Vitamins are any of a group of complex organic substances essential, in small amounts, for the normal functioning of the body. There are 13 vitamins: A, C, D, E, K, B 12 , and seven grouped under the vitamin B complex.
Apart from vitamin D, which the body can synthesize itself, vitamins must be obtained from the diet. A varied diet is likely to contain adequate amounts of all the vitamins, but vitamin supplements may be helpful for: young children, pregnant or breast-feeding women, or those taking drugs that interfere with vitamin function.
Vitamins can be categorized as fat-soluble or water-soluble.
Fat-soluble vitamins (A, D, E, and K) are absorbed with fats from the intestine into the bloodstream and are then stored in fatty tissue (mainly in the liver). Because body reserves of some of these vitamins last for several years, a daily intake is not usually necessary. Deficiency of a fat-soluble vitamin is usually due to a disorder in which intestinal absorption of fats is impaired (malabsorption) or to a prolonged poor diet.
Vitamins C, B12 , and those of the B complex are water-soluble. Vitamin C and B complex vitamins can be stored in the body in only limited amounts and are excreted in the urine if taken in greater amounts than needed. A regular intake is therefore essential to prevent deficiency. However, vitamin B12 is stored in the liver; these stores may last for years.
Function in the body
The role of all the vitamins in the body is not fully understood. Most have several important actions on one or more body systems, and many are involved in the activities of enzymes.
Vitamins and trace elements in detail - technical
Vitamins are diverse, unrelated organic compounds that some higher animals, humans included, cannot synthesize and which play key roles in metabolism, underpinning the most crucial of biological reactions. They are required in small amounts and have diverse functions, e.g. as:
- ◆ activated carriers of biochemical groups—coenzymes (B vitamin derivatives)
- ◆ antioxidants (vitamins A, C and E, carotenoids)
- ◆ precursors of visual pigments (vitamin A)
- ◆ endocrine mediators, especially in calcium and phosphorus metabolism (vitamin D)
- ◆ facilitators of blood clotting (vitamin K)
The clinical importance of vitamins lies not only in overt deficiency syndromes, which develop because of persistent inadequate intake or absorption over different periods of time depending upon body storage, but also in the need for optimal intakes to maintain health. This latter role can be difficult to assess for individual nutrients, but is inferred from knowledge of the beneficial and harmful effects of diets containing particular foods.
The traditional classification of vitamins into water and lipid soluble, and by their associated deficiency conditions, becomes less useful as their biochemical roles are better understood. An inadequate dietary vitamin intake will result in specific cellular failure and even death.
Vitamins as biochemical cofactors
Oxygen is the final electron acceptor when food is oxidized, but the transfer from energy substrate to oxygen is not direct; it occurs via intermediaries, important among which are molecules such as nicotinamide adenine dinucleotide (NAD) or flavin adenine dinucleotide (FAD) derived from the B vitamins niacin and riboflavin respectively.
Other metabolically crucial fragments require activated carriage, e.g. thiamine is a carrier for aldehyde groups, folate for one-carbon groups, and coenzyme A (pantothenate) carries two-carbon (acetyl) fragments into the tricarboxylic acid cycle. Thus a small group of molecules derived from the B vitamins is responsible for diverse biochemical interchanges. The stability of these carrier molecules in the absence of catalysts enables enzymes to control the flow of free energy and reducing power.
Vitamins as antioxidants
Although molecular oxygen is an ideal final electron acceptor, ‘danger lurks in the reduction of O2’. Partial reduction, particularly the transfer of single electrons to form superoxide or two electrons to form peroxide, yields potentially damaging products or reactive oxygen species. White cells use this process to kill pathogens, and most cells are protected from it by antioxidants, particularly the enzyme superoxide dismutase—an enzyme that contains manganese in the enzyme’s mitochondrial form and copper and zinc in the cytoplasmic forms. The antioxidant vitamins C and E are also important in this process, with fat-soluble vitamin E functioning particularly to protect membranes from lipid peroxidation. NADPH generated by glucose-6-phosphate dehydrogenase maintains levels of reduced glutathione.
Intake of vitamins
There is a dose–response effect of vitamin intake, ranging from the physiological through the pharmacological to the toxic. Recommendations for vitamin intake for different ages, needs, and communities are based on dietary intake, bioavailability, steady-state concentrations in plasma and tissue at defined intakes, urine excretion, adverse effects, biochemical and molecular function, and freedom from deficiency. With increasing intake, either orally or as an infusion, a vitamin is distributed through the body fluids and tissues until the saturation point is exceeded.
Some artificial enteral supplements and feeds and most parenteral feeds are deficient in vitamins unless appropriately supplemented: it is the responsibility of the prescriber to ensure that appropriate vitamin intake is maintained during artificial feeding. The prescription of vitamins parenterally, bypassing the absorptive processes, also has dosage implications.
Trace elements (e.g. magnesium, iron, zinc, copper, manganese, fluoride, selenium, molybdenum, chromium, iodine) are essential nutrients that act as cofactors in enzyme oxidation–reduction reactions. They maintain the specific configuration of proteins, are incorporated into the structure of hormones, and play a structural and catalytic role in gene expression and transcriptional regulation.
Deficiency of trace elements can cause a very wide range of clinical problems, including anaemia from iron deficiency and goitre resulting from iodine deficiency, which is endemic in mountainous areas.
Our understanding of the role of vitamins comes from clinical observations, nutritional experiments in animals, and studies using purified preparations of the active principle used to treat deficiency states. Early pioneers differentiated dietary deficiency from infection and other causes of disease. Scurvy was once the scourge of mariners and explorers, but the clinical trials of Lind (1753), confirmed by Captain Cook on his voyages, showed the benefits of citrus fruits. Many years later, Holst and Froelich (1907) produced scurvy in guinea-pigs by dietary deprivation.
Rickets arose in sun-starved urban slums, and Trousseau noted the beneficial effects of cod liver oil (1860). Ejikmann and Grijns fed chickens the same diet as their patients who had beriberi (1897); neuropathy in the chickens resolved when the diet contained whole-grain, rather than polished, rice. In the 1900s, Gowland Hopkins, the discoverer of vitamins, described a fat-soluble, essential growth accessory food factor A in milk. This was differentiated from water-soluble accessory food factor B by McCollum and Davis. Mellanby treated rickets in puppies with a fat-soluble food factor D. In 1931 Lucy Wills described the megaloblastic anaemia of pregnancy, which is now known to be caused by a deficiency of folic acid.
In 1894, Atwater published a table of food composition and dietary standards for the United States of America. The first USA Recommended Daily Allowances was published in 1941. Food rationing in the United Kingdom during the Second World War was a triumph for the science of applied nutrition. The natural development of this work was the emergence of recommended daily intakes that recognized the differing requirements of the young and growing, pregnant and lactating, middle-aged and old, and ill; with this, developed the concept of the optimal intake for optimal nutritional status. The isolation and chemical synthesis of the vitamins and their active principles provided formidable challenges to scientists, rewarded by eight Nobel prizes in medicine and physiology and four in chemistry.
The carotenoids illustrate the complexity of vitamins in physiology and pharmacology, and as toxins. Carotenoids are used by archaebacteria to reinforce cell membranes, their long, rigid carbon backbone acting as a rivet across the membrane. The polyene chain of between 9 and 11 double bonds serves to harvest light energy in plants, and, as the pigment retinal, is a visual pigment in animals. The linear system of conjugated C=C bonds make for a high reducing and antioxidant potential. Carotenoids act as the coloration in plants and to protect egg proteins against the enzymatic activity of proteases. Retinoic acid in animals and abscisic acid in plants act as hormones. When retinoids and carotenoids were used in lung cancer chemoprevention trials, the incidence of cancer increased; this was ascribed possibly to the increase in the oxidized products of β-carotene. A mix of vitamins and antioxidants might prevent such oxidation. Such a mix can be found in fruit and vegetables, which emphasizes the benefit of a good diet containing five portions (60 to 150 g) of fruit and vegetables a day.
An inadequate dietary vitamin intake will result in specific cellular failure and even death. There is a dose–response relationship with vitamin intake from the physiological through the pharmacological to the toxic. Recommendations for vitamin intake for different ages, needs, and communities are based on dietary intake, bioavailability, steady-state concentrations in plasma and tissue at defined intakes, urine excretion, adverse effects, biochemical and molecular function, and freedom from deficiency. With increasing intake, either orally or as an infusion, a vitamin is distributed through the body fluids and tissues until the saturation point is exceeded. The prescription of vitamins parenterally, bypassing the absorptive processes, also has dosage implications.
The traditional classification of vitamins into water-soluble and lipid-soluble and by their associated deficiency conditions becomes less useful as their biochemical roles are better understood, but is still widely used and is adopted here for that reason.
Vitamins as biochemical cofactors
Oxygen is the final electron acceptor when food is oxidized, but the transfer of electrons from energy substrate to oxygen is not direct; it occurs via intermediaries, important among which are molecules such as nicotinamide adenine dinucleotide (NAD) or flavin adenine dinucleotide (FAD) derived from the B vitamins niacin and riboflavin respectively. Other metabolically crucial fragments require activated carriage. For example, coenzyme A, derived from pantothenic acid, carries two-carbon (acetyl) fragments into the tricarboxylic acid cycle. Thiamine is a carrier for aldehyde groups, folate for one-carbon groups, and coenzyme A (pantothenate) for acyl groups. Thus, a small group of molecules derived from the B vitamins is responsible for diverse biochemical interchanges. The stability of these carrier molecules in the absence of catalysts enables enzymes to control the flow of free energy and reducing power.
Vitamins as antioxidants
As Stryer and colleagues have noted, although molecular oxygen is an ideal final electron acceptor, ‘danger lurks in the reduction of O2’. Partial reduction, particularly the transfer of single electrons to form superoxide or two electrons to form peroxide, yields potentially damaging products or reactive oxygen species. White cells use this process to kill pathogens. Most cells are protected from this process by antioxidants, particularly the enzyme superoxide dismutase (EC 18.104.22.168)—an enzyme that contains manganese in the mitochondrial form and copper and zinc in the cytoplasmic forms. The antioxidant vitamins, vitamin C and vitamin E, also are important in this process, with fat-soluble vitamin E functioning particularly to protect membranes from lipid peroxidation. NADPH generated by glucose-6-phosphate dehydrogenase (EC 22.214.171.124) maintains levels of reduced glutathione.
Vitamin C (ascorbic acid)
L-Xyloascorbic acid is the naturally occurring form of vitamin C. The active forms are L-ascorbic acid and L-dehydroascorbate to which it is reversibly oxidized. Further oxidation to 2–3 diketo-L-gulonic acid and oxalate is irreversible. Vitamin C is labile; it is oxidized by removal of two electrons, producing the ascorbate free radical and then dehydroascorbate. The fairly unreactive ascorbate free radical acts as a free radical chain terminator. Importantly, it is widely distributed through the body’s aqueous fluids. In contact with membranes, it maintains vitamin E, which is fat soluble, in its reduced form. The interaction between ascorbate iron and copper is important. Ascorbic acid is a strong antioxidant and reduces ferric (Fe3+) and cupric (Cu2+) ions to ferrous (Fe2+) and cuprous (Cu+), and reduces oxygen to superoxide (O2−) and hydrogen peroxide (H2O2). Paradoxically, Fe2+ and Cu+ act as pro-oxidants by forming OH from superoxide and hydrogen peroxide.
- ◆ is a water-soluble, non-specific radical-trapping antioxidant and reducing agent, which is present in all tissues
- ◆ acts synergistically with vitamin E
- ◆ is needed to maintain the activity of copper-containing hydroxylase enzymes such as prolyl hydroxylase, an enzyme that requires Fe2+ to activate oxygen. (Oxidation of the iron deactivates the enzyme—ascorbate reduces the inactivated enzyme’s Fe3+, reactivating it by functioning as an antioxidant. Collagen formed in the absence of ascorbate is less stable than normal collagen, which explains many of the signs of clinical scurvy, e.g. the bleeding gums.)
- ◆ is involved in carnitine biosynthesis from lysine, which is necessary for transport of long-chain fatty acids from the cytosol into mitochondria
- ◆ is involved in the synthesis of catecholamines
- ◆ is involved in the function of cytochrome P450 microsomal enzymes
Dietary sources of ascorbic acid are fresh fruit and fruit juices, especially blackcurrants, guavas, green leafy vegetables, and fresh milk. Ascorbic acid is readily oxidized during cooking—a process accelerated by traces of copper in alkaline solution. Since humans, guinea-pigs, the Indian fruit-eating bat, the red vented bulbul, and some birds are unable to synthesize ascorbic acid, it represents a vitamin in these species.
