Retinoid Absorption and Storage Is Impaired in Mice Lacking Lecithin:Retinol Acyltransferase (LRAT)

Abstract

Lecithin:retinol acyltransferase (LRAT) is believed to be the predominant if not the sole enzyme in the body responsible for the physiologic esterification of retinol. We have studied Lrat-deficient (Lrat ؊/؊ ) mice to gain a better understanding of how these mice take up and store dietary retinoids and to determine whether other enzymes may be responsible for retinol esterification in the body. Retinoids 3 have important roles in mediating or facilitating many essential physiologic functions within the body (1). In vision, 11-cisretinal serves as the chromophore for the visual pigments present in the rod and cone photoreceptor cells (2, 3). Retinoids are also needed to maintain cell proliferation and normal differentiation, normal immune response, normal reproduction, and normal fetal development (4). It has been suggested in the literature that over 500 different genes may be transcriptionally responsive to retinoids (5). The transcriptional regulatory activities of retinoids are thought to result from the actions of all-trans-and 9-cis-retinoic acid (6 -8). These actions of retinoic acid are mediated through six distinct ligand-dependent transcription factors as follows: three retinoic acid receptors (RAR␣, 4 RAR␤, and RAR␥) and three retinoid X receptors (RXR␣, RXR␤, and RXR␥) (6 -8). Ultimately, all retinoid must be acquired from the diet either as preformed retinoid (primarily as dietary retinol or retinyl ester) or as proretinoid carotenoid (primarily as ␤-carotene) (9, 10). The different dietary retinoid forms are processed within the enterocyte and packaged along with other dietary lipids as retinyl esters in nascent chylomicrons (9, 10). Approximately 66 -75% of dietary retinoid is taken up and stored as retinyl ester in the liver (9, 10), primarily in the nonparenchymal hepatic stellate cells (also called Ito cells, lipocytes, and fat-storing cells) (11). These hepatic stores can be called upon and mobilized into the circulation as retinol bound to plasma retinol-binding protein (RBP) (12, 13). Tissues acquire retinol from the circulating retinol-RBP complex or postprandially from chylomicrons (9, 10, 12-14) and are able to oxidize enzymatically this retinoid to retinal and retinoic acid (14 -16). Most tissues also possess some capacity to esterify and thus store retinol prior to its use for the synthesis of retinal or retinoic acid (9,10,14).It is generally agreed upon in the literature that retinol is esterified primarily through the actions of the enzyme lecithin:retinol acyltransferase (LRAT) (17)(18)(19)(20). LRAT is broadly expressed in tissues and at relatively high levels in the intestine, liver, and eye where it has been proposed to catalyze the transesterification of retinol with an acyl group present in the A1 position of membrane lecithin (17-23). Recently, the gene encoding LRAT was knocked out in mice (20). It was clear from the initial studies of Lrat-deficient (Lrat Ϫ/Ϫ ) mice that LRAT is the preponderant and possibly sole enzyme responsible for retinyl ester formation in the eye and ...

HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues

“…Mice fed a 4 IU vitamin A/g diet had 9.6 nmol/g ROL in liver, less than half that of the 30 IU vitamin A/g stock diet, reflecting the lower level of vitamin A in the diet. Serum and adipose ROL in mice fed a stock diet (serum, 0.9 nmol/g; adipose, 1.8 nmol/g; Table 3) was similar to literature values ranging from 0.6-1.58 nmol/g (22,23,44,48,51) and 1.67-2.9 nmol/ g (22,23), respectively. Serum and adipose values in mice fed a 4 IU vitamin A/g diet were slightly lower and one-third lower, respectively, of the stock diet.…”

“…Serum and adipose values in mice fed a 4 IU vitamin A/g diet were slightly lower and one-third lower, respectively, of the stock diet. Extrahepatic tissue ROL and RE were comparable to those produced by other methods (22,23). Liver RE in mice fed a 4IU vitamin A/g diet was 562 nmol/g, which was comparable to or lower than RE values reported with a stock diet (473-5500 nmol/g) (16,17,22-25,31,32,44,48).…”

“…Liver ROL obtained from mice fed a stock diet (20.7 nmol/g, Table 3) was similar to those fed a comparable copious vitamin A diet, ranging from 6-81.5 nmol/g (16,18,(22)(23)(24)(25)31,32,44,48). Mice fed a 4 IU vitamin A/g diet had 9.6 nmol/g ROL in liver, less than half that of the 30 IU vitamin A/g stock diet, reflecting the lower level of vitamin A in the diet.…”

“…The majority of measurements use the ROL/RE method and the RAL method, which use the same solvents and analytical column, allowing for seamless transition between the two analyses. Both the ROL/RE and the RAL methods have run times of 25 min, including re-equilibration, which is shorter than other methods, for which some separations take >100 min (17,18,(22)(23)(24)(25)30,31,48,51,53,55). Additionally, our limits of detection/quantification and linear working ranges have been characterized rigorously for each analyte, and the analyte concentration is determined from a standard curve spanning the physiological range of the analyte.…”

“…RA exerts additional biological effects through dimerization of RXR with an array of other type II nuclear receptors (13). Various approaches have been used to determine effectors of retinoid metabolism, such as genetic alteration of retinoid-binding proteins (16)(17)(18)(19)(20)(21), enzymes (22)(23)(24)(25), and receptors (12,13,26), dietary manipulation of vitamin A intake (28), and exposure to xenobiotics (29)(30)(31)(32). Quantifying how manipulation of retinoid metabolism effects the flux of retinoids through metabolic paths and/or effects availability of substrate for RA production will provide insight into retinoid homeostasis and metabolism, and thereby function.…”