Pocket Guide to Micronutrients in Health and Disease
3 - FAT SOLUBLE VITAMINS A, D, E AND K
3.1 Vitamin A and provitamin A carotenoids
Vitamin A or retinoids are generic terms for all compounds that exhibit a
“retinol-like” activity including both natural and synthetic molecules. Retinol, the main representative, contains a β-ionone ring with a two-isoprenoid-unit lateral chain containing a final alcohol function. Biologically active vitamin A derivatives are produced in target tissues leading to retinal and/or retinoic acid.
Vitamin A is essential for growth, vision and reproduction. Animal products provide pre-formed vitamin A (liver, dairy products, eggs or fish liver oil) whe-reas provitamin A carotenoids (α-, β-carotene, and β-cryptoxanthin) are mainly provided by fruits and vegetables. Both compounds are fat soluble and absor-bed like lipids. Dietary vitamin A is normally stored in the liver under esterified form. According to body needs, vitamin A is secreted as retinol into blood circu-lation, bound to Retinol Binding Protein (RBP) and transthyretin (TTR). In the tar-get tissues, retinol is then metabolized into active form to exert its biological activity (in visual cycle or in regulation of gene expression).
It is now recommended to use the new retinol equivalent (RE) system rather than the older International Unit (IU) system (using retinol as standard activity).
To take into account carotenoid activity, conversion factors of 1/6 for β
-caro-tene and 1/12 for the other provitamin A carotenoids are applied. It should, however, be pointed out that these factors are approximate and depend on various considerations, especially those affecting the bioavailability of carote-noids.
3.1.1 Nutritional status
Vitamin A status can be estimated by measuring dietary intakes or blood level. However, the blood level is strictly regulated and is useful only for severe deficiency or hypervitaminosis. Vitamin A liver stores can be qualitatively esti-mated by using the relative dose-response test (RDR) and/or the modified RDR test (MRDR). The best method of quantitative measurement is determination of vitamin A level in liver biopsies following stable isotope dilution.
Precise data on vitamin A status have been obtained from two recent epide-miological studies (ASPCC, RIGAUD et al., 1997, and SU·VI·MAX, HERCBERG et al., 1998) showing that hypovitaminosis A is virtually not existent in France, even if marginal status might be observed during disease events in children or elderly. However, it must be pointed out that vitamin A intakes exceeded the safety limits for 5 percent of the population.
3.1.2 Requirements, toxicity and ANCs
Estimation of requirements in Man is based on two old studies using vitamin A deficiency and repletion phases (HUME and KREBS1949), and utiliza-tion rate of radioactive vitamin A (SAUBERLICHet al., 1974). Both concluded that minimal requirements were 400-600 RE and that the suggested allowances for vitamin A storage might be 750-1200 RE. On the basis of physiological and bio-chemical parameters, OLSON(1987) suggested that levels were overestimated and requirements were re-evaluated to 600-700 RE. When a 30% factor is applied, to take into account inter-individual differences (the use of standard deviation is meaningless in the context), the ANCs are established at 800 RE for an adult male.
Based on energy requirements, corrections may be applied to calculate ANCs of specific subgroups of the population, such as adolescents and the elderly. It is not necessary to modify ANCs during pregnancy, but lactating women should be supplied with an extra 350 RE·d–1, corresponding to the vita-min A exported in milk, which increases the ANCs of lactating mothers up to 950 RE·d–1. Finally, since 60% of vitamin A is usually provided by carotenoids, we recommend a dietary intake of 2.1 mg β-carotene equivalent to 350 RE·d–1.
Chronic liver damage has been observed after consumption of 7500 mg RE·day–1 for months. Vitamin A teratogenicity may occur during the first two months of pregnancy and from intakes of 7500 µg RE·d–1. Neither liver toxicity nor teratogenicity have been observed after high levels of carotenoid supplementation.
3.2 Vitamin D
To propose optimal dietary intakes for vitamin D is even more difficult than for the other vitamins. Indeed, in temperate regions like France and under usual professional and living conditions, a substantial part of the body pool derives from the skin production of vitamin D after exposure to UV solar light (STAMPet
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al., 1977; MCKENNA, 1992; HOLICK, 1994, 1995; GARABÉDIANet al., 1999). Dietary needs thus depend on geographical, environmental, social, cultural, and genetic factors that may limit the access of the individuals to optimal solar irradiation (290-315 nm).