Ascorbic acid is rapidly absorbed from the small intestine. The plasma concentration (5% as dehydroascorbate) and dietary intake are in a sigmoidal relationship (80 µmol/litre or 100 mg/day) which plateaus at 1000 mg/day intake. The body pool size is 900 mg (5 mmol/litre in the normal adult): approximately 3%, irrespective of pool size, is degraded each day and excreted in the urine as free ascorbic acid, dehydroascorbate, or diketogulonate. High tissue concentrations at birth steadily decline with increasing age.
A shortfall in vitamin C intake without clinical scurvy may be associated with a reduction in the body’s water-soluble antioxidant capacity, the consequences of which are still being debated. A large-scale prospective study among a representative sample of people aged 75 to 84 demonstrated blood ascorbate’s strong predictive power of mortality over a 4.4 year follow-up. In this British study, 232 of 1175 subjects had plasma ascorbate levels of less than 17 µmol/litre, the lowest quintile. Young people and elderly people are particularly at risk of scurvy, which results from an inadequate intake of ascorbic acid. Clinical scurvy appears after 4 weeks on an ascorbic acid-deficient diet when the body pool is less than 300 mg (1.7 mmol/litre). There is a failure of connective tissue collagen synthesis, and cartilage, bone, and dentine growth are all compromised. Tissues bleed readily, and heal poorly due to defective intracellular linkages between the endothelial cells and capillary basement tissue. Individuals are initially lethargic and irritable. Characteristic livid-coloured, spongy, bleeding gingivitis of the gums develops, and scurvy buds appear in the papillae between the teeth. Large or microscopic haemorrhages occur in the gums, as well as in the eyes (especially bulbar conjunctiva), subcutaneous tissues, synovia of joints, and beneath the periosteum of bones. Perifollicular bleeding occurs in the dependent parts of the body, later becoming more generalized. Fatal haemorrhages may also occur in the brain or heart muscle. Keratin-like material heaps on the surface of hair follicles, through which a deformed corkscrew hair projects. Other signs include dependent oedema, oliguria, depression, megaloblastic or normoblastic anaemia, and superinfection. Infants present with irritability, tender legs, and pseudoparalysis. Scurvy buds, but not gingivitis, occur in edentulous infants. Large subperiosteal haemorrhages develop over the long bones, especially the femur.
Diagnosis requires an awareness of the condition, a careful dietary history, and a clinical examination. The patient can improve on hospital diet alone. Ascorbic acid is measured in plasma or whole blood. While leucocyte or buffy-coat vitamin C concentrations reflect tissue concentrations, this is complicated in disease by different leucocytes types, which vary in number and ascorbic acid content, but a lower limit of 15 µg/108 cells is frequently accepted as an indicator of deficiency. Plasma vitamin C is more sensitive to recent change in intake with values less than 11 µmol/litre(0.2 µg/100 ml) indicative of depletion.
The optimum dietary intake of ascorbic acid has yet to be defined. The body can be saturated with 1 g/day for 5 days. Recommendations for dietary intake range from 40 to 200 mg per day. An upper limit of intake is recommended at 1 to 2 g/day, based upon body saturation figures rather than toxicity. The dietary reference values for adults of both sexes over the age of 19 are 10 mg/day (lower reference nutrient intake), 25 mg/day (estimated average requirement) and 40 mg/day (reference nutrient intake). During pregnancy and lactation, intake should be increased by 10 mg/day and 30 mg/day, respectively. The ascorbic acid content of breast milk varies between 30 and 80 mg/litre, which provides 25 mg/day; clinical scurvy has not been observed in fully breastfed infants. The pharmacological use of ascorbic acid is extensive and imaginative.
The first two B vitamins to be considered, riboflavin and niacin, function as fundamental electron carriers into and within the mitochondrion. There are four sites of entry of electrons into the electron transport system: one for NADH (complex 1) and three for FADH2 (complex II). Flavoproteins are major components of mitochondrial complexes I and II, which transfer electrons through flavin mononucleotide (FMN) and iron–sulphur complexes to ubiquinone. Ubiquinone transfers electrons to complex III, a cytochrome/iron/sulphur enzyme complex. We shall return to iron later in the chapter. The flow of electrons to oxygen to form water, pumps protons out of the mitochondrion and produces a proton gradient which enables synthesis of ATP.
Storage of B vitamins in the body is poor, with the exception of B12, and deficiencies occur early in the presence of reduced intake. Biochemical, mixed deficiencies of B vitamins are common among patients admitted as an emergency to hospital and also among alcoholics.
Riboflavin (vitamin B2)
Riboflavin is a substituted alloxazine ring linked to ribotol, an alcohol derived from the pentose sugar ribose. It is light sensitive. Riboflavin links with phosphoric acid as FMN (or riboflavin 5′-phosphate), which, with adenosine monophosphate (AMP), forms flavin adenine dinucleotide (FAD). FMN and FAD are the prosthetic groups of the flavoprotein enzymes. Flavoproteins are involved in redox processes involving the hydrogen transfer chain in the mitochondria and the production of ATP. FAD/FMN acts as a coenzyme in oxidation–reduction reactions, electron transport, oxidative phosphorylation (e.g. succinic dehydrogenases), and β-oxidation of fatty acids.
Dietary sources are liver, milk, cheese, eggs, some green vegetables, and beer. Other sources are yeast extracts (e.g. Marmite) and meat extracts (e.g. Bovril). Riboflavin in the diet exists in either the free form or the phosphorylated coenzyme form. Riboflavin is absorbed from the upper gastrointestinal tract, there is no specific storage tissue, and it is excreted in the urine either free or in small amounts of hydroxylated products. Chronic infection can affect urinary riboflavin excretion. A deficiency of riboflavin causes cheilosis, angular stomatitis, superficial interstitial keratosis of the cornea, and nasolabial seborrhoea. Riboflavin deficiency may impair iron absorption. No toxic effects have been shown for riboflavin.
Riboflavin status can be estimated from the urinary riboflavin:creatinine ratio, which is insensitive at low intakes. The erythrocyte glutathione reductase activation coefficient (EGRAC) is a functional test, which measures tissue saturation.
Adults, including elderly people, need between 1 and 1.5 mg of riboflavin per day. The average riboflavin content of breast milk in Britain is approximately 0.3 mg/litre, which is dependent upon maternal intake. Intakes should increase by 0.3 mg/day during pregnancy and 0.5 mg/day during lactation. Recommended intakes for children range from 0.4 mg/day for infants up to 3 months of age and 1.0 mg/day thereafter.
Niacin: nicotinic acid and nicotinamide (vitamin B3)
The term ‘niacin’ includes nicotinic acid and niotinamide. It occurs in food as nicotinic acid, as a pyridine nucleotide coenzyme derivative (NAD and NADP), as an amide, nicotinamide (niacinamide), or as a nicotinoyl ester, niacytin, in maize. Nicotinic acid is synthesized from tryptophan, catalysed by kynureninase and kynurenine hydroxylase, which are dependent on vitamin B6 and riboflavin. A deficiency of either vitamin B6 or riboflavin may aggravate niacin deficiency. Some 60 mg of dietary tryptophan generates 1 mg of nicotinic acid. The nicotinic acid equivalent is the dietary nicotinic acid content plus 1/60th of the dietary tryptophan. Nicotinamide is a component of the coenzymes nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) NAD coenzymes are biological carriers of reducing equivalents, i.e. electrons, during metabolic oxidation to NADH or NADPH. NAD+ is a major electron carrier in the oxidation of fuel molecules during which it becomes reduced to NADH. The oxidation of NADH generates ATP. NADPH, the reduced form of NADP+, is the electron donor in most reductive biosyntheses.
Nicotinic acid is found in meat, poultry, fish, wholemeal cereals, pulses, and coffee. The various conjugated forms of niacin are hydrolysed and absorbed in the upper gastrointestinal mucosa as the free acid. Niacytin from maize is neither hydrolysed nor absorbed. In Central America, maize is eaten as tortillas, in which lime water hydrolyses the nicotinoyl ester component. Niacin and tryptophan deficiencies occur in poor populations dependent upon maize for their protein intake. Zein, the principal maize protein, is deficient in tryptophan. Protein energy malnutrition, dietary amino acid imbalances (e.g. an excess leucine intake), anaemia, and other vitamin deficiencies worsen the problem. Dietary fortification with other proteins is necessary.
There is no apparent tissue storage of this vitamin. It is excreted in the urine as nicotinuric acid, nicotinamide-N-oxide, and 5′-methylnicotinamide. An inadequate dietary intake of niacin lasting for 1 to 2 months leads to significant tissue depletion and pellagra, which, if untreated, progresses to death. Pellagra is characterized by dermatitis, diarrhoea, and dementia; the disease is chronic, with a seasonal periodicity. Individuals suffer weight and stamina loss, which is worsened by secondary bacterial and parasitic infections. Erythematous dermatitis is symmetrically distributed on skin exposed to sunlight and mechanical irritation. In chronic pellagra, the skin looks sunburnt. In severe cases, gastrointestinal disturbances occur with diarrhoea. There can be glossitis, angular stomatitis, cheilosis, an inflamed tongue, and secondary infection of the mouth. Mild mental changes include anxiety and irritability progressing to manic depressive illness. There may be paraesthesia in the lower limbs, with loss of vibration sense and proprioception leading to ataxia and spasticity.
Pellagra can occur in alcoholism, malabsorption syndromes, and Hartnup disease. Mild cases rapidly improve on treatment with niacin or a suitable dietary protein supplementation. Oral nicotinamide (100 mg every 4 h) results in symptom resolution within 24 h, although mental symptoms, especially dementia, may be unresponsive to treatment. Nicotinic acid may cause unpleasant flushing and burning sensations. A supplementary diet containing good-quality protein and all of the vitamins is important. Nicotinic acid, but not nicotinamide, is used therapeutically for hyperlipidaemias at a dose of 2 to 6 g/day, but it can be hepatotoxic.
Microbiological methods provide the most sensitive means of measuring niacin and nicotinamide in serum, urine, and food. Alternatively, urinary nicotinamide, n-methyl nicotinamide (NMN), is measured over a defined time or as a ratio of creatine in urine. Urinary N-methyl-2-piridone-5-carboxamide (2-pyridone) excretion is a more sensitive measurement in borderline cases of nicotinamide deficiency, and is expressed in relation to urinary creatinine excretion.
These are related to dietary tryptophan intake. A protein intake of between 60 and 85 g/day contains approximately 13 mg tryptophan/g, equivalent to between 13 and 18 mg/day of niacin. The recommended intake as niacin equivalents is 6.6 mg (54 µmol/litre)/4185 J (4.185 J = 1 kcal). Hormonal changes during pregnancy affect tryptophan metabolism, so that 30 mg of tryptophan is equivalent to 1 mg of dietary niacin. Breast milk should provide not less than 3.5 mg preformed niacin/4185 J. Mature human milk provides preformed niacin (2.7 mg/litre), therefore an increment of 2 mg/day niacin for the nursing mother is suggested.
Nicotinic acid is used pharmacologically in doses of 1.5 to 3.0 g/day as an adjunct to statins in dyslipidaemias. It lowers both cholesterol and triglyceride plasma concentrations by inhibiting synthesis and increases high density lipoprotein (HDL) concentrations. Caution is required in the clinical circumstances of unstable angina, acute myocardial infarction, diabetes mellitus, gout, history of peptic ulceration, hepatic impairment, renal impairment; and pregnancy. The dominant side effect is cutaneous flushing, with variable severity among patients; in addition, infrequent hepatic toxicity, hyperglycemia, gout, and rare retinal macular oedema have been reported Side effects can include diarrhoea, nausea, vomiting, abdominal pain, dyspepsia; flushing; pruritus, rash; less commonly tachycardia, palpitation, shortness of breath, peripheral oedema, headache, dizziness, increase in uric acid, hypophosphataemia, prolonged prothrombin time, and reduced platelet count; rarely hypotension, syncope, rhinitis, insomnia, reduced glucose tolerance, myalgia, myopathy, and myasthenia; very rarely anorexia, rhabdomyolysis. Niacin is experiencing renewed interest because it is the most effective currently available agent for raising HDL cholesterol and increasing lipoprotein particle size, it is the only lipid drug that lowers lipoprotein(a), and it is comparable with fibrates in achieving striking reductions in triglyceride levels.
The β-oxidation of fatty acids depends upon initial linkage to coenzyme A to form an acyl-CoA before being degraded to acetyl-CoA. Coenzyme A and carnitine shuttle acyl groups into the mitochondrion where acyl-CoA is degraded by a recurring sequence of reactions: oxidation by FAD, hydration, oxidation by NAD+, and thiolysis by coenzyme A. In this way, the fatty acid chain length is shortened by two carbons and FADH2, NADH, and acetyl-CoA are formed—an interplay of B vitamin functions. Niacin and riboflavin have already been discussed. A major component of coenzyme A is phosphopantetheine, a direct derivative of pantothenic acid.
