The effect of vitamin A on cellular differentiation and mucous glycoprotein synthesis in long-term rat tracheal organ cultures.

Rat tracheal explants maintained as organ cultures exhibited a normal mucocillary epithelium for at least 46 days in the presence of retinyl acetate. In the absence of vitamin A the explant epithelium became quiescent or underwent a metaplastic change to a keratinizing squamous epithelium. This process was accelerated if explants were derived from vitamin A-deficient animals. Autoradiographic examination showed that [3H]glucosamine label accumulated in various cell types in the explant, but especially in the epithelium. It was found that the explants secreted mucous glycoproteins into the medium and that the production and biochemical characteristics of a specific mucin fraction were dependent upon the vitamin A status of the explant.     

The Effect of Vitamin A on Cellular Differentiation and Mucous Glycoprotein Synthesis in Long-Term Rat Tracheal Organ Cultures

Rat tracheal explants maintained as organ cultures exhibited a normal mucociliary epithelium for at least 46 days in the presence of retinyl acetate. In the absence of vitamin A the explant epithelium became quiescent or underwent a metaplastic change to a keratinizing squamous epithelium. This process was accelerated if explants were derived from vitamin A-deficient animals. Autoradiographic examination showed that [³H]glucosamine label accumulated in various cell types in the explant, but especially in the epithelium. It was found that the explants secreted mucous glycoproteins into the medium and that the production and biochemical characteristics of a specific mucin fraction were dependent upon the vitamin A status of the explant.     

Vitamin adeficiency and keratin biosynthesis in cultured hamster trachea

Summary Tracheas from vitamin A-deficient hamsters in organ culture in vitamin A-free medium developed squamous metaplasia. Addition of retinyl acetate to the medium prevented squamous metaplasia and a mucociliary epithelium was maintained. Indirect immunofluorescent staining with antikeratin antibodies AE1 and AE3 indicated positive reactions with epithelium of tracheas either cultured in vitamin A-free or retinyl acetate (RAc)-containing medium. The “stratum corneum”-like squames in metaplastic tracheas were strongly stained by AE3. Immunoprecipitation of cytoskeletal extracts from [³⁵S]methionine labeled tracheas with a multivalent keratin antiserum indicated that the concentration of keratins synthesized in tracheas cultured in vitamin A-free medium was greater than that observed in tracheas cultured in the presence of RAc. In addition, new species of keratin were expressed in tracheas cultured in RAc-free medium. Alterations in the program of keratin synthesis were clearly detectable after 1 d in vitamin A-free medium, even though squamous metaplasia was not yet obvious. Squamous tracheas were shown by immunoblot analysis to contain keratins of 50, 48, 46.5, and 45 kilodalton (kd) detected with AE1; and 58, 56, and 52 kd detected with AE3. Immunoblot analysis with monospecific antimouse keratin sera also demonstrated the presence of 60, 55, and 50 kd keratins in the metaplastic tracheas. All these various species of keratins were either absent or present in much reduced quantity in mucociliary tracheas in RAc-containing medium. Interestingly, the induction of squamous metaplasia in tracheal epithelium did not result in the expression of the 59 and 67 kd keratins which are characteristically expressed in the differentiated layers of the epidermis. Therefore, this study shows that squamous metaplasia of tracheas due to vitamin A-free cultivation is accompanied by an increase in keratin synthesis as well as by the appearance of keratin species not normally present in mucociliary tracheal epithelium.     

Peptides of human bronchial mucus glycoproteins. Size determination by electron microscopy and by biosynthetic experiments.

