Liquid-gel partition chromatography of vitamin A compounds; formation of retinoic acid from retinyl acetate in vivo

The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoli cells accumulated [3H]retinol in a time- and temperature-dependent manner. At 32 degrees C, the rate of retinol accumulation was biphasic. Accumulation was linear for approximately 1 h, but then accumulation continued at a linear but decreased rate for 23 h. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/micrograms of cellular DNA. This amount of retinol was approximately equal to the cellular content of cellular retinol-binding protein (CRBP). Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [3H]retinol-RBP for [3H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [3H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 microM, suggesting that any change in the normal circulating retinol-RBP level (approximately 2 microM) would directly affect the rate of retinol accumulation. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. In addition, energy inhibitors and lysosomal poisons had no effect on [3H]retinol accumulation, indicating that RBP delivery of retinol to Sertoli cells did not occur by endocytosis of the retinol-RBP complex. Competition studies indicated, however, that protein recognition is important in the retinol uptake process.(ABSTRACT TRUNCATED AT 250 WORDS)

mentioning

confidence: 99%

Characteristics of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells

Shingleton

Skinner²,

Ong³

1989

“…Knowledge of testicular retinoid metabolism is limited to analytical surveys of whole organ extracts (Ito et al, 1974;Napoli & McCormick, 1981;Bhat & Lacroix, 1983) and metabolic studies utilizing extracts obtained from testis injected with [3H]retinyl acetate (Chaudhary & Nelson, 1985). Evidence of the distribution of retinoids has been inferred from the histological examination of testicular tissue for labeled retinol and retinyl esters by autoradiography (Ahluwalia et al, 1975(Ahluwalia et al, , 1980Shakuntala et al, 1982) and by immunohistochemical analysis of CRBP and CRABP (Porter et al, 1985).…”

mentioning

confidence: 99%

Uptake and metabolism of retinol in cultured Sertoli cells: evidence for a kinetic model

Bishop¹,

Griswold²

1987

When cultured Sertoli cells derived from 20-day-old weanling rats were supplied [3H]retinol bound to serum retinol binding protein-transthyretin complex, [3H]retinol was rapidly incorporated and [3H]retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied [3H]retinol in culture under identical conditions likewise incorporated [3H]retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular [3H]retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of [3H]retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of [3H]retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of [3H]retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of [3H]retinol. During the time course the specific activity of [3H]retinyl palmitate eventually exceeded that of intracellular [3H]retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells.

“…Extraction of the Pigments. Carotenoids and retinoids were extracted from the lyophilized samples (Ito et al, 1974) with anhydrous methanol and «-hexane with added BHT (0.005% w/v) at 4 °C. Three extraction steps each comprising 6 mL of solvent with alternating order of solvent addition were used.…”

Section: Methodsmentioning

confidence: 99%

Retinal is not formed in vitro by enzymic central cleavage of .beta.-carotene

Hansen¹,

Maret²

1988

Rat intestinal mucosa was prepared and incubated with beta-carotene by the procedure of Goodman and Olson [Goodman, DeW. S., & Olson, J.A. (1969) Methods Enzymol. 15, 462-475] to determine beta-carotene cleavage activity. A new detection system for the reaction products of the described enzyme beta-carotene 15,15'-dioxygenase (EC 1.13.11.21) employs solvent extraction of retinoids and carotenoids followed by high-performance liquid chromatography separation and photometric detection of the pigments. It has not detected any newly formed retinal or other retinoids in the intestinal protein preparations from normal or vitamin A deficient rats. The latter were chosen as a possible source of more active enzyme preparations. With corresponding blank samples subjected to identical conditions of incubation but without added protein, small amounts of beta-apocarotenals could be detected. They were previously reported as cleavage products of beta-carotene [Ganguly, J., & Sastry, P.S. (1985) World Rev. Nutr. Diet. 45, 198-220] but are clearly not formed as a result of an enzymatic reaction. The failure to detect in vitro enzymatic central or random cleavage of the beta-carotene molecule in extracts of rat intestinal mucosa emphasizes the need to reevaluate the existing theory of conversion of beta-carotene into vitamin A.