Ординатура / Офтальмология / Английские материалы / Handbook of Nutrition and Ophthalmology_Semba_2007

Fig. 4. Structure of carotenoids and vitamin A.

3.2.2. DIGESTION AND ABSORPTION OF VITAMIN A IN FOODS

In the stomach, foods containing vitamin A and carotenoids undergo proteolysis and are released from protein. The presence of fat in the small intestine stimulates the secretion of cholecystokinin, which stimulates the secretion of bile from the gall bladder and the secretion of pancreatic enzymes. Retinyl esters and carotenoids are insoluble in water and within the small intestine are solubilized within bile acid micelles. The presence of fat in the small intestine improves the absorption of retinol and carotenoids by increasing the size and stability of the micelles. In the gut lumen, dietary retinyl esters are hydrolyzed to retinol (138) and then undergo reesterification during absorption (139). The pancreatic enzyme, pancreatic triglyceride lipase, plays an important role in hydrolysis of retinyl esters (140). The brush border membrane in the gut also has hydrolytic activity (141,142) that has been attributed to intestinal phospholipase B (143). Carboxylester lipase was usually considered to be involved in hydrolysis of retinyl esters, but recent work in the carboxylester lipase knockout mouse shows that dietary retinyl ester digestion is not affected by absence of this pancreatic enzyme (144).

Retinol is absorbed in the intestine by both a saturable, carrier-mediated process and a nonsaturable, simple passive diffusion process (145–147). It has been hypothesized that a retinol transporter, not yet characterized, exists on the luminal side of the enterocyte (148). In the cytoplasm of the enterocyte, free retinol appears to be sequestered by cellular retinol-binding protein (CRBP) II, a 16-kDa polypeptide with a single retinoid binding site (149). CRBP II will bind all-trans-retinol, 13-cis-retinol, and all-trans-retinal, but not other cis isomers of retinol (150). CRBP II is found in high concentrations in the small intestine (151,152), and upregulation of CRBP II mRNA occurs during pregnancy and lactation (153) and during retinoid deficiency in the rat model (154). CRBP I is also present in enterocytes, but at a much lower concentration than CRBP II (155).

The uptake of β-carotene by enterocytes appears to occur by simple passive diffusion (156), and a saturable, carrier-mediated process has also been proposed (150). Within the enterocyte, β-carotene is either absorbed intact and passes into the portal circulation, undergoes enzymatic cleavage by β-carotene 15,15'-dioxygenase to retinal (157–160), or

is converted to retinoic acid (161,162). β-carotene can be cleaved centrally via β-carotene 15,15'-dioxygenase or may undergo excentric cleavage in which β-apocarotenals are produced (163–165). In the rat model, central cleavage appears to be the dominant form of oxidation of β-carotene, although small amounts of β-apocarotenals (8', 10', 12', and 14') were detected (166). In the presence of oxidative stress, β-carotene 15,15'-dioxygenase may be downregulated with more formation of excentric cleavage products (167,168). Retinal is reduced to retinol by retinal reductase activity that is present in microsomes (169).

The two sources of retinol in the enterocyte (retinol that is absorbed directly by the enterocyte and retinol generated through cleavage of β-carotene and subsequent reduction of retinal to retinol) have the same metabolic pathway of esterification with longchain fatty acids and release into the lymphatic circulation. In the enterocytes, lecithinretinol acyltransferase (LRAT) and acyl-CoA-retinol acyltransferase (ARAT) are thought to be involved in the esterification of retinol, with retinyl esters formed by LRAT targeted for secretion in chylomicrons and retinyl esters formed by ARAT targeted for storage (148). The four main retinyl esters are produced in a proportion of about 8:4:2:1 for retinyl palmitate, retinyl stearate, retinyl oleate, and retinyl linoleate (170,171). The amount and type of fatty acids ingested with preformed vitamin A in the diet can modulate the pattern of retinyl esters that are secreted in chylomicrons (172). Chylomicrons are spherical lipoprotein particles of 75–450 nm in diameter that are composed of triacylglycerols, unesterified and esterified cholesterol, phospholipids, apolipoproteins, unesterified and esterified retinol, carotenoids, and other fat-soluble vitamins. The assembly of chylomicrons occurs in the endoplasmic reticulum and requires apoB48, microsomal triglyceride transfer protein, phospholipids, and triglycerides (173,174). The secretion of retinyl esters is dependent on the secretion of chylomicrons and is one of the last steps in the molecular assembly of chylomicrons (175). Intraluminal factors such as pH, bile, and fatty acids modulate the secretion of retinyl esters into the lymphatic and portal circulation (176). Chylomicrons are secreted by exocytosis from the basolateral surface of the enterocyte into the mesenteric and then thoracic duct lymphatic circulation and on into the general circulation.