Secreted human bronchial mucins, directly collected from macroscopically healthy bronchial mucosa, were prepared in the presence of six proteinase inhibitors, and analysed by electron microscopy. These mucins were similar in length distribution to molecules prepared from sputum [Slayter, Lamblin, Le Treut, Galabert, Houdret, Degand & Roussel (1984) Eur. J. Biochem. 142, 209-218], although they were a little longer, their lengths ranging up to about 1,650 nm. This length corresponds to an extended mucin peptide of about 450 kDa. In order to compare these peptide lengths with the molecular size of biosynthetic precursors, an antiserum raised against trifluoromethanesulphonic acid-treated highly glycosylated regions of human bronchial mucins was used to isolate mucin precursors synthesized in explants of human bronchial mucosa during pulse-labelling with [3H]threonine or [3H]glucosamine. A main precursor labelled with [3H]threonine and with an apparent molecular mass of about 400 kDa was detected by fluorography following SDS/polyacrylamide-gel electrophoresis. This band was observed as early as 20 min; it was more intense after a 40 min chase and had disappeared after a chase period of 280 min in unlabelled medium, presumably owing to glycosylation. Much fainter bands at about 200 kDa and between 200 and 400 kDa, also labelled with [3H]threonine, were observed mainly after a 40 min chase and had disappeared after a 280 min chase. None of these bands was labelled with [3H]glucosamine, nor did they disappear after multiple treatments with immobilized lectins. After a 280 min chase, [3H]threonine-labelled material appeared in the stacking gel, which also contained [3H]glucosamine label. The results indicate that the 200-400 kDa species are mucin precursors, whose size is comparable with that obtained by electron microscopy for respiratory mucins collected directly from the macroscopically healthy bronchial mucosa.     

Analysis of mucin-derived oligosaccharides by medium-pressure gel-permeation and amino-plate thin-layer chromatography conducted in conjunction

Oligosaccharides present in mucin were labeled by reduction with NaB3H4 and separated by gel-permeation chromatography with a Toyopearl HW-40S column using 0.1 M pyridine acetate, pH 5.0, as the solvent. Each fraction was further analyzed by thin-layer chromatography (TLC) on a Funagel AMP plate, a glass plate precoated with 3-aminopropyl-bonded silica. Acetonitrile/10 mM triethylamine acetate (3/2, by volume) served as the solvent. The sites of oligosaccharides on the TLC plate could be determined according to size, anionic charge, and sugar composition. They could thus be "mapped" on the plate. In this manner, the distribution of oligosaccharides on bovine submaxillary mucin and rat gastric mucin was determined. Each radiolabeled oligosaccharide in newly synthesized rat gastric mucin, metabolically labeled with [14C]glucosamine or [35S]sulfate, was also identified by this method.     

The murine sublingual and submandibular mucins. Their isolation and characterization.

From the mouse sublingual and submandibular glands high-molecular weight glycoproteins (mucins) were isolated. These mucins appeared to be homogeneous in polyacrylamide gel electrophoresis and in the analytical ultracentrifuge. S20,W values of 10.9 and 5.5 were found for the sublingual and submandibular mucin respectively. With sodium dodecyl sulfate or N-acetylcysteine no subunits could be detected. Both mucins consisted for about 1/3 of protein and 2/3 carbohydrate. Their mucin character was also denoted by the high content of serine plus threonine. Respectively 42 mol% and 34 mol% of the protein core of the sublingual and submandibular mucins consisted of these amino acids. The main sugars in these mucins were sialic acid, galactosamine, galactose, glucosamine and mannose. The molar ratio for the sublingual and submandibular mucin being 1.00 : 1.03 : 1.08 : 0.26 : 0.23 and 1.00 : 0.71 : 1.10 : 0.65 : 0.53, respectively. The sialic acid content of both mucins was about 25%. Fucose and sulfate, on the other hand, were less than 1%. The presence of sulfate was also indicated by preliminary studies in vivo on the incorporation of [35SO4] sulfate.     

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.     

Retinoids increase the incorporation of D-[3H]galactose into epidermal glycoproteins

All-trans retinoic acid increased the incorporation of D-[³H]galactose into particulate and soluble glycoproteins in the epidermis of cultured pig skin slices nearly two-fold. Increased incorporation of D-[³H]galactose was not blocked by tunicamycin. This effect was specific for D-[³H]galactose since the incorporation of D-[³H]glucosamine and L-[¹⁴C]leucine into epidermal glycoproteins was unaffected by all-trans retinoic acid. All-trans retinoic acid and 13-cis retinoic acid had quantitatively similar effects on D-[³H]galactose incorporation. All-trans retinyl acetate and an aromatic retinoic acid analogue (‘Etretinate’) were less effective. SDS polyacrylamide gel electrophoresis and fluorography showed increased incorporation of D-[³H]galactose into all epidermal glycoproteins in the presence of all-trans retinoic acid. There was no evidence for synthesis of new glycoproteins such as mucins.     

