Serum content of vitamin A binding proteins (prealbumin and retinol binding proteins) in normal and pathological conditions

Blood PA and RBP content were estimated in 100 normal subjects (50 men and 50 women). Mean values of 28.2 and 3.75 mg/dl were ascertained for PA and RBP, respectively. The molar ratio RBP/PA was calculated as 0.31 +/- 0.05. Serum concentrations of PA and RBP are seen to be sex-dependent (20% higher in males). Hyperthyroid subjects exhibit a decrease in the two vitamin A-binding proteins whereas no modification could be revealed for hypothyroid states. In patients on maintenance hemodialysis mean serum PA and RBP concentrations were remarkably higher than normal, whereas liver cirrhotic subjects showed a highly significant depression of mean serum RBP and PA values.     

Tissue distribution and subcellular localization of retinol-binding protein in normal and vitamin A-deficient rats.

Levels of retinol-binding (RBP), the plasma transport protein for vitamin A, were measured by radioimmunoassay in sera and in a large number of tissues from both normal and vitamin A-deficient rats. The tissues included liver, kidney, fat, muscle, brain, eye, salivary gland, thymus, lung, heart, intestine, spleen, adrenal, testes, thyroid, and red blood cells. The RBP levels in tissues other than serum, liver, and kidneys varied from 12 mug/g of tissue for normal spleen to an undetectable level in red blood cells. Much of the RBP in the tissues with low levels may have been due to residual serum in the samples. In general, except for liver, RBP levels were lower in tissues from vitamin A-deficient rats than in those from normal rats. In normal rats, the liver, kidney, and serum levels were 30 plus or minus 4 (mean plus orminus SEM), 151 plus or minus 22, and 44 plus or minus 3 mug/g, respectively. In vitamin A-deficient rats, the liver RBP level was about three times the normal level whereas the kidney and serum levels were about one-fifth the normal values. When normal liver homogenates were fractionated by centrifugation, 67% of the RBP was recovered in the microsomal fraction and only 9% was found in the soluble 105,000 g supernate. In contrast, 76% of the RBP in homogenates of normal kidneys was in the soluble fraction. Similar results were obtained with deficient livers and kidneys. Incubation with deoxycholate released the liver RBP into the soluble fraction. RBP is produced in the liver and removed from the blood by the kidneys. The levels of RBP in normal and deficient liver, serum, and kidney appear to reflect the relative rates of RBP secretion and turnover.     

Plasma values of vitamin A and its binding proteins (retinol binding proteins and prealbumin), carotene, total cholesterol and HDL-cholesterol in subjects with breast carcinoma

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were determined in 33 subjects with breast cancer and compared to those of a group of healthy subjects previously described. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Mean plasma values of vitamin A, carotene and HDL-cholesterol were lower (P less than 0.01) than in the control group, the same applies to the RBP and PA mean levels (P less than 0.05). On the contrary, the mean value of total cholesterol was higher (P less than 0.01) in the patients than in the control group. Vitamin A plasma levels were significantly related to RBP and PA. No significant statistical correlation was found between clinical stage and vitamin A plasma levels.     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and HDL-cholesterol in patients with carcinoma of the cervix

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol of 40 subjects with carcinoma of cervix uteri were determined and compared to those of the healthy female subjects described in our previous research. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Plasma mean values of carotene, vitamin A, RBP and HDL-cholesterol were lower (P less than 0.01) than in the control group, and the same applies to the PA mean plasma level (P less than 0.05). On the contrary, total cholesterol mean value of the patients resulted to be higher (P less than 0.01) than in the control group. Vitamin A plasma levels were significantly related (P less than 0.01) to RBP and PA. No significant statistical correlation was found between the clinical stage and the histological grading and vitamin A plasma levels.     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and cholesterol-HDL in patients with dysplasia of the uterine cervix

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were determined in 19 female subjects with cervical dysplasia and compared to those of the healthy female subjects described in our previous research. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion and HDL- and total cholesterol by enzymatic colorimetry. Plasma mean values of vitamin A and HDL-cholesterol were lower (P less than 0.05 and P less than 0.01, respectively) than in the control group. On the contrary total cholesterol was higher (P less than 0.01) in the patients than in the control group. Vitamin A plasma levels were significantly related (P less than 0.01) to RBP and PA. No significant statistical correlation was found between the severity of the dysplasia and vitamin A plasma levels.     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and HDL-cholesterol in healthy adults of the female sex

Plasma vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were examined in healthy adult females. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Vitamin A and carotene mean values resulted as 43.0 +/- 8.2 micrograms/100 ml and 231.9 +/- 69.0 micrograms/100 ml, respectively. RBP and PA values averaged as 4.2 +/- 1.1 mg/100 ml and 29.4 +/- 6.1 mg/100 ml, respectively; whereas HDL-and total cholesterol were 179 +/- 16 mg/100 ml and 57 +/- 8 mg/100 ml. Vitamin A plasma levels were shown to be significantly related (P less than 0.01) to RBP and PA, but not to the other parameters examined (carotene, HDL-and total cholesterol).     