Pantothenic acid (vitamin B5)
Pantothenic acid functions as a crucial acyl (including acetyl) group carrier. Pantothenic acid is the dimethyl derivative of butyric acid joined by a peptide linkage to α-alanine. The active form, 4′-phosphopantetheine, is present in all tissues. 4′-Phosphopantetheine is a constituent of both coenzyme A and acyl carrier protein. As a major constituent of coenzyme A and acyl carrier protein. Pantothenic acid derivatives play a central role in metabolism. As acetyl Co A, it carries two carbon groups into the carboxyxlic acid cycle, thereby forming the final common pathway of the metabolism of fats, sugars, and most amino acids, As part of both coenzyme A and acyl carrier protein, it is also intimately linked with fatty acid synthesis.
It is widely available in foods of animal origin, especially liver, although cereals and legumes are also sources. Pantothenic acid is found in food as the coenzyme CoA or acyl carrier protein form and is hydrolysed by a pancreatic enzyme before absorption. It is not stored in the body. Urinary excretion is in the free acid form. Experimental pantothenic acid deficiency in humans results, within a few weeks, in symptoms including personality changes, fatigue, malaise, sleep disturbances, numbness, paraesthesiae, and muscle cramps. There is impaired motor coordination with an abnormal gait. Gastrointestinal complaints include nausea, abdominal cramp, occasional vomiting, and increased passage of flatus. Pantothenic acid deficiency may occur as part of the overall problem in people who are severely malnourished. No toxic intakes have been recorded. There is no biochemical method for measuring pantothenic acid status in humans.
British diets provide a median intake of 6.1 mg/day (adult men) and 4.4 mg/day (adult women). A safe and adequate intake is between 3 and 7 mg per day, including during pregnancy and lactation. Infants require 1.7 mg/day; human milk provides 2.6 mg/day. Infant formula milk should contain at least 2 mg/litre.
Thiamine (vitamin B1)
Thiamine functions principally (in the form of thiamine diphosphate), as a carrier of aldehyde groups. Its major importance is in carbohydrate metabolism, most notably in the pyruvate dehydrogenase system that generates acetyl-CoA from pyruvate. It is needed also as a cofactor in the oxidative phosphorylation of α-ketoglutarate, the common portal of entry into the tricarboxylic acid cycle of many amino acids. It is needed for the carboxylation of the ketoacids of the branched chain amino acids, so that it is important in the metabolism of nearly all amino acids as well as carbohydrates/sugars. Its involvement in the pentose phosphate pathway (hexose monophosphate shunt), at the transketolase stage, links it closely to the supply of NADPH and the synthesis of ribose 5-phosphate; this pentose and its derivatives are components of RNA and DNA, as well as ATP, NADH, FAD, and coenzyme A.
Thiamine hydrochloride consists of a substituted pyrimidine ring linked by a methylene group to a sulphur-containing thiazole ring. Thiamine may also play a role in neural excitation mechanisms.
All animal and plant tissues contain thiamine, usually in the phosphorylated form. The important sources are plant seeds and cereal germ, nuts, peas, beans, pulses, and yeast. Losses occur with cooking and alkaline pH. Absorption is from the upper gastrointestinal tract, followed by phosphorylation to the active diphosphate form. There is no body store and the only reserve is the vitamin functionally bound to enzymes. Multiple end products are excreted in the urine. Beriberi is caused by dietary thiamine deficiency, a disease that was endemic in east Asia as a result of the ingestion of polished rice which is deficient in the vitamin. Carbohydrate metabolism is impaired by a deficiency of thiamine pyrophosphate, a coenzyme necessary for the decarboxylation of pyruvate to acetyl-CoA. Pyruvic and lactic acid accumulate in the body.
The clinical presentations of thiamine deficiency are:
- ◆ wet beriberi (high-output cardiac failure)
- ◆ dry beriberi (polyneuropathy)
- ◆ infantile beriberi
- ◆ neuropathy and cardiomyopathy in chronic alcoholism
- ◆ Wernicke–Korsakoff syndrome
- ◆ lactic acidosis associated with artificial feeding
Initially, the symptoms are of nonspecific malaise and evidence of early cardiac failure and neuropathy. Wet beriberi is characterized by left- and right-sided high-output cardiac failure, cardiomegaly, hypotension, rapid deterioration, and death. Dry beriberi is a polyneuropathy affecting motor and sensory nerves. Initially, there is paraesthesia progressing to painful muscle wasting and polyneuritis. Total sensory loss occurs and patients become immobile and emaciated; they are at a high risk for the development of Wernicke–Korsakoff encephalopathy. Infantile beriberi occurs in breastfed infants of thiamine-deficient mothers, usually when they are between 2 and 5 months old. In the acute form, the child is restless and distressed with evidence of high-output cardiac failure; convulsions may develop, and the child becomes comatose. In the chronic form, the child is fretful, sleeps poorly, and the muscles may be flaccid. Cardiac failure, gastrointestinal symptoms, and sudden death are common. Alcoholic neuropathy presents with a sensory and motor neuropathy sometimes complicated by cardiomyopathy. Sensory nerve dysfunction includes paraesthesia and severe nerve pain (‘causalgia’). Motor nerve lesions are of both upper and lower motor neuron type. A patient with Wernicke–Korsakoff syndrome is disorientated and apathetic. Nystagmus, ataxia, and confabulation are not infrequent consequences of lesions in the brainstem, diencephalon, and cerebellum.
Treatment is with intramuscular thiamine 25 mg twice daily for 3 days, and thereafter 10 mg two or three times daily. The reversal of the wet type of beriberi is rapid. Improvement is slow for dry beriberi, especially for the neurological abnormalities. In infantile beriberi, both mother (10 mg thiamine twice daily) and the infant (thiamine intramuscularly 10 to 20 mg/day for 3 days, and thereafter 5 to 10 mg twice daily) are treated. Beriberi can be prevented by eating thiamine-containing foods, unmilled or thiamine-fortified rice, or thiamine supplements.
Long-term intakes in excess of 50 mg/kg body weight per day are toxic, leading to headaches, irritability, insomnia, rapid pulse, weakness, contact dermatitis, pruritus, and even death. Thiamine status can be measured by urinary thiamine and the thiamine:creatinine ratio with or without a loading dose, or by the reactivation of the cofactor-depleted red-cell enzyme transketolase in vitro.
Thiamine requirements are related to energy and carbohydrate metabolism. The average requirement for adults, normal pregnancy or lactation, and children is 0.4 mg/4185 J and not less than 0.8 mg/day for adults with a supplement of 0.6 mg/4185 J during pregnancy and lactation. Human breast milk contains the equivalent of 0.3 mg/4185 J. Older people may require 1 mg/day.
Pyridoxine (vitamin B6)
Pyridoxine is to be found in several forms: pyridoxal, pyridoxamine, and pyridoxine. Pyridoxal phosphate enzymes are involved in a wide range of amino acid transformations—transamination, decarboxylations, deaminations, racemizations, and aldol cleavages—so pyridoxal 5′-phosphate is a coenzyme that plays a major role in the intermediary metabolism of amino acids. Pyridoxal 5′-phosphate acts as a coenzyme with glycogen phosphorylase in muscle, and has a role in the actions on hormones which modulate gene expression.
This family of vitamin B6 compounds is found in many foods: cereals, meat (particularly liver), fruits, and leafy and other vegetables. The free form is common in plants, the phosphorylated form, pyridoxamine phosphate, in animal tissues. Vitamin B6 is absorbed in the free form and phosphorylated for use in enzymes. There is no specific storage in tissues and it is excreted in urine largely as 4-pyridoxic acid.
Primary dietary deficiency has not been reported in adults, largely because of the wide availability of the vitamin, but deficiency has been seen in infants exposed to formula milk with antivitamin properties. These infants suffered seizures that responded to pyridoxine replacement. Experimental restriction of pyridoxine intake causes fatigue and headaches as early symptoms. A biochemical deficiency occurs commonly in alcoholics and in patients admitted to hospital as an emergency. Plasma concentrations tend to decrease with age. Patients taking isoniazid may develop a pyridoxine deficiency and neuropathy.
Clinical signs of deficiency include glossitis, stomatitis, peripheral neuropathy, microcytic hypochromic (sideroblastic) anaemia, It can be difficult to know how specific these signs are or whether they relate to combined deficiencies of other B vitamins.
Hyperhomocystinaemia is a result of reduced pyridoxine intake.
Evidence that vitamin B6 supplementation can improve premenstrual symptoms and premenstrual depression exists but is limited by the quality of the trials performed.
High-dose (gram) intakes of pyridoxine have been reported to produce ataxia and glove–stocking sensory neuropathy, largely reversible on withdrawal of supplementation.
There is no single marker that is sensitive at all levels of dietary intake. Biochemical markers include plasma pyridoxal phosphate concentrations, red-cell aspartate aminotransferase activation, and the urinary excretion of vitamin B6 degradation products. Metabolic loading tests also measure vitamin B6 status, including the tryptophan- and methionine-load tests.
The total body pool of vitamin B6 is 15 µmole (4 mg)/kg, 80% of which is in muscle, with a half-life of 33 days. Adults, pregnant and lactating women, and older people require a daily intake of 13 µg/g of protein (c.4 mg/day). The vitamin B6 content of human breast milk is low at between 40 and 100 µg/l (or 3–8 µg vitamin B6/g protein). Infants under 3 months of age require 6 µg/g protein, increasing to up to 13 µg/g protein at 7 to 10 years.
Folate (folic acid, folacin; vitamin B9)
Folates are derivatives of folic acid (pteroylglutamic acid) including the folylpolyglutamate found in foods. Folic acid is a pterin ring (2-amino, 4-hydroxypteridine) attached to p-aminobenzoic acid conjugated to L-glutamic acid (PteGlu). Variants include:
- ◆ di-(7,8-tetrahydrofolic acid) (DHF) and tetra-(5,6,7,8-tetrahydrofolic acid) (THF) reduced forms of the pteridine ring
- ◆ one-carbon substitution (methyl, formyl, methenyl, methylene, or formimino) at positions N5 or 10: 5-formyl-THF, 10-formyl-THF, 5-formimino-THF, 5,10-methenyl-THF, 5,10-methylene-THF, and 5-methyl-THF
- ◆ a chain of 4 to 6 glutamates attached to the l-glutamate
Folic acid gives and receives one-carbon groups on the N5 or N10 position in nucleic acid and amino acid biosynthetic reactions.
Sources of folate include liver, yeast extract, and green leafy vegetables. Most dietary folate is in the polyglutamyl form, which is hydrolysed to monoglutamate before being absorbed from the duodenum. A brush-border glutamyl carboxypeptidase is inhibited by alcohol, which is of relevance in alcoholic folic deficiencies. Folate bound to a milk protein is absorbed from the ileum. Folic acid is stored in the liver. Plasma folates are mainly 5-methyl-THF monoglutamate. Within cells, 5-methyl-THF is converted to THF polyglutamates, the main cellular forms of folic acid.
Folate polyglutamates do not readily cross cell membranes. The polyglutamate form has two functions: storage; and as a coenzyme for normal one-carbon metabolism (for which it is the most efficient coenzyme). The 5-methyl group is transferred to homocysteine (creating methionine and THF); the enzyme is the vitamin B12-dependent methionine synthase, wherein methionine, folic acid, and vitamin B12 interlink.
Reactions in which folate is involved include:
- ◆ methylation of amino acids
- ◆ serine reversibly interconverting with glycine
- ◆ methionine interconverting with homocysteine (Methionine is the precursor of S-adenosyl-l-methionine (SAM), a methyl donor in the methylation of lipids, hormones, DNA, cell division, and proteins.)
- ◆ thymidine and purine synthesis
Folic acid deficiency may arise:
- ◆ as a dietary deficiency
- ◆ in malabsorption syndromes
- ◆ where there are excessive demands, as with increased cell proliferation (e.g. in leukaemias and haemolytic anaemias)
- ◆ where drugs interfere with folic acid metabolism
- ◆ in the rare inborn errors of folic metabolism
Folic acid deficiency is an important cause of megaloblastic anaemia, never to be confused with vitamin B12 deficiency. This anaemia was first described in poor Indian textile workers during pregnancy by Lucy Wills and reflected the increased demands for folate during pregnancy.