Role of sulfation in post-translational processing of gastric mucins.

1. Gastric mucosal segments were incubated in MEM supplemented with various sulfate concentrations in the presence of [3H]glucosamine, [3H]proline and [35S]Na2SO4, with and without chlorate, an inhibitor of 3'-phosphoadenosine-5'-phosphosulfate formation. 2. Incorporation of glucosamine and sulfate depended upon the sulfate content of the medium and reached a maximum at 300 microM sulfate. Introduction of chlorate into the medium, while having no effect on protein synthesis as evidenced by proline incorporation, caused, at its optimal concentration of 2 mM, a 90% decrease in mucin sulfation and a 40% drop in glycosylation. 3. At low sulfate content in the medium and in the presence of chlorate, the incorporation of sulfate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulfate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulfation in its carbohydrate chain. 4. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and that sulfate availability is essential for the formation of the high molecular-weight mucin polymer.     

Retinoylation of proteins in a macrophage tumor cell line J774, following uptake of chylomicron remnant retinyl ester

Following uptake of chylomicron remnant retinyl esters by the macrophage cell line J774, the retinyl esters are hydrolyzed to retinol before retinol is further metabolized to retinal and the various retinoic acid isoforms. One hour after the addition of chylomicron remnant [³H]retinyl esters to the cells, the percentage of cell-associated radioactivity in the retinyl ester fraction had decreased from approximately 90% to approximately 40%, whereas the radioactivity in the retinol fraction increased correspondingly. After 4 hours of incubation, more than 79% of the radioactive retinyl esters had been hydrolyzed to retinol. When we measured incorporation of radioactivity in the protein fraction, we observed that the level of [³H]retinoylated proteins increased rapidly the first 4 hours, and then continued to increase at a lower rate up to 24 hours, when approximately 0.6% of the cell-associated radioactivity was covalently bound to protein. These data suggest that approximately 0.18% of all the cellular proteins might be retinoylated under such conditions. In summary, in the present study we have demonstrated that retinoids taken up by a macrophage cell line as chylomicron remnant retinyl esters, a physiologic plasma transport molecule for vitamin A, might be covalently linked to proteins. Such retinoylation might be relevant both for normal function, as well as for the toxic and teratogenic effects of vitamin A.     

Altered glycoprotein synthesis in mouse epidermal cells treated with retinyl acetate in vitro

Retinyl acetate alters glycoprotein synthesis in mouse epidermal cells in culture. Epidermal glycoproteins were enzymatically digested to glycopeptides and separated on DEAE Sephadex A50 columns using different concentrations of LiCl. There was a two-fold increase in incorporation of fucose and glucosamine in the 0.2 M LiCl fraction from cells treated for 3 weeks with 12.5 μg/ml retinyl acetate and 1.25% DMSO as compared with DMSO controls. No changes were noted in other fractions. The glycopeptide from A treated cells isolated on 0.2 M LiCl had a higher molecular weight than glycopeptides from that same fraction eluted by control cells. This isolated newly synthesized glycopeptide from vitamin A treated cells appears to be a single product by rechromatography on DEAE Sephadex A50 and Sephadex G100 columns.     

The murine sublingual and submandibular mucins their isolation and characterization

From the mouse sublingual and submandibular glands high-molecular weight glycoproteins (mucins) were isolated. These mucins appeared to be homogeneous in polyacrylamide gel electrophoresis and in the analytical ultracentrifuge. S_20,w values of 10.9 and 5.5 were found for the sublingual and submandibular mucin respectively. With sodium dodecyl sulfate or N-acetylcysteine no subunits could be detected.Both mucins consisted for about 1/3 of protein and 2/3 of carbohydrate. Their mucin character was also denoted by the high content of serine plus threonine. Respectively 42 mol% and 34 mol% of the protein core of the sublingual and submandibular mucins consisted of these amino acids. The main sugars in these mucins were sialic acid, galactosamine, galactose, glucosamine and mannose. The molar ratio for the sublingual and submandibular mucin being 1.00 : 1.03 : 1.08 : 0.26 : 0.23 and 1.00 : 0.71 : 1.10 : 0.65 : 0.53, respectively.The sialic acid content of both mucins was about 25%. Fucose and sulfate, on the other hand, were less than 1%. The presence of sulfate was also indicated by preliminary studies in vivo on the incorporation of [³⁵SO₄] sulfate.     