Two main vitamin D forms are present in food: vitamin D2, or ergocalciferol, produced by vegetables, and vitamin D3, or cholecalciferol, present in foods of animal origin. Both vitamin D forms are active in humans at grossly similar concentrations. Mean daily dietary intakes of vitamin D in France amount to 2 to 4 µg, 80-160 IU·d–1, with a 1 to 9 µg·d–1range for 95 percent of the adult popu-lation (ESVITAF, 1986; HERCBERGet al., 1994). But few foods contain significant amounts of vitamin D, mainly sea fish like salmon, herring, trout, sardine (10-20 µg per 100 g), and, to a lesser extent, mackerel, eel, halibut and tuna fish (3-7 µg per 100 g). In contrast, other vitamin D-containing foods like eggs, meat and meat products, milk and dairy products contain less than 0.2-2 µg per 100 g (FAVIERet al., 1995).
Vitamin D is almost totally (80%) absorbed in the small intestine. Both, the exogenous and the skin produced calciferols are metabolized by liver cells into 25-(OH) D (for review: BOUILLONet al., 1995; GARABÉDIANet al., 1998). This 25-hydroxylated reserve form has a half-life in blood of approximately 1 month. It is further transformed into several metabolites including 1,25-dihydroxyvitamin D, the main active form of vitamin D. This metabolite is produced by kidney cells of the convoluted proximal tubule at an average rate of 0.3 to 1 µg per day in adults. The rate of renal 1,25-(OH)2D production depends on calcium and phos-phate needs, and is highest when dietary intakes of calcium are low, when there is phosphate deficiency, and during fetal and postnatal growth. Other cell types may be additional sources of 1,25-(OH)2D, mainly cells of the foeto-placental unit during normal pregnancy, or macrophages and activated T lymphocytes in pathological conditions like granulomatosis. Because of the short half life of 1,25-(OH)2D in blood, 10-12 hours, physiological and pathological conditions that increase 1,25-(OH)2D production also accelerate vitamin D catabolism and aggravate the risk for vitamin D deficiency.
3.2.1 Deficiency and populations at risk for vitamin D deficiency in France Features of vitamin D deficiency classically include defective skeletal minera-lization (bone deformations, rickets and osteomalacia), muscular hypotonia, neu-romuscular signs of hypocalcemia, and possibly hematological alterations. The biological signs include low serum calcium and phosphate concentrations, low urinary calcium excretion, increased serum alkaline phosphatase activity, increa-sed serum levels of parathyroid hormone, and low serum levels of 25-(OH)D.
Populations at risk are those who cumulate several of the following risk fac-tors (MC KENNA, 1992; HOLICK, 1994,1998; CHAPUYet al., 1997; SHARLA, 1998;
GARABÉDIANet al., 1999):
- insufficient exposure to solar light (winter-spring, pollution, heavy cloud cover, clothing habits);
- low ability of the skin to produce vitamin D (heavy skin pigmentation, elderly);
- low vitamin D intakes (reduced intakes, vegetarian, vegetalian, macrobiotic diets);
- increased vitamin D needs ( pregnancy, growth, low calcium diets…).
Three age groups are thus especially at risk for vitamin D deficiency:
- Pregnant and lactating women;
- Neonates, infants;
- Elderly, especially when institutionalized.
In addition, several pathological conditions may increase the risk for vitamin D deficiency because they alter vitamin D absorption or production (intestinal malabsorption, liver diseases, extensive skin lesions) or they activate the catabolism of vitamin D (anticonvulsant drugs, granulomatosis).
3.2.2 Markers of vitamin D status
Aside from the clinical signs of overt vitamin D deficiency (rickets, osteoma-lacia, clinical signs of hypocalcemia) or vitamin D intoxication (nephrolithiasis, nephrocalcinosis, clinical signs of hypercalcemia), several biological markers for vitamin D status are available.
The best marker of vitamin D status is the circulating level of 25-(OH)D, the reserve form of vitamin D. Levels below 5 ng·mL–1(12 nmol·L–1) indicate a state of vitamin D deficiency, whether clinical or subclinical. Less severely decreased levels (5-12 ng·mL–1, 12-30 nmol·L–1) indicate high risk for vitamin D deficiency in pre-gnant women, neonates, infants and elderly (MC KENNA, 1992; CHAPUYet al., 1997;
GARABÉDIANet al., 1998; SCHARLA, 1998). At other ages, they reflect low vitamin D status but not necessarily vitamin D deficiency, unless they are associated with hypocalcemia or elevated circulating levels of parathyroid hormone. Inversely, vita-min D overload is evidenced by 25-(OH)D levels above 80-100 ng·mL–1 (200-250 nmol·L–1), or by levels at the upper limit of normal range (40-80 ng·mL–1, 100-125 nmol·L–1) when they are associated with hypercalcemia or hypercalciuria.
3.2.3 Requirements and recommended dietary intakes
3.2.3.1 Requirements
Vitamin D daily requirements for children and adults have been estimated to average 10 µg per day, 400 IU·d–1 (HOLICK, 1994,1998; GLERUP et al., 2000).