Neural tube defects are congenital deformities of the spinal cord and brain: spina bifida, anencephaly, encephalocele, and iniencephaly. Folic acid is involved in the aetiology of these defects. The precise mechanism is unclear, but there may be an underlying genetic predisposition involving a variant of 5,10-methyl-THF reductase (EC 126.96.36.199). Closure of the neural tube occurs early in pregnancy, thereby making aetiological studies difficult. It is clear from a major randomized controlled trial that folate replacement in pregnancy reduces the incidence of neural tube defects by about 72%. Food fortification with folate has been in place in the United States of America since 1998 and has now been recommended in the United Kingdom, though concerns have been expressed that it might have an adverse effect in older people in whom there is a high prevalence of subclinical vitamin B12 deficiency. Concern has also been expressed over the links of folic acid with bowel cancer.
Folic acid supplementation for 3 years in 50- to 70-year-old members of the general population may improve domains of cognitive function that tend to decline with age, but the evidence does not yet provide adequate evidence of an effect of vitamin B6 or vitamin B12 or folic acid supplementation, alone or in combination, on cognitive function testing in people with either normal or impaired cognitive function.
Epidemiological studies suggest an increased risk of vascular disease associated with hyperhomocysteinaemia. Homocysteine is reversibly methylated to methionine, a step which involves folate, vitamin B12, and vitamin B6. Supplementation of these vitamins has been proposed to reduce the putative dangers of hyperhomocysteinaemia. Folic acid supplementation is beneficial in primary prevention of stroke and is associated with reduction in plasma homocysteine concentrations, but has not yet been confirmed as effective in preventing cardiovascular diseases.
Folic acid supplementation does not reduce the risk of colonic adenoma formation, and may increase it.
Folate status is measured by the folate concentration in serum and red cells. Red-cell folate levels reflect body stores. A coincidental measurement of serum vitamin B12 is important.
Children and adults, including elderly people, need a folate intake of 200 µg/day. Women planning a pregnancy should increase their intake of folic acid to 0.4 mg/day by capsule supplement. If a previous pregnancy has been affected by a neural tube defect, then 5 mg of folic acid/day before conception is suggested. A problem is that some pregnancies are unplanned. Dietary supplementation is impractical. Total folic acid excretion in breast milk averages 40 µg/day, and an additional maternal intake of 60 µg per day is required.
Cobalamin (vitamin B12)
Vitamin B12 is a cobalt-containing corrinoid—four linked pyrrole rings (corrin) coordinating with a central cobalt atom. The cobinamides necessary for human well-being are methylcobalamin, adenosylcobalamin, hydroxycobalamin, and cyanocobalamin. Microorganisms, including colonic flora, synthesize cobalamin. Yeast is a source of cobalamin, primarily as adenosyl- and hydroxocobalamin. Methyl cobalamin is found in egg yolk, cheese, and cow’s milk. A vegan diet carries a risk of vitamin B12 deficiency.
The reactions requiring vitamin B12 include:
- ◆ isomerization of methylmalonyl-CoA to succinyl-CoA (methyl malonic acid concentrations increase in cases of vitamin B12 deficiency
- ◆ methyltransferase reactions, e.g. homocysteine to methionine, i.e. the transfer of a methyl group from 5-methyl-TFH to homocysteine, which converts homocysteine to methionine
Vitamin B12 has an important role in the maintenance of myelin. Deoxyadenosyl B12 is essential for propionyl-CoA reactions by transmutation of methylmalonyl-CoA to succinyl-CoA.
Vitamin B12 binds to food proteins and is released by saliva, acid pH, and pepsin, depending upon the mode of cooking and type of food protein. Vitamin B12 at stomach pH forms complexes with glycoproteins, transcobalamin, haptocorrin, and intrinsic factor. In the duodenum, cobalamin is released by pancreatic enzymes and alkaline pH and binds solely to intrinsic factor. The vitamin B12–intrinsic factor complex is absorbed from the ileum through a specific receptor. Cobalamin is released from intrinsic factor, converted (80% to methyl and also adenosyl and hydroxy forms) and carried in the blood by transcobalamins I, II, and III. Of these, transcobalamin II releases vitamin B12 to the tissues to be stored in the adenosyl form. The total body cobalamin content in adults is between 2 and 5 mg, most of which is stored in the liver. Turnover is 0.1% of the body pool each day. There is efficient conservation by the kidneys and the enterohepatic circulation. Most vitamin B12 is excreted in urine and small amounts in faeces, including unabsorbed bacterially synthesized vitamin B12. The relationship between dietary intake and serum concentrations is not linear because the body stores of vitamin B12 are largely in the liver.
Vitamin B12 deficiency is common in older people and results in megaloblastic anaemia and neurological disorders, especially in the posterolateral columns of the spinal cord. Causes of deficiency are dietary (vegans), lack of intrinsic factor (Addison’s pernicious anaemia, gastric resection), intestinal colonization by bacteria and parasites (e.g. tapeworms), and ileal resection. The megaloblastic anaemia is due to a lack of vitamin B12 for methionine synthase, insufficient methionine regeneration, and 5,10-methylene-THF and deficient thymidylate synthesis. In vitamin B12 deficiency, odd number (15- and 17-carbon) fatty acids and branched chain fatty acids are synthesized and incorporated into an unstable myelin nerve sheath. An inability to regenerate methionine from homocysteine, for the S-adenosylmethionine generation necessary for myelin proteins, leads to demyelination.
The efficiency of vitamin B12 absorption is measured by the Schilling test or by a whole-body scanner.
A dietary intake of between 1 and 2 µg/day for adults of all ages is protective. In pregnancy there is a compensatory increased absorption so that 1.5 µg/day is sufficient. During lactation, an increment of 0.5 µg/day should ensure an adequate supply in breast milk (0.2–1.0 µg/litre). The requirement for infants is of the order of 0.1 µg/day and for children aged 3 to 10 years, 0.5 to 1.0 µg/day. Vitamin B12 has extremely low toxicity and as much as 3 mg/day may be taken.
Biotin (vitamin B7)
Biotin contains a ureido group in a five-membered ring fused with a tetrahydrothiophene ring with a five-carbon side chain terminating in a carboxyl group. Dietary biotin is found in yeast, bacteria, liver, kidney, egg yolks, cooked cereals, pulses, nuts, chocolates, and some vegetables, and biotin is synthesized by intestinal flora. Biotin is a carrier for activated CO2 and a cofactor for the acetyl-CoA, propionyl-CoA, and pyruvate carboxylase systems involved in the incorporation of bicarbonate as a carboxyl group (activated CO2/carboxyphosphate) into substrates in fatty acid synthesis and gluconeogenesis. Biotin is absorbed from the upper gastrointestinal tract. Raw egg white contains the glycoprotein avidin (molecular weight 68 000 kDa), which binds biotin with a high affinity and, in large amounts, prevents biotin absorption. Biotin is transported in plasma to the liver for storage. It is metabolized before excretion in the urine as biotin, bisnorbiotin, and biotin sulphoxide.
Biotin deficiency results in fatigue, depression, sleepiness, nausea, loss of appetite, muscle pain, hyperaesthesia and paraesthesia, hallucinations, alopecia, dermatitis, conjunctivitis, smooth tongue, and dry skin. It has been described in the joint contexts of (deficient) parenteral nutrition and short bowel and occurs rarely in individuals unable to absorb the vitamin due to a genetic deficiency of biotinidase. Individuals receiving treatment for epilepsy are at risk of biotin deficiency. There are no indications that excess biotin is toxic. Body stores are measured by plasma biotin concentrations, and lymphocyte propionyl-CoA carboxylase (PCC) and its activation index (ratio of enzyme activity incubated with and without biotin) or urinary 3-hydroxyisovalerate. Egg white feeding results in lower than normal biotin status as judged by PCC within 14 days and activation coefficient is increased by 28 days.
The dietary requirement of biotin is not known with certainty. The average intake of a British man is 39 µg/day, ranging from 15 to 70 µg/day, and for women it averages 26 µg/day, ranging between 10 and 58 µg/day. This prevents deficiency. In infants, preterm to 5 years, an intake between 5 and 25 µg/day is suggested.
Vitamins and coronary heart disease
Homocysteine metabolism by vitamin B6, vitamin B12, and folate
Elevated levels of homocysteine are associated with increased prevalence of coronary heart disease. Dietary supplementation with folic acid and vitamin B12 reduces plasma homocysteine levels. Homocysteine is derived from methionine. Conversely, in remethylation it accepts a methyl group from methyl tetrahydrofolate to form methionine. In trans-sulphuration, homocysteine combines with serine to form cystathionine catalysed by a vitamin B6-dependent enzyme, cystathionine synthase. After adjustment for known risk factors for coronary heart disease, reduction in blood homocysteine by 25% resulted in a decrease of 11% in coronary heart disease and of 19% in stroke.
Interest has focused on coronary heart disease, stoke, depression, cognitive function, and depression. Randomized controlled trials are currently being assessed together to increase their power; however, to date, no evidence for reduction in coronary heart disease or stroke has emerged, though reductions of 10% for myocardial infarction and 20% for stroke cannot be excluded.
Large trials have established either no or an adverse effect on coronary heart disease in patients given these vitamins as supplements, despite effective lowering of plasma homocysteine concentrations. Meta-analysis has demonstrated reductions in stroke with folic acid supplementation. No effect on cognitive function has yet emerged in meta-analysis of the vitamins used singly or in combination.
The vitamin A family (retinoids) are related to the plant pigment carotene. Dietary vitamin A comes in two forms: preformed vitamin A fatty acid esters from animal sources, or provitamin A carotenoids from plant sources. β-Carotene, a provitamin A carotenoid, consists of two retinol molecules. Retinol is vitamin A alcohol, a hydrocarbon chain with a β-ionone ring at one end and an alcohol group at the other, usually esterified with a fatty acid (retinyl esters) as the all-trans stereoisomer. The cis configuration isomer (11 or 13 position) is less potent. Retinol can be oxidized to an aldehyde (retinal) or acid (retinoic acid). Retinol and carotene are readily oxidized and are protected by vitamin E.
Retinol is present in dairy products, liver, and fatty fish liver oils. Carotenes are found predominantly in green vegetables as well as in yellow and red fruits and vegetables. Vitamin A is essential for the maintenance of epithelial tissue, visual function, and the immune system. Most actions of vitamin A in development, differentiation, and metabolism are mediated through retinoid receptors of the nuclear steroid receptor family of proteins that bind retinoic acid and regulate gene expression.
The photopigment rhodopsin is formed by the protein opsin and 11-cis retinal. A photon of light converts the cis retinal to the trans form, which reversibly dissociates from the opsin and is seen as light. Retinyl esters are hydrolysed by pancreatic hydrolases and the enteric mucosa. β-Carotene is cleaved to two retinols by β-carotene 15,15′-deoxygenase, and carotenoids oxidatively cleave to retinal and apocarotenoids. Retinal is reduced to retinol, esterified with long-chain fatty acids, and transported to the liver as retinyl long-chain fatty esters in chylomicrons through the lymph. Retinol is a major liver storage form and circulates to tissues, bound to retinol-binding protein. There is an enterohepatic circulation of retinoids.
Vitamin A deficiency is a major worldwide cause of blindness, due to a poor dietary intake of green vegetables, fruit, and dairy produce. Malabsorption is a less common cause. Vitamin A deficiency results in reduced rhodopsin in the retinal rods resulting in loss of vision. Xerophthalmia causes blindness in 500 000 young children each year, especially amongst bottlefed infants and breastfed infants with vitamin A-deficient mothers. Protein calorie malnutrition compounds the problem during weaning. Vitamin A deficiency aggravates damage from other causes of keratoconjunctivitis, e.g. measles. Epithelial surfaces undergo squamous metaplasia, followed by corneal ulceration and irreversible visual damage. Clinical forms are:
- ◆ conjunctival xerosis (Bitots’s spots are white plaques of thickened conjunctival epithelium indicative of vitamin deficiency in the young)
- ◆ corneal xerosis
- ◆ keratomalacia, leading to blindness
- ◆ night blindness, an early symptom with or without xerophthalmia
- ◆ xerophthalmia fundi
- ◆ corneal scars
Vitamin A is important in epithelial metabolism. Deficiency leads to epithelial metaplasia and inappropriately keratinized epithelium in the mucous membranes of the respiratory, gastrointestinal, and genitourinary tract. Sebaceous glands become blocked, thereby causing follicular keratosis.
Prophylaxis demands education in the eating of dark green vegetables. Where xerophthalmia is endemic, vitamin A is given prophylactically in capsule form or by food fortification. Frank deficiency is treated by high-potency Vitamin A: 200 000 IU for 2 days, and a third dosage at least 2 weeks later. Thereafter, improved diet and supplementation are obligatory. Corneal ulceration is treated by antibiotics, and the response is rapid. Vitamin A deficiency is a putative risk factor for childhood morbidity and death, especially for the underweight and premature infant.
