International Research Conference on Food, Nutrition, and Cancer

New Aspects in Vitamin A Metabolism: the Role of Retinyl Esters as Systemic and Local Sources for Retinol in Mucous Epithelia1
Hans K. Biesalski2 and Donatus Nohr
Department of Biological Chemistry and Nutrition, University of Hohenheim, Stuttgart, FRG
ABSTRACT Vitamin A and its active metabolites are important for growth and differentiation of a variety of cells, mainly in mucosa-associated epithelia, where they exhibit a wide spectrum of activities. Vitamin A, stored as retinyl esters (REs), is delivered from liver stores into the bloodstream as retinol bound to retinol binding protein. This process is regulated homeostatically, ending up in a more or less constant plasma retinol level. In situations of a high vitamin A demand (e.g., inammation, diseases, prenatal period), this supply can be insufcient because of delayed production of retinol binding protein, leading to local deciencies and impairment of structure and function in the respective tissues. This delay may be overcome by cellular RE stores. Several cell types, including buccal mucosa cells, can take up RE. Retinyl palmitate is taken up when it is applied topically to either metaplastically mutated rat vaginal epithelium (as a gel) or to human meta- and dysplastic bronchial epithelia (via inhalation) that have a vitamin A deciency. In rats and humans, the modied epithelia can be normalized, at least in part. In conclusion, topically applied retinyl esters may be a promising therapy for local retinol deciencies and may reverse the morphological alterations of the epithelium in tissues that are vitamin A decient. J. Nutr. 134: 3453S3457S, 2004. KEY WORDS:

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Vitamin A metabolism

lung cancer prevention

mucous epithelia

lung development

In this article we review data showing that the delivery of retinol (ROH)3 via the bloodstream to target cells is not the only way to ensure a sufcient cellular supply of ROH. Vitamin A is stored in target tissues as retinyl esters (REs), which serve as an additional source of the vitamin. The steady-state ROH plasma level is sufcient to ensure cellular vitamin A supply under normal conditions, but in a disease state with enhanced demand, adjustment may be primarily achieved via local tissue-specic RE stores. Uptake and distribution of vitamin A Vitamin A and its active metabolites are important for growth and differentiation of a variety of cells, mainly in mucosa-associated epithelia, where they exhibit a wide spectrum of activities (1). Vitamin A is taken up by mammals
1 Published in a supplement to The Journal of Nutrition. Presented as part of the International Research Conference on Food, Nutrition, and Cancer held in Washington, DC, July 1516, 2004. This conference was organized by the American Institute for Cancer Research and the World Cancer Research Fund International and sponsored by BASF Aktiengesellschaft; Campbell Soup Company; The Cranberry Institute; Danisco USA Inc.; DSM Nutritional Products, Inc.; Hills Pet Nutrition, Inc.; Kellogg Company; National Fisheries Institute; The Solae Company; and United Soybean Board. An educational grant was provided by The Mushroom Council. Guest editors for this symposium were Helen A. Norman, Vay Liang W. Go, and Ritva R. Butrum. 2 To whom correspondence should be addressed. E-mail: biesal@uni-hohenheim.de. 3 Abbreviations used: BPD, bronchopulmonary dysplasia; COPD, chronic obstructive pulmonary disease; LRAT, lecithin:retinol-acyltransferase; RA, retinoic acid; RBP, retinol-binding protein; RE, retinyl ester; ROH, retinol; RP, retinyl palmitate; TTR, transthyretin.

either as RE from meat sources or provitamins from plants. It is generally accepted that under physiological conditions, RE is hydrolyzed to ROH, taken up by the intestinal mucosa, reesteried into RE, coupled to chylomicrons, and released via the thoracic duct to the blood stream. The chylomicron remnants containing most RE are taken up by the liver, mediated by the apolipoprotein E receptor. RE is stored in the liver and forms ROH after hydrolysis. The liver thus forms the biggest vitamin A pool (50 80%) of the organism. Serum vitamin A levels are normally kept constant through homeostatic regulation from the liver stores. ROH has to be bound intrahepatically to retinol-binding protein (RBP) before being released and further coupled to transthyretin (TTR) to avoid renal clearance. Cellular uptake of ROH may occur either mediated by a proposed holo-RBP receptor (e.g., on retinal pigment epithelial cells) with consecutive internalization of the complex by endocytosis and release of ROH into the cytosol or via a receptor-independent mechanism of lipid membranes. Both mechanisms seem to operate with different cells and under different conditions. In the cell, ROH is bound to cellular RBP, oxidized to retinoic acid (RA), or reesteried to RE. RE is stored in various tissues in addition to the liver (Table 1). It is argued that the RE stores in extrahepatic tissues are mainly formed after intracellular esterication of ROH derived from the bloodstream (1). In addition, no mechanism for converting RE to RA, or vice versa, is known, indicating that RE has to be rst hydrolyzed into ROH before being oxidized to RA. As an alternative, orally provided RA can be taken up directly

0022-3166/04 $8.00 2004 American Society for Nutritional Sciences.

