Metabolic events in the intestine Esterification of retinol

Within enterocytes retinol becomes bound in a 1:1 molar ratio to CRBP-II, which is present exclusively and abundantly in these cells. CRBP-II binds all-trans and 13- cis retinol with high affinity; it also binds retinaldehyde, but not retinoic acid. The protein-bound retinol is esterified with saturated long-chain fatty acids, preferentially palmitic acid. The esterifica-tion uses a different pool of fatty acids and hence different enzymes than are used for the synthesis of triglycerides. Two microsomal enzymes are involved in the esterification of retinol, namely acyl coenzyme A:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT). The substrates for the ARAT-catalysed reaction are free retinol and acyl-CoAs; retinol bound to CRBP-II is not a substrate for ARAT. LRAT is an unusual enzyme in that it utilizes a membrane phospholipid, phosphatidylcholine (lecithin), as an endogenous donor of fatty acids for esterifica-tion. The enzyme activity shows positional selectivity as only the fatty acid from position 1 of the phos-pholipid is transferred to retinol. Position 1 is usually occupied by a saturated fatty acid (palmitic or stearic acid), which explains the predominance of saturated fatty acids in the retinyl esters. Unlike ARAT, LRAT

can utilize CRBP-II-bound retinol as well as free retinol. However, because of the abundance of CRBP-II in enterocytes, the majority of retinol will be bound and thus restricted to esterification by LRAT.

Whether ARAT or LRAT is involved in retinol es-terification depends on the amount of available reti-nol. LRAT is responsible for the esterification of retinol-CRBP-II when normal loads of vitamin A are ingested. In contrast, ARAT activity is important if the amount of retinol absorbed exceeds the saturation level of CRBP-II. It seems that CRBP-II functions both to direct retinol to the microsomes for esterifica-tion by LRAT and to prevent retinol from participating in the ARAT reaction.

Conversion of provitamin carotenoids to retinoids

Both central and excentric (asymmetric) oxida-tive cleavage of provitamin carotenoids have been proposed for the biosynthesis of retinaldehyde in enterocytes (Fig. 7.4). In the central cleavage reaction, molecular oxygen reacts with carbon atoms 15 and 15' of the polyene chain, after which the central double bond is cleaved. This reaction would be expected to generate two molecules of retinaldehyde from one molecule of P-carotene (or one molecule of retinal-

Fig. 7.4 Intestinal metabolism of P-carotene. The enzyme P-carotenoid-15,15'-dioxygenase forms retinaldehyde directly. Cleavage at other double bonds forms P-apocarotenals (e.g. 8'-CHO), which can be shortened to retinaldehyde. P-Apocarotenals may be oxidized to P-apocarotenoic acids (e.g. 8'-COOH), which can form retinoic acid. Retinol is esterified, incorporated in chylomicrons together with some intact P-carotene, and secreted into lymph. Retinoic acid enters portal blood accompanied by other polar metabolites. Reprinted from Biochimica et Biophysica Acta, Vol. 486, Sharma et al., Studies on the metabolism of P-carotene and apo-P-carotenoids in rats and chickens, pp. 183-94, © 1977, with permission from Elsevier.

Fig. 7.4 Intestinal metabolism of P-carotene. The enzyme P-carotenoid-15,15'-dioxygenase forms retinaldehyde directly. Cleavage at other double bonds forms P-apocarotenals (e.g. 8'-CHO), which can be shortened to retinaldehyde. P-Apocarotenals may be oxidized to P-apocarotenoic acids (e.g. 8'-COOH), which can form retinoic acid. Retinol is esterified, incorporated in chylomicrons together with some intact P-carotene, and secreted into lymph. Retinoic acid enters portal blood accompanied by other polar metabolites. Reprinted from Biochimica et Biophysica Acta, Vol. 486, Sharma et al., Studies on the metabolism of P-carotene and apo-P-carotenoids in rats and chickens, pp. 183-94, © 1977, with permission from Elsevier.

dehyde in the case of other provitamin carotenoids). However, the reaction fails to produce the theoretical amount of retinoids in vivo because of incomplete absorption of P-carotene from the intestinal lumen and (in humans) inefficient conversion in the mucosa. Metabolism of carotenoids to retinoic acid as a result of excentric cleavage may account for some of the discrepancy in humans.

In the excentric cleavage of P-carotene described by Glover (1960) one molecule of P-carotene ultimately yields one molecule of retinaldehyde. The initial reaction is cleavage of the terminal 7'-8' double bond to produce P-apo-8'-carotenal (Fig. 7.5). The stepwise degradation of this compound is postulated to take place by a P-oxidative-type enzyme system. All of the P-apocarotenals formed from P-carotene can be shortened to retinaldehyde.

There is good evidence for the existence of both central and excentric cleavage of carotenoids (Wolf, 1995). The enzyme responsible for central cleavage, P-carotenoid-15,15'-dioxygenase, is found in both intestine and liver. However, because of lability during attempts to purify it, the pure enzyme has not yet been isolated. Bile salts have been found to be essential for P-carotene cleavage. Enzyme(s) seem to be involved in excentric cleavage: amounts of P-apocarotenals and retinoids were markedly reduced when NAD+ was replaced by NADH and their formation was completely inhibited by an inhibitor of sulphydryl-containing enzymes (Wang et al., 1991). However, no enzyme(s) specifically responsible for excentric cleavage has yet been found. It is not known whether different specific dioxygenases cleave the different double bonds of the polyene chain or whether the P-carotenoid-15,15'-di-oxygenase is rather non-specific and can attack other double bonds also.

Most of the retinaldehyde formed from carotenoids becomes bound to CRBP-II and reversibly reduced to retinol by retinaldehyde reductase - a relatively nonspecific aldehyde reductase which does not appear to be zinc-dependent (Fidge & Goodman, 1968). The resulting retinol-CRBP-II complex is then used as a substrate for esterification by LRAT.

The bioconversion of provitamins to retinoids may be regulated both up and down at the level of the intestinal cleavage enzyme. Using a dioxygenase assay, van Vliet et al. (1992) found a 130% higher cleavage activity in hamsters fed a low vitamin A diet compared with normally fed controls. This up-regulation was confirmed in rats by van Vliet et al. (1996) who also found that a high intake of either retinyl ester or P-carotene down-regulated (decreased) cleavage activity.

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