Vitamin A – Tolerance Extends Longevity

Abstract

Vitamin A is a family of essential fat-soluble dietary compounds, three of which—retinol, retinal and retinoic acid—play significant roles in the human body, with each compound performing functions the others cannot. Retinol is the major transport and storage form of vitamin A; retinal is essential for vision, and retinoic acid acts like a hormone, binding to nuclear-hormone receptors and affecting the expression of more than 500 genes. Vitamin A is required for vision, immune function, skin health, regulation of cell growth and bone metabolism, thus symptoms of vitamin A insufficiency include night blindness; increased susceptibility to infection and loss of immune tolerance (environmental and food allergies, autoimmune diseases); hyperkeratosis, dry skin, brittle nails, hair loss; epithelial cancers and bone loss.

Due to the perceived risk of hypervitaminosis A, provitamin A carotenoids have been recommended as the preferred source of this nutrient for women of reproductive age and are its primary source for vegetarians and its only source for vegans. While it has long been recognized that vitamin A and provitamin A differ significantly in bioavailability and therefore biologic activity, recently published research reveals that a significant number of individuals are “low-responders” unable to absorb and/or convert provitamin A carotenoids to vitamin A, and that even in individuals who would normally be able to metabolize provitamin A to vitamin A, numerous factors can effectively impede this conversion. Research showing large individual variations in the actual absorption of pro-vitamin A from foods and in its conversion to retinol has demonstrated that current data on retinol activity equivalents (RAEs) for vitamin A sufficiency is highly misleading, and that a significant percentage of the American population may be vitamin A insufficient.

Retinol and its provitamin A precursors are converted into active metabolites in tissue-specific patterns. Conversion of retinol to retinal and then to all-trans-retinoic acid, the predominant isomer of vitamin A, requires two successive oxidation reactions, first by alcohol dehydrogenases (which contain zinc), and then by either aldehyde dehydrogenase or retinal dehydrogenase (which contains the riboflavin-dependent enzyme, flavin-adenine dinucleotide [FAD]).⁴

In addition to the aforementioned research, this article reviews recent discoveries regarding the key roles played by vitamin A in promoting the development of regulatory T cells that favor oral and self- tolerance, and in both potentiating and balancing the effects of vitamin D. Symptom and lab assessment of vitamin A status is discussed.

Healthy centenarians are consistently found to have vitamin A levels comparable to those in healthy young adults, in contrast to lower levels of vitamin A seen in typical older control subjects.

Aging is characterized by a peculiar chronic inflammatory status for which researchers have recently coined the term, “inflammaging,” a key aspect of which is the age-dependent expansion of effector T cells with pro-inflammatory cytokine production potential. Particularly in light of vitamin A’s emergence as a pivotal inducer of oral and self-tolerant immune function, vitamin A sufficiency should be recognized as a key contributing factor to longevity.

Introduction to Vitamin A

Vitamin A is a family of essential fat-soluble dietary compounds that contain a retinyl group. Three different forms of vitamin A, collectively referred to as retinoids, are active in the body: retinol (alcohol), retinal (aldehyde) and retinoic acid (acid). All natural retinoids have a least one β-ionone ring linked to a side chain of conjugated carbon-carbon double bonds that may exist in trans or cis conformation.^{1 2 3}

Conversion of retinol to retinal is reversible, but the further conversion of retinal to retinoic acid is not—a significant fact since each form of vitamin A performs functions the others cannot. (Please see Conversion of Vitamin A Compounds chart above.)

Most (80%) of the body’s vitamin A reserves (around 450 mg in healthy vitamin A-replete adults) are stored in the liver (500 µg/g wet tissue), an amount that can cover vitamin A requirements for several months. High alcohol intake, however, can rapidly deplete vitamin A stores.

Retinoic acid and other vitamin A metabolites can be conjugated to glucuronide by CYP26 and excreted with bile; however, the high efficiency of intestinal absorption for most retinoids minimizes losses and maintains extensive enterohepatic recycling. In addition, megalin, a particularly large member of the LDL-receptor family, binds retinol binding protein 4 (RBP4) and mediates its uptake by endocytosis. Despite the considerable size of the retinol carrying RBP4-transthyretin complex, it has been shown to proceed through the epithelial cell and return intact into circulation. Oxidation of retinoic acid is the only known inactivating catabolic pathway; all retinoids, however, are highly susceptible to oxidation.⁴