They may be higher (10-15 µg, 400-600 IU·d–1) for pregnant women, neonates and infants, as shown by the beneficial effects of a daily 10-15 µg supplements on the calcium homeostasis of pregnant women and neonates as well as on intra-uterine and post-natal growth (for review GARABÉDIANet al., 1998).
3.2.3.2 Recommended daily intakes for healthy children and adults
As mentioned above, it is difficult to estimate the level of dietary vitamin D intakes that should be recommended for children and adults, as the endoge-nous production of this vitamin covers a significant part of the vitamin D requi-rements. Based on the biological follow-up of healthy populations normally exposed to sunlight, skin production covers 50-70 percent of the vitamin D requirements. On such a basis, one may recommend a daily dietary intake of 5 µg·d–1(200 IU·d–1), that is 50% of the total daily needs, for healthy popula-tions normally exposed to sunlight. An identical value has been proposed as reference value for food labeling by the Commission of the European Communi-ties (SCF, 1993), and is now considered to be adequate for adults and children in the USA (HOLICK, 1998).
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3.2.3.3 Recommended daily intakes for infants, pregnant women, elderly, and other populations at risk for vitamin D deficiency
These populations are those most at risk for insufficient exposure to solar light. In addition, they may have a low ability of the skin to produce vitamin D (heavy skin pigmentation, elderly), and/or low dietary intakes (malnutrition, diets not including fish or meat). Finally, they may have increased needs for vitamin D (pregnancy, growth). For these reasons, one may recommend dietary intakes of 10 µg·d–1(400 IU·d–1), which will cover the total daily needs of individuals not exposed to sunlight. Because such intakes are higher than usual intakes in France (9 µg·d–1for the 95thpercentile of adult healthy populations), a systema-tic prophylaxis of vitamin D deficiency by oral supplements remains mandatory for pregnant women (80,000-100,000 IU during the 6th-7th months of pre-gnancy), infants (daily supplements of 1,000-1,500 IU including the vitamin D present in infant formula, semestrial doses of 200,000 IU or trimestrial doses of 100,000 IU) and elderly not exposed to the sun during summer and fall. This recommendation can be extended to at risk individuals in other age groups.
3.2.4 Beyond the needs
Clear clinical signs of vitamin D overload (loss of weight, growth retardation, vascular hypertension, polyuria-polydipsia, renal insufficiency, extra skeletal cal-cifications, fetal anomalies) have been observed after:
– chronic administration of 10,000 to 50,000 IU·d–1(250-1250 µg·d–1) in adults;
– several administration of 600,000 IU (15 mg) in pregnant women;
– chronic administration of trimestrial doses of 600,000 IU (15 mg) or of daily doses of 4,500 to 30,000 IU·d–1(112-750 µg·d–1) in infants.
In contrast, prolonged administration of 2,000 IU·d–1(50 µg·d–1) to neonates does not alter infantile growth and does not lead to excessive 25-(OH)D levels.
Based on these observations, a daily security limit of 1,000 IU (25 µg) in addition to the vitamin D present in food has been proposed for children and adults in France normally exposed to sunlight. A higher tolerable level, 2,000 IU (50 µg) has been proposed for infants under two years of age (CSHPF, 1996).
3.3 Vitamin E
Plants contain 8 vitamin E congeners formed with a saturated (tocopherols) or unsaturated (tocotrienols) lateral chain and a chromanol ring substituted with 1, 2 or 3 methyl groups (α, β, γ, δcompounds). Synthetic tocopherol is a mixture of 8 stereoisomers called all-rac-α-tocopherol (DELBAL, 1992). Vegetal oils and secondary products are the richest dietary sources of vitamin E contributing at 50-70% dietary supplies. Fruits and vegetables, and animal products are res-pectively the second and the third sources of vitamin E.
Biological equivalence of vitamin E congeners is first based on their capacity to restore gestation in rats, but the antioxidant capacity has been recently proposed as equivalence parameter. Because the antioxidant value is largely dependent on the test used and on the bioavailability of the different vitamin E compounds, all-rac-α-tocopherol acetate was chosen as a reference (1 mg = 1 IU). Some authors have proposed the tocopherol equivalence unit (1 TE = 1 mg RRR-α-tocopherol),
but we recommend to express vitamin E equivalence as mg of compound whene-ver it is possible.
Free tocopherol absorption is closely related to that of lipids. Vitamin E is incorporated into chylomicrons, secreted in blood circulation via the lymph, to extrahepatic tissues. Vitamin E is mainly distributed in adipose tissue and adre-nals but it can also be absorbed by other tissue through apo B/E membranes receptors.
The main property of vitamin E is its capacity to scavenge peroxyl free radi-cal and consequently to prevent propagation of polyunsaturated fatty acid (PUFA) peroxidation (DEVARAJet al., 1997). Vitamin E is a safe compound and no side effects were observed after 40-1000 mg·d–1supplementation, but it is suggested to limit such interventions for medical investigations.
