DeWitt S. Goodman

Retinol-binding protein: the transport protein for vitamin A in human plasma

Vitamin A circulates in human plasma as retinol bound to a specific transport protein. This protein differs from the known low and high density plasma lipoproteins and has a hydrated density greater than 1.21. In order to study this protein, volunteers were injected intravenously with retinol-15-(14)C. Plasma was collected 1-3 days later, and the purification of retinol-binding protein (RBP) was monitored by assaying for (14)C and also by following the fluorescence of the protein-bound retinol. Purification of RBP was effected by the sequence: Cohn fractionation, chromatography on columns of Sephadex G-200 and diethylaminoethyl (DEAE)-Sephadex, preparative polyacrylamide gel electrophoresis, and finally chromatography on Sephadex G-100. These procedures resulted in a preparation of RBP which was at least 98% pure and which had been purified more than 1500-fold. Purified RBP has alpha(1) mobility on electrophoresis and has a molecular weight of approximately 21,000-22,000. There appears to be one binding site for retinol per molecule of RBP. Solutions of RBP are fluorescent (characteristic of retinol) and have ultraviolet absorption spectra with peaks at 330 mmu (resulting from the bound retinol) and at 280 mmu. There are no fatty acid or fatty acyl chains present in purified RBP. The usual concentration of RBP in plasma is of the order of 3-4 mg/100 ml. In plasma, RBP apparently circulates as a complex, together with another, larger protein with prealbumin mobility on electrophoresis. The RBP-prealbumin complex remains intact during Cohn fractionation and during chromatography on Sephadex and on DEAE-Sephadex columns. The complex dissociates during gel electrophoresis, permitting the isolation and subsequent purification of each of the components. The complex is again formed by mixing together solutions of the separated RBP and of prealbumin. Retinol transport in plasma thus appears to involve both a lipid-protein (retinol-RBP) interaction and a protein-protein (RBP-prealbumin) interaction.

Plasma retinol-binding protein.

Vitamin A is mobilized from liver stores and transported in plasma in the form of the lipid alcohol retinol, bound to a specific transport protein, retinol-binding protein (RBP). A great deal is known about the chemical structure, metabolism, and biological roles of RBP. RBP is a single polypeptide chain with molecular weight close to 20,000. RBP interacts strongly with plasma prealbumin, and normally circulates in plasma as a 1:1 molar RBP-prealbumin complex. Both the primary and the tertiary structure of prealbumin are known, and the primary structure of RBP has recently been reported. Much information is available about the protein-protein and protein-ligand interactions that are involved in this transport system. Many clinical studies have examined the effects of a variety of diseases on the plasma levels of RBP and prealbumin in humans. Plasma RBP levels are low in patients with liver disease and are high in patients with chronic renal disease. These findings reflect the facts that RBP is produced in the liver and mainly catabolized in the kidneys. Delivery of retinol to extra-hepatic tissues appears to involve specific cell surface receptors for RBP. Vitamin A mobilization from the liver, and delivery to peripheral tissues, is highly regulated by factors that control the rates of RBP production and secretion. Retinol deficiency specifically blocks the secretion of RBP, so that plasma RBP levels fall and liver RBP levels rise. Injection of retinol into vitamin A-deficient rats stimulates the rapid secretion of RBP from the liver into the plasma. The cellular and molecular mechanisms that mediate these phenomena are under investigation. Elucidation of these mechanisms should help define the basic mechanisms that control the mobilization, transport, and delivery of vitamin A.

The effects of diseases of the liver, thyroid, and kidneys on the transport of vitamin A in human plasma.

The effects of diseases of the liver, the thyroid, and the kidneys on the retinol-binding protein (RBP)-prealbumin (PA) system responsible for the transport of vitamin A in plasma were examined, using a radial gel diffusion immunoassay for PA and the previously described radioimmunoassay for RBP. Measurements were made on plasma samples from 118 normal subjects, 31 patients with cirrhosis, 5 with chronic active hepatitis, 27 with acute viral hepatitis, 14 patients with hyperthyroidism, 7 with hypothyroidism, and 26 patients with chronic renal disease of varying etiologies. In the patients with liver disease, the levels of vitamin A, RBP, and PA were all markedly decreased and were highly significantly correlated over a wide range of concentrations. Serial samples were available in 19 patients with acute hepatitis; as the disease improved the plasma concentrations of vitamin A, RBP, and PA all increased. In patients with acute hepatitis RBP concentrations correlated negatively with the levels of plasma bilirubin, glutamic-oxaloacetic transaminase, and alkaline phosphatase. In the hyperthyroid patients both RBP and PA concentrations were significantly lower than normal; in hypothyroidism, neither protein showed levels significantly different from normal. In both hyper- and hypothyroidism and in liver disease, the molar ratios of RBP:PA and of RBP:vitamin A were not significantly different from normal.Patients with chronic renal disease had marked abnormalities in the plasma concentrations of RBP and vitamin A and in the molar ratios examined. In renal disease the levels of both RBP and vitamin A were greatly elevated, while the PA levels remained normal. The molar ratios of RBP:PA and of RBP:vitamin A were both markedly elevated. In many patients RBP was present in molar excess as compared with PA. The presence of a relatively large proportion of free RBP, not complexed to PA, in some patients with chronic renal disease was confirmed by gel filtration. The free RBP, present in molar excess, was capable of forming a complex with additional purified PA added to the plasma. The kidneys appear to play an important role in the normal metabolism of RBP.