Retinol

Retinol is the major transport and storage form of vitamin A. Retinol is absorbed from the proximal small intestine in a process that requires concurrent fat absorption and the formation of mixed micelles, which are transferred into chylomicrons and then exit enterocytes to be exported into portal blood and taken up by the liver. Retinol is released from the liver within retinol-binding protein 4 (RBP4), which engulfs the retinol molecule, shielding it from the hydrophilic environment in circulation or inside cells. After its release from the liver, RBP4 combines with transthyretin, forming a complex that transports retinol in the blood. A surface receptor on many peripheral cells binds RBP4, mediating retinol uptake. In vitamin A deficiency, the liver’s production and release of RBP4 increases. About 70-90% of ingested retinol is absorbed, but only 3% or less of carotenoids. Carotenoids, which also must be incorporated into mixed micelles and then into chylomicrons for absorption, are exported into lymph, appearing in the blood about 8 hours after a challenge meal. Chylomicrons rapidly lose triglycerides, but not carotenoids, and the depleted chylomicron remnants are taken up into heptaocytes and extrahepatic cells via diverse lipoprotein receptors.

Retinal

Retinal serves as the intermediate in the conversion of retinol to retinoic acid and is involved in vision as an integral component of the pigment molecules in the retina, rhodopsin, which are composed of one molecule of opsin bonded to one of retinal. When light passes through the cornea and strikes the retina, the retinal in rhodopsin shifts from a cis to a trans configuration, which cannot remain bonded to opsin. Opsin changes shape, generating an electrical impulse that transmits visual information to a nerve cell and thence to the brain.

While most of the retinal converts back to its cis isoform and recombines with opsin to regenerate rhodopsin, some is oxidized to retinoic acid. Thus visual activity results in continuous small losses of retinal, which must be constantly replenished from retinol stores, food or supplements.

Retinoic acid

Vitamins A and D are notably distinct from other vitamins in that their respective bioactive metabolites, retinoic acid and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), have hormone-like properties. Both 1,25(OH)2D3 and retinoic acid are synthesized from their vitamin precursors by different tissues and cells in the body and exert their effects on remote target cells by binding to nuclear-hormone receptors.⁵ Retinoic acid regulates normal endodermal differentiation, morphogenesis, embryonic and childhood development; and sustains balanced cell proliferation, differentiation, and apoptosis in adulthood. Many of the underlying events involve the binding of retinoid receptors to specific binding elements in nuclear DNA and control of the expression of associated genes. More than 532 genes are recognized as either directly or indirectly regulated by retinoic acid.^{4 6 7}

Two groups of retinoid receptors have been identified. The first includes the retinoic acid receptors (RAR) α, β, and γ, which bind both the trans and cis isoforms of retinoic acid. The second group is comprised of the retinoic X receptors (RXR) α, β, and γ, with the cis isoform of retinoic acid as the main activating ligand. The RXR group is of special interest since its binding is necessary for the actions of numerous other nuclear receptors. The list of RXR-dependent nuclear binding proteins includes the RAR, vitamin D receptor (VDR), thyroid receptors (TR), peroxisome proliferation activating receptors (PPAR), pregnane X receptor (steroid and xenobiotic receptor, SXR/PXR), liver X receptors (LXR), famesoid X-activated receptor (FXR), and benzoate X receptor (BXR). The importance and diversity of retinoic acid’s gene regulating effects are evidenced by the wide ranging diversity of these receptors.⁸

Functions of Vitamin A and Related Symptoms of Deficiency

Since vitamin A is essential for vision, skin health, immune function/oral tolerance (discussed below), regulation of cell growth, and bone metabolism, symptoms of vitamin A insufficiency include:

• Impaired vision—initially most evident in reduced light (e.g., night blindness, due to lack of vitamin A at the back of the eye, the retina)
• Xerophthalmia—total blindness resulting from lack of vitamin A at the front of the eye, the cornea
• Xerosis—abnormal drying of the skin and mucous membranes
• Dry skin
• Dry hair
• Pruritus
• Easily broken or ridged fingernails
• Hyperkeratosis—goose bump-like appearance of the skin caused by excessive production of keratin, which blocks hair follicles. Skin becomes dry, scaly and rough, initially on the forearms and thighs. In advanced stages, the entire body is affected, causing hair loss.
• Skin lesions—actinic keratoses, increased susceptibility to basal and squamous skin cancers
• Epithelial cancers (lung, colon, breast, prostate cancers).
• Viral infections, most notably measles, chicken pox, pneumonia, and respiratory syncytial virus (RSV—the most common cause of bronchiolitis and pneumonia in children under 1 year of age and now being recognized as an important cause of respiratory illness in adults).
• Infections of the respiratory, GI, and urinary tracts, the vagina, and possibly the inner ear.
• Fungal infections, e.g., nail fungal infections, athlete’s foot
• Environmental and food allergies
• Inflammatory bowel disease
• Autoimmune disease
• Bone loss

Vitamin A Toxicity – a Question of Balance?