Synthesis of mucous glycoproteins by rabbit tracheal cells in vitro. Modulation by substratum, retinoids and cyclic AMP.

One function of airway epithelium is the secretion of mucins, which comprise an important component of the mucous lining layer. We demonstrate that rabbit tracheal epithelial cells grown in primary culture incorporate [3H]glucosamine into material released into the medium which is characterized as mucin by the following criteria: high Mr, monosaccharide composition, ion-exchange behaviour different from that of glycosaminoglycans and oligosaccharides attached via N-acetylgalactosamine. The production of mucin by the cells requires growth on a substratum of collagen gel and is enhanced by retinoids in the extracellular medium. In the presence of retinoids, 8-bromo cyclic AMP and factors present in medium from 3T3 fibroblasts each further stimulate mucin production. These results indicate that an isolated epithelial-cell culture system, in the absence of nervous, mesenchymal or other tissue types, can be used to answer questions about the regulation of mucin production at the cellular level.     

Role of sulfation in the processing of gastric mucins.

The role of sulfation in the processing of mucus glycoprotein in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in MEM at various medium sulfate concentrations in the presence of [35S]Na2SO4, [3H]glucosamine and [3H]proline, with and without chlorate an inhibitor of PAPS formation. The results revealed that the mucin sulfation attained maximum at 300 microM medium sulfate concentration. Introduction of chlorate into the incubation medium, while having no effect on the protein synthesis as evidenced by [3H]proline incorporation, caused at its optimal concentration of 2 mM a 90% decrease in mucin sulfation and a 40% drop in mucin glycosylation. Evaluation of mucin molecular forms distribution indicated the predominance of the high molecular mucin form in the intracellular fraction and the low molecular mucin from in the extracellular fraction. Increase in medium sulfate caused an increase in the high molecular weight mucin form in both fractions, and this effect was inhibited by chlorate. Also, higher medium sulfate concentrations led to a higher degree of sulfation in the high molecular weight mucin form, the effect of which was inhibited by chlorate. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and is required for mucin polymer formation. Hence, the disturbances in mucin sulfation process could be detrimental to the maintenance of gastric mucus coat integrity.     

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.     

Purification and characterization of a human pancreatic adenocarcinoma mucin.

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.     

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.     

Regulation of terminal differentiation of cultured human keratinocytes by vitamin A

Vitamin A is known to exert an important influence on epithelial differentiation. The fetal calf serum supplement of cell-culture medium contains enough of the vitamin to affect the differentiation of cultured keratinocytes derived from epidermis and from other stratified squamous epithelia. The cellular and molecular properties of the cultures are altered when the medium is supplemented with serum from which the vitamin A has been removed by solvent extraction (delipidized serum). Cell motility is reduced, the adhesiveness of cells increases and pattern formation is prevented. In both epidermal and conjunctival keratinocytes, removal of vitamin A leads to the synthesis of a 67 kd keratin characteristic of terminally differentiating epidermis and to much reduced synthesis of the 52 kd and 40 kd keratins typical of conjunctiva. These changes, both cellular and molecular, are reversed by the addition of retinyl acetate to the medium containing delipidized serum. Cell motility and pattern formation are restored, and detachment of the most mature cells from the surface of the stratified epithelium is promoted. Synthesis of the 67 kd keratin is prevented and the synthesis of the 40 and 52 kd keratins is stimulated. The nature of the keratins synthesized is regulated by the concentration of vitamin A, and each cell type adjusts its synthesis differently at a given vitamin concentration.     

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,′-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.