Early variations of the disialoganglioside GD3 in chicken embryonic brain support its role in cell migration

In the present study a primary culture system of chicken embryo brain neurons was used in the early period of chicken brain development from day 6 until day 8, which was shown to be a suitable model of neuritogenesis, cell migration and reaggregation. Dissociated chicken optic tectum cells from embryonic stage 31 were cultured on polylysine-coated dishes under serum-free conditions up to 3 days. Freshly dissociated neurons developed short processes, which contacted one another and formed fasciculated bundles. Cell somata migrated along the neurite bundles, similar to migrating neurons in vivo, forming three-dimensional tissue-like clusters. This system was used to study the possible functions of the disialoganglioside GD3 for these neuronal differentiation steps. GD3 represents the predominant ganglioside of embryonic neurons before neuritogenesis in vitro and in vivo. Its biosynthesis is followed during day 6 until day 8 of embryonic brain development. Incubation of dissociated neurons with the monoclonal antibody R-24, recognising the GD3 on the cell surface, led to a total blocking of neurite outgrowth. Accordingly, neither cell migration nor reaggregation could be found. These results indicate that the disialoganglioside GD3 plays a central role in neuronal differentiation and development in the embryonic chicken brain.     

The retinol-binding protein of the expanding pig blastocyst: molecular cloning and expression in trophectoderm and embryonic disc.

Retinol-binding protein (RBP) is a major secretory product of the porcine conceptus. Using an oligonucleotide probe corresponding to a highly conserved region of all known mammalian RBP, we have isolated an apparently full-length cDNA clone for porcine conceptus RBP from a cDNA library constructed from pig conceptuses collected between days 13-17 of pregnancy. The cDNA was 937 base-pairs in length and coded for a protein whose inferred amino-terminal sequence was identical to that reported for both porcine conceptus RBP and porcine serum RBP. Its length was consistent with the size (approximately 1 kilobase) of the RBP message in porcine conceptuses. Porcine conceptus RBP and human serum RBP share 91% amino acid sequence identity. The inferred differences in sequence were evenly distributed throughout the length of the polypeptide. RBP mRNA was detectable within the trophoblast of day 11 porcine conceptuses by in situ hybridization with a 618-basepair 35S-labeled probe corresponding to the 3' end of porcine RBP. Silver grain density was distributed relatively uniformly over the trophoblast and the inner cell mass. Western blot analysis of conceptus culture medium demonstrated that the conceptuses of cattle (on day 19) and sheep (on day 15) as well as pigs secrete RBP during early pregnancy. Secretion of large quantities of RBP by the trophoblast of preimplantation pig conceptuses suggests important roles for vitamin A and RBP near the time of conceptus elongation.     

Development of antioxidant capacity in tissues of the chick embryo

The concentrations of vitamin E, vitamin A, selenium, reduced glutathione and the activities of glutathione peroxidase and superoxide dismutase were determined in the yolk, yolk sac membrane, liver and brain of the developing chick embryo. The changes in the concentrations of vitamins E and A in the yolk and liver during development were consistent with the occurrence of a preferential transfer of vitamin A from the yolk to the embryo before day 14 of incubation, whereas the main period of vitamin E transfer occurred later, during the last week of incubation. The concentrations of reduced glutathione in the yolk sac membrane, liver and brain were similar at all developmental stages studied. However, the levels of the other measured antioxidant systems were very much higher in the liver than in the brain. Thus, in the newly hatched chick, the levels of vitamin E, vitamin A, selenium, glutathione peroxidase and superoxide dismutase were, respectively, 58.0-, 174.7-, 3.6-, 4.0- and 4.7- fold higher in the liver than in the brain when expressed on the basis of tissue fresh weight.     