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TABLE 1
Distribution of retinyl esters in biological samples of guinea pig
Concentration of retinyl ester Sample Retinyl palmitate Retinyl stearate Retinyl oleate Retinyl palmitoleate Retinyl linoleate

g/g dry weight

Liver Kidney Testicle Epididymis Spermatic cord Trachea Lung Nasal mucosa Tongue Pigment epithelium Organ of corti 105.3 31.5 1.31 0.65 5.72 4.2 2.35 0.94 3.90 1.55 0.18 0.11 0.42 0.31 0.58 0.40 1.81 0.68 21.92 6.35 4.80 0.3 45.6 9.2 0.72 0.3 2.88 1.95 0.81 0.44 1.15 0.37 0.13 0.07 0.23 0.11 0.11 0.10 0.97 0.46 7.89 2.1 1.60 0.1 12.7 2.8 0.11 0.08 0.71 0.43 0.27 0.11 0.31 0.2 0.11 0.05 0.19 0.12 0.14 0.08 0.28 0.21 1.10 0.32 0.80 0.4 4.8 0.9 0.02 0.01 0.06 0.02 n.d. n.d. n.d. n.d. n.d. 0.08 0.06 0.08 0.05 n.d. 16.7 5.5 0.15 0.06 1.22 0.75 0.72 0.51 1.95 0.69 0.09 0.04 0.21 0.09 0.17 0.11 0.14 0.11 0.91 0.37 0.04 0.01

n.d. not determined [data from (2)]. Downloaded from jn.nutrition.org by guest on July 5, 2012

by the cells and bound to cellular RA-binding protein. RA adsorbed to albumin can pass through the membrane (3) and bind to the nuclear RA receptors RAR and RXR or their 30 known subtypes to exert its specic effects. The role of intracellular RE as a source for ROH and RA independent from ROH delivery from the blood is not really known. Intracellular concentrations of ROH and RA regulate the metabolic pathway from RE, leading to a controlled intracellular concentration of RA. Regarding the strict control of RA formation, the concentration of RE is metabolically inert, which enables the cell to store high concentrations of vitamin A without affecting regulatory steps and cellular vitamin A activity. We assume that 1) REs within target tissues are important short-term stores in case demand suddenly increases (e.g., under inammatory conditions ROH delivery can become inadequate because of a delay or impairment of RBP synthesis) and 2) part of the RE in cells is derived from circulating chylomicrons or from LDL. As a consequence, the supply of RE to target cells (either via chylomicrons or topical application) may be benecial when increased demand causes local vitamin A deciency. Importance of cellular retinyl esters as sources for ROH and RA ROH bound to RBP and TTR is released from the liver into the bloodstream under strict homeostatic control. This control is mainly due to a continuous but limited RBP synthesis and transfer of ROH to the RBP binding site. After delivering ROH to the target cells, the remaining RBP without retinol (apo-RBP) is degraded in the kidney. An increase of circulating apo-RBP is a sign of higher cellular demand and in renal dysfunction (impaired catabolism of apo-RBP) will stimulate RBP synthesis and as a consequence lead to an increase in the circulation of the ROH-RBP-TTR complex (4). With a sudden increased demand of ROH, however, an increase of ROHRBP does not appear within minutes. The delay is due to the process of RBP expression and may be up to 24 h after the increased demand. When vitamin A deciency begins with decreasing ROH blood levels but not in the normal vitamin A statesupplementation with 3000 IU vitamin A results in a sudden increase (within 5 h) of the ROH in blood. This increase

