The postrun data were analyzed with the real-time detection system software of the thermal cycler iCycler iQ ver. Briefly, the threshold value C T was selected in the log phase of the amplification curve. The copy numbers of the samples were deduced by plotting the C T values of unknown samples on the standard curve. Melting curve analysis was included in every run, to confirm the specificity of the PCR reaction.
Protein concentration was then determined Dc Protein assay system; Bio-Rad. Peroxidase-conjugated anti-mouse IgG was used as the secondary antibody. Afterward, the detection blots were stripped and probed with actin antibody as an internal control. Enzyme preparation and BCO activity assays were performed as described by During et al. Protein isolated from Caco-2 TC7 cells was used as a positive control in assay reactions.
The reaction mixture was extracted three times with n -hexane. Results are expressed as nanograms of all- trans -retinal per milligram of protein per hour. After the incubation, the cells were washed three times with phosphate-buffered saline PBS , scraped with a cell scraper, and pelleted down by centrifuging at g for 5 minutes.
Vitamin A metabolites were extracted as described elsewhere. The hexane phase was separated by centrifuging at g for 1 minute and transferred into a fresh tube. The hexane extraction was repeated two times, and the phases were pooled and dried under a stream of nitrogen gas. Chromatograms were recorded and analyzed Star Chromatography Workstation Software, ver.
Caco-2 TC7 is a clone of Caco-2, which is a human colon cancer cell line. Total protein was isolated from confluent cultures of D and Caco-2 TC7 cell lines. This preamplification step minimized inter- and intra-assay variations and increased the efficiency of the PCR method, in accordance with other studies in which a comparable protocol was used for accurate quantification of changes in the expression of genes with low-abundance mRNA levels.
D cells were incubated with 0. BCO protein expression had increased twofold at 2 and 4 hours but then decreased after 24 and 48 hours Fig. CYP26A1 is an enzyme involved in retinoic acid catabolism, which converts retinoic acid into polar metabolites. To elucidate the possible role of RA in the regulation of BCO expression, cells were incubated with all- trans -retinoic acid—supplemented medium for a fixed time of 6 hours.
BCO protein expression was upregulated by onefold at 0. The cells were incubated with 0. BCO protein was upregulated over time with a maximum fivefold increase at 24 hours in 0.
BCO protein also increased with incubation time, to a maximum of fourfold at 24 hours Fig. Expression of both mRNAs was strongly induced by retinoic acid in a concentration- and time-dependent manner data not shown. RXR-a expression increased by fourfold at 2 hours and twofold at 4 hours and then declined to basal level at 6 hours and 24 hours Fig. To demonstrate BCO at the functional level, we performed tests for enzymatic activity in total protein extracts from D cells and Caco-2 TC7 cells.
Enzyme activity was observed by the formation of 4. In extracts from Caco-2 TC7 cells, 7. All- trans -retinal was the only product detected in the in vitro test system. In addition, we performed HPLC profiling for retinol and retinal at different time points. In humans, provitamin A carotenoids are the major dietary precursors for vitamin A. In the photoreceptor outer segments, rhodopsin exists in millimolar concentrations.
Therefore, eyes have a very high demand for vitamin A, to maintain the visual process. Bernstein et al. Recently, Yan et al. This cell line is an immortal human RPE line that retains many of the metabolic and morphologic characteristics of RPE cells found in vivo, even after several passages, and it has a long survival time.
CYP26A1 is known to be induced by retinoic acid and its receptors through a transactivation of a retinoic acid—responsive element RARE located in its promoter. In D cells, we found significant amounts of retinol, but retinoic acid was below the detection limit during the initial periods and was detectable only after 24 hours. Nevertheless, traces of retinoic acid are sufficient to activate its nuclear receptors. In addition, an increase in cellular retinoic acid levels rapidly induces its catabolism through CYP26 monooxygenases.
Retinoic acid regulated BCO expression in a bidirectional manner by inducing expression at lower concentrations and inhibiting it at higher concentrations. Therefore, a role of 9- cis -retinoic acid in the observed effects on the regulation of BCO expression in D cells can most probably be excluded. Bachman et al. Further investigations are necessary to gain a mechanistic understanding of the regulation of BCO expression via RA and its nuclear receptors in different cell types and organisms.
Bhatti et al. This may explain the differences between the two studies, because PCR sensitivity varies between different primer sets. Recently, expression of BCO has been reported in epithelia of several tissues in human including glandular cells in the prostate, endometrium, mammary tissue, kidney tubules, keratinocytes of the squamous epithelium of skin; steroidogenic cells in testis, ovary, and adrenal gland; and skeletal muscle cells.
A most interesting finding in two siblings with impaired retinol transport caused by mutations in the RBP gene was that only mild clinical vitamin A deficiency symptoms such as night blindness and modest retinal dystrophy were present. Our study provides evidence for a tissue-specific vitamin A synthesis in the RPE. Submitted for publication January 25, ; revised May 24, ; accepted August 25, Disclosure: G. Food and Nutrition Board, Institute of Medicine.
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Retinol is esterified to palmitic acid and delivered to the blood via chylomicrons. Finally the retinol formed is stored in the liver as retinyl esters. This is why cod liver oil used to be taken as a vitamin A supplement.
It is also why you should never eat polar bear liver if you run out of food in the Arctic; vitamin A is toxic in excess and a modest portion of polar bear liver contains more than two years supply! Beta-carotene, on the other hand, is a safe source of vitamin A. The efficiency of conversion of beta-carotene to retinol depends on the level in the diet. If you eat more beta-carotene, less is converted, and the rest is stored in fat reserves in the body. So too much beta-carotene can make you turn yellow, but will not kill you with hypervitaminosis.
Introduction The long chain of alternating double bonds conjugated is responsible for the orange color of beta-carotene. Vitamin A Vitamin A has several functions in the body.
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