the release into plasma of retinol that had been cleared by liver and lung as CM retinyl esters; stimulated the uptake of retinol from plasma holo-retinol binding protein into carcass; and decreased the retinol turnover out of the liver. Overall, neonatal VA trafficking differed from that previously described for adult animals, with a larger contribution of extrahepatic tissues to CM clearance, especially after VA supplementation, and a significant amount of VA distributed in extrahepatic tissues.     

Vitamin A is a key regulator for cell growth, cytokine production, and differentiation in normal B cells.

In the present paper we demonstrate that retinol-retinol-binding protein and chylomicron remnant retinyl esters in concentrations normally found in human plasma inhibit growth of normal human B lymphocytes. Physiological concentrations of retinoic acid (about 30 nM) were less active than physiological concentrations of retinol (about 3 microM). Pharmacological concentrations of retinol and retinoic acid were more active than the concentrations normally found in plasma. Retinol (3 microM) inhibited anti-IgM-mediated DNA synthesis as measured by [3H]thymidine uptake at 72 h by 78%. Furthermore, we found that the cells were blocked in the mid-G1 phase of the cell cycle. Thus, neither MYC up-regulation measured at 3 h nor the expression of the early activation antigen 4F2 was reduced by retinol, whereas the late activation markers (transferrin receptor expression and actinomycin D staining at 48 h of stimulation) were markedly inhibited. Retinol reduced the interleukin 6 production induced by anti-IgM and interleukin 4 after 48 h, whereas the induction of interleukin 6 and tumor necrosis factor by O-tetradecanoylphorbol-13-acetate and ionomycin was less affected. We also noted that the retinoids reduced the formation of plaque-forming cells (i.e. Ig synthesis). These data imply that vitamin A present in human plasma is a normal modulator of B cell function.     

Lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine. Evidence for different pathways

The lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine has been studied. When rats were cannulated in the intestinal lymph duct and given an intraduodenal bolus of [3H]retinol and 14C-labelled vitamin D-3, 14C-labeled vitamin D-3 appeared later in the intestinal lymph than [3H]retinol and the rate of absorption of vitamin D-3 was still maximal at a time when that of retinol had declined. Both vitamins were absorbed via the lymphatic route in association with chylomicrons. Almost all the retinol was esterified, while vitamin D-3 appeared in the chylomicrons as free vitamin D-3. In vitro incubations and in vivo studies using hepatectomized and normal rats showed that the retinyl ester was a relatively nonexchangeable component of the chylomicrons and their remnants. Hence, all the vitamin A followed the remnants in their clearance from plasma. In contrast, significant amounts of vitamin D-3 were transferred from the chylomicrons to other plasma fractions. Therefore, only a fraction of this vitamin may be removed in association with the chylomicron remnants.     

Characteristics of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells

The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoli cells accumulated [3H]retinol in a time- and temperature-dependent manner. At 32 degrees C, the rate of retinol accumulation was biphasic. Accumulation was linear for approximately 1 h, but then accumulation continued at a linear but decreased rate for 23 h. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/micrograms of cellular DNA. This amount of retinol was approximately equal to the cellular content of cellular retinol-binding protein (CRBP). Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [3H]retinol-RBP for [3H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [3H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 microM, suggesting that any change in the normal circulating retinol-RBP level (approximately 2 microM) would directly affect the rate of retinol accumulation. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. In addition, energy inhibitors and lysosomal poisons had no effect on [3H]retinol accumulation, indicating that RBP delivery of retinol to Sertoli cells did not occur by endocytosis of the retinol-RBP complex. Competition studies indicated, however, that protein recognition is important in the retinol uptake process.(ABSTRACT TRUNCATED AT 250 WORDS)