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.     

Dietary vitamins A and E influence retinyl ester composition and content of the retinal pigment epithelium

Experiments were conducted to determine the influence of dietary levels of vitamin A and alpha-tocopherol on the amounts and composition of retinyl esters in the retinal pigment epithelium of light-adapted albino rats. Groups of rats were fed diets containing alpha-tocopherol and either no retinyl palmitate, adequate retinyl palmitate, or excessive retinyl palmitate. Other groups of rats received diets lacking alpha-tocopherol and containing the same three levels of retinyl palmitate. Retinoic acid was added to diets lacking retinyl palmitate. After 27 weeks, the animals were light-adapted to achieve essentially total visual pigment bleaches, and the neural retinas and retinal pigment epithelium-eyecups were then dissected from each eye for vitamin A ester determinations. Almost all of the retinyl esters were found in the retinal pigment epithelium-eyecup portions of the eyes, mainly as retinyl palmitate and retinyl stearate. Maintaining rats on a vitamin A-deficient, retinoic acid-containing diet led to significant reductions in retinal pigment epithelial retinyl ester levels in rats fed both the vitamin E-supplemented and vitamin E-deficient diets; contrary to expectations, the effect of dietary vitamin A deficiency was more pronounced in the vitamin E-supplemented rats. Vitamin A deficiency in retinoic acid-maintained animals also led to significant reductions in retinyl palmitate-to-stearate ester ratios in the retinal pigment epithelia of both vitamin E-supplemented and vitamin E-deficient rats. Excessive dietary intake of vitamin A had little, if any, effect on retinal pigment epithelial retinyl ester content or composition. Vitamin E deficiency resulted in significant increases in retinal pigment epithelial retinyl palmitate content and in palmitate-to-stearate ester ratios in rats fed all three levels of vitamin A, but had little effect on retinal pigment epithelial retinyl stearate content. In other tissues, vitamin E deficiency has been shown to lower vitamin A levels, and it is widely accepted that this effect is due to autoxidative destruction of vitamin A. The increase in retinal pigment epithelial vitamin A ester levels in response to vitamin E deficiency indicates that vitamin E does not regulate vitamin A levels in this tissue primarily by acting as an antioxidant, but rather may act as an inhibitor of vitamin A uptake and/or storage. The effect of vitamin E on pigment epithelial vitamin A levels may be mediated by the vitamin E-induced change in retinyl palmitate-to-stearate ratios.     

Effect of vitamin A nutritional status on the growth of oestrogen-primed chick oviduct.

Growth and development of the oestrogen-primed oviduct of immature chicks in terms of weight, length, total protein, total RNA, total DNA and total phospholipids are markedly arrested on deprivation of vitamin A; supplementation with retinyl acetate reversed the effect of the deficiency almost fully, whereas retinoic acid was only partly effective.     

Effect of vitamin A deprivation on the cholesterol side-chain cleavage enzyme activity of testes and ovaries of rats (Short Communication)

The cholesterol side-chain cleavage enzyme activity is decreased considerably at the mild stage of vitamin A deficiency in rat testes and ovaries and the decrease in activity becomes more pronounced with progress of deficiency. Supplementation of the deficient rats with retinyl acetate, but not retinoic acid, restores the enzyme activity to normal values. The cholesterol side-chain cleavage enzyme of adrenals is not affected by any of the above treatments.     

5,6-epoxyretinoic acid is a physiological metabolite of retinoic acid in the rat.

5,6-Epoxyretinoic acid was detected in small intestine, kidney, liver, testes and serum of vitamin A-deficient rats 3 h after a single physiological dose of [3H]retinoic acid. The maximum concentration of 5,6-epoxide in intestinal mucosa was observed 3 h after intrajugular administration of retinoic acid. However, at 7 h post administration, no 5,6-epoxyretinoic acid was detected in mucosa, demonstrating the rapid intestinal metabolism or excretion of this metabolite. No 5,6-epoxy[3H]retinoic acid was detected in mucosa, liver or serum of retinoic acid-repleted rats 3 h after administration of 2 micrograms of [3H]retinoic acid.     

Recent evidence for the participation of vitamin A in glycoprotein synthesis.