Early gestational expression of retinol-binding protein mRNA by the ovine conceptus and endometrium

Communication between the mother and the early developing embryo is mediated by a variety of signals secreted by either the uterus or the embryo to elicit a response from the other. These signals include prostaglandins, proteins, and steroids. Recently, retinol-binding protein (RBP) has been described as a product of both the conceptus and endometrium in several species. Utilizing a cDNA clone to bovine RBP, we have described RBP mRNA expression in the endometrium, early conceptus, and extraembryonic membranes of sheep. Endometrial RBP mRNA expression did not differ between samples collected on day 13 of the estrous cycle and early pregnancy. In cyclic animals, RBP mRNA expression decreased two-fold between days 13 and 16, presumably a result of luteal regression and the consequential withdrawal of progesterone. In pregnant animals, endometrial RBP mRNA expression likewise decreased between days 13 and 16 and remained at this reduced level through day 30, despite the presence of a functional corpus luteum. Initiation of embryonic RBP expression appeared to coincide with early stages of blastocyst elongation at day 13. Levels of expression increased dramatically with conceptus development, peaked on day 23, and declined afterwards. Results from restriction enzyme analysis of genomic DNA indicated that RBP was encoded by a single gene per haploid genome. Differences in the temporal and tissue-specific expression of the protein, despite the apparent utilization of a single gene, suggest complex regulation of RBP gene expression. © 1994 Wiley-Liss, Inc.     

An essential ligand-binding domain in the membrane receptor for retinol-binding protein revealed by large-scale mutagenesis and a human polymorphism

Plasma retinol-binding protein (RBP), the principal carrier of vitamin A in the blood, delivers vitamin A from liver, the site of storage, to distant organs that need vitamin A, such as the eye, brain, placenta, and testis. STRA6 is a high-affinity membrane receptor for RBP and mediates vitamin A uptake in these target organs. STRA6 is a 74-kDa multi-transmembrane domain protein that represents a new class of membrane transport protein. In this study, we used an unbiased strategy by analyzing >900 random mutants of STRA6 to study its structure and function, and we identified an essential RBP-binding domain in STRA6. Mutations in any of the three essential residues in this domain can almost completely abolish binding of STRA6 to RBP and its vitamin A uptake activity from holo-RBP without affecting its cell surface expression. We have also functionally characterized the mutations in human STRA6 that cause severe birth defects as well as several human polymorphisms. All STRA6 mutants associated with severe birth defects have largely abolished vitamin A uptake activity, consistent with the severe clinical phenotypes. In addition, we have identified a human polymorphism that significantly reduces the vitamin A uptake activity of STRA6. Interestingly, the residue affected by this polymorphism is located in the RBP-binding domain we identified, and the polymorphism causes decreased vitamin A uptake by reducing RBP binding. This study identifies an essential functional domain in STRA6 and a human polymorphism in this domain that leads to reduced vitamin A uptake activity.     

Effect of renal and liver failure on blood levels of vitamin A, its precursor (beta-carotene) and its carrier proteins (prealbumin and retinol binding protein)

Plasma beta-carotene and retinol assay was performed by high pressure liquid chromatography (HPLC) in subjects with chronic renal failure or liver cirrhosis. In the same subjects blood prealbumin (PA) and retinol binding protein (RBP) were determined by immunological technique. A considerable increase of retinol and in a lesser extent of beta-carotene was noted in the blood of patients with renal insufficiency. In cirrhotic patients it was shown a marked decrease both of beta-carotene and retinol plasma concentrations. PA and RBP there were greatly increased in renal failure and decreased in liver cirrhosis. This results suggest that kidney and liver chronic failure interfere with vitamin A metabolism throughout their action on metabolic processes of synthesis and elimination of PA and RBP.     

Assay of human transthyretin-bound holo-retinol-binding protein with reversed-phase high-performance liquid chromatography

We describe a reversed-phase high-performance liquid chromatographic method for the determination of vitamin A-transporting (holo) transthyretin-bound (TTR) retinol-binding protein (RBP) concentrations in serum or plasma. Holo-TTR—RBP and free retinol derived primarily from free RBP are consistently observed with this chromatographic method. Holo-TTR—RBP concentrations determined by this method are highly correlated to holo-TTR—RBP concentrations measured by chromatography. This method has the advantage of using less expensive columns and having peak areas which are more proportional to their true concentrations in plasma, as determined by comparison to purified protein spectrophotometry and radial immunodiffusion. The percentage of RBP circulating as holo-TTR—RBP decreased significantly as the total concentration of RBP or retinol increased. Because purified holo-TTR—RBP did not dissociate under these chromatographic conditions, this suggests that more vitamin A circulates as holo-free RBP or free retinol in the blood of people with high serum RBP.     

Availability of avidin-bound biotin to the chicken embryo.

