Retinoic acid tissue concentrations

JL Napoli State University of New York (SUNY) at Buffalo, NY Determine whether 2,3,7,8-TCDD causes functional vitamin A abnormalities by altering the steady-state concentrations of retinoic acid and/or RAR/RXR in male and female rat and male Syrian golden hamster tissues... 

CRABP-GST is immobilized onto a glutathione-containing resin at a concentration that saturates the resin. A complex lipid mixture was prepared by combining a concentrated lipid extract derived from mouse tissue (brain) [5] (DMSO stock) with exogenously added retinoic acid (RA, positive control) and 13C-oleic acid (negative control). Analysis of this complex lipid mixture by LC-MS prior to incubation with immobilized CRABP demonstrated that the mixture contained RA (added), phospholipids, acylglycerols, cholesterol esters, and cholesterol. A portion of this mixture (corresponding to 1 nmol of RA) was added to PBS (DMSO concentration should not exceed 5%) and incubated with the CRABP-GST bound beads for 1 h. 

Oxidation of retinol produces retinoic acid tretinoin). The reaction is irreversible. Retinoic acid enters the portal blood, is transported bound to albumin, and is not stored to any great extent. The concentration of retinoic acid in plasma is normally 3-4 ng/mL. A biologically active metabolite, 5,6-epoxyretinoic acid, has been isolated from the intestinal mucosa of vitamin A-deficient rats following administration of H-retinoic acid. Several tissues have specific cellular retinoic acid-binding proteins (CRABPs). 

In certain instances, it may be critical to know the relative amount of retinoid released from individual tissues or dissected regions of the embryo. To obtain a quantitative measure of retinoids released from tissues, we developed a reporter cell line using luciferase as the reporter molecule. When luciferase oxidizes its substrate luciferin, a photon is emitted that can be detected and quantified by use of a luminometer or scintillation counter. By relating known concentrations of retinoids to the amount of luciferase activity that they induce, the concentration of retinoids released by tissue samples can be determined. For quantifying retinoid release, a standard curve relating known concentrations of released retinoids to induced luciferase activity must be established. Known concentrations of released retinoids can be achieved by the use of ion-exchange beads that are loaded with retinoic acid and act as a point source of retinoid released onto reporter cell monolayers. 

Prepare cell or tissue extracts by standard procedures (see Note 3). Wash cultured cells or tissues well with PBS prior to preparation of extract to remove serum components (albumin binds to retinoic acid). Buffers containing high salt concentrations (0 3-0.6 M KCl) are usually used to ensure removal of receptor from DNA. Determine protein concentrations of all samples, store in aliquots at -80°C, and avoid freeze-thawing more than once. 

It is now well established that retinoic acid is present in low eoncentrations as an endogenous retinoid in a number of tissues (Ito et al.., 1974 DeLuca, 1979 Saari et al., 1982). Retinoic acid has been reported to be present as an endogenous compound in human plasma at concentrations of approximately 3-4 ng/ml (10- M) (De Leenheer et al., 1982). 

In the plasma, vitamin A is transported from its major depots in the liver to tissues in several forms, primarily as a 1 1 molar complex of all-frans-retinol with RBP. Low concentrations of dll-trans- and 13-ds-retinoic acid, probably bound to albumin, and retinyl and retinoyl p-glucuronides, are also present. Holo-RBP also interacts strongly with transthyretin in the plasma. All of these forms of vitamin A can be taken up by various tissue cells. Several tissues besides the parenchymal cells of the liver can also synthesize RBP, as evidenced by the presence of mRNA for RBP within such cells. Thus, the extensive recycling of retinol between the liver and peripheral tissue cells may well occur as complexes with RBP. Another possibility is that retinyl ester, which is synthesized in essentially all cells of the body, might be carried back to the liver in lipoproteins. 

Table 1. Tissue all-rran -retinoic acid concentrations and percentage of all-rra 5-retinoic acid present in the tissue contributed from the circulating all-rran -retinoic acid pool in rats ...

---