Recent evidence supports the concept that vitamin A plays some role in glycoprotein synthesis in a large-variety of tissues examined. Its involvement may be through participation of a retinol-linked sugar, mannosyl retinyl phosphate (MRP). Upon injection of [3H]retinol and [14C]mannose into rats, [14C, 3H]MRP could be isolated from liver and intestinal mucosa, and identified by chromatographic and hydrolytic experiments. The enzyme system that forms MRP from GDP-mannose and retinyl phosphate was located primarily in rough endoplasmic reticulum of fractionated liver cells, with some activity also in smooth membranes and Golgi apparatus. Vitamin A deficiency resulted in depressed synthesis of the rat serum glycoprotein alpha 1-macroglubin (alpha 1-MG), as shown by a decline in labeling. Analysis of the labeled alpha 1-MG from serum of normal and vitamin A-deficient rats showed this to be the result of a defect in glycosylation. The specific activity ratio (deficient:normal) of the alpha 1-MG of serum declined progressively with development of the deficiency, as a result of underglycosylation. Complete carbohydrate analysis of the alpha 1-MG of normal and deficient serum revealed a sugar loss in this glycoprotein as a result of vitamin A deficiency.     

Cyclic AMP dependent protein kinase and its endogenous inhibitor protein: tissue distribution and effect of vitamin D status in the chick

1. Vitamin D deficiency in the chick leads to decreased (to 55% of normal) cyclic AMP-dependent protein kinase activity in the kidney but does not alter calcium-dependent phospholipid-sensitive protein kinase activity. 2. Decreased cyclic AMP-dependent protein kinase activity in response to vitamin D deficiency was not observed in other tissues including pancreas, brain, liver, intestinal mucosa, or heart. 3. Vitamin D deficiency leads to elevated levels of the endogenous inhibitor protein of cyclic AMP-dependent protein kinase in kidney, but not heart, muscle, pancreas, or brain.     

Impaired vitamin A-mediated mucosal IgA response in IL-5 receptor-knockout mice.

To clarify actions of vitamin A on mucosal immunity associated with interleukin-5 (IL-5), we examined effects of vitamin A on mucosal IgA level in IL-5 receptor alpha-chain-knockout (IL-5Ralpha(-/-)) mice. Daily supplementation of retinyl acetate (1 mg/mouse) increased Th2 cytokine levels and a number of their positive cells in the small intestinal mucosa of IL-5Ralpha(-/-) mice, as observed in wild-type or IL-5Ralpha(+/-) mice. Wild-type and heterozygous mice increased the IgA level and a number of IgA-containing cells in the mucosa in response to the vitamin A treatment, but not in IL-5Ralpha(-/-) mice. Retinyl acetate increased anti-cholera toxin (CT) IgA level in the mucosa of wild-type mice, improving their survival rate after an exposure to 0.4 mg of CT. However, retinyl acetate failed to induce resistance to CT toxicity in IL-5Ralpha(-/-) mice. Our results suggest that IL-5 may play an important role in an action of vitamin A on mucosal IgA system.     

Effects of retinoic acid on the concentrations of radioactive metabolites of retinol in tissues of rats maintained on a retinol-deficient diet

The effects of feeding retinoic acid for 2 and 6 days on the metabolism of labeled retinol in tissues of rats maintained on a vitamin A deficient diet was studied. The metabolites of retinol were analyzed by high performance liquid chromatography. Feeding retinoic acid for 2 days significantly reduced the blood retinol and retinyl ester levels without affecting the vitamin A content of the liver. In intestine and testis the content of labeled retinoic acid was decreased significantly by dietary retinoic acid. Addition of retinoic acid to the diet for 6 days resulted, in addition to decreased blood retinol and retinyl ester values, in an increase in the retinyl ester values in the liver. The accumulation of retinyl ester in the retinoic acid fed rat liver was accompanied by an absence of labeled retinoic acid. Kidney tissue was found to contain the highest levels of labeled retinoic acid, retinol, and retinyl esters; dietary retinoic acid did not alter the concentrations of these retinoids in the kidney during the experimental period. Since kidney retained more vitamin A when the liver vitamin A was low and also dietary retinoic acid did not affect the concentrations of radioactive retinoic acid in the kidney, it is suggested that the kidney may play a major role in the production of retinoic acid from retinol in the body.     

Influence of vitamin A status and DDT on vitamin A-dependent protein mannosylation in rat liver

Male Wistar rats of different vitamin A status (total depletion to moderate deficiency) were administered DDT (5 mg/kg/day) or vehicule (corn oil) i.p. daily for 14 days. Vitamin A-dependent protein mannosylation was measured either by in vivo incorporation of [3H]mannose into liver glycoprotein or by in vitro assay of incorporation of [14C]mannose into mannosylretinyl phosphate. Vitamin A deficiency resulted in a significantly impaired in vivo incorporation of mannose in liver glycoprotein but had no effect on the in vitro transport of mannose via retinyl phosphate. Although DDT induced an increase synthesis of liver proteins in smooth endoplasmic reticulum and caused a diminution of the hepatic vitamin A content, it did not affect vitamin A-dependent protein mannosylation.     