Excessive retinol intake (UL = 3,000 µg/day [10,000 IU/day]) may increase bone fracture risk in older people; however, recent studies indicate retinol, which promotes osteoblast differentiation and activation, helps prevent bone loss in combination with vitamin D,^{9 10 11 12 13} suggesting imbalance between vitamins A and D is the real issue. Two recently published studies in which the relationship between vitamin A intake and fracture risk was evaluated support this hypothesis.

A study involving 3,113 postmenopausal women living at higher latitudes in the UK found that retinol derived from cod liver oil showed no detrimental associations with markers of bone health. Retinol from other vitamin supplements was initially associated with lower BMD; however, a trend for lower bone resorption was noted at the follow-up visit, and less bone loss occurred over 6 years. Retinol from food was associated with increased bone resorption, leading study authors to note that it appears “retinol from supplements and food have different effects, which may in part be due to whether the source of retinol also provides vitamin D.¹⁴

The second study, which involved 75,747 women from the Women’s Health Initiative Observational Study, found a modest risk in total fractures associated with increased total vitamin A or retinol intakes; however, this association was observed only among women with low vitamin D intakes. In addition, no association between vitamin A intake and the risk of hip fracture was observed. Researchers speculated, “A deleterious effect of vitamin A on bone may operate through its antagonistic relation with vitamin D.¹⁵

We would characterize the relationship between vitamin A and vitamin D as one of maintaining balance rather than antagonism.

Retinoic acid and 1,25(OH)2D3 compete for the same nuclear receptor partners; both the RAR and the VDR must form heterodimers with RXRs to be able to bind to response elements and initiate transcription. For this reason, 1,25(OH)2D3 and retinoic acid naturally mitigate against each other’s uncontrolled effects.¹⁶

In addition, vitamin A optimizes the body’s use of vitamin D. Recent research has shown that 9-cis-retinoic acid, a derivative of vitamin A, increases the affinity of VDR/RXR to its DNA recognition site, induces recruitment of coactivators by the DNA-bound heterodimer, and potentiates vitamin D-dependent transcriptional responses—all of which suggests that vitamin A, when properly balanced with vitamin D, promotes bone health.^{17 18}

Vitamin A intake >3,000 µg/day [>10,000 IU supplemental pre-formed vitamin A] during early pregnancy may increase risk of birth defects.^{4 3 1} Since such negative outcomes may be related to vitamin D insufficiency, maintaining balance between vitamins A and D (indicated by lab values in normal range for both nutrients) is especially important for women who might conceive.

Food Sources of Vitamin A

Only animal-derived foods contain retinol. Highest concentrations of retinol are found in liver (106µg/g), butter (6.84µg/g), hard and cream cheeses, e.g., Swiss cheese (2.5 µg/g), cheddar (2.8µg/g) and regular (not low fat) cream cheese (3.8µg/g). Eggs contain 1.9µg/g, and cow’s milk 0.6 µg/g.

Good sources of provitamin A include carrots (24.6 µg RAE [retinal activity equivalent]/g), sweet potatoes (16.4µg RAE/g), spinach (8.2µg RAE/g), kale (7.4 µg RAE/g), and broccoli (1.4µg RAE/g). Cantaloupe (1.69µg/g), mangos (.38 μg/g), and many other dark green or orange-yellow colored fruits and vegetables are also good sources of provitamin A. In the case of mangoes, however, up to 64% of the total β-carotene is present as the cis-isomer, which is not taken up or transported as efficiently as the trans-isomer.¹

Many at Risk of Vitamin A Insufficiency Due to Increased Intake of Vitamin D and High Variability in β-carotene Absorption and Conversion to Retinol

Bioavailability of pre-formed vs. pro-vitamin A

Vitamin A and provitamin A differ significantly in bioavailability and therefore biologic activity. The retinol activity equivalent (RAE) was adopted in 2001 to account for the much lower biological activity of provitamin A carotenoids compared to pre-formed vitamin A. In RAE units, 1 µg RAE is equivalent to 1µg of pure all-trans-retinol, 2 µg of all-trans-β-carotene in oil (a highly absorbable form), 12µg of food-based all-trans-β-carotene, or 24 µg of other, food based, all-trans provitamin A carotenoids. Of the 600 carotenoids discovered so far, only β-carotene, α-carotene and β-cryptoxanthin possess vitamin A activity.¹