β-Carotene is not toxic, but high intakes lead to a yellow appearance sparing the eyes (hypercarotenaemia). Polar bear liver, rich in retinol, is toxic; ingestion can cause drowsiness, headache, vomiting, and excess peeling of the skin. Large intakes of retinol are teratogenic. Pregnant women should be careful not to exceed the recommended intake of vitamin A in the first trimester.
Plasma retinol is an insensitive indicator of vitamin A status. The relative dose–response (RDR test), which measures retinol transport by the retinol-binding protein, is used as a functional test for calculating retinol stores. The concentration of plasma carotenoids reflects short- to medium-term intakes. The following are equipotent to 1 µg of all-trans-retinol equivalents/day: 3.33 IU vitamin A, 3.5 nmol retinol or retinyl ester, or 6 µg all-trans-β-carotene.
Adults require 500 µg retinol equivalents/day; infants, 250 to 350 µg retinol equivalents/day; children, 350 µg retinol equivalents/day; pregnancy, an increment of 100 µg retinol equivalents/day, particularly during the third trimester. Lactation requires an increment of 300 µg retinol equivalents/day. Breast milk vitamin A concentration should exceed 1.5 mmol/litre.
Insertion the term ‘vitamin D’ as a keyword into Medline retrieves in excess of 30 000 references. The vitamin D family of sterols includes vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Cholecalciferol is to be regarded as a hormone rather than a vitamin, and is produced by the ultraviolet irradiation of dietary 7-dehydrocholesterol (provitamin D3) in the skin. The extent of exposure to sunlight determines production. Cholecalciferol is also available in fatty fish (e.g. cod), eggs, and chicken liver. Ergocalciferol is also the result of the exposure of ergosterol (provitamin D2) to ultraviolet light. Ergocalciferol differs from cholecalciferol in having an extra methyl group at C-24 and a double bond at C-22,23. Although not a steroid, vitamin D acts similarly in that it binds to steroid-like receptor and forms a complex which, as a transcription factor, regulates gene expression through ‘zinc fingers’. 1,25-dihydroxycholecalciferol vitamin D (1,25(OH)2D, calcitriol) regulates calcium and phosphate absorption, metabolism, and export into the bloodstream. Such regulation is through steroid:thyroid hormone nuclear receptors. Calcitriol is also a developmental hormone inhibiting proliferation and promoting differentiated function in cells.
Though hitherto vitamin D has been regarded principally in the context of bone metabolism its broader roles are emerging. Vitamin D supplementation is demonstrated in meta-analysis to reduce overall mortality rates. Brain, prostate, breast, colon, and immune cells have a vitamin D receptor and respond to calcitriol. Calcitriol controls more than 200 genes, including genes responsible for regulation of cell proliferation, differentiation, apoptosis, and angiogenesis. 25-Hydroxyvitamin D (calcidiol) reduces cell proliferation of normal cells and cancer cells and favours terminal differentiation. Vitamin D deficiency inhibits innate immunity (monocytes and macrophages). Calcitriol increases insulin production, inhibits renin synthesis, and enhances myocardial function. Vitamin D deficiency has been associated with increased risks of Hodgkin’s lymphoma, colon, prostate, and breast cancer. Vitamin D deficiency is associated with insulin resistance and with diabetes mellitus. As with the epidemiology of these cancers, living at high latitude is associated with an increased risk of diabetes, multiple sclerosis, and Crohn’s disease. Vitamin D supplementation has been reported to reduce risks of multiple sclerosis, rheumatoid arthritis, osteoarthritis, and type 1 diabetes. Supplemental vitamin D in a dose of 700-1000 IU a day reduces the risk of falling among older individuals. The latter seems to be related to the ability of vitamin D to reduce islet cell antibody production.
Dietary vitamin D is absorbed in the small intestine, as a lipid, transported to the liver bound to α-globulin (trans-calciferol) in chylomicrons. Both vitamin D2 and vitamin D3 are inactive. They are converted in the liver by a P450 inducible microsomal enzyme into 25-hydroxyvitamin D (calcidiol), which has modest biological activity, before plasma transport on a specific globulin. The active form of vitamin D is calcitriol, formed in the kidney by a mitochondrial D-1-αhydroxylase acting on calcidiol. Production of calcitriol is tightly regulated by plasma parathormone, calcium, and phosphorus concentrations. The half-life of calcidiol is less than 24 h. All forms of vitamin D are stored in fat. Vitamin D is 24-hydroxylated and inactive calcitroic acid formed and excreted.
The standard measure of vitamin D status is the plasma calcidiol concentration, and deficiency is defined by plasma levels of less than 20 ng/ml (50 nmol/litre). Levels of calcidiol are inversely related to parathormone levels until the parathormone level reaches a minimum at calcidiol levels of 75 to 100 nmol/litre (30–40 ng/ml). Hypomagnesaemia results in a blunting of the response of parathormone to vitamin D and calcium deficiency. 30 ng/ml (75 nmol/litre) is regarded as evidence of vitamin D sufficiency when optimal absorption of calcium transport is achieved.
The prime consequence of vitamin D deficiency is rickets, caused by a failure to mineralize the bony skeleton. The epiphyseal cartilage replacement is defective, leading to an overgrowth of subperiosteal osteoid tissue and poor mineralization of the bone matrix, resulting in soft bones. The type of bony abnormalities depends on the age of onset and the weight-bearing bones involved. The appearance of the rachitic child is deceptive; the child may appear quite well or be restless with hypotonic muscles and twisted limb postures. The abdomen is swollen and the child suffers from diarrhoea, respiratory infection, and delayed tooth development. In the infant, the commonest abnormality is enlargement of the end of the radius and the costochondrial junction, termed the ‘ricketic rosary’. Later there is bossing of the frontal and parietal bones and delayed closure of the anterior fontanelle. So-called ‘pigeon chest’ can occur, which is an undue prominence of the sternum and a transverse depression from the costal margins towards the axillae. All are due to pressure on the soft bones when the child is supine. In the walking child, the weight-bearing bones bend, and kyphosis of the spine and bowing of the lower ends of the femur, tibia, and fibula result. Pelvic deformity can make delivery difficult in subsequent pregnancies. Tetanic spasm can occur, whereby spasm of the hands, feet, and vocal chord result in high-pitched cries and breathing problems.
Diagnosis is based on the clinical appearance and measurements of plasma alkaline phosphatase (although interpretation of the results is difficult in the growing child) and plasma calcidiol levels. There are several risk factors:
- ◆ inadequate exposure to sunlight (dependent on latitude; in the United Kingdom 30- to 90-min exposure of the face and legs per day will restore calcidiol concentrations)
- ◆ strict vegetarianism
- ◆ a vitamin D-deficient mother breastfeeding her baby
- ◆ high melanin content in the skin, which screens the metabolically active skin sites
- ◆ malabsorption
Prevention of vitamin D deficiency requires a supplement of 10 μg of vitamin D daily or regular exposure to sunlight in well-nourished individuals.
Osteomalacia may present with muscular weakness and a waddling gait. Bone pain, tetany, and spontaneous fractures may develop. Radiographic features include bone rarification, pseudofractures, and Looser’s zones at points of compression stress. Renal disease, from many causes, may be associated with impaired renal synthesis of calcitriol. A failure of 25-hydroxylation of vitamin D can occur in hepatic disease. Dietary vitamin D intake may be important in immunity to tuberculosis, and calcidiol deficiency may contribute to the occurrence of tuberculosis. Hypervitaminosis occurs during infant supplementation and replacement therapy. Plasma calcium concentrations increase with tetany, ECG changes with resultant convulsions, and occasionally death. Vitamin D in milligram amounts is poisonous, and is used as a rodenticide.
The best measure of the vitamin status in humans is the plasma calcidiol concentration.
No minimum dietary intake has been identified for adults exposed to ample sunlight. However, 10 µg/day vitamin D is recommended for those with poor sun exposure. Vitamin D concentrations in breast milk are low (0.25–1.25 µg/litre) and are reduced in the winter. Vitamin D intakes are a problem for the 6- to 12-month-old baby dependent upon modestly fortified weaning foods. The diet thereafter expands, and the plasma calcidiol concentrations are usually satisfactory. Pregnant and lactating women should receive supplementary vitamin D at 10 µg/day. Where older people are insufficiently exposed to the summer sun, their stores may be reduced and a supplement of 10 µg/day vitamin D is recommended.
Supplementation of people over the age of 50 with calcium and vitamin D increases bone mineral content and reduces fracture risk.
Vitamin K, a naphthoquinone, occurs in two forms in human nutrition: vitamin K1 and vitamin K2. Vitamin K1 of plant origin is a phytylmenaquinone (also known as phylloquinone or phytylmenadione) and consists of 2-methyl-1,4-naphthoquinone (menadione or menaquinone) attached to a 20-carbon phytyl side chain. Vitamin K2 is one of several homologues produced by bacteria with 4 to 13 isoprenyl units in the side chain (menaquinone-4 to -13). Vitamin K1 is present in fresh green vegetables (e.g. broccoli, lettuce, cabbage, and spinach) and beef. Vitamin K is involved in the synthesis of proteins central to blood coagulation, namely prothrombin and factors VII, IX, and X. Vitamin K is necessary for the post-translational carboxylation of glutamic acid in the coagulation proteins. γ-Carboxyglutamate allows the binding of calcium and phospholipids in the formation of thrombin.
Vitamin K is absorbed as a lipid, and is transported from the intestine in the blood in chylomicrons as β-lipoproteins. Vitamin K2 of bacterial origin is absorbed from the colon. When there is vitamin K deficiency, the blood clotting time is prolonged and factor VII, IX, and X activities are reduced. Deficiency is uncommon in adults. However, in infants, deficiency results from a sterile intestinal tract and the inadequate vitamin K content of human and cow’s milk. The problem is compounded by the immature liver of the infant being slow to synthesize prothrombin. Acquired deficiencies occur as a result of any cause of lipid malabsorption and after bowel sterilization with broad-spectrum antimicrobial agents. Vitamin K deficiency may also result from the regular ingestion of liquid paraffin, since the vitamin partitions preferentially into this nonabsorbed, nonpolar hydrocarbon oil and is excreted rather than absorbed. Liquid paraffin is still used in many parts of the world as a regular aperient, principally by older people.
Natural vitamin K preparations are free from toxic effects. There are naturally occurring vitamin K antagonists—e.g. spoilt sweet clover produces a dicoumarol that prolongs the prothrombin time of the cow, thereby causing a bleeding condition. Drugs designed to prolong prothrombin time were developed as a result of this observation. Vitamin K deficiency can be detected by the prothrombin time test, which measures prolongation of clotting time.
The children and adult dietary requirements of phylloquinone are between 0.5 and 1.0 µg/kg body weight per day. Vitamin K in human breast milk is as the phylloquinone and the concentration varies between 1 and 10 µg/litre. An adequate intake for breastfed infants is 8.5 μg phylloquinone/day. Vitamin K is given as supplements in malabsorption syndromes and prophylactically for haemorrhagic disease of the newborn.
The vitamin E family consists of fat-soluble biologically active tocopherols and tocotrienols. The tocopherols are the most potent, their activity depending upon the position and number of methyl substitutions. α-Tocopherol, is the most potent; γ-tocopherol and γ-tocotrienol have activity of 48% and 20%, respectively, when compared to α-tocopherol.
The free-radical scavenging properties of vitamin E are a function of the fused chroman ring system; the phytyl side chain facilitates entry into the hydrophobic environment of the membrane. Ascorbic acid may reduce tocopheroxyl radicals formed by the scavenging of free radicals during metabolism. This enables a molecule of tocopherol to scavenge many radicals.
Vegetable oils—wheat germ, sunflower seed, cottonseed, safflower, palm, rape seed, and other oils—are abundant sources of vitamin E. The absorption of the vitamin is incomplete and varies between 20% and 80%. Vitamin E enters the systemic circulation in chylomicrons and very low-density lipoproteins (VLDL) and coincidentally protect the polyunsaturated fatty acids (PUFA), which are also transported. Lipoprotein lipase controls uptake by the liver or transfer to other lipoproteins. The normal lipoprotein concentrations of vitamin E as α-tocopherol range from 11 to 37 µmol/litre. α-Tocopherol forms 90% of the vitamin E found in tissues, including all cell membranes where it inhibits the nonenzymatic oxidation of PUFA by molecular oxygen.
