Vitamin A-sufficient Rats Research Articles

Roles of Vitamin a and Macula Flava in Maintaining Vocal Folds

Vitamin A plays important roles in development, growth, and regeneration. Vitamin A-storing stellate cells have been identified in several organs. The functional roles of vitamin A in the vocal folds are still unknown, although vitamin A-storing vocal fold stellate cells have been observed in the macula flava of human and rat vocal folds. The purpose of this study was to investigate the roles of vitamin A in vocal folds. Vitamin A-deficient rats were generated, and the vocal folds were examined histologically. Messenger RNA was extracted from the vocal folds and analyzed by real-time polymerase chain reaction. Immunohistochemical analysis of normal vocal folds revealed expression of retinoic acid receptor a in vocal fold stellate cells. The cells in the macula flava of vitamin A-deficient rats showed a larger nucleus/cytoplasm ratio than did those of vitamin A-sufficient rats, but messenger RNA expression of major extracellular matrix components in the macula flava of vitamin A-deficient rats did not present a remarkable change except for procollagen type I. Expression of hyaluronic acid, collagen types I and III, and elastin did not show a significant change in vitamin A-deficient rat vocal folds. These results indicate that vitamin A is not essential to maintaining the extracellular matrix of normal adult vocal folds, although vocal fold stellate cells participate in vitamin A storage.

Vitamin A deficiency promotes bronchial hyperreactivity in rats by altering muscarinic M(2) receptor function.

Vitamin A deficiency (VAD) remains an important health problem among children in developing countries. Children living in these areas have a higher mortality from respiratory infections, which likely results in part from suboptimal nutrition, including VAD. Bronchial hyperreactivity can follow viral respiratory infections and may complicate the recovery. To investigate whether VAD promotes bronchial hyperreactivity, we have assessed methacholine-induced bronchoconstriction in VAD and vitamin A-sufficient rats. Bronchial constriction developed at lower concentrations of inhaled methacholine in VAD than in vitamin A-sufficient rats. This did not result from an increase in the bronchial wall thickness or the clearance of a small molecule (with a size similar to methacholine) from the air space. The function and abundance of the muscarinic M(2) receptors in bronchial tissue were reduced in VAD rats, suggesting that this receptor may contribute to these animals' diminished ability to limit cholinergic-mediated bronchoconstriction. A similar reduction in muscarinic M(2) receptor function has been observed in asthma. Vitamin A (retinol) and its congeners (retinoids) may be required to regulate bronchial responsiveness in addition to maintaining a normal bronchial epithelium.

Cellular expression of retinal dehydrogenase types 1 and 2: effects of vitamin A status on testis mRNA.

We examined expression of retinal dehydrogenase (RALDH) types 1 and 2 in liver and lung, and the effect of vitamin A status on testis expression by in situ hybridization. Liver expressed RALDH1 and RALDH2 only in stellate cells and hepatocytes, respectively. Lung expressed RALDH1 and RALDH2 throughout the epithelia of the airways, from the principal bronchi to the respiratory bronchiole. Vitamin A-sufficient rats expressed RALDH1 in spermatocytes, with less intense expression in spermatogonia and spermatids, and expressed RALDH2 in interstitial cells, spermatogonia, and spermatocytes. Neither Sertoli nor peritubular cells showed detectable RALDH1 or RALDH2 mRNA. Vitamin A deficiency produced a sevenfold increase in RALDH1 and a 70-fold decrease in RALDH2 mRNA in testis. In each case, the net change reflected extensive loss of germ cells, increased intensity of expression in residual germ cells, and expression in Sertoli and peritubular cells. Low-dose RA relatively early during vitamin A depletion supported spermatogenesis and affected expression of both RALDHs, but did not reinstate "vitamin A normal" expression patterns. These results show that: RALDH1 and RALDH2 have distinct mRNA expression patterns in multiple cell types in three vitamin A target tissues; RALDH expression occurs in cell types that express cellular retinol-binding protein and retinol dehydrogenase isozymes (except stellate cells, for which retinol dehydrogenase expression remains unknown); vitamin A deficiency and RA supplementation affects the loci and intensity of RALDH mRNAs in testis; and low-dose RA does not substitute completely for retinol. Overall, these data provide insight into the unique functions of RALDH1 and RALDH2 in retinoid metabolism.

Dietary restriction alters retinol and retinol-binding protein metabolism in aging rats.