The RDAs for vitamin A are extrapolated from the estimated average requirement (EAR), which is equal to the intake that meets the estimated nutrient needs of half of the individuals in a group. To this amount is factored in the considerations that 0.5% of vitamin A stores are lost daily, and minimum acceptable liver vitamin A storage reserves are 20µg. Thus, the EAR for men >18 is 625 and the RDA is 900, while for women the EAR is 500 and RDA is 700 µg RAE/d.1

While these amounts appear to include a reasonable safety margin, the RDAs are based on assumptions regarding the amount of β-carotene absorbed and converted to retinol that do not take into account a number of food and host-related factors that significantly impact carotenoid bioavailability, absorption and conversion to retinol.¹⁹

Factors affecting carotenoid absorption and metabolism to retinol

Current evidence indicates that large variations in the concentration of provitamin A carotenoids in foodstuffs occur—even in the same type of food—due to varietal differences, stage of maturity, climatic conditions, processing and/or cooking.

Efficiency of carotenoid absorption is affected not only by the amount of carotenoid ingested, but by processing and/ or cooking of the food, other dietary ingredients that stimulate absorption (e.g., the type and amount of dietary fat [carotenoids must be incorporated into mixed micelles and then into chylomicrons for absorption] or inhibit it [fiber, especially pectins], food matrix effects, and interactions between carotenoids [lutein and β-carotene appear to inhibit one another’s absorption]).²⁰

Other factors affecting the individual’s ability to utilize carotenoids as a source of retinol include:

• Current nutritional status: protein and nutrient-cofactors are necessary for function of the enzymes involved in carotenoid metabolism to retinal, including 15,15΄monoxygenase, the brush border retinal ester hydrolases, and retinal dehydrogenase [RALDH]1
• Inadequate bile flow: affected by acute and chronic liver diseases, including non-alcoholic steatohepatitis related to insulin resistance / metabolic syndrome, which is estimated to affect ~30% of the US population²¹, and by alcoholism. Alcohol dehydrogenase—the enzyme that catalyzes conversion of retinol to retinaldehyde, which is then oxidixed to retinoic acid—has an affinity for ethanol.
• Imbalanced gut ecology: due, for example, to parasitic infestation, food allergies such as gluten intolerance, celiac disease (U.S. incidence 1 in 133)²², lipid malabsorption, hydrochloric acid insufficiency (30% of the US population >60 is estimated to have atrophic gastritis)20, infection with H.pylori, (prevalence of H.pylori infection is 50% worldwide, 40% in the US²³ ) or Clostridium difficile (annual incidence of C.difficile infection increased from 49.2 to 101.6 per 100,000 population from 2001-2005²⁴)
• Genetic inheritance: discussed immediately below

Single nucleotide polymorphisms (SNPs) have recently been identified that significantly lessen the activity of 15,15′-monoxygenase, the key enzyme responsible for the conversion of β-carotene to retinal, and can therefore greatly impact an individual’s ability to derive adequate levels of vitamin A from carotenoid-rich foods. These SNPS (R267S and A379V) are common, with variant allele frequencies in the population of 42 and 24%, respectively. In vitro biochemical characterization of the 267S + 379V double mutant revealed a 57% reduction in catalytic activity of 15,15′-monoxygenase. Assessment of the responsiveness of female volunteers to a pharmacological dose of β-carotene confirmed that carriers of both the 379V and 267S + 379V variant alleles have a greatly reduced ability to convert β-carotene, indicated by reduced retinyl palmitate: beta-carotene ratios in the triglyceride-rich lipoprotein fraction of -32% and -69% , respectively, and increased fasting beta-carotene concentrations of +160% and +240% , respectively.²⁵

Beta-carotene 15,15′-monoxygenase, which converts the provitamin A carotenoids to retinal mainly in the intestinal mucosa (and, to a lesser extent, in testes, liver and kidneys), also requires bile acids and iron for its three-step activity (epoxidation at the 15,15′-double bond, hydration to the diol, and oxidative cleavage). Retinal resulting from carotenoid cleavage is metabolized, mainly to retinol, by several tissue specific enzymes. In testes, a specific isoform of lactate dehydrogenase is closely associated with beta-carotene I5, 15′-dioxygenase, and the reduction of newly generated retinal is thought to be its main physiological function. In most other tissues, class III alcohol dehydrogenase may be more important. Co-factors required by these enzymes are riboflavin, niacin, and zinc.⁴

The activity of each of these dehydrogenase enzymes is also dependent on sufficient protein in the diet and is surely affected by SNPs as well. Although the research directly connecting various dehydrogenase SNPs with vitamin A metabolism is still forthcoming, researchers have recently identified six alcohol dehydrogenase SNPs, several of which appear to confer protection against aerodigestive cancers, suggesting a connection to retinol metabolism.²⁶ While these SNPs appear to promote enzyme activity, it seems only reasonable to assume that other SNPs result in its decrease.

