John Edgar Smith

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.

Case Report Vitamin A Toxicity and Hypercalcemia

A patient hospitalized with hypercalcemia and a history of chronic vitamin A ingestion was studied in order to investigate the rarely reported association between elevated serum calcium and vitamin A toxicity. The clinical presentation marked by profound weight loss, a psychiatric disturbance, total body alopecia, erosive dermatitis, and liver disease, was compatible with hypervitaminosis A. The diagnosis of vitamin A toxicity was established by elevated total vitamin A levels and the component due to retinyl esters. Other etiologies for hypercalcemia were excluded. In view of these results and the well-known effects of vitamin A on bone metabolism, it is concluded that the most likely etiology of the hypercalcemia in this patient was vitamin A toxicity.

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

Abstract 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, MH 1 C 1 and H 4 II EC 3 (H 4 ), 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 Tytells Serumless Medium (NTS medium), while the MH 1 C 1 cells also synthesized RBP in Hams 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.

Rat liver retinyl palmitate hydrolase activity. Relationship to cholesteryl oleate and triolein hydrolase activities

Studies were conducted to explore relationships in rat liver between retinyl palmitate hydrolase activity and the hydrolytic activities against cholesteryl oleate and triolein. Previous studies have shown positive correlations between these three lipid ester hydrolase activities. In order to extend this work, the hydrolase activities were further purified and characterized. The activities against cholesteryl oleate and triolein resembled retinyl palmitate hydrolase activity in showing great variability from rat to rat as assayed in vitro. The relative levels of the three activities were highly correlated with each other over a 50-fold range of activity in a series of 66 liver homogenates. Partial purification (approx. 200-fold) in the absence of detergents was achieved by sequential chromatography of an acetone powder extract of liver on columns of phenyl-Sepharose, DEAE-Sepharose and heparin-Sepharose. The three hydrolase activities copurified during each of these chromatographic steps. The properties of the three copurifying activities were similar with regard to stimulation of activity by trihydroxy bile salts, pH optimum (near 8.0), and observance of Michaelis-Menten-type saturation kinetics. The three activities were different in their sensitivity towards the serine esterase inhibitors diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and in their solubility properties in 10 mM sodium acetate, pH 5.0. Thus, triolein hydrolase activity was much less sensitive than the other two activities to the two inhibitors. In addition, the activity against cholesteryl oleate could be separated from the other two activities by extraction of an acetone powder with acetate buffer, pH 5.0. These results indicate that the three lipid hydrolase activities are due to at least three different catalytically active centers, and at least two distinct and separable enzymes. It is likely that three separate but similar enzymes, that appear to be coordinately regulated, are involved.

Association of cellular retinol-binding protein and several lipid hydrolase activities with a vitamin A-containing high-molecular-weight lipid-protein aggregate from rat liver cytosol.

Abstract A fluorescent, high-molecular-weight, lipid-protein aggregate was partially isolated from the cytosol fraction of rat liver by gel filtration on columns of Sepharose 4B or 6B. This aggregate was composed of approximately equal parts of protein and of lipid (mainly triglycerides), and was found to contain approximately 19% of the total liver vitamin A (predominantly as retinyl esters). Most of the liver cellular retinol-binding protein (CRBP) was found associated with the fluorescent, lipid-protein aggregate, along with much of the retinyl palmitate hydrolase activity present in the liver cytosol. The lipid-protein aggregate, and its several vitamin A-related components, all displayed an apparent hydrated density between 1.052 and 1.090 in the ultracentrifuge. CRBP in association with the lipid-protein aggregate was not immunoreactive in the CRBP radioimmunoassay. CRBP was, however, released from this aggregate and rendered immunoreactive by addition of detergents (e.g., Triton X-100). Three other lipid hydrolytic activities were also found in association with the lipid-protein aggregate, namely, triolein, cholesteryl oleate, and dipalmitoyl phosphatidylcholine hydrolase activities. These several hydrolytic activities were all found to be stimulated optimally by the addition of either sodium cholate or bovine serum albumin. With the information available, it is not clear whether this lipid-protein aggregate is formed in vitro , during liver homogenization, or whether it represents a specific lipoprotein with a significant functional role that exists in vivo in the liver cell.

Structure-function relationships of retinoids in their effects on retinol-binding protein metabolism in cultured H4II EC3 liver cells☆

Studies were conducted to explore the structural features of retinoids that may be required to stimulate the secretion or production of retinol-binding protein (RBP) by H4II EC3 rat hepatoma cells in culture. Sixteen retinoids, that differed from all-trans-retinol in the cyclohexene ring, the polyene side chain, and/or the functional end group, were each incubated with H4II EC3 cells, and RBP secretion and accumulation were determined by radioimmunoassay. A number of retinoids, in addition to retinol, effectively stimulated RBP secretion. The results suggest that an intact cyclohexene ring may be necessary for the stimulation of RBP secretion. In contrast the system did not exhibit much specificity with regard to either the structure of the side chain or the nature of the end group. No relationship was found between the ability of a retinoid to stimulate RBP secretion and production and its biological activity. The biologically active retinoid, 13-cis-retinoic acid, was inactive in the present system, whereas the biologically inactive perhydromonoeneretinol was moderately effective in stimulating both RBP secretion and accumulation. In contrast, there appeared to be some relationship between the ability of different retinoids to stimulate RBP secretion and their ability to bind to RBP. In general, retinoids that had previously been shown to bind to RBP produced a greater stimulation of RBP secretion than those that did not bind to RBP. The secretion of RBP obtained with a given retinoid was not well correlated with the net accumulation of RBP. For example, retinoyl amide did not stimulate RBP secretion but was moderately effective in stimulating RBP accumulation. Thus, the secretion of RBP does not appear to be necessary for the stimulation of the net accumulation of RBP.