Recent studies reported that retinoid metabolism was influenced by long-term dietary restriction (DR) in rats. Because plasma retinol was decreased in rats subjected to DR, it was thought that this dietary manipulation may have an effect on retinol-binding protein (RBP) metabolism. Thus, the aim of this study was to assess retinoids, RBP, and transthyretin (TTR) levels in plasma and liver of young (3 months), adult (12 months), and old (22 months) female Sprague-Dawley rats fed ad libitum (AL) or subjected to a 40% DR, enriched (DR+), or not (DR), with vitamins and minerals. Results indicate that hepatic total retinoid concentrations and content increased with age in all the groups. DR+ rats showed higher hepatic retinoid concentrations than age-matched AL and DR rats. Adult and old DR and DR+ rats exhibited significantly lower plasma RBP-retinol and higher total retinoic acid levels than corresponding controls, although these parameters were not influenced by aging. Liver RBP levels were also decreased in DR and DR+ rats when compared to respective AL controls. There was a slight age-related decline in plasma TTR levels in DR and DR+ rats which was not associated with modifications in liver TTR levels. Hepatic gene expression of RBP and TTR, as evaluated by Northern blot hybridization, did not change with age or diet, suggesting that the lower levels of plasma RBP-retinol and liver RBP in vitamin A-sufficient rats subjected to DR may reflect post-transcriptional alterations and/or accelerated degradation of hepatic RBP. The elevated plasma levels of retinoic acid may represent an adaptive mechanism developed by DR rats to maintain retinoid-dependent functions.

Retinol and retinyl ester concentrations in rat tissues during total parenteral nutrition

We have previously observed that continuous total parenteral nutrition (TPN) that supplies retinyl palmitate induces a strong decrease of the circulating retinol, which is associated with an impaired hepatic production of retinol-binding protein. We have investigated the effect of 7 days of TPN on the retinol and retinyl ester concentrations in rat tissues, relative to the vitamin A status [ n = 30 for vitamin A-sufficient (A +) and n = 30 for deficient (A −)]. Rats were cannulated for intravenous feeding ( n = 12 for TPN-A +, n = 12 for TPN-A −) and were compared with their per os pair-fed counterparts ( n = 12 for O-A + and n = 12 for O-A −). Retinol and retinoic acid in serum and retinol and retinyl ester concentrations in liver, kidney, lung, heart, and testis were measured by high performance liquid chromatography. TPN induced a dramatic decrease in circulating retinol of A + rats, whereas retinoic acid concentration in serum was unchanged. When TPN was given to A − rats, retinol concentration in serum remained low. Lower retinol and retinyl ester concentrations were measured in the livers of TPN rats compared with orally pair-fed rats, no matter the initial vitamin A status ( P < 0.02 for A + and P < 0.01 for A −). By contrast, in extrahepatic tissues, retinol and retinyl ester concentrations were similar in TPN rats compared with orally pair-fed rats. Our results indicate that TPN induced a decrease of retinol in serum and retinyl esters in liver. However, TPN was able to maintain retinol and retinyl ester concentrations in extrahepatic tissues of vitamin A-sufficient rats and to restore retinol and retinyl ester concentrations in extrahepatic tissues of vitamin A-deficient rats, albeit a low circulating retinol.

N-(4-Hydroxyphenyl)-Retinamide Increases Lecithin:Retinol Acyltransferase Activity in Rat Liver

N-(4-Hydroxyphenyl)-retinamide (4-HPR; Fenretinide) is a synthetic retinoid which is undergoing investigation as a cancer chemopreventive agent. However, 4-HPR alters vitamin A kinetics and reduces the concentration of plasma retinol. We have conducted studies to examine the effects of 4-HPR on the activity of the enzyme lecithin:retinol acyltransferase (LRAT). This enzyme is implicated in the absorption and storage of vitamin A and is regulated, in liver, by vitamin A nutritional status. To determine whether 4-HPR, like retinoic acid, is able to induce liver LRAT activity, vitamin A-deficient rats having negligible liver LRAT activity were treated with single doses of 4-HPR (0.02–2.5 mg) and liver homogenates were assayed for LRAT activity using 3H-retinol bound to the cellular-retinol binding protein, CRBP, as substrate. Treatment with 4-HPR resulted in a dose- and time-dependent increase in liver LRAT activity which reached a maximum at 24 h. The activity of LRAT assayed in vitro and of hepatic 13 H-retinyl ester content determined after an in vivo pulse of 3H-retinol were highly correlated (r = 0.802, P < 0.0002). When vitamin A-sufficient rats were fed a 4-HPR-supplemented diet for 30 d, LRAT activity differed significantly from control values in the liver (P < 0.0001) but not the small intestines. Changes in hepatic retinol metabolism which favor the esterification of vitamin A may be related to the mechanism by which 4-HPR alters vitamin A kinetics in vivo.