Environmental toxicants affect retinol storage and metabolism

Numerous hepatotoxicants, e.g., carbon tetrachloride, reduce retinol storage and/or alter retinoid metabolism. In 2008, a study of common cleaning products found the presence of carbon tetrachloride in “very high concentrations” (up to 101 mcg per cubic meter) as a result of manufacturers’ mixing of surfactants or soap with sodium hypochlorite, i.e. chlorine bleach.²⁷

So, while on the basis of central enzymatic cleavage alone, one molecule of β-carotene provides two molecules of retinol (so 1 mg β-carotene in a food is theoretically equal to 1 mg retinol), in vivo, the above factors affecting carotenoid absorption greatly impact how much of the provitamin ingested in the diet is actually presented to and absorbed by an individual’s intestinal mucosa.

Research confirms provitamin A may not supply adequate retinol

Several studies have demonstrated the impact of the above-noted factors on carotenoid absorption and metabolism to retinol. In one study in which stir fried vegetables or supplements of β-carotene enriched wafers were given to Indonesian breast feeding women with low hemoglobin, no significant changes were seen in the vegetable group in serum retinol, β-carotene or other serum carotenoids, or breast milk retinol. In the enriched wafer group, serum retinol, serum β-carotene, and breast milk retinol all increased 0.32 nmol/L (38%), 0.73 nmol/L (390%) and 0.59 nmol/L (67%), respectively. The authors concluded that the assumption that provitamin A-containing vegetables can be relied upon to prevent or combat vitamin A deficiency should be re-examined.²⁸

Although the processing and/or cooking of vegetables may cause a change from the all-trans to the cis isomer of carotenoids¹⁹, which results in less efficient production of retinol²⁹, partially because up to 95% of the cis isomer is first converted back to trans-β-carotene before absorption³⁰, studies indicate that up to 50% of carotenoids may be absorbed from cooked vegetables in comparison to less than 10% from raw vegetables. Other research shows that absorption of β-carotene and other carotenoids from vegetables is only 5-30% of that absorbed from synthetic supplements, due to the food matrix of fiber and/or protein that must first be broken down by mastication, gastric acid and bile acids.²⁰

In another study, a prepared meal containing a pharmacological dose of 120 mg of β-carotene in oil was fed to 79 healthy male volunteers. Response was highly variable (coefficient of variation [CV] =61%) due to inter-individual differences in the efficiency of intestinal absorption of β-carotene and in chylomicron metabolism. The inter-individual variability of the apparent vitamin A activity of β-carotene ingested was also high, although the higher the amount of β-carotene absorbed, the higher the amount of retinol palmitate secreted into chylomicrons. The authors concluded that the dietary equivalence of β-carotene and retinol varies greatly among individuals.³¹

Variability in the conversion of beta-carotene to vitamin A has also been shown in a recent study at UC Davis in which 11 healthy women were given 30 micromol retinyl acetate orally, followed one week later by an approximately equimolar dose of 37 micromol β-carotene. Although mean absorption of β-carotene was 3.3%, only 6 of the 11 subjects had measurable plasma β-carotene and retinol concentrations. Mean absorption of β-carotene in the 6 women with measureable levels was 6.1%, and their conversion ratio was 1.47 mol for retinol to 1 mol β-carotene. The remaining 5 subjects were “low responders” with

In Germany, pregnant women or those considering becoming pregnant are generally advised to avoid the intake of liver due to concerns about the teratogenic potential of hypervitaminosis A, a concern that may be misguided (as noted above and in the following section, vitamins A and D mitigate and balance one another’s effects). As a result, β-carotene is their primary source of vitamin A. A clinical study in pregnant women with short birth intervals or multiple births showed that, regardless of a high to moderate socio-economic background, 27.6% of the women had plasma retinol levels below 1.4 micromol/l, corresponding to a borderline deficiency (or worse.) Despite high total carotenoid intake of 6.9 +/- 3.6 mg/d, 20.7% of mothers showed plasma levels <0.5 micromol/l β-carotene, indicating frank deficiency. Vitamin A is recognized by the American Pediatric Society as one of the most critical vitamins during pregnancy and the breastfeeding period, especially in terms of lung function and maturation. If the vitamin A supply of the mother is inadequate, her supply to the fetus will also be inadequate, as will be the supply of vitamin A in her breast milk. The developmental effects of these inadequacies cannot be reversed by postnatal supplementation and may be a contributing factor to the escalation in the incidence of allergic diseases, including asthma, and autoimmune diseases, including type 1 diabetes, seen in the western world.^{33 34 35 36 37}