3.2.3. UPTAKE AND STORAGE OF VITAMIN A IN THE LIVER AND OTHER TISSUES

In the general circulation, chylomicrons are converted to chylomicron remnants in a process that involves both the hydrolysis of the chylomicron triacylglycerols by lipoprotein lipase, an enzyme located on the luminal surface of capillary endothelial cells (177), and the transfer of apolipoproteins, phospholipids, and carotenoids to other lipoproteins or cell membranes. Most of the retinyl esters contained in the chylomicrons do not transfer to other lipoproteins and are largely cleared by the liver (178,179). Other tissues take up retinyl esters, such as the mammary gland during lactation (180–182), muscle, adipose tissue, and kidneys (183), lung (184), and blood leukocytes (185). Chylomicron remnants are rapidly removed from the circulation by the liver. In normal healthy humans, it is generally thought that the liver contains about 90% of the body stores of vitamin A; however, the distribution of vitamin A stores during deficiency and disease states is unclear (186). The liver absorbs most of the retinyl esters in chylomicrons (187,188). Chylomicron remnants are removed by the liver between the endothelial cells and hepatocytes within the space of Disse (189). The chylomicron remnants bind to receptors on the hepa-

tocytes such as low-density lipoprotein (LDL) receptor and apo E (190), or chylomicron remnant receptor (189).

Retinyl esters are hydrolyzed by retinyl ester hydrolase (191), and within the hepatocyte, retinol is found within endosomes (192). Retinol is transferred to the endoplasmic reticulum, where it binds to retinol-binding protein (RBP). The retinol and RBP complex is translocated to the Golgi complex and then secreted from the hepatocyte (186). Retinol is transferred from hepatocytes to stellate cells (193), and most of the vitamin A stored within the liver is found in stellate cells (194–196), where it is stored as retinyl esters in lipid droplets (186). Some retinol bound to RBP is not taken up by stellate cells and enters the blood (186). Circulating retinol may leave and enter the liver many times in a process known as retinol recycling (197). Two enzymes, LRAT and CRAT, can esterify retinol in stellate cells, of which LRAT is considered the more important enzyme for retinol esterification (198). In vitamin A-deficient rats, LRAT activity is low in the liver but is rapidly upregulated after an oral dose of retinol, suggesting that low LRAT activity may be a mechanism by which retinol is made available for secretion to plasma and delivery to target tissues during a state of vitamin A deficiency (198,199).

Carotenoids are also delivered to tissues via chylomicrons, and carotenoids accumulate in high concentrations in adipose tissue. Higher doses of β-carotene were associated with higher serum concentrations of all-trans retinoic acid in rabbits (200). β-carotene can be converted to all-trans retinoic acid in the intestine, testes, lung, and kidney without retinol as an obligatory intermediate (162,201). The processing of carotenoids from chylomicron remnants by the liver has not been well characterized.