Biochemical deficiency may occur as a result of gastrointestinal malabsorption and in premature infants. Vitamin E deficiency has been implicated in peripheral neuropathy associated with malabsorption syndromes and often accounts for the acanthocytosis, retinitis pigmentosa, and neurological features in Bassen–Kornzweig disease (abetalipoproteinaemia). Patients with fat malabsorption due to cystic fibrosis, coeliac disease, prolonged cholestasis, and after massive small intestinal resections are particularly at risk from vitamin E deficiency. Supplements prevent and may slowly ameliorate the neurological deficit. Homozygosity for inactivating mutations in the α-tocopherol transfer protein is a cause of isolated vitamin E deficiency and ataxia, which closely resembles the spinocerebellar ataxia of Friedreich’s disease. Patients with Friedreich’s ataxia in the absence of frataxin mutations should be investigated for vitamin E deficiency and defects in the tocopherol transfer protein; vitamin E supplements may slowly improve this condition. Longstanding profound deficiency of vitamin E is associated with progressive spinocerebellar ataxia, visual loss due to retinitis pigmentosa, haemolysis (with red cell acanthocytosis), upward visual gaze palsies, dementia, and muscle weakness. If detected, vigorous long-term vitamin E supplementation is indicated as well as attention to the primary cause of the deficiency. There appears to be no adverse effects from large doses of vitamin E up to 3200 mg/day.
Vitamin E status can be measured from the plasma tocopherol concentration, or expressed as a ratio of total blood lipids or vitamin E:cholesterol. A functional test of vitamin E status is the hydrogen peroxide haemolysis test (erythrocyte stress test).
The average intake in Britain is 6 mg/day, most of which is derived from fats, oils, and cereals. The dietary requirement is determined by the PUFA content of membranes and tissues and the PUFA content of the diet. The relationship between PUFA intake and vitamin E requirements is not a simple linear relationship. Intakes of 4 mg and 3 mg of α-tocopherol equivalents per day, respectively, for men and women have been regarded as adequate, but may be too low. Alternatively, and better, would be 0.4 mg α-tocopherol equivalents per gram of dietary PUFA/day, thereby increasing the recommendation to 7 mg. This formula might also be used for infants. Human breast milk contains 10 mg of α-tocopherol equivalents/litre in colostrum, reducing to 3.2 mg/litre at 12 days and thereafter.
Trace elements are required in small amounts ranging between milligram amounts (magnesium, iron, zinc, copper, manganese, fluoride) and microgram amounts (selenium, molybdenum, chromium, iodine). They are essential nutrients and, as with vitamins, artificial feeding must contain sufficient amounts of them. They act as cofactors in enzyme oxidation–reduction reactions. Trace elements maintain the specific configuration of proteins; are incorporated into the structure of hormones; and play a structural and catalytic role in gene expression and transcriptional regulation of genes.
The trace elements contained in soil and drinking water vary from area to area, which determines the variation in intake seen in different communities. The amount and chemistry of dietary constituents eaten with the trace elements affects the absorption efficiency of the essential elements. The absorption of calcium and trace metals (e.g. zinc) can be inhibited by dietary phytate. Copper absorption is reduced by competitive interactions affecting its solubility. A mild degree of iron depletion increases not only iron absorption but also that of lead, zinc, cadmium, cobalt, and manganese.
Some properties of trace elements are listed in Table 1.
|Table 1 Properties of trace elements|
|Element||Atomic weighta||Valency||Natural isotopesb||Abundance (%)c|
|Chromium||52||2, 3||50, 52, 53, 54||0.033|
|Copper||64||1, 2||63, 65||0.010|
|Iodine||127||1||127||6 × 10–6|
|Magnesium||24||2||24, 25, 26||1.94|
|Molybdenum||96||2, 3, 4||92, 94, 95, 96, 97, 98, 100||7 × 10–4|
|Nickel||59||2, 3||58, 60, 61, 62, 64||0.018|
|Selenium||79||2, 4||74, 76, 77, 78, 80, 82||8 × 10–5|
|Silicon||28||4||28, 29, 30||25.8|
|Sulphur||32||2, 4||32, 33, 34, 36||0.048|
|Zinc||65||2||64, 66, 67, 68, 70||0.02|
a Rounded to the nearest integer.
b Atomic numbers of natural isotopes.
c Natural abundance in Earth’s crust as a percentage of the total.
Sources are wholemeal flour and seafoods. Cobalt’s role is as a component of vitamin B12. Uncomplexed cobalt can be absorbed and subsequently excreted in urine. Intakes of cobalt are approximately 0.3 mg or 5 µmol/day and the total body content is 1.5 mg (7.5 µmol). In Quebec, a cobalt-containing beer improver (15 µmol of cobalt/litre) proved to be toxic; its best customers developed severe cardiomyopathy.
Chromium potentiates the action of insulin and may participate in lipoprotein metabolism, in maintaining the structure of nucleic acids, and in gene expression. The cationic trivalent form is biochemically active. Chromium is present in most foods especially meat, brewer’s yeast, wheat germ and whole grains, legumes, and nuts. Dietary intake in adults vary between 13 and 49 µg/day, more in older people, but absorption is meagre at 1%. Requirements are calculated to be about 23 µg (0.38 µmol/day) and a safe intake of above 25 µg/day has been given for adults. The plasma concentration of chromium is 0.3 µg/ml bound to transferrin, and it is excreted in urine. Deficiency increases the risk of type 2 diabetes and cardiovascular disease. The recommended adult intake is 0.5 µmol/day and between 2 and 19 nmol/kg per day for children and adolescents. The adult body contains 100 to 200 µmol. The chromium content of human milk is 0.06 to 1.56 ng/ml. Chromium in high dosage is well tolerated. Chromium deficiency has been reported in patients on long-term unsupplemented parenteral nutrition in whom glucose intolerance developed.
Copper is a component of mitochondrial complex IV, cytochrome c oxidase and other oxidase enzymes, and cuproenzymes involved in the synthesis of haem. It is also a component of many other enzymes, including superoxide dismutase. Cuproenzymes are involved in the synthesis of a range of neuroactive amines. Copper is required by the immune, nervous, and cardiovascular systems for skeletal development, iron metabolism, and red-cell formation.
Good sources of copper include green vegetables, fish, oysters, and liver. Between 35% and 70% of ingested copper is actively absorbed, but this is affected by age and the chemistry of accompanying food. Copper is concentrated in the liver, excreted in bile, and lost in faeces. In plasma, copper is bound to caeruloplasmin and albumin. The total amount of copper in an adult is approximately 2 mmol (50–120 mg). Copper accumulates in the fetal liver for early extrauterine life; premature birth results in depleted copper stores. Copper deficiency in adults results in anaemia and neutropenia, and a myelopathy presenting with a spastic gait and sensory ataxia, which is similar to the subacute combined degeneration of B12 deficiency. There may be hyperzincaemia even in the absence of excess zinc intake. Patients taking large doses of zinc, or the chelator penicillamine for cystinuria and as a second-line agent in rheumatoid arthritis, are at risk from copper deficiency, as are patients post-gastrectomy or with malabsorption. Copper has been implicated as related to the development of Alzheimer’s disease. Poisoning with copper presents with haemolysis and brain and hepatocellular damage. The requirement for adults (including during pregnancy) is 1.5 to 3 mg/day, with a proposed reference nutrient intake (RNI) of 1.2 mg (19 µmol)/day. Children range 0.6 to 1.5 mg/day with RNI of 36 µg (0.6 µmol)/kg per day at 9 months to 17 µg (0.3 µmol)/kg per day at age 15 years.
Iodine is present in modest amounts in most food and drinking water. Seafood, milk, and meat are good sources. Iodine is required for thyroxine 3,5,3′,5′-tetraiodothyronine (T4) and 3,5,3′-tri-iodothyronine (T3). Selenium and zinc are important in the conversion of T4 to the active T3, catalysed by selenium-dependent iodothyronine deiodinase. Dietary iodine from food and water is absorbed as inorganic iodide and transported to the thyroid gland. The body content of iodine is between 20 and 50 mg (160–400 µmol).
Goitre results from iodine deficiency and is endemic in mountainous areas. Some 800 million people are at risk of iodine deficiency, of whom 190 million may develop goitres and more than 3 million are cretinous. These populations have an iodine intake less than 25 µg/day—the required intake being 80 to 150 µg/day. Iodine replacement is essential for these populations and may be added to food, salt, or water or by the direct administration of iodine. Goitre may also arise through eating plants containing goitrogens (e.g. thiocyanate in cassava, maize, bamboo shoots, etc.) Maternal iodine deficiency is associated with perinatal death, stillbirths, spontaneous abortions, endemic cretinism, and congenital abnormalities. Thyroxine is essential for brain development during the first 2 years of life. A modest increase in the incidence of hyperthyroidism occurs following the ingestion of iodized salt preparations in individuals over 40 years of age.
Thyroid hormone and urinary iodine measurements reflect iodine status. Adults require 140 µg/day, babies 40 µg/day, infants 50 µg/day, and children 50 to 140 µg/day. Intake should increase to 175 µg/day during pregnancy and 200 µg/day with lactation. Breast milk contains 44 to 93 µg/litre, an adequate iodine intake.
Magnesium is present in most foods, particularly chlorophyll-containing vegetables (it is necessary for photosynthesis). Magnesium is said to be absorbed by a saturable transport system and passive diffusion, but recent studies have shown no change in magnesium fractional intestinal absorption over the typical range of dietary magnesium intakes. Excretion through the kidneys is the primary mechanism for magnesium regulation. This regulation occurs in response to plasma magnesium concentrations in the distal tubule and the ascending loop of Henle. Magnesium reabsorption is also governed by potassium depletion, the rate of salt and water excretion, parathyroid hormone, calcitonin, glucagon, vasopressin, and acid–base changes. Magnesium homeostasis includes reabsorption of endogenous magnesium from enteric secretions. The plasma concentration varies between 0.6 and 1.0 mmol/litre, and adult whole-body magnesium is 1 mol or 25 g—two-thirds in bone with phosphate and calcium, the remainder being complexed with ATP. Magnesium is the second most prevalent intracellular cation (after potassium).
Magnesium is a cofactor for cocarboxylase and is involved in the replication and transcription of DNA and translation of RNA. Restriction enzymes require magnesium for catalytic activity, and it is essential to the function of restriction endonucleases.
Magnesium is also important in energy metabolism. Nucleoside monophosphate kinases catalyse the transfer of a phosphoryl group from a nucleoside triphosphate such as ATP to the phosphoryl group on a nucleosyl monophosphate, e.g. AMP. These enzymes require divalent metal ions such as magnesium or manganese for activity, which bind to the nucleotide (e.g. ATP). Essentially, all nucleoside triphosphates are present as magnesium complexes. Magnesium assists in catalysis by ‘induced fit’ in which induced structural changes bring two substrates (e.g. AMP and ATP) in close apposition. Hexokinase, which catalyses the transfer of phosphoryl from ATP to six-carbon hexoses, is another important example of a magnesium- (or manganese-) dependent enzyme. Deficiency in dietary magnesium may predispose to type 2 diabetes.
Magnesium has an important role in skeletal development and the maintenance of electrical potential in nerve and muscle membranes.
Magnesium deficiency is manifested by progressive muscle weakness, failure to thrive, neuromuscular dysfunction, arrhythmias, hallucinations, positive Chvosteck’s and Trousseau’s signs, coma, and death. It results in glucose intolerance and hyperinsulinaemia. Cardiovascular abnormalities associated with hypomagnesaemia include widening of the QRS complex, prolongation of the P–R interval, inversion of the T wave, ventricular arrythmias, and increased sensitivity to cardiac glycosides. Hypomagnesaemia is commonly associated with hypocalcaemia and hypokalaemia, and correction of the magnesium depletion may be necessary to restore plasma levels of the other two ions. Magnesium supplementation leads to a rise in parathormone level under these circumstances.
Hypomagnesaemia results most commonly from gastrointestinal losses in diarrhoeal states, malabsorption, and primary intestinal hypomagnesaemia, and renal losses in volume overexpansion, hypercalcaemia, and osmotic diuresis. It may result from drugs such as diuretics, alcohol, aminoglycosides, cisplatin, amphotericin, ciclosporin, foscarnet, and pentamidine. Primary renal tubular magnesium wasting occurs in two conditions: one is characterized by hypercalciuria, nephrocalcinosis, and a tubular acidification defect; the other, Gitelman’s syndrome, is associated with hypocalciuria and a defect in the gene encoding for the thiazide-sensitive sodium–chloride cotransporter. A low plasma magnesium concentration results in a state of functional hypoparathyroidism with parathormone resistance. Hypomagnesaemia can occur after parathyroidectomy, phosphate depletion, correction of chronic acidosis, obstructive nephropathy, and the diuretic phase of acute tubular necrosis. It may also occur during the refeeding of the malnourished patient as part of the refeeding syndrome.