Effects of acute inflammation on plasma retinol, retinol-binding protein, and its mRNA in the liver and kidneys of vitamin A-sufficient rats

The acute inflammatory response to tissue injury and infection is associated with low concentrations of plasma retinol and its specific transport proteins, retinol-binding protein (RBP) and transthyretin (TTR). To examine the kinetics and mechanism of hyporetinemia, we have induced acute inflammation with lipopolysaccharide (LPS, from Pseudomonas aeruginosa) in rats with adequate stores of vitamin A. Twenty-four h after treatment with LPS (50 micrograms i.p. per 100 g body weight) or saline and food withdrawal, plasma retinol equalled 0.72 +/- 0.06 mumol/L (mean +/- SEM) in five LPS-treated rats versus 1.35 +/- 0.1 mumol/L in five saline-treated rats (P < 0.01). Plasma, liver, and kidney RBP and TTR concentrations were also significantly reduced, but liver and kidney retinol concentrations did not differ between treatment groups. The relative abundance of RBP mRNA in liver (LPS treatment compared to saline treatment) was reduced as early as 12 h (0.44 +/- 0.15, n = 4 pairs, P < 0.02), and continued to be reduced at 24 h (0.57 +/- 0.12, n = 5 pairs, P < 0.02). In the kidney this ratio did not change significantly due to LPS treatment. The relative abundance of cellular retinol-binding protein (CRBP) mRNA in liver and kidney also was not affected by LPS treatment. We infer from these data that inflammation-induced hyporetinemia results from a reduction in the hepatic synthesis of RBP and secretion of the retinol-RBP complex. Moreover, the results imply that plasma retinol concentration is a poor indicator of vitamin A status during inflammation.

THE EFFECT OF RETINOIC ACID AND DEXAMETHASONE ON THE mRNA EXPRESSION OF THE RETINOIC ACID RECEPTOR-β IN THE RAT. † 1833

Retinol is essential for normal epithelial cell differentiation and maintenance of epithelial cell integrity. Retinoic acid (RA), the active metabolite of retinol, influences gene expression via binding to nuclear retinoic acid receptors (RAR). In vitamin A-deficient adult rats, lung RAR-β gene expression is induced by retinoic acid (Proc Natl Acad Sci 1991; 88:8272). Recently, RAR-β gene expression has been shown to be decreased by dexamethasone (DEX) in neonatal rats (Pediatr Pulmonol 1995; 20:234). We hypothesized that RA and DEX interact to influence RAR-β gene expression. To test this hypothesis, adult, female, vitamin A-sufficient rats were treated with RA 100 μg. DEX 2 μg/g body weight, both RA and DEX, or cottonseed oil or saline as controls. The animals were sacrificed 6 hours after treatment; lung and liver tissue were then obtained for northern blot RNA analysis using 32P labeled cDNA probes specific for RAR-β (two isoforms, β1 = 3.3 kb andβ2 = 3.0 kb) and G3PDH. Densitometry was performed and results are expressed in arbitrary densitometry units. The results (mean ±SD) are shown in the table.

All-trans-retinoyl β-glucuronide: new procedure for chemical synthesis and its metabolism in vitamin A-deficient rats

All-trans-retinoyl beta-glucuronide (RAG) was chemically synthesized in high yields (up to 79%) by a new procedure involving the reaction of the tetrabutylammonium salt of glucuronic acid with all-trans-retinoic acid (RA) via the imidazole or triazole derivative. When RAG was fed orally to vitamin A-deficient rats, RA was identified as the major metabolite in the serum within hours of administration of RAG. Very little or no RAG was detected in the serum. Thus RAG, which was not appreciably hydrolysed to RA in vitamin A-sufficient rats [Barua and Olson (1987) Biochem. J. 263, 403-409], was rapidly converted into RA in vitamin A-deficient rats.