occurs because of storage of RBP in the liver that does not occur under normal conditions (5). Reduced ROH plasma levels often occur during infections and are counted among the main complications of a poor vitamin A supply in developing countries. In addition, the serum vitamin A level continues to drop during infectious diseases (e.g., measles and, particularly, diseases of the respiratory tract). In RBP knockout mice, RE levels of the lung need 5 wk to be depleted (data not shown). This can be explained by the increased metabolic demand, which is not adjusted for reasons mentioned above, or an increased and perhaps additional renal elimination of ROH and of RBP during acute infections (6). If ROH blood levels drop, this cellular delivery gap might be compensated via RE in target tissues. Depending on the concentration of RE in the tissues, specically in the lung, the demand may be covered for a couple of days. A good example is children with measles, where ROH in the blood is decreased. Children with a low vitamin A intake have a higher risk for respiratory tract infections than do children with sufcient vitamin A intake (7). The above-mentioned hypothesis that REs are important sources for vitamin A delivery especially during increased demand is well documented during lung development (8,9). Importance of REs in the respiratory epithelium A disease discussed in connection with vitamin A supply is bronchopulmonary dysplasia (BPD) in preterm children. The pathogenesis of BPD depends on a multitude of factors. Some of the observed morphological changes are very similar to those seen in vitamin A deciency in humans and animals. In particular, there is focal loss of ciliated cells with keratinizing metaplasia, necrosis of the bronchial mucosa, and an increase of mucous secreting cells (Figs. 1 and 2). This alteration is similar to changes found during vitamin A deciency. Vitamin A deciency leads to typical signs and symptoms, starting with impaired dark adaptation and the development of xerophthalmia leading to blindness in 5 million children each year worldwide. Long before these clinical signs appear, an increased incidence of respiratory tract infections becomes evident; these infections are the main reason for the high mortality (10). The underlying mechanism, besides an impair-

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FIGURE 1 High resolution of respiratory epithelium under vitamin A deciency. Ciliated cells that usually cover the whole surface are replaced by secretory goblet cells.

FIGURE 3 Concentration of retinyl esters in pre- and postnatal lungs of the rat. [Modied from (10).]

ment of the immune system, is a typical alteration of the respiratory epithelium (Fig. 1). Loss of ciliated cells and an increase of mucous-secreting cells favor the adherence of bacteria and, as a consequence, infections of the respiratory tract. Focal keratinizing metaplasia as occurs in vitamin A deciency strengthens the assumption of impairment of the differentiation occurring at the level of the gene expression. Because vitamin A regulates the expression of different cytokeratins and therefore inuences terminal differentiation, it seems obvious to suppose common mechanisms for both. However if the vitamin A status of the mother is poor, the risk for BPD increases (11). The neonate, and especially the premature neonate, depends on a sufcient supply of vitamin A to ensure regulation of the cellular differentiation of the respiratory and lung epithelium during maturation. The developing lung, especially late in maturation just before birth, is a good example of sudden increased demand and the importance of an

intracellular RE source. RE levels in the fetal rat lung peak at embryonic day 17 and then decrease until birth (Fig. 3). The parallel increase of ROH levels indicates that the obvious high vitamin A demand during that period cannot be fullled from fetal liver stores but has to be delivered from lung intrinsic RE stores (Fig. 4). Postnatally, RE levels start to increase again, ensuring a continuous supply of ROH and RA. The earlier a child is born before the due date, the lower its serum ROH levels are. Because a further decrease of the serum ROH and RBP levels occurs postnatally, the plasma value at the time of birth is considered to be a critical value for lung development, especially if RE levels in the lung are low. Serum ROH and RBP levels in premature infants are signicantly lower than in full-term neonates (12). The liver of a premature neonate might not sufciently synthesize RBP and as a consequence the increased demand of tissues, especially the lung, might not be fullled. Consequently, lung RE storage sites that are formed before delivery are of great importance for late lung development. If the site concentrations are low, they may not sufciently supply the maturing cell with ROH and RA, the latter of which controls the expression of several genes involved in differentiation and function (surfactant proteins, laminin, midkine genes, etc.). In a study in rats, Masuyama et al. (8) showed that an ROH deciency in the mothers for 28 d had several consequences in

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FIGURE 2 Amount of ciliated and goblet cells in the tracheal and bronchial epithelium of guinea pigs without (black bars) and with (gray bars) vitamin A deciency.

FIGURE 4 Concentration of retinyl palmitate (left) and retinol (right) in the lung of rats from the last days of pregnancy to postnatal day 1. [Modied from (9).]