3.2.4. VITAMIN A IN THE CIRCULATION

The major form of circulating retinol in the blood is as retinol bound to RBP (202), and about 95% of plasma RBP is associated with transthyretin (TTR) in a one-to-one molar ratio. RBP is a single-polypeptide chain with a molecular weight of about 21,000 and a binding site for one molecular of all-trans retinol (203). In addition to retinol, other forms of retinoids found in plasma include all-trans retinoic acid (204), 13-cis-retinoic acid, 13- cis-4-oxoretinoic acid (205,206), all-trans retinyl β-glucuronide, and all-trans retinoyl β- glucuronide (207). The relative concentrations of various retinoids in human plasma are retinol (1–2.5 μmol/L), retinyl esters (0.03–0.3 μmol/L), retinoic acid (0.003–0.03 μmol/ L) and retinyl β-glucuronide (0.002–0.01 μmol/L) (207,208). Other retinoids such as 9- cis-retinoic acid, 9,13-di-cis-retinoic acid, and 14-hydroxy-4,14-retro-retinol have been described in human plasma after consumption of liver (209). Retinoic acid also circulates bound to albumin (210). Some insight into the importance of RBP transport of retinol has come from a study of two siblings who had two point mutations in the RBP gene, and normal growth and development but extremely low concentrations of plasma retinol, plasma RBP, and night blindness (211). These observations suggest that retinyl esters in chylomicrons, retinoic acid bound to albumin, and other circulating forms of vitamin A may provide a sufficient supply of vitamin A to most tissues except the retina (211).

Retinol is released from the liver into the circulation in the form of holo-RBP (all-trans retinol bound with RBP in a 1:1 molecular complex). Under normal circumstances, plasma retinol concentrations are usually maintained at what is considered a normal homeostatic level, or “set-point” for individuals (212). This level may vary from individual to individual, but is usually in a concentration of about 1–3 μmol/L in healthy adults. Plasma retinol

concentrations will decrease when hepatic retinol stores become inadequate, and this usually occurs when there is insufficient dietary intake of vitamin A for a prolonged period. Plasma retinol concentrations will increase to high levels above the normal range when vitamin A intake is excessive. Various disease states can decrease plasma retinol concentrations, including protein-energy malnutrition (213,214), hypothyroidism (215), zinc status (216), and inflammation and infection (217), or increase plasma retinol concentrations, such as renal disease (218).

Retinol is recycled between the liver and peripheral tissues, as shown by kinetic studies (219). In rats, about 90% of retinol that left the circulation was recycled and not irreversibly metabolized (219). The recycling of retinol appears to be tightly regulated and dependent on the amount of hepatic vitamin A, and extrahepatic tissues contribute a large proportion to the retinol that circulates in plasma (219,220). The half-life of holoRBP is about 12 h (221). As noted earlier, a large portion of dietary carotenoids pass into the circulation intact within chylomicrons. It is estimated that there are more than 40 dietary carotenoids that may be absorbed, metabolized, and/or utilized by the human body (222). The main dietary carotenoids found in human plasma include α-carotene, β-caro- tene, β-cryptoxanthin, lutein, zeaxanthin, and lycopene, but other carotenoids and their oxidation products have been identified in human plasma (223). Most carotenoids in the plasma are transported by LDLs, and carotenoids can be delivered to peripheral cells that express the LDL receptor (224).

3.2.5. VITAMIN A UPTAKE BY TISSUES

Retinol is taken up by peripheral tissues in a processs that has not been well characterized. Some cells, such as retinal pigment epithelial cells and parenchymal and stellate cells of the liver, have specific surface receptors for RBP (225). Circulating all-trans retinoic acid is taken up by cells, as it is able to move through cell membranes in an uncharged state and enter cells. In steady-state tracer studies in rats, the percentage of all- trans retinoic acid derived from the plasma was high for the liver and brain, but lower for the spleen and eyes (226). All-trans retinoic acid may act on cells through retinoylation, or the acylation of all-trans retinoic acid by protein (227–229). All-trans retinoic acid may be incorporated into proteins of cells through posttranslation modification in which a retinoyl-CoA intermediate is formed and is followed by the transfer and covalent binding of the retinoyl moiety to protein (230).

