Absorption and Metabolism of Retinol and Retinoic Acid

About 70% to 90% of dietary retinol is absorbed, and, even at high intakes, this falls only slightly. Retinyl esters are hydrolyzed by pancreatic lipase and carboxyl ester lipase in lipid micelles in the intestinal lumen, and also by one or more retinyl ester hydrolases in the intestinal mucosal brush border membrane. At physiological levels of intake, retinol uptake into enterocytes is by facilitated diffusion from the lipid micelles. When the transport protein in the intestinal mucosal brush border cells is saturated, there is also passive uptake of retinol.

Within the enterocyte, retinol is bound to cellular retinol binding protein (CRBP II) and is esterified by lecithin:retinol acyltransferase (LRAT), which uses phosphatidylcholine as the fatty acid donor, mainly yielding retinyl palmitate, although small amounts of stearate and oleate are also formed. At unphysiologically high levels of retinol, when CRBP II is saturated, acyl coenzyme A (CoA):retinol acyltransferase (ARAT) esterifies the free retinol that accumulates in intracellular membranes. Then the retinyl esters enter the lymphatic circulation and then the bloodstream (in chylomicrons), together with dietary lipid and carotenoids (Norum et al., 1986; Olson, 1986; Blomhoff et al., 1991; Green et al., 1993; Harrison and Hussain, 2001).

A small proportion of dietary retinol is oxidized to retinoic acid, which is absorbed into the portal circulation and bound to serum albumin. Some retinyl esters are also transferred into the portal circulation. Patients with abeta-lipoproteinemia, who are unable to synthesize chylomicrons, can nevertheless maintain adequate vitamin A status if they are provided with relatively high intakes of retinol.

2.2.1.1 Liver Storage and Release of Retinol Tissues can take up retinyl esters from chylomicrons, but most is left in the chylomicron remnants that are taken up into the liver by endocytosis. The retinyl esters are hydrolyzed at the hepatocyte cell membrane, and free retinol is transferred to the rough endoplasmic reticulum, where it binds to apo-RBP. Holo-RBP then migrates through the smooth endoplasmic reticulum to the Golgi and is secreted as a 1:1 complex with the thyroid hormone binding protein, transthyretin (Section 2.2.3).

Studies in vitamin A replete animals suggest that most of the retinol is transferred from hepatocytes to the perisinusoidal stellate cells of the liver. Here, it is again esterified by LRAT to form mainly retinyl palmitate (76% to 80%), with smaller amounts of stearate (9% to 12%), oleate (5% to 7%), and linoleate (3% to 4%). The stellate cells contain 90% to 95% of hepatic vitamin A, as cytoplasmic lipid droplets that consist of between 12% to 65% retinyl esters (Batres and Olson, 1987). Studies with [13C]retinyl palmitate show that much of the recently ingested retinol appears more or less immediately in the circulation, bound to RBP, and is only sequestered in the liver reserves subsequently. This suggests that there may be little or no direct transfer of retinol from hepatocytes to stellate cells, but rather retinol is cleared from the circulation into stellate cells for storage. LRAT is induced by retinoic acid (and by dietary vitamin A) and is down-regulated in vitamin A depletion, when the need is to transfer vitamin A to tissues rather than to store it in the liver (Zolfaghari and Ross, 2000; Wolf, 2001).

Release of retinol from stellate cells into the circulation may occur either directly, as free retinol bound to RBP, or indirectly as a result of the transfer of retinol from stellate cells to hepatocytes. The release of retinol from stores is impaired in iron deficiency, as is the absorption of dietary vitamin A (Jang et al., 2000).

The concentration of retinol in most tissues is between 1 to 5 ^mol per kg; in the liver, the mean concentration is 500 ^mol per kg, with a very wide range of individual variation. In a number of studies of postmortem tissue, between 10% to 30% of the population of the United States had liver retinol below 140 ^mol per kg, and about 5% had reserves in excess of 1,700 ^mol per kg. Five percent to 10% of samples analyzed in Canada showed undetectably low liver reserves of retinol, although similar studies in Britain did not show any significant proportion of the population with extremely low liver reserves of vitamin A (Sauberlichetal., 1974; Huque, 1982). Abnormally low liverreserves of retinol may result not only from prolonged low intake, but also from the induction by barbiturates of cytochrome P450, which catalyzes the catabolism of retinol (Section 2.2.1.2). Chlorinated hydrocarbons, as in many agricultural pesticides, also deplete liver retinol by effects on the metabolism of RBP (Section 2.2.3).

