Vitamin A deficiency reduces uptake of beta-carotene by brush border membrane vesicles but does not alter intestinal retinyl ester hydrolase activity in the rat

Vitamin A deficiency has been reported to result in mild structural and functional changes within the small intestine. The objective of this study was to measure the impact of vitamin A deficiency in the rat on several functional aspects of beta-carotene uptake and intestinal retinyl ester hydrolysis. These included uptake of (14)C-beta-carotene by brush border membrane vesicles (BBMV) and in vitro activity of intrinsic retinyl ester hydrolase (REH). Rats (n = 33) were randomly assigned to receive one of three dietary treatments: vitamin A deficient (-VA), vitamin A sufficient pair-fed (PF), or vitamin A sufficient free access-fed (FA). Liver, serum retinol, and growth data were used to verify clinical vitamin A deficiency. Rats in the -VA group were clinically vitamin A deficient by Day 56 on a vitamin A-free diet and, at that point, all rats were randomly assigned to one of two experimental treatments: BBMV studies or REH activity assays. Uptake of (14)C-beta-carotene by BBMV was significantly suppressed (P < 0.05) in -VA rats when compared to both PF and FA control rats during early passive uptake equilibration (10-20 sec). Uptake was also significantly decreased by BBMV isolated from -VA rats compared to PF controls, but not FA controls, after a 10-min incubation (P < 0.05). In vitro activity of REH was not impacted by vitamin A deficiency in rats, although a trend for greater activity from -VA rats was noted. These data suggest that vitamin A deficiency impairs enterocyte membrane uptake of beta-carotene without altering the enzymatic activity of intrinsic REH.     

Vitamin D metabolites specific binding by rat intestinal cytosol

The intestine of ricketic rats, a recognized target tissue of vitamin D, contains s soluble macromolecule capable of specific in vitro binding of both 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol. Its sedimentation behavior on linear 5–20% sucrose gradients suggests as a molecular weight of approximately 100 000. The binding is specific for sterols possessing both an open B ring and 25-hydroxyl group, and is destroyed by pre-incubation with trypsin. The binding affinity for 25-hydroxycholecalciferol (K_A = 2 · 10⁹ 1/mole) was 2.8 times that for 1,25-dihydroxycholecalciferol.     

A reactive hydroxymethyl sulfate ester formed regioselectively from the carcinogen, 7,12-dihydroxymethylbenz[alpha]anthracene, by rat liver sulfotransferase

The carcinogen, 7,12-dihydroxymethylbenz[alpha]anthracene (DHBA), was regioselectively conjugated in the presence of 3'-phosphoadenosine 5'-phosphosulfate by male rat liver cytosolic sulfotransferase to DHBA 7-sulfate. The sulfate ester was highly reactive and showed a potent, intrinsic mutagenicity toward Salmonella typhimurium TA 98.     

Vitamin A metabolism in cultured somatic cells from rat testis

Sertoli and peritubular myoid cells, the somatic cells of the seminiferous tubule, support growth and differentiation of developing germ cells. This action strictly depends on the availability of in situ synthesized retinoic acid and we have previously documented the ability of Sertoli, but not peritubular cell extracts, to support the oxidation of retinol to retinoic acid. Using primary cultures of somatic cells treated with a physiological concentration of free retinol, we show here that the same is essentially true also for whole cultured cells. Sertoli cells are capable of producing not only retinoic acid, but are also the major site of retinyl ester (mainly, retinyl palmitate) formation. Compared with retinyl palmitate accumulation, retinoic acid synthesis was both faster and positively influenced by prior exposure to retinol. This increase in retinoic acid synthesis was further augmented by treatment with the retinoic acid catabolic inhibitor liarozole, thus indicating that enhanced synthesis, rather than reduced catabolism, is responsible for such an effect. Myoid cells had a higher capacity to incorporate exogenously supplied retinol, yet retinoic acid synthesis, and even more so retinyl palmitate formation, were considerably lower than in Sertoli cells. Retinoic acid synthesis in myoid cells was not only depressed, but also very little influenced by prior retinol exposure and totally insensitive to liarozole. These data further support the view that myoid cells are involved in retinol uptake from the blood and its transfer to other cells, rather than in metabolic interconversion or long-term storage of vitamin A, two processes that mainly take place in Sertoli cells.     

Study of Vitamin C ester synthesis by immobilized lipase from Candida sp.

Oleoyl ester of -ascorbic acid was synthesized by using immobilized lipases from Candida sp. A series of solvents, such as ethanol, tetrahydrofuran, pyridine, butanol, tertiary amyl alcohol (t-amyl alcohol), hexanol, octanol and hexane (log P from −0.24 to 3.5) were investigated for the reaction, and t-amyl alcohol was found to be the most suitable from the standpoint of the substrate concentration and the enzyme activity. And the equilibrium of the reaction was affected by the addition of the molecular sieves and the temperature. Reaction carried out at 55 °C and with 50 g/l of 4 Å molecular sieves is good for the enzyme to keep its activity and for making the equilibrium go to the product. The kinetic model was studied and the result showed that the reaction can be described by Ping-Pong mechanism. Parameters value of V_m and K_m′ were obtained. Last, the pure products of the reaction were attained and determined by IR spectra, mass spectrometry and ¹H NMR spectra.     

