Frank Chytil

9 – Cellular Retinoid-Binding Proteins

Publisher Summary This chapter discusses cellular retinol-binding proteins (CRBP). Retinoid-binding proteins are found in cytosols of many tissues from a number of species. CRBP is important in the physiological function of retinol in animals. The absorption spectrum of purified human CRBP complexed with retinol is dominated by the absorbance of bound retinol with λmax at 350 nm. This spectrum of retinol is altered from its spectrum in organic solvents, having λmax red shifted 25 nm and with the introduction of fine structure. The spectrum of all-trans-retinol in organic solution is atypical for a conjugated pentaene, owing to the steric hindrance in the molecule that prevents coplanarity of the ring and side chain and, consequently, full conjugation of the double bond of the ring with the four double bonds of the side chain. The chapter describes the amino terminal amino acid sequences of CRBP, isolated from rat liver, human liver, and bovine retina.

Vitamin A Status of Neonates with Bronchopulmonary Dysplasia

ABSTRACT: We prospectively assessed and compared the vitamin A status of two groups of preterm neonates (<1500 g birth weight, <32 wk gestation), one who developed clinical and radiographic evidence of bronchopulmonary dysplasia (BPD) (n = 10), and the other (control) who developed no significant lung disease (n = 8). The infants with BPD in this study required prolonged mechanical ventilation and supplemental O2 therapy, and had a higher incidence of cardiorespiratory complications when compared to controls. Their mean plasma vitamin A concentrations were significantly lower than those of controls at four sampling times in the 1st postnatal month. In contrast to the controls, infants with BPD showed a substantial decline in their plasma vitamin A concentrations from the initial values, and a high percentage of individual values of plasma vitamin A concentration in these infants were <10 μg/dl during the 8-wk postnatal period of observation. Delayed establishment of gastrointestinal feeding and a lower vitamin A intake in these infants relative to controls may have accounted for this decline. Our data show that preterm neonates who develop BPD have suboptimal plasma vitamin A concentrations for extended periods of time postnatally. We speculate that the necrotizing bronchiolitis and squamous metaplasia of conducting airways associated with vitamin A deficiency could influence the orderly repair of lung injury in susceptible neonates who are mechanically ventilated and could contribute to the pathophysiology of BPD in these infants. Further studies are needed to determine whether more efficient management of these neonates with regard to their vitamin A nutrition and early achievement of vitamin A sufficiency could result in a decrease in the incidence and severity of BPD.

Transfer of retinoic acid from its complex with cellular retinoic acid-binding protein to the nucleus☆

Cellular retinoic acid-binding protein (CRABP), a potential mediator of retinoic acid action, enables retinoic acid to bind in a specific manner to nuclei and chromatin isolated from testes of control and vitamin A-deficient rats. The binding of retinoic acid was followed after complexing [3H]retinoic acid with CRABP purified from rat testes. The binding was specific, saturable, and temperature dependent. If CRABP charged with nonlabeled retinoic acid was included in the incubation, binding of radioactivity was diminished, whereas inclusion of free retinoic acid, or the complex of retinol with cellular retinol binding protein (CRBP) or serum retinol binding protein had no effect. Approximately 4.0 X 10(4) specific binding sites for retinoic acid were detected per nucleus from deficient animals. The number of binding sites observed was influenced by vitamin A status. Refeeding vitamin A-deficient rats (4 h) with retinoic acid lowered the amount of detectable binding sites in the nucleus. CRABP itself did not remain bound to these sites, indicating a transfer of retinoic acid from its complex with CRABP to the nuclear sites. Further, CRBP, the putative mediator of retinol action, was found to enable retinol to be bound to testicular nuclei, in an interaction similar to the binding of retinol to liver nuclei described previously.

Retinoids in lung development.

The “retinoid revolution” has had considerable impact on research activity in the field of lung development. Postnatal lungs are very sensitive to dietary retinol (R) deprivation. The trachea and the bronchopulmonary tree in R‐deficient animals show a striking change in morphology. The columnar epithelium undergoes keratinizing metaplasia, which is reversed when R is supplied. The fetal lung develops postnatally to become one of the most complex organs, characterized by 40 different cell types. The fetal lungs can accumulate retinyl esters. Before birth an unknown signal causes the lowering of these esters, which may be the source of retinoic acid (RA). The administration of glucocorticoids speeds up this process, which in turn is followed by acceleration of lung maturation, ending postnatally. It is not clear what role, if any, the cellular retinoid binding proteins play in this phenomenon. Fetal lung branching leading to the development of the alveolar tree is accelerated by RA, and so is the expression of some fetal genes coding for surfactant proteins and the enzymes that produce their lipid components. It is not clear whether RA influences the development of pulmonary epithelium by interacting directly with the epithelial cells or whether its effects require interaction with other cells like mesenchymal cells. Nuclear RA receptors are intimately involved in lung development. Despite the wealth of evidence on the effect of retinoids on lung development, the application of vitamin A to clinical work has been limited to prematurely delivered infants who have immature lungs. Here attempts are made, by improving their R status, to aid in the development and healing of lungs injured by oxygen therapy.—Chytil, F. Retinoids in lung development. FASEBJ. 10, 986‐992 (1996)