Amyloid fibril protein in familial amyloidotic polyneuropathy, Portuguese type. Definition of molecular abnormality in transthyretin (prealbumin).

Amyloid fibril protein in patients with familial amyloidotic polyneuropathy is known to be chemically related to transthyretin (TTR), the plasma protein that is usually referred to as prealbumin. A genetically abnormal TTR may be involved in this disease. Studies were conducted on amyloid fibril protein (AFp) isolated from tissues of two Portuguese patients who died with familial amyloidosis, and on TTR isolated from sera of patients with this disease. AFp, purified by affinity chromatography on retinol-binding protein linked to Sepharose, resembled plasma TTR in forming a stable tetrameric structure, and in its binding affinities for both thyroxine and retinol-binding protein. The structural studies included: (a) comparative peptide mappings by reverse-phase high performance liquid chromatography (HPLC) after trypsin digestion; (b) cyanogen bromide cleavage studies; and (c) amino acid microsequence analysis of selected tryptic and CNBr peptides. On the basis of the known amino acid sequence of TTR, comparative tryptic peptide maps showed the presence of a single aberrant tryptic peptide (peptide 4, residues 22-34) in AFp as compared with TTR. This aberrant peptide contained a methionine residue, not present in normal tryptic peptide 4. CNBr cleavage of AFp produced two extra peptide fragments, which were demonstrated, respectively, by HPLC analysis and by sodium dodecyl sulfate-gel electrophoresis. Sequence analyses indicated the presence of a methionine-for-valine substitution at position 30 in AFp as compared with TTR. Thus, the purified amyloid fibril protein comprised a TTR variant with a methionine-forvaline substitution at position 30. A single nucleotide change in a possible codon for valine 30 could explain the substitution. The variant TTR was also present in the TTR isolated from the pooled sera of amyloidoses patients, together with larger (four- to six-fold) amounts of the normal TTR. Thus, in these patients, the variant TTR was circulating in plasma, along with larger amounts of normal TTR. We suggest that the variant TTR represents the specific biochemical cause of the disease, and that this abnormal form of TTR selectively deposits in tissues as the amyloid characteristic of the disease.

Vitamin A Transport in Human Vitamin A Toxicity

The plasma retinol transport system was studied in three patients with chronic hypervitaminosis A. The toxic state in each was associated with increased plasma concentrations of total vitamin A, and particularly of retinyl esters. The concentrations of plasma retinol-binding protein and prealbumin were, in contrast, non to retinol-binding protein. These limited clinical data support conclusions from detailed studies with hypervitaminotic rats, which suggest that vitamin A toxicity occurs when excessive amounts of vitamin A are presented to cell membranes in association with plasma lipoproteins, rather than specifically bound to retinol-binding protein. Retinol-binding protein may not only regulate the supply of retinol to tissues but also protect tissues from the surface-active properties of the vitamin.

Transthyretin: A choroid plexus‐specific transport protein in human brain: The 1986 S. Weir Mitchell Award

Plasma transthyretin (TTR, formerly called prealbumin) is a 55-kd protein that participates in the plasma transport of both thyroxine and retinol (vitamin A). TTR concentrations are disproportionately high in human ventricular CSF, suggesting that TTR is either selectively transported across or synthesized de novo within the blood-CSF barrier. To address this question, we adopted a molecular genetic approach; after isolating a cDNA clone encoding human TTR, we previously demonstrated specific TTR messenger RNA (mRNA) synthesis in rat choroid plexus. We have now extended these investigations to the human brain. Northern analysis of postmortem brain homogenates revealed abundant TTR mRNA in choroid plexus, but not in cerebellum or cerebral cortex. Choroid plexus mRNA was readily translated into TTR preprotein in an in vitro translation system. An immunocytochemical survey of human postmortem brain sections revealed the presence of TTR protein specifically and uniquely in the cytoplasm of choroid plexus epithelial cells; these results were corroborated at the mRNA level by an extensive survey of whole rat-brain sections by in situ hybridization. Therefore, within the mammalian CNS, TTR is the first known protein synthesized solely by the choroid plexus, suggesting a special role for TTR in the brain or CSF. Whether this function differs from its established plasma transport functions is presently unknown.