Vitamin A-dependent fucose-glycopeptide from rat tracheal epithelium

Tracheas from normal and vitamin A-deficient rats were incubated in the presence of l-[³H]fucose and d-[¹⁴C]glucosamine to label epithelial and mesenchymal glycoproteins. The epithelium and the cartilage were separated by incubation with testicular hyraluronidase and processed separately for the preparation of glycopeptides. One major epithelial glycopeptide was doubly labeled. In vitamin A deficiency the amount of l-[³H]fucose was reduced to 33% that of the normal. The tracheal fucose-glycopeptide, in molar ratios, contained fucose 1.0, mannonse 1.0, galactose 0.4, hexasamine 4.7, and sialic acid 3.5. Sulfate was absent.     

Pyrroline-5-carboxylate synthesis from glutamate by rat intestinal mucosa

The mitochondria of rat intestinal mucosa were found to have an enzymatic activity that converts radioactive glutamate to pyrroline-5-carboxylate (P5C) in the presence of ATP, NADPH, and MgCl2. The product of this enzyme was identified as P5C by the fact that it was converted to proline by chemical reduction with NaBH4 or by enzymatic reduction with NADH in the presence of purified yeast P5C reductase. The product was demonstrated to be P5C rather than pyrroline-2-carboxylate by thin layer chromatography. The presence of the activity in mitochondria prepared from intestinal mucosa of germ-free rats proved that this activity is of mammalian origin. Omission of either ATP, NADPH, or MgCl2 from the reaction mixture resulted in little or no activity. The optimal pH appeared to be about 7.0 under the conditions used. Substrate saturation curves in the presence of an ATP and an NADPH regeneration system gave apparent Km values of 2.5 mM for glutamate, 0.19 mM for ATP, and 6.5 microM for NADPH in the presence of 20 mM MgCl2. The mitochondrial preparation usually produced P5C at a rate of 1.2 to 1.6 nmol/mg/min at 20 degrees C when incubated with 1 mM glutamate, 3 mM ATP, 0.2 mM NADPH, and 20 mM MgCl2.     

Accumulation and retention of micellar beta-carotene and lutein by Caco-2 human intestinal cells

Despite the interest in the diverse roles of dietary carotenoids in human health, little is known about the transfer of these plant pigments from foods to micelles during digestion and their subsequent transfer across the intestinal epithelium. We conducted this study to characterize the intestinal uptake of micellarized carotenoids using monolayers of differentiated Caco-2 human intestinal cells. Crystalline beta-carotene (BC) and lutein (LUT), solubilized in mixed micelles for delivery to cells, were stable in a tissue culture environment for 20 hours. Cellular accumulation of micellar BC and LUT was proportional to the media content of carotenoids at /=18 micromol/L. There was no indication that high levels of BC in medium or within cells adversely affected micellar LUT accumulation. These data support the use of the Caco-2 human cell line as a model for studying the intestinal uptake, absorption, and possible interactions of dietary carotenoids.     

Retinoic acid can be produced from excentric cleavage of beta-carotene in human intestinal mucosa.

The hypothesis that retinoic acid (RA) is produced from the excentric cleavage of beta-carotene was tested in human intestinal homogenates in vitro. Significant amounts of RA were identified by HPLC and derivatization after incubation of intestinal mucosal homogenates with retinal, beta-carotene, or beta-apocarotenals at 37 degrees C for 60 min. RA formation was inhibited, in a dose-dependent fashion, when retinal was incubated in the presence of 0.1-3.0 mM citral (3,7-dimethyl-2,6-octadienal) under identical experimental conditions. The formation of RA from both beta-carotene and beta-apocarotenals was dose and time dependent and RA was the major metabolite of both beta-apo-8'-carotenal and beta-apo-12'-carotenal after the incubation. However, citral (0.1 to 4 mM) did not inhibit the formation of beta-apocarotenals and RA from 2 microM beta-carotene (P greater than 0.05), which proves the existence of an excentric cleavage mechanism for beta-carotene conversion into retinoids. Furthermore, RA formation from both beta-apo-8'-carotenal and beta-apo-12'-carotenal in human intestinal homogenate occurred in the presence of citral, which demonstrates that RA can be produced from excentric cleavage of beta-carotene via a series of beta-apocarotenals as intermediates.     

Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferase 4-4, the most active among these enzymes, had a Vmax of 615 nmol X min-1 X mg protein-1 at 30 degrees C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3-4 which had a Vmax of 160 nmol X min-1 X mg-1 under the same conditions. Transferases 1-1, 1-2, 2-2 and 3-3 had at least 30 times lower Vmax values than transferase 4-4.     

Formation of leukotriene B4-coenzyme A ester by rat liver microsomes

When leukotriene B4 (LTB4) was incubated with rat liver microsomal fraction in the presence of coenzyme A (CoA) and ATP, a more polar product (compound I) was detected on reverse-phase high-performance liquid chromatography (RP-HPLC). The product was identified as LTB4-CoA ester on the basis of ultraviolet spectrometry, alkaline hydrolysis followed by RP-HPLC, and fast atom bombardment mass spectrometry (FAB-MS). The activity forming LTB4-CoA ester was localized in the microsomal fraction. The reaction was proportional to the concentration of the microsomal protein with an optimal pH of 7.5-8.0 and completely dependent on CoA and ATP. Palmitic acid and myristic acid significantly inhibited the formation.