Metabolism of all-trans-retinoic acid and all-trans-retinyl acetate. Demonstration of common physiological metabolites in rat small intestinal mucosa and circulation

The kinetics and metabolism of physiological doses of all-trans-retinoic acid were examined in blood and small intestinal mucosa of vitamin A-depleted rats. A major portion of intrajugularly injected retinoic acid is rapidly (within 2 min) sequestered by tissues; subsequently 13-cis-retinoic acid and polar metabolites are released into circulation. All-trans-retinoic acid appears in small intestinal epithelium within 2 min after dosing and is the major radioactive compound there for at least 2 h. Retinoyl glucuronide and 13-cis-retinoic acid are early metabolites of all-trans-retinoic acid in the small intestine of bile duct-cannulated rats. Retinoyl glucuronide, the major metabolite of retinoic acid intestinal epithelium, in contrast to other polar metabolites, was not detected in circulation. An examination of [3H]retinyl acetate metabolites under steady state conditions in vitamin A-repleted rats demonstrates the occurrence of all-trans-retinoic acid and 13-cis-retinoic acid in circulation and in intestinal epithelium, in a pattern similar to that found after injection of retinoic acid into vitamin A-depleted rats. Our data establish that all-trans-retinoic acid, 13-cis-retinoic acid, and retinoyl glucuronide are physiological metabolites of vitamin A in target tissues, and therefore are important candidates as mediators of the biological effect of the vitamin.     

Vitamin A metabolism: α-Tocopherol modulates tissue retinol levels in vivo,and retinyl palmitate hydrolysis in vitro

The steady-state concentrations of retinol in rat tissues varied as a function of dietary α-tocopherol. The liver, kidney, and intestinal retinol concentrations increased in animals fed an α-tocopherol-deficient diet despite a decrease (liver) or no change (kidney and intestine) in the concentrations of total vitamin A. In contrast, in lung the concentrations of both retinol and total vitamin A decreased. α-Tocopherol inhibited retinyl palmitate hydrolase in vitro in liver, kidney, and intestine; had minimal effect on the testes hydrolase; and stimulated the lung hydrolase. Fifty percent inhibition of the liver hydrolase was provided by an α-tocopherol concentration (100 μm), close to that reported in livers of rats fed a purified diet, constituted with moderately low amounts of α-tocopheryl acetate. Phylloquinone (vitamin K₁) inhibited the retinyl palmitate hydrolase in vitro in all tissues tested, and was about fivefold more potent than α-tocopherol. The effects of phylloquinone and α-tocopherol on the liver hydrolase were additive, not synergistic. The antioxidant N,N′-diphenyl-p-phenylenediamine, the most effective synthetic vitamin E substitute known, had little effect on the hydrolase. These data show that α-tocopherol effects vitamin A metabolism in several tissues, and suggest that it may be a physiological effector of tissue retinol homeostasis.     

Effect of iron and/or vitamin A re-supplementation on vitamin A and iron status of rats after a dietary deficiency of both components

Iron and vitamin A deficiency are common nutritional problems in developing countries. From animal experiments and intervention studies, growing evidence is pointing to a possible influence of iron on vitamin A metabolism. We assessed the affects of an oral supplementation of vitamin A and/or iron on the recovery of rats from vitamin A and iron deficiency. Weanling male Wistar rats were kept for four weeks on an iron and vitamin A deficient diet. Thereafter, rats were repleted with iron 35 mg/kg feed, with vitamin A 4500 IU/kg feed both, or with iron 35 mg/kg and vitamin A 4500 IU/kg for five weeks. Retinol and retinyl esters in plasma and tissues were determined by HPLC. Iron was determined by atomic absorption spectrophotometry. The determination of haematological parameters showed that rats developed an anaemia during depletion. This was reversed by the re-supplementation with iron but not vitamin A alone. The simultaneous supplementation of vitamin A was of no additional benefit. When rats were resupplemented with iron alone a substantial further decrease in plasma retinol (P < 0.002) and liver vitamin A (P < 0.05) was observed. A similar but less pronounced decrease in plasma retinol was observed in the rats re-supplemented with vitamin A alone, despite a substantial increase in liver vitamin A (P < 0.002). Despite lower liver vitamin A levels compared to the group re-supplemented with vitamin A lone, the group re-supplemented with iron and vitamin A had substantial higher plasma levels compared to the one supplemented with iron alone (P < 0.002). In conclusion, the study supports an interaction of iron and vitamin A on the level of retinol transport in plasma. Despite a comparable availability of vitamin A as indicated by the comparable liver levels only the re-supplementation of both iron and vitamin A can normalize the retinol level in plasma. This might be of nutritional consequence in developing countries with regard to the supplementation regime of both nutrients iron and vitamin A to prevent a functional deficiency of vitamin A despite sufficient dietary availability.     

