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Isoprenoids: 2. Retinoids (Vitamin A)

That a dietary factor was involved in visual acuity was known to the ancient Egyptians and Greeks, but it was the 1930s before the importance of carotenes was recognized and β-carotene and retinol were characterized. The research into what was then termed 'vitamin A' continued apace, and a share in the Nobel Prize for Medicine in 1967 was awarded to George Wald, who had shown how retinol derivatives constituted the chemical basis of vision. Now, it is recognized that retinol, retinoic acid and their many metabolites have innumerable other functions in human metabolism from embryogenesis to adulthood, including growth and development, reproduction, cancer and resistance to infection. They are important natural antioxidants with benefits to health, although some potentially harmful properties have been reported. Other fat-soluble vitamins tocopherols (vitamin E), vitamin K and vitamin D are discussed in separate web pages.

1.  Occurrence and Basic Metabolism

Formula of all-trans-retinolThe term ‘vitamin A’ is used to denote retinol or all-trans-retinol together with a family of biologically active retinoids derived from this ('vitamers'). These are only found in animal tissues, where they are essential to innumerable biochemical processes. However, they cannot be synthesised de novo in animals and their biosynthetic precursors are plant carotenoids (provitamin A), of which β-carotene is most efficient and occurs in the green parts of plants and in seed oils. In the human diet in the developed world, plant sources tend to be less important than those from dairy products, meat, fish oils and margarines. In the U.K., for example, all vegetable spreads must be supplemented with the same level of vitamin A (synthetic retinol or β-carotene) as is found in butter.

The biosynthesis of plant carotenoids via isopentenyl diphosphate and dimethylallyl diphosphate has much in common with that of the plant sterols, but this is too specialized a topic to be treated at length here (but see the reading list below).

In animals, dietary carotenoids, including β-carotene, are absorbed in the intestines in intact form in humans, facilitated by a specific receptor protein. Conversion to retinoids leading ultimately to retinol esters occurs in the intestines, where dietary β-carotene is subjected to oxidative cleavage, the first step of which is catalysed by a cytosolic enzyme β-carotene-C15,15'-oxygenase-1, at its centre to yield two molecules of all-trans-retinal, which is reversibly reduced by a retinol reductase to retinol. Dietary retinol and retinol esters are absorbed similarly, but the latter are first hydrolysed. Next, the retinol is esterified to form retinyl palmitate by transfer of fatty acids from the position sn-1 of phosphatidylcholine, mainly via the action of a lecithin:retinol acyltransferase but also by lesser acyl-CoA dependent pathways, including an acyl CoA:retinol acyltransferase and even the enzyme diacylglycerol acyltransferase 1 (DGAT1); esterification is facilitated by binding to cellular retinol-binding protein type II (CRBP2). Any unchanged β-carotene is carried with the retinol esters to the liver in chylomicrons for uptake and metabolism by the hepatocytes.

Retinoid metabolism

Both retinol and retinoic acid are precursors of a number of metabolites (retinoids), which are required for specific purposes in tissues, by enzymatic modification of the functional groups and geometrical isomerization of the polyene chains. In the liver, activation of the retinol pathway involves first mobilization of the ester, followed by hydrolysis by retinol ester hydrolases, which includes carboxylesterase ES-10. Then, reversible oxidation of retinol to retinal is effected by one of several enzymes that include dehydrogenases and various cytochrome P450s, before some retinal is oxidized irreversibly to retinoic acid by enzymes with retinal dehydrogenase activity. On demand, conversion of retinol to retinoic acid occurs by the same mechanisms in other tissues, although for vision, retinol esters serve directly as the substrate for the formation of the visual chromophore 11-cis-retinal (see below). Retinyl-β-D-glucoside, retinyl-β-D-glucuronide, and retinoyl-β-D-glucuronide are naturally occurring and biologically active metabolites of vitamin A, which are found in fish and mammals. Indeed, the last has similar activity to all-trans-retinoic acid without any of the unwanted side effects in some circumstances.

Cleavage of β-carotene at double bonds other than that in the centre or of other carotenoids, for example by an isoenzyme β-carotene-oxygenase-2, leads to the formation of similar molecules, i.e. β-apocarotenals and β-apocarotenones of variable chain-length, which may exert distinctive biological activities in their own right. However, there is evidence that they can also be metabolized to form retinal.

A relatively small proportion of the cellular retinoids is located in membranes in tissues. Rather, retinol esters, mainly retinyl palmitate, are the main storage form of vitamin A, and they occur in many different organs, including adipose tissue and testes, but chiefly in stellate cells of the liver which comprise only 5 to 8% of all liver cells. How the retinol is directed specifically to these cells and enters them prior to esterification is not known. Although hepatic stellate cells are much smaller and less abundant than hepatocytes, they are characterized by lipid droplets that contain 90-95% of the hepatic retinoids (and 80% of the body pool) in addition to other non-retinoid lipids; the lecithin:retinol acyltransferase is the only retinol ester synthase in this instance. In addition, specialized cells in the eye store retinoids essential for vision in the form of lipid droplets. When the supply of retinol in the diet is limited, hepatic stores of retinol esters are mobilized as retinol ester hydrolases are activated to maintain constant circulating retinol levels; this also occurs in response to certain forms of liver damage such as alcoholic liver cirrhosis.