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the fetal rats: body and lung weights were reduced and concentrations of retinyl palmitate (RP) and phosphatidylcholine were lower in the lungs. The RA receptors were also affected; the expression of RAR was increased and that of RAR was decreased (favoring AP1 activation) whereas RAR was left unchanged. Importance of vitamin A for the mature lung and the risk for lung cancer Chronic obstructive pulmonary disease (COPD) is a disease of the lung found almost exclusively in smokers. The disease is characterized by a progressive replacement of the normal respiratory epithelium with goblet cells and ciliated cells by a squamous metaplastic epithelium comparable with that seen in BPD due to vitamin A deciency (Fig. 5) and possibly induced by the inhaled components of cigarette smoke. As a consequence, the mucociliary clearance becomes more reduced, leading to increased exposure of the local epithelium to the cigarette smoke, resulting in a vicious cycle. The risk for COPD increases with decreasing vitamin A serum levels (13,14); daily oral doses of 25,000 IE vitamin A for 30 d attenuated symptoms (15). Although smokers with COPD do not have systemic vitamin A deciency, a local deciency occurs as a result of exposure to cigarette smoke; the immune response subsequent to exposure is assumed to be one reason for the deciency. This deciency will nally lead to lung cancer via COPD-associated metaplasia and dysplasia. Epidemiologic studies can show that the relative risk for lung cancer is signicantly increased in smokers with COPD as compared with nonsmokers. The Second National Health and Nutrition Examination Survey (16) documents that the relative risk of smokers to develop COPD is strongly and dosedependently decreased by the intake of vitamin A. Thus, low vitamin intake in smokers may lead to COPD and thus increase the risk for lung cancer. If, as speculated for COPD, areas of local vitamin A deciencies exist in the tracheobronchial tract, this might lead to a disturbance of differentiation and at least to a formation of metaplasia, which are frequently found in COPD. Biesalski and Stofft (17) showed that, despite nearly normal serum vitamin A levels in smokers, concentrations of ROH as well as of REs were strongly reduced in metaplastic as compared with normal epithelia of the same COPD patients. This reduction indicates that local vitamin A deciencies in the lung (and other organs) with all their consequences may develop much earlier than can be determined by routinely measuring serum vitamin A levels. Local deciencies in COPD of smokers may be due to the exposure of the epithelium to irritants contained in cigarette smoke. Data from our laboratory from studies with human bronchial epithelial cells indicate that benzo[a]pyrene increases the activity of RP hydrolase, resulting in an increase of cellular ROH (Fig. 6), and decreases the activity of lecithin:retinol-acyltransferase (LRAT), leading to an impaired production of RP from

FIGURE 6 Activity assay for retinyl palmitate hydrolase (RPH) in normal human bronchial epithelial cells that were untreated (left bar), treated with benzo(a)pyrene (BaP; middle bar), or BaP and phytomenadione (RPH-antagonist). 1U 2500 pmol ROH mg protein1 min1. [Data from (18).]

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ROH (18). As a consequence, ROH accumulates and CYP26, the RA metabolizing enzyme, is activated, resulting in a continuous depletion of cell RE accompanied by an increased turnover of ROH and RA. Finally, a cellular vitamin A decit may result from the enhanced metabolism of RA to polar metabolites and their subsequent excretion. In conclusion, local vitamin A deciency may occur as a result of continuous contamination with cigarette smoke condensate. This exposure disturbs differentiation and leads to formation of a squamous phenotype in the respiratory epithelium. If local depletion of RE leads to an impairment of cellular vitamin A metabolism, which at least may result in a downregulation of RAR (8), this may lead to neoplastic transformation. In kidney cancer cells, a local vitamin A deciency was accompanied by a downregulation of LRAT and RAR (19,20). If REs, as discussed above, are indeed important sources for the epithelia of the tracheobronchial tract, a delivery of RE either from high oral doses or a topical application should result in a cellular accumulation that might help to overcome such local deciencies and reverse the squamous phenotype to a normal epithelium. Such a reversal was frequently documented in trachea explants from vitamin A decient hamsters (21). Topical application of REs To investigate whether RE can be taken up by epithelial cells, we performed a controlled study with healthy young volunteers using RP-enriched toothpaste for 8 wk. After 7 d a signicant uptake of RP into the buccal mucosa cells (analyzed by HPLC) was accompanied by a slight increase of intracellular ROH (22). The question arose whether the vitamin A taken up by epithelial cells also has a functional consequence. This can be demonstrated in a model of vitamin A decient ovariectomized rats subsequently expressing continuous and estrus phaseindependent squamous metaplastic changes in the vaginal epithelium. Topical application of vitamin A in a gel twice per day for 2 d reversed the metaplasia to an almost normal vaginal epithelium, indicating a normalization of the vitamin A metabolism including the regulation of differentiation and proliferation of the cells. This leads

FIGURE 5 Changes of the respiratory epithelium in various stages of vitamin A (retinoic acid: RA) deciency. Most of the effects are reversible by adequate administration of vitamin A. GC: goblet cells; CC: ciliated cells.