Cellular Vitamin A Binding Proteins

Publisher Summary This chapter elaborates the different aspects of cellular vitamin A binding proteins. The mechanism of uptake of retinol by the cell from the circulating complex of retinol–retinol-binding protein (RBP)-prealbumin is of importance because the plasma membrane is very likely one of the points of regulation of vitamin A action. The data suggest complexity of the receptor site, containing a carboxypeptidase responsible for inactivation of RBP by removing the terminal arginine, which renders it incapable of interacting with retinol, prealbumin, and the receptor. RBP not only appears to be important for transport of retinol in blood but is also the indispensable entity for the recognition by the target cell and consequently for penetration of retinol through the plasma membrane. Incubation of serum with radioactive retinol and subsequent sucrose gradient centrifugation gives rise to a single peak in the 5 S region of the gradient. Excess nonradioactive retinol, retinal, or retinoic acid does not abolish the peak, indicating that retinol binds to this component nonspecifically. It is shown that when α-retinyl acetate is added directly to serum-free organ cultures of hamster trachea with keratinized squamous lesions induced by vitamin A deficiency, it is virtually as effective as all-trans-retinyl acetate in reversing the metaplasia.

Liver Vitamin A Reserves of Very Low Birth Weight Neonates

ABSTRACT: This study assessed the liver vitamin A concentrations at birth in a group of very low birth weight neonates (n = 25) (<1500 g birth weight, <32 wk gestation), dying within 24 h of birth, prior to possiblechanges in vitamin A status induced by postnatal intervention. Serum concentrations of vitamin A and retinol-binding protein were also measured in 16 of these neonates. The mean (± SD) liver vitamin A concentration was 30.0 ± 12.9 Mg/g (range 2.0-49.0 μg/g)- The mean (± SD) serum vitamin A concentration was 13.0 ± 4.7 μg/dl (range 6.7-22.8 μg/dl). The mean (± SD) serum retinol-binding protein concentration was 2.2 ± 0.8 mg/dl (range 1.5–4.8 mg/ dl). Liver vitamin A, serum vitamin A, and serum retinolbinding protein concentrations did not correlate significantly with gestational age or birth weight. Linear regression analysis did not show a significant correlation between liver vitamin A, and serum vitamin A or retinol-binding protein concentrations. This study provides reference values for vitamin A concentrations at birth in very low birth weight neonates, which may be helpful in future studies designed to evaluate postnatal changes in the vitamin A status of these high-risk neonates.

European Consensus Statement on Lung Cancer: Risk factors and prevention. Lung Cancer Panel

This article is based on discussions of the lung cancer panel at the Hohenheim Consensus Meeting organized by the World Health Organization and the German Ministry of Health in November 1996. Panel members were international experts in the field of diet and cancer who discussed specific questions relating to lung cancer risk factors and prevention.

Cellular retinoic acid-binding protein and the role of retinoic acid in the development of the chick embryo

The distribution of cellular retinoic acid-binding protein (CRABP) in four stages of chick development is described using an affinity-purified antibody against rat CRABP. CRABP is the protein to which retinoic acid (RA) binds when it enters cells and may reflect the requirement of those cells for RA. We found several discrete cell populations which showed high levels of immunoreactivity. Some were in the neural tube such as the commissural neurons and the dorsal roof plate. Some were of neural crest origin such as the dorsal root ganglia, sensory axons, sympathetic ganglia, and enteric ganglia. The remaining populations were certain connective tissue cells, limb bud cells, and the myotome. These results suggest that certain organ systems, particularly the nervous system, have a requirement for RA during development and they may further our understanding of the teratogenic effects of retinoids on the embryo.

Vitamin A and cancer.

Publisher Summary This chapter discusses the aspects of the relationship between vitamin A and cancer. The lower intake of vitamin A leads to an elevated susceptibility of the animal to cancerogenic insult. The rats receiving a vitamin A-deficient diet has a higher frequency of spontaneous gastric carcinomas. The use of chemical carcinogens to produce experimental tumors with high frequency in laboratory animals shows the existence of a relationship between vitamin A and cancer. An inadequate supply of retinol (vitamin A alcohol) or its esters brings about a higher frequency of tumors. The demonstration of hyperplastic lesions of epithelial linings in vitamin A deficiency, possible alteration of vitamin A metabolism in tumor bearing patients, the effect of carcinogens on vitamin A metabolism, and occasional experiments that the dietary vitamin A level may influence the growth and incidence of experimental cancer are evident. Many tissues in vitamin A-deficient animals develop altered states of differentiation and increased rates of proliferation, which may make any carcinogen more potent.

Cellular retinol-binding protein

1. A protein which binds retinol in vitro with high affinity and specificity was detected by sucrose gradient centrifugation or by gel filtration after preincubating rat tissue cytosols with all-trans-[3H]retinol. This protein sediments in the 2 S region of sucrose gradients. Molecular size determination by gel filtration indicates a molecular weight of 16 000. 2. Competition studies revealed that only all-trans-retinol, not retinal or retinoic acid, competes for binding. The binding of radioactive retinol is reversible. 3. This protein was detected in cytosols of rat liver, lung, spleen, brain, testis, ovaries, uterus and intestinal mucosa whereas heart or gastrocnemius muscle seem to lack this protein. 4. The cellular retinol binding protein was found in fetuses as early as day 12 of the gestation period and possessed the same specificity for the ligand as the one in adult tissues. 5. This binding component was not detected in cytosols prepared from Novikoff hepatoma, ascites hepatoma AS-30D, mouse Ehrlich ascites tumor and mouse pituitary tumor cell line AtT 20. 6. The cellular retinol binding protein seems to be different from that described to be present in the serum as suggested by difference in size and by the inability of the antisera against the serum retinol binding protein to remove the cellular binding protein from the cytosol preparations.