Percutaneous absorption, excretion and metabolism of all-trans-retinoyl beta-glucuronide and of all-trans-retinoic acid in the rat.

The purpose of these studies was to compare directly the percutaneous absorption,excretion and metabolism of all-trans-retinoyl beta-glucuronide (RAG), a nontoxic retinoid, with all-trans-retinoic acid (RA) in the rat. Previously, it was demonstrated that topical treatment of human acne with either RAG or RA in cream resulted in a significant reduction of lesions. Whereas 0.1% RA showed adverse effects, concentrations of RAG up to 2.4% did not cause any adverse reactions. In the present studies, radiolabeled RAG or RA, dispersed in a water-based cream, was applied to the shaved dorsal skin of vitamin A-sufficient rats. Both RAG and RA were absorbed from the skin in a similar way. In both cases, radioactivity peaked in the plasma within 2-4 h and within the liver at 4-12 h. During a 7-day period, the overall excretion of radioactivity derived from RA and RAG in the feces and urine were similar, e.g. 17 and 12%, respectively. it is concluded that: (1) the transport, metabolism and excretion of topically applied radioactive RA and RAG are similar, although not identical, in the rat and (2) the toxic skin manifestations induced by RA but not by RAG cannot be attributed to major differences in their overall absorption, metabolism and excretion.

Immunologic memory is established in nursling rats immunized with tetanus toxoid, but is not affected by concurrent supplementation with vitamin A

Experiments were conducted to determine whether nursling rats immunized with tetanus toxoid (TT) are able to produce a specific antibody response and whether oral treatment with retinyl palmitate, concurrent with immunization, affects the magnitude of the anti-TT response. When rats aged 8-15 d and nursed by vitamin A-sufficient dams were immunized with TT, no primary anti-TT immunoglobulin (Ig) M or IgG response was detected. However, nursling rats formed immunologic memory to TT because, when they were reimmunized at 40 d of age, their secondary anti-TT IgG response exceeded the primary response of 40-d-old vitamin A-sufficient rats (P < 0.02). Provision of retinyl palmitate (equal to 37.5 or 150 micrograms retinol equivalents) by mouth with early primary immunization did not change the magnitude of the secondary anti-TT IgG response. However, the age of nursling rats at first immunization significantly affected the magnitude of their secondary anti-TT IgG response, because rats first immunized at 15 d of age and reimmunized at 40 d of age produced a secondary response that was nearly fivefold greater than that of rats immunized at 8 and 40 d of age. In conclusion, nursling rats immunized with TT formed immunologic memory, which was affected significantly by the timing of the primary immunization. However, the administration of retinyl palmitate concurrent with early primary immunization did not significantly affect the development of memory to TT.

Retinoic Acid Repletion Restores the Number of Leukocytes and Their Subsets and Stimulates Natural Cytotoxicity in Vitamin A-Deficient Rats

Previously we reported that vitamin A-deficient rats have a low number of natural killer (NK) cells in their blood and spleen. The current studies were designed to address whether other cells of the immune system are also affected and whether dietary retinoic acid is able to reverse the changes caused by a deficiency of retinol and its metabolites. Total white blood cells, differential counts and spleen cell numbers were compared in vitamin A-sufficient rats (controls) and rats deficient in vitamin A, and lymphocyte and NK cell populations were identified and enumerated by flow cytometry. In comparison with control rats, the blood of deficient rats had three times the number of granulocytes, and fewer B lymphocytes (73% of control) and NK cells (38% of control). The numbers of splenic B cells (OX12+), CD5+ (OX19+) and CD4+ (W3/25+) T lymphocytes and NK cells (NKR-P1+) were also significantly reduced. When vitamin A-deficient rats were fed a retinoic acid supplement (4.2 mg all-trans retinoic acid/kg diet) for 28 d, the numbers of blood granulocytes and NK cells equaled those of control rats and NK cell cytotoxicity was significantly elevated. Blood lymphocyte number was increased 40% due to increases of B cells and T cells of the CD5+, CD4+ and CD8+ subsets. These data indicate that vitamin A deficiency affects a number of cells of the immune system and that repletion with retinoic acid effectively reestablishes the number of circulating lymphocytes. In addition, retinoic acid may stimulate NK cell function.

The antibody response of vitamin A-deficient rats to pneumococcal polysaccharide is enhanced through coimmunization with lipopolysaccharide.