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to a rebuilding of the impaired epithelial barrier of the vaginal mucosa. In addition, this shows that the relative short topical application of RE results in the formation of a sufcient cellular vitamin A source lasting for a couple of days. The permanence of the benecial effect was signicantly dependent on the dose applied and the duration of the treatment (23). This nding may have important clinical consequences, as a functioning epithelial barrier not only in the vagina is a prerequisite for the best possible circumvention of intruding viruses or other antigens, as documented in HIV infection (24). Inhalation of vitamin A To further evaluate whether topical application of vitamin A helps to induce a normal phenotype in cases of squamous metaplasia in COPD patients, we carried out a pilot study with 11 subjects including 9 smokers and 2 exsmokers (25). Inhaling retinyl palmitate (18,000 IU/d) for 3 mo led to a remission of the metaplasia in 44% and to a partial remission in 12% of patients, whereas the metaplasias remained unaffected in 24% of the other patients and showed a further progression in 18%. Conclusions In conclusion, RE serves as an important cellular pool for vitamin A. The formation of the pool depends on the ROH demand of the cell, the delivery of ROH from the bloodstream, the activity of the esterifying enzyme LRAT (for unused ROH), and the delivery of RE from chylomicrons (depending on the postprandial load). This RE pool does not interfere with the controlled RA formation and is consequently an inert storage site that delivers ROH and at least retinoic acid (Fig. 7). Delivery depends on the activity and regulation of the hydrolyzing enzyme that is controlled via cellular demand of ROH and RA. The topical application of RE via direct administration (gel or aerosol) to mucous membranes results in an accumulation of RE in target cells. This accumulation contributes to the cellular delivery of ROH and RA, which helps to overcome the distur-

bance of cellular differentiation when there is a local deciency. Topical application may be of special importance for cigarette smokeinduced metaplasias, which can be treated with inhaled RE LITERATURE CITED
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M., Li, Z., Heber, D., Liu, C. & Livingston, E. H. (2002) Vitamin A deciency in a newborn resulting from maternal hypovitaminosis A after biliopancreatic diversion for the treatment of morbid obesity. Am. J. Clin. Nutr. 76: 426 429. 12. Ambalavanan, N., Wu, T. J., Tyson, J. E., Kennedy, K. A., Roane, C. & Carlo, W. A. (2003) Comparison of three vitamin A dosing regimens in extremely-low-birth-weight infants. J. Pediatr. 142: 656 61. 13. Morabia, A., Sorenson, A., Kumanyika, S. K. & Abbey, H. (1989) Vitamin A, cigarette smoking and airway obstruction. Am. Rev. Resp. Dis. 140: 13121316. 14. Morabia, A., Menkes, M. J., Comstock, G. W. & Tockman, M. S. (1990) Serum retinol and airway obstruction. Am. J. Epidemiol. 132: 77 82. 15. Lorenz, J. & Biesalski, H. K. (1993) Vitamin A-Mangel und Bronchialkarzinom: Perspektiven der Chemoprevention bronchialer Tumoren. Pneumologie 47(12): 657 665. 16. NHANES II (1991) Plan and Operation of the Second National Health and Nutrition Examination Survey 1976 80. http://www.cdc.gov/nchs/data/series/sr_01/sr01_015.pdf [accessed October 1, 2004]. 17. Biesalski, H. K. & Stofft, E. (1992) Biochemical, morphological, and functional aspects of systemic and local vitamin A deciency in the respiratory tract. Ann. N.Y. Acad. Sci. 669: 325331. 18. Sobeck, U. (2003) Thesis. University of Hohenheim, Stuttgart, FRG. 19. Guo, X., Nanus, D. M., Ruiz, A., Rando, R. R., Bok, D. & Gudas, L. J. (2001) Reduced levels of retinyl esters and vitamin A in human renal cancers. Cancer Res. 61: 2774 2781. 20. Gudas, L. J. (2004) Aberrant vitamin A metabolism in cancer cells. J. Nutr. 134: 0000S 0000S. 21. Zhang, X. M. & McDowell, E. M. (1992) Vitamin A deciency and inammation: the pivotal role of secretory cells in the development of atrophic, hyperplastic and metaplastic change in the tracheal epithelium in vivo. Virchows Arch. B Cell Pathol. Incl. Mol. 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FIGURE 7 Interaction between the liver and peripheral tissues and their interchange of ROH. We propose an alternative mechanism to the normal supply of the target tissue with ROH bound to RBP via the bloodstream from liver stores. RE is supplied either by chylomicrons via the bloodstream or by topical application of RE directly to the respective tissue (e.g., vaginal or respiratory epithelium). This RE pool does not interfere with the controlled RA formation and is consequently an inert storage site that delivers retinol and at least retinoic acid. [Modied from (26).]