In the aqueous environment within cells, as well as in plasma, retinol, retinal and retinoic acid are bound to retinoid-binding proteins (RBP), which solubilize, protect and in effect detoxify them. These proteins also have a role in facilitating retinoid transport and metabolism; some are present only in certain tissues, and many are specific for particular retinoids and metabolic pathways. To prevent infiltration through the kidneys, retinol and holo-RBP form an association in blood with a protein termed transthyretin (TTR), which also serves as a thyroid hormone carrier and is essential for secretion. Normally, vitamin A circulates in plasma as a retinol:RBP:TTR complex with a 1:1:1 molar ratio. Unesterified retinol is the main form of the vitamin that is exported from the liver upon demand, and it is transported in blood in this bound form in VLDL, LDL and HDL (see our web page on lipoproteins), with some directly from the diet in the chylomicrons and their remnants. Peripheral tissues have specific receptors to take up what they require, probably after hydrolysis of any esters to retinol by means of the enzyme lipoprotein lipase. Then, retinol dissociates from the protein as it forms a complex with a receptor (STRA6) at a target cell and diffuses through the plasma membrane, a process driven by retinol esterification.

The RBP-TTR complex does not bind to retinal and retinoic acid, although these do bind to RBP on its own, and most of the low levels of retinoic acid transported in blood are bound to albumin. Local levels of retinoic acid are the result of an interplay between synthesis, binding and catabolism enzymes. For example, within cells retinoic acid binding proteins (CRABP1 and CRABP2) bind to the newly synthesised retinoic acid, increase its rate of metabolism and protect cells from an excess.

Catabolism. As all-trans-retinoic acid formation is irreversible, as a first step in catabolism, the excess is cleared by conversion to more polar metabolites through oxidation by various enzymes of the cytochrome P450 family. Secondly, the water-soluble retinoic acid metabolites, including 4-hydroxy-, 4-oxo- and 18-hydroxy-retinoic acids, conjugate with glucuronic acid and then can be rapidly removed from circulation and eliminated from the body via the kidney.

2.  Retinoids and Vision

Formula of 11-cis-retinalIt has long been know that retinoids are essential for vision, and there is now a good appreciation of how this works at the molecular level. Retinal rod and cone cells in the eye contain membranous vesicles that serve as light receptors. Roughly half of the proteins in these vesicles consist of the protein conjugate, rhodopsin, which consists of a protein – opsin – with the retinoid 11-cis-retinal. However, the process by which light is converted to a signal recognized by the brain, sometimes termed the 'retinoid (visual) cycle', begins in the retinal pigment epithelium, the pigmented cell layer just outside the neurosensory retina that nourishes retinal visual cells. Each step in the process requires specific binding or transport proteins.

All-trans-retinol is first converted to its ester by the enzyme lecithin:retinol acyltransferase as described above, and the products coalesce into lipid droplets, i.e. dynamic organelles termed 'retinosomes'. The next step involves a dual purpose enzyme, which cleaves the O-alkyl bond (not a conventional hydrolysis reaction) in the retinol ester and at the same time effects a change in the geometry of the double bond in position 11 of retinol from trans to cis. The 11-cis-retinol is then oxidized to 11-cis-retinal by 11-cis-retinal dehydrogenase.

Retinol and the visual cycle

The final part of the cycle occurs in the photoreceptor, where first the 11-cis-retinal is reacted with opsin to produce the protein conjugate rhodopsin in a protonated form. When rhodopsin is activated by light, the cis-double bond in the retinoid component is isomerized non-enzymatically to the 11-trans form with a change of conformation that in turn affects the permeability of the membrane and influences calcium transport. This results in further molecular changes that culminate in the release of opsin and all-trans-retinal, which is the trigger that sets off the nerve impulse so that the light is perceived by the brain. The all-trans-retinal is removed from the photoreceptor by a specific transporter (ABCA4), which provides phosphatidylethanolamine for conversion to the Schiff-base adduct, i.e. N-retinylidene-phosphatidylethanolamine, which it flips from the lumen to the cytosolic leaflet of the disc membrane. The effect is to remove potentially toxic retinoid compounds from the photoreceptors. The adduct then dissociates and the retinal is reduced back to all-trans-retinol by the cytoplasmic retinol dehydrogenase to complete the cycle.

Formula of retinylidene-phosphatidylethanolamine

As a side-reaction, some troublesome bis-retinoid adducts may be produced that accumulate with age and can affect vision (see our web page on phosphatidylethanolamine).