3.3. Retinoic Acid Receptors and Gene Regulation

Vitamin A exerts its effects via retinoic acid and retinoid receptors, which are found in the nucleus of the cell. Retinol is converted to all-trans-retinoic acid and 9-cis retinoic acid in the cytoplasm. Retinoic acid influences gene activation through specific receptors which belong to the superfamily of thyroid and steroid receptors (231). Retinoic acid receptors (RARs) act as transcriptional activators for many specific target genes. The RAR is expressed as isoforms, referred to as RAR α, β, and γ, and retinoid-x receptor (RXR) is also expressed as isoforms, referred to as RXR α, β, and γ (232). All-trans retinoic acid is a ligand for RARs, whereas 9-cis retinoic acid is a ligand for both RARs and RXRs. 9-cis-retinoic acid is functionally distinct from all-trans-retinoic acid, and inter-conver- sion may exist between the two isomers. Each RAR and RXR has a specific DNA-binding domain by which these nuclear receptors may effect transcriptional activity.

The DNA sequences which interact with RAR and RXR are known as retinoic acid response elements (RAREs). RAR and RXR receptors form heterodimers, which bind to DNA and control gene expression. In addition, RXR receptors also can form heterodimers with the thyroid hormone receptor, vitamin D3 receptor, peroxisome proliferator-activated receptors, and a number of “orphan receptors.” Most RAREs seem to occur in the regulatory region of genes. In the presence of 9-cis retinoic acid, RXR/RXR homodimers may form and recognize a subset of RAREs or inhibit the formation of certain heterodimers. Orphan receptors such as chicken ovalbumin up-stream promoter transcription factor (COUP-TF) (233), ARP-1, TAK1 (234), RVR (235), RZR (236), and thymus orphan receptor (TOR) (237) may repress or modulate the induction of genes by retinoic acid. The three-dimensional structures of several different DNA-binding complexes of RXR have been elucidated (238–240). RARs and RXRs may interact with multiple transcriptional mediators and/or corepressors, adding an enormous level of complexity to regulation of retinoic acid responses. Other vitamin A metabolites in the retroretinoid family may support biological functions via a pathway that is distinct from the retinoic acid pathway. 14-hydroxy-4,14-retro retinol supports whereas anhydroretinol inhibits cell growth (241,242). In addition, the oxoretinoids may play a role as RAR ligands (243).

Recently described nuclear receptor-associated proteins, such as SMRT/N-CoR, Sin3, and histone deacetylases (HDACs)-1 and -2 and histone acetylases (CBP/p300 and P/ CAF), appear to function as corepressors and coactivators of transcription. A binary paradigm has emerged as an attempt to explain how these proteins work. Unligated receptors bind to response elements of target genes and repress transcription through recruitment of a repressor complex containing corepressors, and transcription is repressed (244,245). Under these conditions, DNA is compact and packaged into chromatin. Ligand binding causes the dissociation of corepressor proteins and promotes the association of coactivators with liganded receptors. Coactivators associated with complexes displaying histone acetyltransferases (HAT), methultransferase, and kinase or ATP-dependent remodeling (SWI/SNF) activities lead to changes that reduces the compact nature of chromatin (245). The corepressors then dissoaciate, and a mediator complex known as the Srb and mediator protein-containing complex (SMCC) assembles. SMCC mediates the entry of the transcriptional machinery to the promoter, with resulting initiation of transcription (245).

3.4. Dietary Requirements for Vitamin A

The Food and Nutrition Board of the Institute of Medicine has made new recommendations of vitamin A intake by life stage and gender group (Table 2) (246). These Dietary Reference Intakes (DRIs) are reference values that are quantitative estimates of nutrient intakes to be used for planning and assessing diets in apparently healthy people and include Recommended Dietary Allowances (RDAs), Estimated Average Requirement (EAR), and Adequate Intake (AI) (246). The RDA is defined as “the dietary intake level that is sufficient to meet the nutrient requirement of nearly all (97 to 98 percent) healthy individuals in a particular life stage and gender group.” The EAR is defined as “a nutrient intake that is estimated to meet the requirement of half of the healthy individuals in a life stage and gender group.” AI is defined as “a recommended intake value based on observed or experimentally determined approximations or estimates of nutrient intake by a group (or groups) of healthy people that are assumed to be adequate—used when an RDA cannot be determined” (246).