Avidin, an exceptionally stable protein in egg white, binds the vitamin biotin with very high affinity and can induce biotin deficiency when fed to animals. To determine if biotin bound to avidin is available to the chicken embryo, the fate of [3H]biotin complexed to avidin was monitored during embryonic development. The majority (greater than 85%) of the [3H]biotin was extraembryonic until the day before hatching, when embryos swallow egg white and withdraw the yolk sac into their abdomen. Thus, biotin in the egg white of chicken eggs contributes little to the biotin status of the chick prior to hatching. After hatching, much of the [3H]biotin was assimilated. About 30% of the total was found in the liver and kidneys by 4 days of age. The biotin in liver was associated with large proteins and not with avidin. In a separate experiment, biotin injected into the egg white of biotin-deficient eggs failed to increase embryonic development or hatchability. Both experiments suggest that biotin in egg yolk is the primary and virtually sole source of biotin for the chicken embryo.     

PHOSPHOCHOLINE DIGLYCERIDE TRANSFERASE ACTIVITY DURING DEVELOPMENT OF THE CHICKEN BRAIN

Abstract— The activity of chicken brain phosphocholine diglyceride transferase was followed during pre- and postnatal development. The specific activity of this enzyme increases from the 10th day of embryonic life, reaches a maximum at hatching and decreases thereafter. Total brain activity increases in parallel with the increase of brain lecithins. The apparent K _m of the enzyme for CDP choline is 1.5 × 10^-4 m before the 10th day of embryonic life, 2.5 × 10^-5 m between the 13th day of the embryo and the 10th day after hatching, and finally 1.3 × 10^-4 m after the 38th day of postnatal life. These data suggest the existence of isoenzymes, one of which appears at the beginning of myelination.     

Regulation of retinol-binding protein metabolism in cultured rat liver cell lines

Retinol-binding protein (RBP), the plasma transport protein for vitamin A, is synthesized and secreted by the liver. In vitamin A deficiency, RBP secretion is blocked, leading to low serum and high liver levels of RBP. Administration of retinol to the intact rat stimulates a rapid secretion of RBP from liver into serum. We explored the use of a liver cell culture system to study the regulation of the synthesis and secretion of RBP. We found two lines of differentiated rat hepatoma cells, MH₁C₁ and H₄ II EC₃ (H₄), that synthesized RBP during culture in vitro. The net synthesis of RBP was a function of the number of cells per dish and the duration of incubation. Both cell lines synthesized RBP when incubated in Neuman and Tytell's Serumless Medium (NTS medium), while the MH₁C₁ cells also synthesized RBP in Ham's F-12 medium with added serum. A relatively large proportion (14–56%) of the RBP was retained within the cells when they were incubated in the vitamin A-free NTS medium alone. Addition of serum to NTS medium stimulated the release of RBP from the cells into the medium and also increased the net synthesis of RBP. These effects were not due to the increased adhesion of the cells to the petri dish. Addition of retinol (at levels of 0.35 or 3.5 nmole/ml) to the NTS medium resulted in the stimulation of RBP secretion from the cells into the medium and an increase in the net synthesis of RBP. By contrast, retinol had no effect on either the net synthesis or the cell-to-medium distribution of rat serum albumin. The data from these cell lines in culture suggest that retinol has a specific regulatory effect on RBP metabolism. These cells thus resemble the normal rat liver cell in vivo in regard to the known regulation of RBP metabolism.     

Detection of nerve growth factor mRNA in the developing chicken embryo

Nerve growth factor (beta NGF) is a protein supporting sympathetic and sensory innervation in the peripheral tissues as well as cholinergic innervation in the brain. A DNA probe derived from a genomic clone coding for chicken NGF was used to study NGF mRNA levels during development. NGF mRNA was detected in the chicken embryo as early as day 3.5 of incubation. The level of NGF mRNA in total embryo increased four-fold until day 8, remained high until day 12, and subsequently decreased. No corresponding peak in NGF mRNA expression was found in heart and brain measured separately. Instead these organs showed increased NGF mRNA levels after hatching. The highest levels of NGF mRNA in the day-8 embryo were found in skin and eye (in particular cornea, but also iris, sclera-choroid and neural retina) suggesting a correlation between sensory innervation and this early peak of NGF expression.     

A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication-stimulating activity for embryo fibroblasts

A polypeptide fraction with multiplication-stimulating activity for chicken and rat embryo fibroblasts was partially purified from serum-free medium conditioned by the growth of a line of rat liver cells. The specific multiplication-stimulating activity of this fraction was 27,000 times that of serum. The rat liver cell multiplication-stimulating activity had a molecular weight of approximately 10,000 daltons and was inactivated by mercaptoethanol and dithiothreitol. It had sulfation factor and non-suppressible insulin-like activities, but did not have anti-trypsin activity. The rat liver cell multiplication-stimulating activity resembled both multiplication-stimulating activity from calf serum and somatomedin.