3.  Other Functions of Retinoids in Health and Disease

It is now realized that retinoids also have essential roles in growth and development, reproduction and resistance to infection. They are particularly important for the function of epithelial cells in the digestive tract, lungs, nervous system, immune system, skin and bone at all stages of life. They are required for the regeneration of damaged tissues, including the heart, and they appear to have some potential as chemo-preventive agents for cancer and for the treatment of skin diseases such as acne. Cirrhosis of the liver is accompanied by a massive loss of retinoids, but it is not clear whether this is a cause or a symptom, and there appears to be confusion as to when supplementation may be helpful in this and other diseases of the liver. In addition to retinol and retinoic acid, the metabolite 9-cis-retinoic acid has valuable pharmaceutical properties.

Carotenoids in general and retinoids in particular are efficient quenchers of singlet oxygen and scavengers of other reactive oxygen species. This is believed to be important in terms of general health, but it is not clear how much these physical properties contribute to specific biochemical processes in comparison to the effects of retinoids on signalling and gene transcription.

Many of the retinol metabolites function as ligands to activate specific transcription factors for particular receptors in the nucleus of the cell, and thus they control the expression of a large number of genes (>500), including those essential to the maintenance of normal cell proliferation and differentiation, embryogenesis, for a healthy immune system, and for male and female reproduction. Retinoic acid and its 9-cis-isomer are especially important in this context, and they are often considered the most important retinoids in terms of function other than in the eye. In essence, retinoic acid moves to the nucleus with the aid of small intracellular lipid-binding proteins (CRABP2 and FABP5), which channel it to specific nuclear receptors, the retinoic acid receptors (RAR) of which there are three, RAR-α, β and γ. These are ligand-dependent regulators of transcription and they function in vivo as heterodimers with retinoid X receptors (RXR) to process the retinoic acid signal. Similarly, 9-cis-retinoic acid and 9-cis-13,14-dihydroretinoic acid are high-affinity ligands for RXR. Together with retinoic acid, these are also ligands for the farnesoid X receptor (FXR), which forms a heterodimer with RXR; this receptor complex is involved primarily in bile acid homeostasis; conversely, there are suggestions that bile acids may have regulatory effects upon vitamin A homeostasis.

Scottish thistleIn addition, other nuclear receptors, such as the peroxisome proliferator-activated receptor PPARγ forms a heterodimer with the retinoid X receptor and is activated by retinoic acid to recruit cofactors. This complex in turn binds to the peroxisome proliferator response element (PPRE) gene promoter, leading to regulation mainly of those genes involved in lipid and glucose metabolism, including some involved in inflammation and cancer. To add to the complexity, retinoic acid has extra-nuclear, non-transcriptional effects, such as the activation of protein kinases and other signalling pathways.

It has also become evident that many of the functions of retinoids are mediated via the action of specific binding proteins (as discussed briefly above), which control their metabolism in vivo by reducing the effective or free retinoid concentrations, by protecting them from unwanted chemical attack, and by presenting them to enzyme systems in an appropriate conformation. With some tissues, retinol-bound RBP in blood is recognized by the membrane protein STRA6, which transports retinol into cells where it binds to an intracellular retinol acceptor, cellular retinol-binding protein 1 (CRBP1), and is then able to activate a signalling cascade that targets specific genes. In addition, a specific retinol-binding protein secreted by adipose tissue (RPB4) is involved in the development of insulin resistance and type 2 diabetes, possibly by affecting glucose utilization by muscle tissue, with obvious application to controlling obesity. In the eye, the activity of retinoic acid during development is controlled by binding to apolipoprotein A1.

All-trans-retinoic acid has been shown to be effective against many different types of human cancers, especially in model systems but also in some clinical trials, because of its specific effects on cell proliferation, differentiation, and apoptosis (as well as having low toxicity). For example, it induces complete remission in most of cases of acute promyelocytic leukaemia, when administered in combination with other chemotherapy techniques. Similarly, 13-cis-retinoic acid has been used successfully in the treatment of children with high-risk neuroblastoma to reduce the risk of recurrence and increase long-term survival. However, the efficacy of similar treatments against other types of acute myeloid leukaemia and solid tumours appears to be poor. It is hoped that current efforts to obtain a better understanding of the mechanism of the anticancer activities will lead to improved treatments. Synthetic analogues of retinoic acid, termed rexinoids, which activate retinoic X receptors, also hold promise as anti-cancer agents.

Vitamin A deficiency in children and adult patients is usually accompanied by impairment of the immune system, leading to a greater susceptibility to infection and an increased mortality rate, often with growth retardation and congenital malformations. However, one of the main effects of vitamin A deficiency in malnourished children, and seen too often in the underdeveloped world, is blindness. This is doubly tragic in that it is so easily prevented. Unfortunately, it is not always easy to distinguish between the effects of vitamin A deficiency and primary defects of retinoid signalling.

Recommended Reading

Lipid listings Credits/disclaimer Updated: April 3rd, 2017 Author: William W. Christie LipidWeb icon
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