Fig. 5. Night blindness in a public health poster from Indonesia.

classified in order of severity from night blindness (XN) to corneal ulceration and keratomalacia that involves one-third of the cornea or greater (X3B). A corneal scar (XS) is not a sign of active vitamin A deficiency. Xerophthalmic fundus (XS) is usually considered to be a rare condition.

4.1.1. NIGHT BLINDNESS

Vitamin A is the biochemical precursor to rhodopsin, which is essential to the visual cycle in rod photoreceptors and night vision. The earliest clinical manifestation of vitamin A deficiency is often night blindness. Vision is normal during the day, but the vitamin A-deficient individual may have difficulty distinguishing objects at night. A typical history may involve a child who bumps into furniture or objects at night and is afraid to come to the mother when called. In areas where vitamin A deficiency is endemic, it is not uncommon that the condition is well recognized by local people and has a specific name,

such as kwak moin “chicken blindness” in Cambodia, or buta ayam “chicken eyes” in Indonesia (chickens lack rod photoreceptors and have poor night vision). This phenomenon is shown in a public health poster from Indonesia (Fig. 5).

4.1.2. CONJUNCTIVAL XEROSIS

Vitamin A is essential for the maintenance of normal mucosal epithelia, including the conjunctiva. The normal conjunctiva consists of nonkeratinized columnar epithelium with mucin-secreting goblet cells. During vitamin A deficiency, there is loss of mucin and goblet cells, increased keratinization, and a transition to stratified squamous epithelium. The bulbar conjunctiva may appear dry and roughened, and on oblique illumination may have a pebble-like or bubbly appearance (Fig. 6). The surface of the epithelium may not easily be wetted by tears. Conjunctival xerosis is often associated with night blindness, an observation that was made as early as 1855 (25). Conjunctival xerosis is not considered a good diagnostic indicator of vitamin A deficiency, as it is often overdiagnosed in nutrition surveys. Bitot spots are a more well demarcated area of conjunctival xerosis and are considered separately under Subheading 4.1.3.

The pathological changes in the conjunctiva are related to the role that vitamin A plays in the regulation of mucin and keratin expression. In rats, membrane-spanning mucin ASGP (rMuc4) and secretory mucin rMuc5AC were downregulated during vitamin A deficiency (248). The alterations in mucin gene expression occurred in goblet cells and stratified epithelium of the cornea and conjunctiva, and once severe keratinization occurred, mRNA for rMuc5AC and ASGP were no longer detectable (248). In the rabbit model, vitamin A deficiency is associated with keratinization of the conjunctiva, with upregulation of AE1 (56.5 kd), AE3 (65–67 kd), and AE2 (56.5 and 65–67 kd) keratins (249). Following treatment with vitamin A, the conjunctiva changes from keratinized, stratified squamous epithelium without goblet cells to normal columnar epithelium with goblet cells (250,251). The loss of keratinization following vitamin A therapy appears to precede the restoration of goblet cells, which appear more slowly in response to treatment (251).

Around the beginning of the nineteenth century, the terms “xerosis,” “xerophthalmia,” and “xerophthalmos” were often used to describe dryness and associated changes of the eye that were considered to be related to conditions such as exposure, scrofula, syphilis, trachoma, and dryness of the eyes in older adults (252–256). The term “xerophthalmia” was applied in relationship to night blindness, corneal ulceration, and keratomalacia after further clinical observations and the development of ideas in the 1860s (20,27).

4.1.3. BITOT SPOT

A well demarcated patch of keratinized, squamous metaplasia of the bulbar conjunctiva, known as a Bitot spot, is considered pathognomonic for vitamin A deficiency. Pierre Bitot’s original description from 1863 (20) is worth repeating:

“La forme de cette tache diffère non-seulement selon les sujets, mais encore aux deux yeux d’un même individu. En général, elle est triangulaire, à sommet externe; la base, voisine de la cornée, est un peu concave. Dans quelques cas, elle était circulaire ou ovalaire; dans d’autres, simplement linéaire. Le plus souvent, les particules qui la composent sont agglomérées de façon à constituer une surface ponctuée, chagrinée; d’autres fois ces particules se disponsent en séries ou lignes flexueuse, parallèles, qui donnent à la tache l’aspect d’une surface ondulée ou ridée.”