Opinions differ as to what constitutes an adequate concentration of retinol in the liver. When the concentration rises above 70 ^mol per kg, there is increased catabolism of retinol (Section 2.2.1.2). Estimates of requirements based on the fractional catabolic rate of whole body retinol and using liver reserves of 70 ^mol per kg as a basis for calculation are generally in agreement with estimates based on the very few depletion/repletion studies that have been performed (Section 2.4). However, from the observed range of liver reserves in healthy subjects, it can be argued that a more appropriate level is 140 ^mol per kg, which gives a higher estimate of requirements (Sauberlich et al., 1974; Hodges et al., 1978; Olson, 1987a).

Although the major storage of vitamin A is in the liver (50% to 80% of the total body content), adipose tissue may contain 15% to 20% of total body vitamin A. Much of this is taken upfrom chylomicrons; retinyl esters are hydrolyzed by lipoprotein lipase (Blaner et al., 1994), but some vitamin A is also taken up from circulating vitamin bound to RBP. Release of retinol from adipose tissue is by hydrolysis of stored retinyl esters, catalyzed by (cAMP-stimulated) hormone-sensitive lipase, bound to RBP, which is synthesized by both white and brown adipose tissue (Wei et al., 1997).

A variety of other tissues synthesize RBP; this provides a mechanism for return to the liver of retinol in excess of requirements that has been taken up from chylomicrons by the action of lipoprotein lipase. Because these tissues do not synthesize transthyretin, the binding of holo-RBP to transthyretin must occur in the circulation after release.

2.2.1.2 Metabolism of Retinoic Acid Retinoic acid is the normal major metabolite of physiological amounts of retinol. However, it is not a catabolic product of retinol, but the ligand for nuclear retinoid receptors involved in modulation of gene expression (Section 2.3.2). It may be formed in the liver, although there is no hepatic storage, and is then transported bound to serum albumin rather than RBP. Other tissues are also able to form retinoic acid from retinol. The rate-limiting step is the dehydrogenation of retinol to retinaldehyde; the Km of the dehydrogenase is high, so that a major determinant of the rate of formation of retinoic acid will be the concentration of retinol in the cell (Napoli, 1996).

Cytosolic alcohol dehydrogenases only act on free retinol, not retinol bound to CRBP, so they are unlikely to be involved in formation of retinaldehyde and retinoic acid. Furthermore, inhibition of cytosolic alcohol dehydrogenases does not inhibit the oxidation of retinol to retinoic acid (Boerman and Napoli, 1996). CRBP-bound retinol is a substrate for at least three microsomal NADP+-dependent dehydrogenases; but, given the intracellular NADP+:NADPH ratio (0.01, compared with an NAD+:NADH ratio of the order of 103), it is likely that these microsomal enzymes will act mainly to reduce retinaldehyde to retinol and not to oxidize retinol.

A microsomal retinol dehydrogenase catalyzes the oxidation of CRBP-bound all-frans-retinol to retinaldehyde; it also acts as a 3a-hydroxysteroid dehydrogenase. A similar enzyme catalyzes the oxidation of 9-cis- and 11-cis-retinol, but not all-frans-retinol; again, it has 3a-hydroxysteroid dehydrogenase activity. In the eye, the major product of this enzyme is 11-cis-retinaldehyde, whereas in other tissues it is 9-cis-retinaldehyde, which is then oxidized to 9-cis-retinoic acid (Section 2.3.2.1; Chen et al., 2000; Duester, 2000, 2001; Gamble et al., 2000; Napoli, 2001). Although there is known to be an isomerase in the eye for the formation of 11-cis-retinaldehyde as a substrate for the dehydrogenase, there is no information concerning an iso-merase in other tissues to produce 9-ds-retinol (Wang et al., 1999; Gamble et al., 2000; McBee et al., 2000).

Intracellular concentrations of retinoic acid are controlled not only by the rate of synthesis, but also by catabolism. At least in culture, prior exposure of cells to retinoic acid induces the enzymes of retinoic acid catabolism (Chytil, 1984; Napoli and Race, 1987). The major metabolite of retinoic acid is the glucuronide (Section 2.2.1.3).