Studies of the Release from Human Platelets of the Growth Factor for Cultured Human Arterial Smooth Muscle Cells

Platelets contain a growth-promoting factor for arterial smooth muscle cells (SMC) that may play a major role in atberogenesis. We have studied some of the effects of the platelet-derived growth factor (PDGF) on human arterial SMC in culture and the release of PDGF from human platelets in relationship to other released substances. Material released from platelets was highly potent in stimulating human SMC to proliferate. A substantial portion of the growth-promoting activity of human serum could be attributed to a factor(s) released from platelets. Similar dose-response patterns to PDGF were observed with human SMC and with mouse 3T3 cells. The time-course of release of PDGF and its concentration dependence on human thrombin were determined in comparison with serotonin, ADP, ATP, an acid bydrolase, platelet factor 4 (PF4), and β-thromboglobulln (βJTG). PDGF activity was assayed by stimulation of the incorporation of +-H-thymidine into DNA of 3T3 cells; PF4 and βJTG were measured by newly developed radioimmunoassays. PDGF, PF4, and βJTG were released from platelets by lower concentrations of thrombin than those required for release of the other components. The results suggest that PDGF, PF4, and βTG are localized in the platelet in granules different from either the dense bodies (that contain serotonin, ADP, ATP) or the acid hydrolase-containing granules, possibly in α-granules. The contents of these PDGF- containlng a-granules are actively released during the release reaction and are particularly sensitive to release by low doses of thrombin.

Biosynthesis of Vitamin A with Rat Intestinal Enzymes

Vitamin A is synthesized from β-carotene in cell-free homogenates of rat intestinal mucosa, the biosynthetic enzymatic activity being present in the soluble protein fraction of the homogenate. Also required are a heat-stable factor in the particulate fraction, molecular oxygen, and bile salts. The reaction is stimulated by glutathione. The product, obtained in yields of up to 50 percent, has been identified as vitamin A aldehyde (retinal) by way of its semicarbazone derivative. The reaction mechanism involves the central cleavage of β-carotene into two molecules of retinal.

8 – Plasma Retinol-Binding Protein

Publisher Summary This chapter focuses on the plasma retinol-binding protein (RBP) and describes the structure and chemistry, biochemistry, and metabolism of RBP. Vitamin A is transported normally in postabsorptive plasma as the lipid alcohol retinol bound to a specific transport protein plasma RBP. Human RBP is a single polypeptide chain with a molecular weight close to 21,000, α1-mobility on electrophoresis, and a single binding site for one molecule of retinol. In plasma, most of RBP normally circulates as the retinol-RBP complex. Vitamin A is mobilized from the liver and is delivered to peripheral target tissues as the retinol-RBP complex. Retinol mobilization and delivery are highly regulated processes that are particularly controlled by processes that regulate the rates of synthesis and secretion of RBP by the liver. Solutions of the retinol-RBP complex are highly fluorescent. The RBP molecule is a single polypeptide chain of about 180–185 amino acid residues containing three intramolecular disulfide bonds. RBP is cleaved by cyanogen bromide into five fragments; of these, the carboxy-terminal fragment represents slightly more than half the molecule.

The turnover and transport of vitamin D and of a polar metabolite with the properties of 25-hydroxycholecalciferol in human plasma

Four normal men were injected intravenously with physiological doses (6 mug) of vitamin D(3)-1,2-(3)H. Serial samples of plasma were collected for 50 days. Total lipid extracts were chromatographed on silicic acid columns or thin-layer plates in order to characterize the radioactive components. Labeled vitamin D(3) disappeared rapidly from plasma (initial half-life approximately 12 hr); after 7 days unchanged vitamin D(3) represented less than 1% of circulating radioactivity. Coincident with vitamin D(3) disappearance a more polar labeled metabolite appeared with chromatographic and other properties identical with those of 25-hydroxycholecalciferol. The disappearance of the more polar metabolite was relatively slow with a half-life of 19.6 +/-0.6 days. A similar half-life was seen in a fifth subject, injected with 80 mug of vitamin D(3)-(3)H. Most (approximately 92%) of the plasma total radioactivity was represented by this component throughout the study. Plasma samples collected at various times were adjusted to density (d) 1.21 and were ultracentrifuged to separate plasma lipoproteins from proteins with d > 1.21. In all samples, almost all (mean 94%) of the radioactivity was found in association with proteins of d > 1.21. This observation was confirmed by bioassay, measuring uptake of (45)Ca by intestinal slices. All plasma bioassayable vitamin D was found in association with proteins of d > 1.21; 55% of bioactivity was found in the chromatographic fraction corresponding to 25-hydroxycholecalciferol and 44% in the fractions representing vitamin D(3). Since both vitamin D(3) and its 25-hydroxy metabolite are lipid-soluble sterol derivatives, the finding that these compounds do not circulate in association with the known plasma lipoproteins provides presumptive evidence for the existence of a specific transport protein of d > 1.21. The transport protein for the polar metabolite has been partly characterized by gel filtration on Sephadex G-200 and by electrophoresis on polyacrylamide gel. The protein has an apparent size slightly smaller than plasma albumin (approximate mol wt 50,000-60,000) and an electrophoretic mobility very slightly greater than that of albumin. Studies are in progress to fractionate further and to characterize the transport protein.