| dc.description.abstract | Vitamin A is indispensable for growth, vision and reproduction. A relationship between vitamin A deficiency and a higher incidence of cancer was suggested more than 50 years ago. The role of vitamin A in biochemical processes like nucleic acid, protein and glycoprotein biosynthesis as well as its role in physiological processes like growth, vision and reproduction is reviewed. The plasma transport of vitamin A in several species, especially its transport by RBP–PA complex, the role of cellular binding proteins in utilisation of this vitamin and the metabolism of vitamin A and its derivatives are discussed. The wide variety of biochemical effects the vitamin can bring about in in vitro systems are summarised.
The multitudinous functions of vitamin A in the target cells are made possible by its plasma transport, delivery into and metabolism in the target cells. The preferred mode of transport in most species is by the formation of RBP–PA complex. In order to compare the transport in a single species viz., the avian, attempts were directed towards the isolation of duck RBP and comparison of its properties with those of the chicken protein. The transport and metabolism of the most important analogue of vitamin A i.e., retinoic acid was studied in the avian species using chicken as a model system. The importance of retinoic acid as an inhibitor of neoplastic growth prompted an examination of its role in cell–cell interactions using Con A induced agglutinability as a probe.
The general methods used in this study, such as polyacrylamide gel electrophoresis, molecular weight determination, protein fluorescence spectrophotometry, immunological methods, two phase extraction method used for the preparation of apo RBP, preparation of vitamin A deficient chicken, extraction of vitamin A compounds from the tissues and their chromatography by liquid–gel partition, assay of lectin induced agglutination etc., are described.
The vitamin A transport proteins, RBP and PA were isolated from duck plasma by ion exchange chromatography on PEAB cellulose and DEAE Sephadex, gel filtration on Sephadex G 100 and preparative electrophoresis using 7.5% polyacrylamide gels.
The homogeneity of the protein preparations was established by polyacrylamide gel electrophoresis, Ouchterlony immuno double diffusion and immunoelectrophoresis. The molecular weights of RBP, PA and RBP–PA complex were determined to be 20,000, 55,000 and 75,000 respectively by gel filtration. On SDS polyacrylamide gel electrophoresis, RBP gave a molecular weight of 20,000, whereas PA dissociated into subunits of Mr 13,500 suggesting that RBP was a monomer whereas PA was a homotetramer. Neither of these proteins was a glycoprotein.
Two absorption maxima, one at 280 nm and the other at 330 nm, were observed for RBP as well as PA–RBP complex. The fluorescence spectra displayed two excitation maxima (280 nm and 330 nm) and two emission maxima (330 nm and 470 nm). Thus, the ultraviolet and fluorescence spectra of RBP and RBP–PA complex were characteristic of protein and bound retinol.
The antiserum for RBP did not give any precipitin band with PA. Similarly, the antiserum for PA did not show any cross reactivity with RBP. Both RBP and PA exhibited strong cross reactivity with their counterpart proteins in chicken but not in man or goat.
The addition of RBP or L thyroxine to PA quenched its protein fluorescence indicating that both these ligands were capable of binding to PA.
The elution of a mixture of PA and RBP as a single peak of molecular weight 75,000 on Sephadex G 100 column chromatography (compared to 55,000 and 20,000 for the individual proteins) confirmed that PA was binding to RBP to form a 1:1 molar complex. When PA and apo RBP (i.e., RBP from which retinol was extracted by diethyl ether) were mixed and subjected to gel filtration, PA–apoRBP complex was not formed.
The duck PA–RBP complex was stable at high ionic strength while a low ionic strength buffer, i.e., 2 mM Tris HCl buffer, pH 7.6 completely dissociated it. The complex was stable to changes in pH in the range of 3 to 8, though a very small amount of RBP was found to be dissociated at pH 3. Dissociation of duck PA–RBP complex was also brought about by the presence of 2 M urea in the buffer, by CM Sephadex chromatography at pH 5.4 and by electrophoresis in 7.5% polyacrylamide gels at pH 8.9.
A simple three step procedure for the isolation of RBP involving DEAE cellulose chromatography, CM Sephadex chromatography and gel filtration on Sephadex G 100 was developed.
Heterologous association was observed between duck RBP and PA obtained from goat or chicken. Similarly, duck PA formed a complex with RBP isolated from goat or chicken. The complex formation was monitored by gel filtration on Sephadex G 100.
The interaction of RBP with retinol was studied by fluorescence enhancement as well as by a two phase extraction method. The fluorescence data analysed by a Scatchard plot gave a Ka value for the binding of retinol to apo RBP.
The two phase extraction procedure using diethyl ether and buffer indicated that retinol was slowly released from RBP in about 6 h. The retinol–RBP complex was less stable at alkaline pH and in solutions of low ionic strength or protein concentration.
The specific inhibition by PA of the release of retinol from RBP suggested that the highly labile and water insoluble retinol was further protected by the formation of RBP–PA complex.
Sephadex LH 20 chromatography of the tissue extracts of vitamin A deficient chicken administered radioactive retinoic acid indicated that this compound was transported in the avian plasma by binding to albumin.
The radioactivity of administered retinoic acid was distributed in all the tissues of vitamin A deficient chicken with the highest levels recovered in the liver and small intestine. Serum and kidney contained relatively low amounts while traces were found in large intestine, brain, heart and testis.
Time course experiments suggested that, in the liver, the maximum radioactivity was found 1 h after administration, while in other tissues, the maximum was reached at 4 h. The radioactivity decreased to near zero value in 10–15 h in all tissues.
Sephadex LH 20 chromatography of the tissue extracts revealed that, in addition to retinoic acid, a less polar derivative of retinoic acid identified as an ester and two sets of polar derivatives were present. Retinoic acid was the major compound in most tissues except the small intestine where its concentration was less than that of other metabolites.
The metabolite patterns in liver and kidney were similar, exhibiting three peaks viz., 1a (ester), 3 (retinoic acid) and 8 (polar metabolite). The small intestine showed peaks 1a, 8, 9 and 10. Blood contained, in addition to retinoic acid, three slightly more polar metabolites i.e., peaks B1, B2 and B3 and four metabolites i.e., peaks 8, 9, 10 and 11 in the more polar region. The less polar derivative i.e., peak 1a was absent in the blood.
On increasing the time interval between injection and sacrifice to 8–10 h, the levels of polar metabolites increased with a concomitant decrease in the level of free retinoic acid. In the case of blood, the metabolites B2, B3 and 11 were not detectable after 8–10 h.
Administration of either retinoic acid or retinyl acetate gave similar chromatographic profiles of the metabolites in the small intestine.
Con A induced agglutinability of human erythrocytes was increased by retinoic acid and retinol, while that of chicken erythrocytes was increased by retinoic acid. The increase in agglutinability was not accompanied by the release of either cathepsin D or ninhydrin positive material into the medium. The increase by vitamin A was not affected by aminocaproic acid or hydrocortisone but inhibited by cholesterol, progesterone, testosterone or vitamin E.
These results highlight that
(a) the common mechanism of transport of vitamin A in the avian species is by the formation of RBP–PA complex;
(b) the transport of retinoic acid in the chicken is probably mediated by its binding to albumin;
(c) the metabolism of retinoic acid in the avian species, though similar to that in the rat, has some distinctive differences; and
(d) the vitamin may have a function in cell–cell interactions by mechanisms which are probably not mediated through its well known action on the lysosomes. | |