Intravenous infusion of magnesium is beneficial in atrial fibrillation, and in the management of drug-induced ventricular tachycardia/fibrillation—torsade de pointes. Intravenous and nebulized magnesium salts are beneficial in asthma. Intravenous magnesium has been used in pre-eclampsia.
Urinary magnesium is an approximate measure of dietary intake. Typically, a diet in the United Kingdom contains between 8 and 17 mmol of magnesium/day (200–400 mg). Reference nutrient intakes for adult men and women are 12.3 mmol (300 mg) and 10.9 (270 mg) per day, with an increment during lactation of 50 mg (2.1 mmol). Human breast milk contains 0.12 mmol (2.8 mg)/litre. The lactating mother should increase her magnesium intake by 2.0 mmol/day (50 mg). Babies require 30 mg/day, infants 75 mg/day, and children 80 to 200 mg/day.
Therapeutically, magnesium can be administered intravenously in the acute management of tetany or cardiac arrythmias or orally in more chronic deficiency. Oral preparations can cause diarrhoea and the preparations most commonly used are either magnesium glycerophosphate or magnesium oxide tablets, providing 4 mmol per tablet. Doses of 24 mmol/day or more may be needed. Intravenous magnesium is given as magnesium sulphate, 4 to 8 mmol given over 15 to 60 min, depending on the situation.
Tea, cereals, legumes, and leafy vegetables are good sources of manganese. Manganese absorption is only 3 to 4% efficient. Calcium, phosphorus, fibre, and phytate interact with and reduce manganese absorption. Manganese is a cofactor and enzyme activator. In animals, manganese is present in enzymes, such as hexokinase, mitochondrial superoxide dismutase, and xanthine oxidase. The plasma concentration is between 1 and 2 µg/g bound to transferrin, the body pool contains 0.3 mmol, and excretion is in bile. In children receiving parenteral nutrition, manganese toxicity has been associated with chronic liver disease and cholestasis. Manganese deficiency has not been reported in humans. The average intake in the United Kingdom is 2 to 5 mg/day, half from tea. Safe intakes for adults are more than 1.5 mg (25 µmol)/day and for children and infants more than 16 µg (0.3 µmol)/kg per day. Breast milk contains 15 µg/litre. A syndrome similar to Parkinson’s disease is associated with manganese toxicity and ‘manganese madness’ was the term used to describe the psychiatric syndrome of manganese toxicity (compulsive behaviour, emotional lability, hallucinations).
Important dietary sources are wheat flour and its germ, legumes, and meat. Molybdenum is a cofactor for oxidases important in the metabolism of DNA and sulphites, such as xanthine oxidase, xanthine dehydrogenase, aldehyde oxidase, and sulphite oxidase. Intestinal absorption efficiency is high, at 40 to 100%. Plasma concentration is 1 µg/100 ml, and molybdenum is bound to protein. Storage is in the liver and excretion in urine. There are no clinical reports of molybdenum deficiency in humans, but functional defects responsive to molybdenum occur in adults taking 25 µg/day. Gout has been attributed to high molybdenum intakes of 10 to 15 mg/day. Safe intakes for adults lie between 50 and 400 µg/day (0.5 to 4 µmol/day). Breastfed infants require 0.5 to 1.5 µg/kg per day.
It has not been established whether nickel is essential in humans. Absorption is 3 to 6% of the dietary intake. Plasma concentrations are between 2 and 4 µg/100 ml, some of which is bound to albumin. Nickel is excreted in urine. Nickel deficiency might result in depressed growth and haemopoesis. Requirements are unknown, but intakes in the United Kingdom are about 140 µg/day (2.4 µmol/day).
Phosphorus is present in all natural foods, the usual diet in the United Kingdom providing 1.5 g of phosphorus daily. Phosphorus is an important physiological component: with calcium, of the bony skeleton; of ATP in oxidative phosphorylation; in nucleic acids through phosphorylation of nucleotides; and in enzyme control through phosphorylation by protein kinases. Phosphorus is absorbed as free inorganic phosphorus from the diet (controlled by calcitriol) at both the brush-border and basolateral membranes. The plasma concentration of phosphorus is between 0.8 and 1.4 mmol/litre and it is excreted in both urine and faeces. The bony skeleton contains 80% of the body content of phosphorus as the calcium salt, 19 to 29 mmol (600–900 g). Recommended phosphorus requirements are equimolar to calcium.
Selenium is said be ‘the only trace element to be specified in the genetic code’. Selenocysteine, which has been dubbed ‘the 21st genetically coded amino acid’, is a vital component of 35 or more selenoproteins, some of which are important enzymes. Selenium functions as a redox centre, an example of which is the reduction of hydrogen peroxide and lipid and phospholipid hydroperoxides to nondamaging water and alcohols by the glutathione peroxidases. Functions of this kind help maintain membrane integrity, and protect prostacyclin production. Prevention of the oxidative chain reactions in this way prevents further damage to lipids, lipoproteins, and DNA; hence its antioxidant function helps prevent atheroma and cancer, among other things. Selenium is also a cofactor of certain enzymes (e.g. iodothyronine deiodinase and glutathione peroxidases).
Selenium is found in food as selenoamino acids or selenoproteins, and as selenide, selenite, or selenate. Brazil nuts are a rich source, but the main sources of selenium are cereals, meat, and fish. The selenium content of food depends upon soil content and varies regionally and nationally, with soil levels relatively high in Canada, Venezuela, the United States of America, and Japan, and low in most European countries including the United Kingdom. Soil conditions have been associated with clinical selenium toxicity in animals (selenosis) in some areas with very high selenium content, and with selenium deficiency syndromes in animals and humans in areas of low soil selenium. Soil levels in China vary notoriously, with some being exceptionally high, but low level areas associated with the endemic cardiomyopathy, Keshan disease.
Absorption is efficient at 35 to 85%. Population minimum mean intakes likely to meet basal requirements and prevent overt deficiency disease for adult males and females are estimated to be 21 and 16 µg/day, respectively. The RNI for adult men is 75 µg/day (0.9 µmol/day) and for women 60 µg/day (0.8 µmol/day). This intake optimizes the activity of glutathione in plasma and this optimization occurs at plasma levels of 95 µg/litre (range 89–114 µg/litre). However, current intakes in the United Kingdom are about half the RNI. The plasma concentration of selenium is between 7 and 30 µg/100 ml protein bound. Excretion is in urine and possibly bile. Urinary selenium output, red-cell selenium levels, or glutathione peroxidase activity are markers of recent and medium-term dietary intake. Fertility requires an adequate selenium intake, but pregnant women have no additional dietary selenium requirements. Lactation requires an increase in dietary intake of 15 µg/day (0.2 µmol/day). Breast-fed infants should receive approximately 10 µg/day (0.1 µmol/day) and children 15 to 30 µg/day (0.2–0.4 µmol/day). Breast milk contains 20 to 60 µg/litre of selenium.
Selenium deficiency results in impaired immunity, and supplementation is immune stimulant. Selenium deficiency increases the virulence of some viruses (see Keshan disease in the following paragraphs) by altering their genome to a more virulent one. In veterinary practice, selenium deficiency is well known to result in fetal loss in pregnancy and this may be true in humans too. Selenium is important for male fertility through testosterone synthesis and spermatozoa function. Deficiency may be associated with low mood, increased cognitive decline in older people, and susceptibility to epilepsy. Selenium deficiency exacerbates hypothyroidism in iodine deficiency and may be protective against cardiovascular disease and cancer. It may also be beneficial in a variety of inflammatory conditions by reducing oxidative stress.
Clinical interest in selenium lies in four main areas:
- ◆ overt deficiency syndromes
- ◆ clinical toxicity
- ◆ optimal intakes for health
- ◆ use in critical care as an antioxidant
Keshan disease is a cardiomyopathy associated with enlarged and swollen mitochondria in cardiac muscle. It occurs in selenium-deficient areas of China, and selenium supplementation is beneficial and preventive. It may be the result of an interaction between selenium deficiency and some other factor, perhaps viral (coxsackievirus).
Kashin–Beck disease, first described in 1849, is estimated to affect more than 3 million people worldwide. It is a disabling osteoarticular disease of the epiphyseal growth plate and articular cartilage, which presents at about the age of 5 and involves increasing numbers of joints up the age of 25, following which osteoarthritic degeneration of the affected joints occurs. There is enlargement of the metaphyseal area and shortening of the diaphysis giving rise to arthropathy and growth retardation. Radiologically, the disease affects distal aspects of the limb, especially the lower limb, most with the foot and ankle involved very commonly. The disease is usually bilateral. Its aetiology is probably multifactorial: factors include selenium and iodine deficiency, fungal contamination of grain, and water contamination with organic material. Kashin–Beck disease is endemic in parts of south-eastern Siberia, Tibet, and China.
The acute ingestion of selenious acid is almost invariably fatal, preceded by stupor, hypotension, and respiratory depression. Prolonged (months, years) intakes of selenium of more than 1000 µg/day are potentially toxic and can result in hair and nail brittleness and loss. The breath smells of garlic as a result of the expiration of dimethyl selenide and there may be a rash, nausea and vomiting, irritability, and fatigue.
Optimal intakes for health
If the intake of selenium in many countries in the world, including the United Kingdom and Europe, are naturally depleted such that they do not achieve optimal plateau levels of glutathione peroxidase or selenoprotein P, what intakes should we aim at in order to optimize health? There is growing evidence that selenium supplementation in such populations may reduce risks of cancer, particularly prostate and gastrointestinal cancer, and cardiovascular disease. Putative anticancer mechanisms include the induction of apoptosis, blocking of cell cycle progression, and inhibition of angiogenesis, reduction in DNA damage, and reduction in oxidative stress, particularly in the context of concomitant low intake of vitamin E. Optimal glutathione peroxidase levels can be achieved an oral intakes close to 100 µg/day but to achieve maximal selenoprotein P concentrations may require larger intakes, which are as yet not fully known. Optimization requirements in individuals may depend upon selenoprotein gene polymorphisms.
Sepsis and critical care
Enzymes such as superoxide dismutase, catalase, and gluthathione peroxidase protect against reactive oxygen species. Selenium is a critical cofactor in the activity of gluthathione peroxidase and is also important in the management of peroxynitrite.
The acute-phase response can reduce circulating levels of selenium, via redistribution out of the blood stream. Studies of patients with burns or systemic immune response syndrome (SIRS) have shown that without supplementation selenium levels can be depleted for a period of 10 to 14 days.
Various doses of selenium have been prescribed in clinical trials to see if this can reduce oxidant stress and mortality in critical illness. The 500-µg dose has been shown to significantly reduce the need for haemodialysis in patients with SIRS and reduce pulmonary infections in patients with burns. Meta-analyses of selenium supplementation trials in critical illness have demonstrated reduction in mortality and that studies using higher than the median dose of selenium(500–1000 µg/day) were associated with a trend toward reduced mortality, whereas studies using a dose less than the median(<500 µg/day) were found to have no effect on mortality. However, a Cochrane meta-analysis in 2005 concluded that there was insufficient evidence to recommend the supplementation of critically ill patients with selenium, except in the setting of randomized clinical trials.
Cereal grains and other sources of dietary fibre are important sources of silicon. The role of silicon in human nutrition may be important in cartilage and connective tissue as the human aorta, trachea, lungs, and tendons are rich in silicon. Silicic acid is readily absorbed. The body storage pool is approximately 3 g (1 mol) in a 60-kg man; the plasma monosilicic acid concentration is 500 µg/100 ml. The dietary requirements for silicon are unknown.
Sulphur occurs in proteoglycans; dermatan, chondroitin, and keratin sulphate; glutathione; and coenzymes including coenzyme A. Cysteine, methionine, and disulphide crosslinkage are important in proteins, and sulphate is involved in detoxification processes. Sulphur is absorbed as amino acids, which are subsequently desulphated, and excreted in urine as sulphates. Dietary intake is of the order of 0.7 mg (22 µmol)/day. The dietary requirements for sulphur are unknown.
Zinc is an essential nutrient, whose biological functions include cellular integrity and function, growth, immunity, antiapoptotic effects, antioxidant effects through metallothionein induction, and protection against vitamin E depletion. Zinc is an important component of many metalloenzymes, of which carbonic anhydrase was the first to be described; included are over 200 enzymes such as alcohol dehydrogenase, superoxide dismutase, DNA polymerase, RNA polymerase, and alkaline phosphatase. Zinc is therefore required in many processes including nucleic acid synthesis, cell division, protein synthesis and digestion, carbohydrate metabolism, oxygen transport, protection from free radical damage, dark adaptation. It is essential in immune defence: leucocyte-mediated, antibody-mediated, cell-mediated and delayed immune responses. Zinc finger proteins are important as gene transcriptional regulators. It has long been believed that zinc is important for wound healing.