Vitamin A – dancing with vitamin D to promote balanced immunity

Both the retinoic acid receptor (RAR) and the vitamin D receptor (VDR) must form heterodimers with RXR to signal, and 9-cis-retinoic acid, a derivative of vitamin A, increases the affinity of VDR/RXR to its DNA recognition site, induces recruitment of coactivators by the DNA-bound heterodimer, and potentiates vitamin D-dependent transcriptional responses.

Since not only is 1,25(OH)2D3 recognized to play a protective role in dampening or limiting potential autoimmune responses at the cellular level (vitamin D regulates TGF-β signaling molecules called Smads that affect gene expression, decreasing induction of pro-inflammatory TH1 [type 1 helper T cell] cytokines^{38 39}), but vitamin A, specifically all-trans retinoic acid, has recently been shown to decrease effector T-cell function, while also increasing regulatory T-cell populations and activities (further discussed below), one might predict that insufficiency of either vitamin D or vitamin A could predispose to hypersensitivity or autoimmunity.

Consistent with this hypothesis, both vitamin A and vitamin D have been found to be relatively deficient in adults as well as children with type 1 diabetes. Serum levels of 1,25(OH)2D3 are also often decreased in patients with systemic lupus erythematosus and are inversely correlated with disease activity in patients with rheumatoid arthritis. Insufficiency of both vitamins A and D is found in children with rickets (typically due to insufficient vitamin D), who also have a higher incidence of diabetes than vitamin D-sufficient children, and are more susceptible to infection} ⁴⁰ suggesting a lack of vitamin A for protective immune responses.⁴¹

Research reported in the January 2009 issue of Diabetes provides evidence that all-trans retinoic acid inhibits the development of type 1 diabetes in NOD mice. Spleen cells from diabetic mice were transferred into NOD.scid mice normally resistant to type 1 diabetes development, a protocol through which diabetes is consistently transferred to recipient mice. NOD.scid mice are naturally type 1 diabetes resistant since, although they have the same genetic background as NOD mice, they also carry a mutation rendering them immunodeficient (i.e., no T- or B-lymphocytes).

Treating NOD.scid recipients with all-trans retinoic acid markedly suppressed the transfer of diabetes by diabetogenic splenocytes. All-trans retinoic acid also significantly delayed progression to type 1diabetes in a “late prevention” model in which it was given by intraperitoneal injection to treat 10-week-old NOD mice. These beneficial therapeutic outcomes were found to result from the immunoregulatory effects of all-trans retinoic acid (discussed below).⁴² In an article also published in the January 2009 issue of Diabetes and entitled “Taking a daily vitamin to prevent type 1 diabetes?” Wasserfall and Atkinson note that both vitamin A and D are fat-soluble and found in fish oil supplements, which epidemiological evidence suggests are associated with reduced type 1 diabetes-associated autoantibody conversion, raising “an intriguing possibility that a combination of vitamins A and D, in safe pharmacologically formulated doses rather than the usual daily recommended dose, might be of benefit in the treatment of those at increased risk for type 1 diabetes.⁴¹

Vitamin A’s Effects on Immunity

T Lymphocytes

T lymphocytes (T cells), which activate and direct other immune cells, account for 70-80% of peripheral lymphocytes in the blood and express the surface protein CD4, so are often referred to as CD4+ T cells. Naive T cells originate in bone marrow and mature primarily in the thymus where, after activation by unique cytokines, they differentiate into lineages of effector/memory (TH) and regulatory T (Treg) cells, each of which are characterized by distinct developmental pathways and unique biologic functions.

Meet the new subset of T cells

Until recently, helper T cells that develop into effector T cells were thought to be a binary system, including only TH1 and TH2 effector cell types, but the TH family is now known to include an additional lineage, the TH17 cell. TH1 cells produce IFN-γ and TNF-β, activate marcophages, and are involved in cell-mediated immunity. TH1 cells elicit delayed-type hypersensitivity responses and effectively clear intracellular pathogens. TH2 cells produce IL-4, IL-5, IL-6 and IL-10, partner with B cells, and contribute to humoral immunity. TH2 cells are involved in IgE production, eosinophilic inflammation, and the clearance of helminthic parasite infections.

