Detection of Retinyl Palmitate and Retinol in the Liver of Mice Injected with Excessive Amounts of Retinyl Acetate.

Abstract

ABSTRACT. The transport of subcutaneously injected retinyl acetate (RA, 100,000 IU/mouse, 105,470 nM) was investigated in male ICR mice (10-week-old) at 0, 3,6,12,18,24 and 72 hr after a single injection. The retinol and retinyl palmitate levels of liver homogenates, bile in the gallbladder and serum from peripheral blood were measured by high performance liquid chromatography (HPLC) method. Retinyl palmitate in the lipid droplets of hepatocytes and Ito cells was localized by a modified gold chloride staining method. Accumulation of retinyl palmitate peaked at 12 hr post-injection and decreased thereafter until 24 hr post-injection. Fluorescence microscopy revealed many fluorescent vitamin A-containing lipid droplets in hepatocytes around central veins at 12 hr post-injection, but such droplets were not observed in the vehicle control mice or at in the RA-injected mice after 18 hr of injection. Electron microscopic observation also indicated that many retinyl esters-containing lipid droplets were observed in hepatocytes around the central veins at 12 hr post-injection, but no droplets were seen in the controls or 18 hr post-injection. The retinyl palmitate levels in liver homogenates assessed by HPLC decreased from 12 to 24 hr post-injection and increased significantly in bile, while retinol in liver homogenates and serum markedly increased. One of the morphological alterations was intense vacuolization in hepatocyte cell cords from the portal toward the central vein observed at 24 hr post-injection. Transitional lipid droplets between vacuoles and lipid droplets were identified in those hepatocytes. These results of HPLC analysis of retinol and retinyl palmitate in liver homogenates, serum, and bile, together with the results of gold chloride staining suggested that subcutaneously injected RA was first incorporated in hepatocytes at 12 hr and then partially metabolized through vacuoles, transferred into the blood and secreted into the bile over a 24 hr period. Many retinyl esters-containing lipid droplets were visualized in Ito cells at 72 hr post-injection. Most of vitamin A in the liver homogenates measured by HPLC was retinyl palmitate. Therefore, the contents in those lipid droplets might be retinyl palmitate. -KEY WORDS: gold chloride method, hepatocyte, HPLC, Ito cell, vitamin A.

Liver autofluorescence properties in animal model under altered nutritional conditions

“…The occurrence of an accumulation of vitamin A in intracytoplasmic lipid droplets of hepatocytes has been already reported, as a consequence of alterations of vitamin A homeostasis in rats administered with excessive amounts of retinyl acetate. 31 Upon exposure to 366 nm continuous irradiation, autofluorescence signals of liver samples undergoes a decrease attributable to a strong photobleaching effect mainly involving vitamin A. An almost completed disappearance of vitamin A during the first 20 s of UV irradiation was already exploited to eliminate its undesired signal, when liver energetic metabolic studies based on NAD(P)H emission analysis are performed.…”

Distribution of vitamin A‐storing lipid droplets in hepatic stellate cells in liver lobules—A comparative study

“…The intralobular heterogeneity of vitamin A storage may be related to the number of hepatic stellate cells within each zone, or to the maturation of the hepatic stellate cells themselves (Wake et al, 1991). At the molecular level, this heterogeneity may involve various regulatory factors, e.g., the retinol metabolism rate of hepatocytes (Shintaku et al, 1997), and the mechanism for mobilization of retinol from stellate cells to the plasma retinol pool (Blomhoff et al, 1990). However, regarding the quantification of cell density in each zone, the average density calculated in all the animal species examined in our study did not differ among the zones.…”

High, but not low, dietary retinoids aggravate manifestation of rat liver fibrosis

“…The only suitable approach to induce nutritional vitamin A deficiency is the withdrawal of vitamin A in food over an extended period of more than weeks, as also reported by others 21–25 . To compare these hypovitaminotic animals with hypervitaminotic animals, we decided to induce hypervitaminosis also by chronic feeding of a vitamin A‐supplemented diet, 26 despite the fact that others apply vitamin A and/or other retinoid metabolites more frequently by either oral gavage 18–20 or subcutaneous injections 27,28 . However, we feel that chronic feeding of vitamin A more closely mimicks the human situation with ingestion of massive doses of vitamin A for prolonged periods of time 29,30 …”

“…For the induction of liver fibrosis, we exposed animals subcutaneously to CCl 4 , as described previously by our group 13,31 , 32 and others, 33 with the exception of using vitamin A‐deficient peanut oil instead of olive oil as the vehicle. In experimental studies on the transport and metabolism of retinyl acetate in mice, peanut oil has been used as vehicle for the subcutaneous injection of retinoic acid and was solely applied to treat the respective control animals 28 . Comparably, others used vitamin A‐deficient sunflower (vegetable) oil as the vehicle for CCl 4 to examine the effects of provitamin A on CCl 4 ‐related general and hepatic toxicity in rats 9 .…”