Although the antibody response of vitamin A-deficient rats immunized with pneumococcal polysaccharide (SSS-III) was barely detectable, coimmunization with SSS-III plus 2 or 20 micrograms of Pseudomonas aeruginosa lipopolysaccharide (LPS) resulted in an anti-SSS-III response that was equal to or greater than that of vitamin A-sufficient rats immunized only with SSS-III. Doses of 2 and 20 micrograms of LPS, but not 0.1 and 0.5 micrograms, produced significant adjuvanticity, and LPS functioned as an adjuvant when given concomitantly with SSS-III (day 0) or up to 3 days later. Thus, vitamin A deficiency per se does not preclude the anti-SSS-III response, and LPS or factors released in response to LPS, even after initiation of the response to SSS-III, may substantially enhance antibody production to this thymus-independent, type 2 antigen.

Vitamin A deficiency potentiates carbon tetrachloride-induced liver fibrosis in rats

Earlier studies have shown that retinoid administration suppresses the generation of hepatic fibrosis and stimulates its regression in normal (i.e., vitamin A-sufficient) carbon tetrachloride-treated rats. This study focuses on the possible role of a marginal or deficient vitamin A status on carbon tetrachloride-induced fibrosis. This experimental study in rats shows that vitamin A status, reflected by hepatic retinoid content (retinol and retinyl esters), modulates the development of hepatic fibrosis induced by carbon tetrachloride. In rats with low hepatic retinoid levels (12 +/- 0.9 micrograms/gm liver), carbon tetrachloride-induced liver fibrosis was more pronounced than in rats with sufficient hepatic retinoid levels (1,065 +/- 327 micrograms/gm liver). Enhanced liver fibrogenesis was confirmed both morphologically and by a higher hydroxyproline content of the liver. It was associated with a reduced liver weight and the development of parenchymal regeneration nodules. Furthermore, carbon tetrachloride treatment itself reduced the hepatic retinoid content in rats independently of the liver vitamin A status before treatment and increased serum retinol levels in vitamin A-sufficient rats. The results show that the vitamin A status of the liver plays an important role in hepatic fibrogenesis. Low hepatic vitamin A levels, which can be the result not only of low dietary intake but also of interference with vitamin A metabolism by agents such as ethanol and carbon tetrachloride, may be a risk factor for the development of liver fibrosis. We suggest that retinoids modulate collagen synthesis and deposition irrespective of the degree of hepatocellular necrosis induced by carbon tetrachloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucuronidation of all- trans-retinoic acid in liposomal membranes

Retinoyl β- d-glucuronide is a biologically active metabolite of retinoic acid. The kinetics of UDP-glucuronosyltransferase-catalyzed biosynthesis of retinoyl β- d-glucuronide was examined in rat liver and intestinal native microsomes incubated with [ 3H]retinoic acid incorporated into liposomes. The product was identified by cochromatography with authentic all- trans retinoyl β- d-glucuronide, by hydrolysis with β- d-glucuronidase, and by mass spectrometry. In vitamin A-sufficient rats the apparent K m values for all- trans-retinoic acid were 173 μM and 125 μM, and the apparent V max , 62 and 41 pmol/min per mg, for small intestinal and liver microsomes, respectively. In vitamin A-deficient rats repleted with all- trans-retinyl acetate, the apparent K m (91 μM) and V max (53 pmol/min per mg) for intestinal microsomes were in range of those of vitamin A-sufficient rats. The similarities in the kinetic parameters for UDP-glucuronosyltransferase in small intestinal mucosa and liver suggest that the reactions are catalyzed by the same enzyme. In vitamin A-deficient rats given a large amount all- trans-retinoic acid (1.2 mmol/day for 3 days) the apparent K m was 105 μM and V max , 127 pmol/min per mg of intestinal microsomal protein. We conclude that the kinetics of intestinal retinoic acid glucuronidation are not characteristic of simple detoxification reactions. Retinoyl glucuronide may be important in mediating retinoic acid metabolism and function.