The recently recognized T cells, TH17 cells, which are induced to differentiate from naïve CD4+ T cells by the transcription factor receptor-related orphan receptor-γt (RORγt) in the presence of TGF-β and IL-6, generate IL-17 cytokines that initiate activation of NFκB, which leads to the transcription of multiple target genes involved in innate immunity. TH17 cells appear to be critical for enhancement of host protection against extracellular bacteria and fungi, which are not efficiently cleared by TH1 and TH2 responses, and also have emerged as potent mediators of autoimmune disease.

T regulatory cells

In counterbalance, Treg cells maintain immune tolerance and protect against excessive TH effector activity and autoimmune pathology. Treg cells are characterized by the expression of a DNA binding protein, the forkhead–winged helix transcription factor family member, FOXP3+, which is induced by TGF-β in the presence of IL-2. This process is significantly upregulated by retinoic acid (RA), which enforces the generation of Treg cells and inhibits the differentiation of TH17 cells. FOXP3+ Treg cells secrete IL-10, which suppresses activation of aggressive effector T cells.

So, we now have four functionally unique populations of CD4+ T cells that are directly involved in the regulation of immune responses to pathogens, allergens, and self-antigens: TH1, TH2, TH17 and Treg cells.⁴³

Vitamin A Key to Self and Oral Tolerance

This emerging picture has revealed vitamin A as an important regulator of the TH1/TH17 versus TH2 balance in the immune system. Vitamin A deficiency skews balance in a TH1/TH17 direction, while adequate levels of vitamin A, by promoting the development of Treg cells responsible for self-tolerance and the prevention of autoimmune diseases, favors immune responses characterized by production of anti-inflammatory IL-10 cytokines.^{44 45}

Regulatory T cells develop both constitutively in the thymus (referred to as natural Tregs [nTregs]) and peripherally, as a result of induction by FOXP+3 (referred to as induced Tregs [iTregs]).8 Retinoic acid [RA] promotes induction of FOXP3+ Tregs in the presence of TGF-β by downregulating RORγt, thus also blocking the differentiation of TH17 cells.

Transforming growth factor-β (TGF-β), a family of cytokines with numerous signaling effects, (members of the TGF- β superfamily are involved in cell growth and differentiation and influence immune and endocrine functions⁴⁶) is required for both constitutive nTreg development in the thymus and peripheral induction of iTreg, but TGF-β plays a dual role since, in the presence of inflammatory cytokines such as IL-6, it is also able to promote development of pro-inflammatory TH17 cells. RA thus emerges as a key regulatory factor, modulating immunity by inhibiting the TGF-β/IL-6-driven induction of pro-inflammatory TH17 cells and simultaneously promoting the TGF-β-dependent peripheral differentiation of anti-inflammatoryFOXP3+ iTregs, which are indispensable to prevent excessive and self-destructive immune responses.⁸

In addition to enhancing the TGF-β-driven generation of iTregs in peripheral tissues, RA enhances the production of iTregs in response to antigen-presenting dendritic cells in the gut‑associated lymphoid tissue (GALT) and small intestinal lamina propria, and upregulates gut-homing receptors on iTreg cells and B lymphocytes, targeting both cell types to the gut mucosa. Effector and memory T cells exhibit plasticity in their homing commitment: skin-homing T cells can become gut-homing T cells and vice versa if they are restimulated either with or without RA, respectively. Like T cells, B cells also exhibit plasticity in their homing commitment and can either acquire or lose gut-homing potential when reactivated with or without RA.⁴⁰

Both TGF- β and RA are actively produced by the intestinal epithelium and play important roles in mucosal epithelial cell differentiation and in maintaining the integrity of its barrier function. For example, by actively promoting IgA class switching, TGF-β and RA positively regulate secretory IgA production, important in mucosal barrier function and antibacterial protection.