All-frans-retinoic acid (but apparently not 9-ds-retinoic acid) undergoes microsomal oxidation to yield a variety of polar metabolites. Retinoic acid hydroxylase is a retinoic acid-induced cytochrome P450 (CYP26) - from its amino acid sequence, it appears to represent a novel family of cytochrome P450 . 4-Hydroxyretinoic acid then undergoes further oxidation to yield 4-oxo-retinoic acid. The same enzyme also catalyzes 18-hydroxylation and 5,6-epoxidation of retinoic acid. 4-Hydroxy- and 4-oxo-retinoic acids were originally considered to be inactivation products of all-frans-retinoic acid; however, they bind to, and activate, the RAR and show high activity as modulators of positional specificity in Xenopus embryogenesis. Furthermore, CYP26 is expressed differently through the process of embryogenesis (Sonneveld et al., 1998).

In addition to oxidation of retinol, retinoic acid may be formed by the f-oxidation of apo-carotenals arising from the asymmetric cleavage of f-carotene (Section 2.2.2.1).

Retinoic acid regulates its own synthesis from retinol in a variety of tissues by induction of LRAT; this increases the rate of esterification of re-tinol, thereby decreasing the amount available for oxidation to retinoic acid (Kurlandsky et al., 1996). Retinoic acid also induces the cytochrome P450 that catalyzes oxidation to 4-oxo-retinoic acid, and regulates both its own synthesis and catabolism.

2.2.1.3 Retinoyl Glucuronide and Other Metabolites The main excretory product of both retinol and retinoic acid is retinoyl glucuronide, which is secreted in bile. Some retinoyl taurine is also secreted in bile. This suggests that, in addition to regulated synthesis of retinoic acid for its biological activity, oxidation to the acid is also a significant pathway for catabolism of retinol. Unlike retinoyl glucuronide, which has biological activity, retinoyl taurine seems to be solely an excretory product.

The plasma concentration of retinoyl glucuronide is between 5 and 14 nmol per L, and the activity of retinoic acid UDP-glucuronyltransferase increases in vitamin A deficiency, suggesting that glucuronidation may be important other than as a pathway for inactivation and excretion of retinoic acid (Miller and DeLuca, 1986). Retinoyl glucuronide has biological activity; it is not clear whether or not this is as a result of hydrolysis to retinoic acid. In some experimental systems, the glucuronide appears to act without undergoing hydrolysis, although it binds to neither cellular retinoic acid binding protein nor nuclear retinoid receptors. However, glucuronidases in the liver and kidney hy-drolyze retinoyl glucuronide, and activity of the glucuronidase, like that of the UDP-glucuronyltransferase, increases in vitamin A deficiency (Barua, 1997; Sidell et al., 2000). It has also been suggested that retinoyl glucuronide, rather than retinoic acid, may be the precursor of retinoyl CoA for retinoylation of proteins (Section 2.3.3.1).

Small amounts of a number of other metabolites, including epoxy-retinoic acid glucuronide and a number of products of side-chain oxidation of retinol and retinoic acid, are also formed, some of which are excreted in the urine as well as bile. As the intake of retinol increases, and the liver concentration rises above 70 ^mol per kg, a different catabolic pathway becomes increasingly important for the catabolism of retinol in liver parenchymal cells. This is a microsomal cytochrome P450-dependent oxidation, leading to a number of polar metabolites, including 4-hydroxyretinol, which are excreted in the urine and bile. Thus, there is a catabolic mechanism that allows excretion of excess retinol. However, at high intakes, the microsomal pathway is saturated, and this may be one of the factors in the toxicity of excess retinol, because there is no further capacity for its catabolism and excretion. Stored retinyl esters in the stellate cells of the liver are only slowly released to the parenchymal cells for catabolism, and retinol is chronically toxic (Section 2.5.1). Induction of cytochrome P450 enzymes by chronic administration of barbiturates can result in depletion of liver reserves of retinol and may be a factor in drug-induced vitamin A deficiency (Leo and Lieber, 1985; Olson, 1986; Leo et al., 1989).

Anhydroretinol may arise by nonenzymic isomerization of all-frans-retinol under acidic conditions and can act as a precursor for the synthesis of other biologically active retroretinoids (McBee et al., 2000).

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