Vitamin A is Involved in Maintenance of Epithelial Cells on the Bronchioles and Cells in the Alveoli of Rats

We examined the effects of mild vitamin A deficiency and ozone (O3) exposure on the labeling index, a marker of cell proliferation, of epithelial cells on the bronchiole and cells in the alveoli of rat lungs, to assess the role of vitamin A in maintenance of epithelial cells on the distal airway and alveolus. Three-week-old rats were fed a vitamin A-depleted diet for 4 wk to induce mild vitamin A deficiency. After 2 wk rats were exposed to 16.4 mumol O3/m3 for 1 to 14 d. In vitamin A-sufficient rats, labeling indices of epithelial cells on the bronchiole and cells in the alveolus increased significantly in comparison with those of controls exposed to clean air on d 2 and 3 of O3 exposure. In vitamin A-deficient rats as well, labeling indices were increased by O3 exposure, but the magnitude of increase was significantly smaller than for vitamin A-sufficient rats. These results indicate that vitamin A deficiency resulted in decrease of proliferation of epithelial cells on the distal airway and cells in the alveolus of rats when the proliferation of these cells was stimulated by O3 exposure, suggesting an involvement of vitamin A in maintenance of lung epithelial cells.

Opposing Effects of Retinoic Acid and Dexamethasone on Cellular Retinol-Binding Protein Ribonucleic Acid Levels in the Rat*

Cellular retinol-binding protein (CRBP) is a potential mediator of vitamin A action. To determine whether retinoic acid and dexamethasone administration, alone and in combination, influence CRBP gene expression, adult female vitamin A-sufficient Sprague-Dawley rats randomly received 1) all-trans retinoic acid (100 micrograms) by intragastric intubation, 2) dexamethasone (2 micrograms/g BW) by ip injection, or 3) both all-trans retinoic acid and dexamethasone in the same doses. Control animals received either cottonseed oil by intragastric intubation or saline by ip injection. Six hours after treatment, lung and liver tissue were collected for Northern blot analysis with the radiolabeled cDNA specific for rat CRBP. Retinoic acid administration increased the amount of lung CRBP mRNA only, whereas dexamethasone decreased both lung and liver CRBP mRNA abundance. In animals treated with both retinoic acid and dexamethasone, CRBP mRNA abundance was also reduced. We conclude that CRBP gene expression can be modulated by both retinoic acid and dexamethasone in the vitamin A-sufficient animal. In the whole animal, our findings indicate that dexamethasone not only represses CRBP gene expression, but also opposes the effect of retinoic acid.

Vitamin A in parenteral nutrition: uptake and distribution of retinyl esters after intravenous application

Short-term parenteral application of vitamin A was examined in rats. Retinyl margarinate, which is chemically similar to physiological retinyl esters, was used in vitamin A-depleted rats to study uptake, distribution, and storage of retinyl esters in tissues. Vitamin A-depleted and Vitamin A-sufficient rats were infused with a micellar suspension of retinyl margarinate for 7 h and then killed at different times. Retinyl margarinate was directly taken up by all tissues examined. It appears that infusion of retinyl esters in micellar form provides a direct way to supply vitamin A to peripheral, vitamin A-dependent tissues. Therefore, a short-term infusion of retinyl esters with an emulsifier may be an effective means of preventing development of vitamin A-deficiency during long-term application of TPN, particularly in cases of liver dysfunction.

Lack of biological activity of physiological metabolites of all- trans-retinoic acid on vaginal epithelial differentiation ∗

It has been of interest to determine whether the metabolites of physiological doses of retinoic acid represent active forms of vitamin A. Previous work ( Biochem. J. 206, 33–41, 1982) studied the metabolites produced from 2-μg doses of all- trans-retinoic acid in the vitamin A-deficient rat. Four major metabolites common to all of the tissues studied were discovered. In the present work, three of these metabolites are isolated from vitamin A-deficient rats given physiological doses (5 μg) of all- trans-retinoic acid and from vitamin A-sufficient rats given high doses (1 mg) of all- trans-retinoic acid. Cochromatography on anion-exchange and reverse-phase high-performance liquid chromatography showed that metabolites resulting from high doses of retinoic acid contained the metabolites generated from physiological doses of retinoic acid. Quantities of these metabolites were isolated, purified, and tested for their epithelial-differentiating activity in the vitamin A-deficient rat vagina. The metabolites were inactive at all dose levels tested. These metabolites have less than 10% the biological activity of all- trans-retinoic acid. Therefore, these metabolites appear to be products of the inactivation of all- trans-retinoic acid. Based upon these and previous data, it seems likely that all- trans-retinoic acid or its β-glucuronide derivative is the most likely active form of vitamin A in the maintenance of normal epithelial differentiation.

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 fatstoring 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.