Fig. 6. Conjunctival xerosis. (Courtesy of Task Force Sight and Life.)

“The shape of this patch differs not only from subject to subject, but also differs from eye to eye in the same individual. In general, it is triangular [à sommet externe], the base, which is near the cornea, is slighty concave. In some cases, it was circular or oval; in others, simply linear. Most frequently, the particles that compose it are grouped in such a way as to make up a stippled, distressed surface; in other cases these particles are arranged in a series or in flexible parallel lines that give the patch the appearance of a wavy or wrinkled surface.”

Bitot spots are usually located on the temporal bulbar conjunctiva in the interpalpebral zone of one or both eyes. The spots may have a foamy, “cheesy,” “greasy,” or granular appearance (Fig. 7). If vitamin A deficiency is more severe or longstanding, Bitot spots may also be found on the nasal bulbar conjunctiva on one or both eyes. Thus, any combination of one to four Bitot spots may be found in one individual. The number of Bitot spots generally correlates with the severity of vitamin A deficiency and low serum retinol concentrations (257). It appears that if the Bitot spots are longstanding, there may be a point beyond which the keratinizing squamous metaplasia is irreversible even with vitamin A supplementation. Bitot spots that do not respond to vitamin A therapy have been termed “nonresponsive Bitot spots,” and this phenomenon gave rise to earlier observations of Bitot spots in individuals with normal serum retinol concentrations (258,259). If the spots are rubbed or scraped off the bulbar conjunctiva, they usually reappear after a few days if the patient has not received substantial improvement in vitamin A intake through diet or supplementation.

The histopathology of Bitot spots has been studied extensively and described repeatedly in the scientific literature (20,260–276). The lesion consists of keratinized squamous epithelium with keratohyalin granules in the granular layer. The surface of the lesion may contain desquamated epithelial cells, amorphous debris, and bacteria and/or fungi. Goblet cells are absent. No differences in histopathology have been noted between

Fig. 7. Bitot spot. (Courtesy of Task Force Sight and Life.)

Bitot spots that are responsive or nonresponsive to vitamin A treatment; however, in the latter, the conjunctiva surrounding the Bitot spot is relatively normal (275). Many investigators have described the presence of a Gram-positive bacillus, Corynebacterium xerosis, in Bitot spots. The organism, also known as Bacillus xerosis, Bacterium xerosis and

Bacterium colomatti, and Corynebacterium conjunctivae, received its present designation from Karl Lehmann (1858–1940) and Rudolf Neumann (1868–1952) in 1899 (277). The bacteria appears to be a commensal organism that does not normally produce eye pathology. P. Colomiatti described an organism from Bitot spots taken from young boys in a correctional institution (278). John Elmer Weeks (1853–1949) obtained pure cultures of the “double bacillus” and inoculated a rabbit cornea using a Graefe knife that had been dipped in the bacterial culture. The cornea healed without suppuration. Other experiments were conducted in with inoculations in the conjunctival sac, and the rabbit developed no reaction (261). Studies conducted by Eugen Fraenkel (1853–1925) and Ernst Franke (1857–1925) at the Allgemeinen Krankenhause in Hamburg, showed that xerosis bacillis were nonpathogenic (279). In 1898, Stephenson attempted to induce xerosis in another human subject by inoculation of the conjunctiva with the frothy material from the conjunctiva and by injecting the conjunctiva of another subject with pure cultures of the xerosis bacilli; this inoculation produced no resulting pathology (280).

4.1.3. CORNEAL XEROSIS

With more severe vitamin A deficiency, the cornea may also undergo squamous metaplasia with keratinization, and the cornea appears hazy and rough instead of smooth and transparent (Fig. 8). The earliest change in corneal xerosis is a punctate keratitis characterized by fine pin-point lesions in the corneal epithelium (281–284). The punctate keratitis is more visible with fluorescein staining, and the intensity of the punctate keratitis is