3.3.1 Nutritional status
The vitamin E status can be estimated through recording of dietary intakes or measurement in blood. In normocholesterolemic individuals, the range of plasma vitamin E is around 12 mg·L–1, and concentrations lower than 4-6 mg·L–1could reflect a marginal vitamin E status. Recently, it has been sugges-ted that the ratio of γ and α tocopherol in adipose tissue (HANDELMAN et al., 1994) or the vitamin E levels in platelets and red blood cells (LEHMANet al., 1988), could be better biomarkers.
Vitamin E deficiency is not common in humans. In experimental studies, it produces various damage of nervous cell membranes (VATASSERY, 1997). In adults, vitamin E deficiency can be the consequence of long-term alterations of lipid absorption and metabolism (i.e. low incorporation in VLDLs, familial hypo-β-lipoproteinemia in its homozygous form). Vitamin E status in the French popu-lation was estimated from 5 epidemiological studies showing no marginal vitamin E status (based on blood level measurement) despite of median intakes ranging between 5.6 and 11 mg·d–1. However, it was shown that for 5% of the population daily intake was less than 5 mg.
3.2.2 Requirements and ANCs
Since 1992, the ANCs for adults (12 mg·d–1) has not been modified. The ANCs for the other groups of subjects has been calculated on energy allo-wances basis. In most studies, no side effects were observed after 40-1000 mg·d–1 supplementation and many epidemiological studies reported a positive association between relatively high level of vitamin E (not reachable by dietary means) and the cardiovascular disease risk or other oxidative-related diseases (LECERF, 1997). Data about protective effects of dietary vitamin E intakes are stimulating but have to be confirmed before recommending higher ANCs for vitamin E. Supplementation is recommended for preterm infants because they often exhibit low stores of vitamin E; together with oxygeno-the-rapy, this low status may induce hemolytic anemia.
3.4 Vitamin K
Vitamin K is a generic term for fat-soluble cofactors associated to protein activation (like enzymatic carboxylation of glutamate to γ-carboxyglutamic acid
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in proteins). A well-known example is the activation of blood “clotting” factors (like factors II, VII, IX and X) (SUTTIE, 1995) or proteins involved in bone metabo-lism (like osteocalcin) (SAUPEet al., 1993). All vitamin K molecules contain a 2-methyl-1-4-naphtoquinone group and occur naturally in plants (phylloquinone) and in bacteria (menaquinone) or in synthetic form (menadione).
Dietary vitamin K (mainly found as phylloquinone in cabbages, salad, spi-nach and oils) is incorporated in lipid droplets, transferred in mixed micelles and absorbed by proximal jejunum. Then vitamin K is encapsulated into chylomi-crons and transported up to liver where it is transfered to VLDLs. In blood, it is largely distributed in VLDLs, and at lower levels in LDLs and HDLs. Liver accu-mulates specifically menaquinones and secretes phylloquinones into blood (SHEARERet al., 1988).
3.4.1 Nutritional status
Biological status is estimated through indirect measurements such as clot-ting tests. Determination of γ-carboxyprothrombin with monoclonal antibodies is only limited to clinical measurements (BELLE et al., 1995). Because vitamin K metabolism is quite complex, the best way to assess nutritional status is HPLC measurement at the blood level. In adults, usual phylloquinone concentration ranges between 150 and 1150 ng·L–1(GUILLAUMONTet al., 1998).
As dietary intakes were not determined during recent epidemiological stu-dies (insufficient food composition databases, lack of valid clinical biomarkers), no clear information is yet available.
In humans, vitamin K deficiency is marginal. It can result from very low intakes (parenteral nutrition), an alteration of intestinal absorption (lipid malab-sorption, altered pancreatic functions, antibiotherapy), or anticlotting treatment.
In newborns, vitamin K levels are very low, and consequently, hemorrhagic events can be observed in the gastrointestinal tract and in brain. It is thus recommended to supplement newborns with vitamin K.
3.4.2 Requirements, toxicity and ANCs
Vitamin K is safe, because even after high dosage of vitamin K, no toxicity has been reported. However, long term supplementation is not justified due to the adequacy of dietary supply.
Vitamin K requirements of adults are not precisely known but it is largely admitted that they are extremely low (0.1-1 µg·kg–1body weight·d–1). Recently, it has been suggested to re-evaluate the requirements using complete γ-car-boxylation as endpoint (SHEARER, 1995).
ANCs would be of 45 µg·d–1 for adults and 10 µg·d–1 for newborns and infants. There would be no difficulties for adequate supply of vitamin K, since dietary intakes are estimated around 300-400 µg vitamin K per day. Specially, for newborns under prophylactic treatment against hemorrhagic events, 2 mg dosage per os followed by 2 mg per week during the breast lactation period is recommended.