Vitamin A’s effects on B cells

During the primary immune response, naïve B cells proliferate and differentiate either into IgM-producing plasma cells, followed by hypermutation into plasma cells producing high affinity IgG antibodies, or into long-lived memory B cells, which are characterized by a lower threshold for activation and differentiation. Memory B cells also have a higher density of toll like receptor 9 (TLR9) than naïve B cells. In vitro, TLR 9 binds unmethylated CpG oligonucleotides mimicking the unmethylated DNA typically found in bacteria and viruses. RA has been shown to markedly increase proliferation of memory B cells while inhibiting proliferation of naïve B cells. Thus, RA plays a role in maintaining long-lived humoral memory.⁴⁴

nTregs represent 5–10% of peripheral CD4+ T cells in naive mice and humans. Chronic ablation of Treg cells in adult healthy mice results in their death within 3 weeks, demonstrating how important Treg cell-mediated suppression is for preventing immune pathology throughout the lifespan.⁴⁷ In the intestine, where an improper balance between inflammatory and suppressive immunity can jeopardize mucosal homeostasis and destroy the integrity of the mucosal barrier, FOXP3+ Tregs, induced by RA, play a key role in maintaining the steady-state of tolerance towards innocuous antigens, thus preventing excessive, self-destructive immune responses and the development of inflammatory bowel disease and autoimmunity.

In a recent paper entitled “Vitamin A rewrites the ABCs of oral tolerance,” Strober sums up the situation saying, “Thus, we arrive at the somewhat surprising realization that mucosal unresponsiveness [self and oral tolerance] relies upon the bioavailability of a factor in the food stream.”⁴⁸

Plasma Vitamin A Correlates with Healthy Aging and Longevity

Healthy centenarians are consistently found to have vitamin A levels comparable to those in healthy young adults, in contrast to lower levels of vitamin A seen in typical older control subjects.^{49 50 51 52} Vitamin A’s effects on immune regulation provide a likely explanation for this correlation.

Recent observations indicate that immunosenescence is not accompanied by an unavoidable and progressive deterioration of immunity, but results from a remodeling in which some immune functions are reduced, while others remain unchanged or even increase. Specifically, major age-related changes in immunity include a progressive, age-dependent decline in virgin T cells combined with a progressive age-dependent increase in effector T cells that produce pro-inflammatory cytokines (e.g., IL-2, IFN-γ, TNF-α).^{53 54} These changes support the hypothesis of “inflame-aging,” which suggests that immunosenescence is driven by an increasing lack of oral and self-tolerance that results in a chronically elevated perceived antigenic load, which not only induces enormous expansion of effector T cells, but increases their production of pro-inflammatory cytokines. In older subjects, these cells appear to be equipped with a greater capability to produce IFN-γ and TNF-α, two cytokines, while they amplify, via IFN-γ, the immune response against internal or external pathogens, also, promote an unremitting inflammatory and/or autoimmune processes that negatively correlate with human longevity.⁵⁵

Vitamin A Assessment

The most commonly used indicator of vitamin A status is the serum retinol concentration. If measuring serum retinol, ensure the sample is protected from light and heat. Normal range using HPLC methodology is 24 to 90 µg/dL.^{56 57}

Since retinol is unstable when exposed to heat or light, and HPLC requires costly, complicated lab equipment, making it impractical for field studies, retinol-binding protein has been proposed as a surrogate measure. RBP is significantly more light-and-heat stable than retinol, with which it is released into the circulation from the liver in a 1-1 complex. Despite the fact that RBP concentrations may fall under circumstances of protein malnutrition or the acute phase response (which accompanies inflammatory states), equimolar RBP cutoffs have been shown to predict vitamin A deficiency with high sensitivity and specificity, even in the context of significant infection (HIV-1) and protein malnutrition. RBP cutoffs of 1.05 µmol/L and 0.70 µmol/L identify marginal vitamin A status (retinol < 1.05 µmol/L) and vitamin A deficiency (retinol < 0.70 µmol/L), respectively. Finally, the techniques used to quantify serum RBP are easier and less expensive.⁵⁸

Conclusion

Large individual variations in the actual absorption of β-carotene from foods and its conversion to retinol demonstrate that reliance on current data on retinol equivalents for vitamin A sufficiency is highly misleading and that a significant percentage of the American population may be vitamin A insufficient, despite the fact that foods containing pre-formed vitamin A and β-carotene are generally available. Given recent discoveries regarding the importance of vitamin in balancing the activities of vitamin D and its pivotal role in promoting oral and self- tolerance, thus counterbalancing the tendency to “inflammaging,” vitamin A sufficiency should be considered a key factor in healthy aging.

Aging is characterized by a peculiar chronic inflammatory status for which researchers have recently coined the term, “inflammaging,” a key aspect of which is the age-dependent expansion of effector T cells with pro-inflammatory cytokine production potential. Particularly in light of vitamin A’s emergence as a pivotal inducer of oral and self-tolerant immune function, vitamin A sufficiency should be recognized as a key factor in healthy aging.

References