Liquid-gel partition chromatography of vitamin A compounds; formation of retinoic acid from retinyl acetate in vivo

A clear separation of retinol, retinal, and retinoic acid has been achieved by liquid-gel partition chromatography on Sephadex LH-20 with solvent mixtures of chloroform, Skellysolve B, and methanol. A mixture of retinyl esters, retinol, retinal, and retinoic acid has been resolved on hydroxyalkoxypropyl Sephadex using Skellysolve B and acetone. There is no decomposition of any of the vitamin A compounds during chromatography, and recovery is complete. The combination of mildness and potential for resolution makes liquid-gel partition chromatography a superior tool for the separation of vitamin A compounds. This method has been applied to the study of vitamin A metabolism at physiological levels in the vitamin A-deficient rat. Retinyl palmitate, an ester of retinoic acid, retinal, retinol, retinoic acid, and a polar metabolite have been demonstrated in various tissues of the rat 12 hr after a dose of 2 micro g of [11-(14)C]retinyl acetate.     

Effect of depletion of vitamin A, followed by supplementation with retinyl acetate or retinoic acid, on regeneration of rat liver.

After partial hepatectomy the net increase in tissue weight and in RNA, DNA and proteins in the regenerating liver was markedly less in vitamin A-deleted or retinoic acid-supplemented male rats, compared with the corresponding normal control or retinyl acetate-supplemented ones.     

A quantitative analysis of germ cells and the histone variants in the testes of vitamin A-deficient rats and during subsequent repletion with vitamin A

A quantitative analysis of the different types of germ cells present in the seminiferous tubules of vitamin A-deficient-retinoate maintained rats revealed that the number of pachytene spermatocytes and spermatogonia was greatly reduced in the deficient rats. Spermatids were virtually absent in the deficient tubules which contained mostly spermatogonia and preleptotene spermatocytes along with the Sertoli cells. There was no change in the number of Sertoli cells present in the tubules of deficient rats as compared to that of normal rats. Following supplementation of retinyl acetate to vitamin A-deficient-retinoate maintained rats, there was an immediate thinning of the germinal epithelium resulting from the sloughing off of the damaged spermatocytes which were beyond repair. However, after 12 days of vitamin A supplementation fresh batch of pachytene spermatocytes started appearing while by day 16 round spermatids could be seen. Analysis of the acid soluble proteins from nuclei on different types of Polyacrylamide gel electrophoretic systems has revealed that the levels of the testis specific histone variants Hlt, TH2A and TH2B, synthesized predominantly in the pachytene spermatocytes were greatly reduced in the testes of retinoate maintained rats. Following supplementation of retinyl acetate for either 4 days or 8 days the levels of these histone variants further decreased which correlated with the decrease in the number of pachytene spermatocytes. However, by day 12 of supplementation onwards, their levels started increasing and reached near normal levels by day 24 of vitamin A-supplementation     

Spontaneous transfer of retinoic acid, retinyl acetate, and retinyl palmitate between single unilamellar vesicles

The transfer of retinoic acid, retinyl acetate, and retinyl palmitate between single unilamellar vesicles was studied by resonance energy transfer. The retinoic acid transfers spontaneously between single unilamellar vesicles with a first order rate constant of 9.6 s-1 at 15 degrees C and pH 7.4. At 30 degrees C, the transfer rate was 3.5 times faster than that at 10 degrees C. At pH 7.4, the transfer rate was almost 2 orders of magnitude faster than that observed at pH 1.6. Increasing the concentration of NaCl decreased the retinoic acid transfer rate significantly. Retinyl acetate transfers with a rate constant of 0.15 s-1, but no spontaneous transfer of retinyl palmitate was observed over 60 min. The evidence supports the proposal that retinoic acid and retinyl acetate transfer between single unilamellar vesicles occur via the aqueous phase. In contrast, no spontaneous transfer of retinyl palmitate was observed. However, linear free energy relationships and the thermodynamic parameters for retinyl acetate transfer permit the calculation of rate constant for retinyl palmitate transfer.