Dietary sources are meats, cheese, whole grains and, to a lesser extent, unrefined cereals, legumes and shellfish. Approximately 20% of dietary zinc is absorbed complexed with amino acids, phosphates, and organic acids. The daily intake of zinc in developed countries is around 9 to 12 mg per day and the RNI for men and women respectively is 9.5 mg and 7.0 mg/day. Deficiency is predicted on long-term intakes below the lower reference nutrient intake (LRNI) of 5.5 and 4.0 mg/day in adult men and women respectively. Phytates and oxalates form insoluble complexes, which inhibit absorption. The normal plasma concentration of zinc is between 80 and 110 µg/100 ml, complexed with albumin. The adult body content of zinc is over 2 g (30 mmol). Bone, the prostate, semen, and the choroid of the eye all contain high concentrations of zinc. Loss of zinc from the body is in faeces.
Deficiency of zinc has been recognized for over 30 years as an important component in growth failure and delayed maturation in populations whose diet is marginal. Zinc replacement can enhance growth and immune competence and reduce infant morbidity, e.g. by reducing acute and chronic diarrhoea and respiratory illness. In hospital practice, deficiency is seen in diarrhoeal states, particularly in the context of short bowel when the duodenal secretions of zinc are insufficiently reabsorbed. Zinc deficiency has been reported as a feature in a number of diseases, including the florid deficiency state and skin condition acrodermatitis enteropathica, an autosomal recessive trait leading to selective impairment of zinc uptake by the upper small-intestinal mucosa. It is also reported in incomplete parenteral nutrition and in patients with severe malabsorption due to Crohn’s disease and other intestinal disorders, especially those associated with a loss of inflammatory cells in the gut lumen.
|Table 2 Dietary reference values: RNI (United Kingdom) and USDA goals (United States)|
|Nutrient||Age/gender||LRNI units||EAR (UK)||RNIa||Age/gender||USDA RDA|
|Thiamin||M 11–50+||0.23 mg/1000 kcal||0.3||0.4||M 14–70+||1.2 mg/day|
|F 11–50+||0.23 mg/1000 kcal||0.3||0.4||F 19–70+||1.1 mg/day|
|Riboflavin||M 11–50+||0.8 mg/day||1.0||1.3||M14–70+||1.3 mg/day|
|F 11–50+||0.8 mg/day||0.9||1.1||F 19–70+||1.1 mg/day|
|Niacin||M11–50+||4.4 mg niacin equiv per 1000 kcal||5.5||6.6||M 14–70+||16 mg/day|
|F11–50+||4.4 mg niacin equiv per 1000 kcal||5.5||6.6||F 14–70+||14 mg/day|
|Vitamin B6||M11–50+||11 µg per g protein||13||15||M 50+||1.7 mg/day|
|F11–50+||11 µg per g protein||13||15||M/F 19–50||1.3 mg/day|
|Vitamin B12||M 15–50+||1.0 µg/day||1.25||1.5||M/F 14–70+||2.4 µg/day|
|F 15–50+||1.0 µg/day||1.25||1.5|
|Folate||M 11–50+||100 µg/day||150||200||M/F 14–70+||400 µg/day|
|Vitamin C||M 15–50+||10 mg/day||25||40||M 19–70+||90 mg/day|
|F 15–50+||10 mg/day||25||40||F19–70+||75 mg/day|
|Vitamin A||M 15–50+||300µg retinol equivalent/day||500||700||M 11+||1000 µg retinol equivalents|
|F 11–50+||250µg retinol equivalent/day||400||600||F 11+||800 µg retinol equivalents|
|Vitamin D||M/F 11–50||0||0||0|
|Vitamin E||M||- mg/day||M/F 14–70+||15 mg/day|
|Vitamin K||M/F||-µg/kg per day|
|Calcium||M/F 19–50+||400 (10) mg/day (mmol/day)||525 (13.1)||700 (17.5)||M/F14–70+||1000–1300 mg/day|
|Magnesium||M 15–50+||190 (7.8) mg/day (mmol/day)||250 (10.3)||300 (12.3)||M 14–70+||400–420 mg/day|
|F 19–50+||150 (6.2) mg/day (-mmol/day)||200 (8.2)||270 (10.9)||F 14–70+||310–360 mg/day|
|Phosphorus||Equal to calcium in mmol|
|Sodium||M/F 15–50+||575 mg/day(25 mmol/day)||–||1600 (70)||M/F 14–70+||<2300 mg/day(<101 mmol)|
|Potassium||M/F 15–50+||2000 mg/day (50 mmol/day)||–||3500 (90)||M/F 14–70+||4700 mg/day (121 mmol)|
|Chloride||M/F||Equal to sodium in mmol|
|Iron||M 19–50||4.7 mg/day (80 µmol/day)||6.7 (120)||8.7 (160)||M 14–70+; F 51–70+||8–11 mg/day|
|F 19–50||8.0 mg/day (140 µmol/day)||11.4 (200)||14.8 (260)||F 14–50||15–18 mg|
|M/F 50+||4.7 mg/day (80 µmol/day)||6.7 (120)||8.7 (160)|
|Zinc||M 15–50+||5.3 mg/day (80 µmol/day)||7.3 (110)||9.5 (145)||M14–70+||11 mg|
|F 15–50+||4.0 mg/day (60 µmol/day)||5.5 (85)||7.0 (110)||F 14–70+||8–9 mg|
|Copper||M/F 18–50+||-mg/d (µmol/day)||–||1.2 (19)||M/F 14–70+||890–900 µg/day|
|Selenium||M 15–50+||40 µg/d (0.5 µmol/day)||–||70 (0.9)|
|F 15–50+||40 µg/d (0.5 µmol/day)||–||60 (0.8)|
|Iodine||M/F 15–50+||70 µg/d (0.6 µmol/day)||140 (1.1)|
a UK RNI (units as per LRNI).
The clinical signs are growth retardation, hypogonadism, bullous-pustular dermatitis, paronychia, lethargy, hepatosplenomegaly, and iron-deficiency anaemia, which responds to zinc supplements (15 mg three times daily). Excessive zinc can lead to nausea, vomiting, and fever.
The plasma concentration of unhaemolysed zinc is a measure of a person’s current zinc status. Zinc in the red blood cells and hair gives a long-term assessment of zinc status. Adults of all ages (and during pregnancy and lactation) require between 12 and 15 mg of zinc (110–145 µmol)/day. Infants need between 4 and 5 mg/day; however, human milk is not a rich source of zinc (2–3 mg/litre) and the infant depends very much on the stores obtained during its last 3 months of interuterine life. Children require 10 to 15 mg/day.
Clinical applications of micronutrient mixtures
Immunonutrition and micronutrient supplementation in critical illness and surgery
Clinical controlled trials have used ‘immune enhancing’ enteral feeds, which combine arginine with other ‘nutriceuticals’ such as antioxidants, glutamine, anti-inflammatory fatty acids, and nucleotides. Most of the clinical trials have employed mixtures of these nutrients in enteral feeds. Since these mixtures vary, results can be difficult to analyse. Meta-analyses have been performed, but provide different interpretations dependent upon which trials are included and which trials are taken together. ‘Immunonutrition’ feeds tend to be associated with a lower number of infectious complications in critically ill patients and a significantly shorter length of hospital stay, most notably in interventions using a high content of arginine. However, the same meta-analysis drew attention to the significantly higher mortality associated with immunonutrition in trials with high (good) methodological scores. At present, routine use cannot be recommended in critical care, but there may be a place for limited use in the context of complicated surgery.
Oxidant stress can be viewed as pivotal to a gradual amplification of the generalized immune response, to the point where it becomes harmful and progresses to multiple organ failure. The use of large doses of antioxidants might prevent this. In critically ill surgical patients a combination of α-tocopherol and ascorbic acid reduced the risk of developing multiple organ failure. Other antioxidants which have been employed include N-acetyl cysteine, vitamin A, and selenium. A meta-analysis showed that aggregation of 11 randomized clinical trials demonstrated reduction in mortality but not infectious complications when antioxidants were tested in critical illness. High-dose parenteral selenium appeared to emerge as the most effective. It is of interest to note that selenium could, through glutathione peroxidase activation, enhance the clinical effect of glutamine.
Preventive antioxidant preparations
Should diet be routinely supplemented? A systematic review of the published literature examined the efficacy of multivitamin and mineral preparations in the primary prevention of chronic disease, specifically cancer, cardiovascular disease, hypertension, cataracts, or age-related macular degeneration. Data for other chronic diseases were lacking. Evidence suggested potential benefit in the primary prevention of cancer in persons with poor nutritional status or suboptimal antioxidant intake. Multivitamin and mineral supplementation had no significant effect on the primary prevention of hypertension, cardiovascular disease, and cataracts, but may slow progression of age-related macular degeneration among persons at high risk for advanced stages of the disease.
Dietary reference values
The dietary recommendations in this chapter are derived from the United Kingdom Department of Health Report on Health and Social Subjects No 41, Dietary Reference Values for Food and Energy and Nutrients for the United Kingdom. The estimated average requirement (EAR) is the mean intake which satisfies the needs for an individual nutrient in a normal population of individuals. The requirements are regarded as being normally distributed and the interindividual variability can be expressed as standard deviations of this mean. The reference nutrient intake (RNI) is set two standard deviations above the EAR and represents a level of intake that is very likely to be adequate for this nutrient. The lower reference nutrient intake (LRNI) is set two standard deviations below the EAR and represents a level below which intake of the nutrient is very likely to be inadequate. These can be compared with the United States Institute of Medicine recommendations, the dietary reference intakes (DRIs), which include estimated average requirements (EARs), recommended daily allowances (RDAs), adequate intakes (AI), and tolerable upper intake levels (ULs). The EAR is the average daily nutrient intake level estimated to meet the requirement of half the healthy individuals in a particular life stage and gender group; the RDA is the dietary intake level that is sufficient to meet the nutrient requirement of nearly all (97–98%) healthy individuals in a particular life stage and gender group; the AI is a recommended average daily intake level based on observed or experimentally determined approximations or estimates of mean nutrient intake by groups of apparently healthy people, which is used when the RDA cannot be determined. The UL is the highest average daily nutrient intake likely to pose no risk of adverse health effects for nearly all individuals in a particular life stage or gender group. As intake increases above the UL, the potential risk of adverse effects increases. Table 2 shows EARs and RNIs for the United Kingdom and compares them with the American RDA.
Cox DN et al. (1998). Take five, a nutrition education intervention to increase fruit and vegetable intakes. Br J Nutr, 80, 123–31.
Eastwood M (1997). Principles of human nutrition. Aspen, Gaithersburg, MD.
Panel on Dietary Reference Values of the Committee on Medical Aspects of Food Policy (1991). Report on Health and Social Subjects 41. Dietary reference values for food energy and nutrients for the United Kingdom. HMSO, London.
Powers HJ (1997). Vitamin requirements for term infants: considerations for infant formulae. Nutr Res Rev, 10, 1–33.
Sadler MJ, Strain JJ, Caballero B, (eds) (1999). Encylopedia of human nutrition. Academic Press, San Diego, CA.
Ziegler EE, Filer LJ Jr (eds.) (1996). Present knowledge in nutrition, 7th edition. ILSI Press, Washington DC.
Benzie IFF (1999). Vitamin C: prospective functional markers for defining optimal nutritional status. Proc Nutr Soc, 58, 469–76.
Sauberlich HE (1994). Pharmacology of vitamin C. Annu Rev Nutr, 14, 371–91.
Thiamin, biotin, and pantothenic acid
Bender DA (1999). Optimum nutrition: thiamin, biotin and pantothenate. Proc Nutr Soc, 58, 427–33.
Butterworth CE Jr, Bendich A (1996). Folic acid and the prevention of birth defects. Annu Rev Nutr, 16, 73–97.
McNulty H (1997). Folate requirements for women. Proc Nutr Soc, 56, 291–303.
Scott JM (1999). Folate and vitamin B12. Proc Nutr Soc, 58, 441–8.
Selhub J (1999). Homocysteine metabolism. Annu Rev Nutr, 19, 217–46.
Scott JM (1999). Folate and vitamin B12. Proc Nutr Soc, 58, 441–8.
McClaren DS (1980). Nutritional ophthalmology. Academic Press, London.
Semba RD (1997). Vitamin A and human immunodeficiency virus disease. Proc Nutr Soc, 56, 459–69.
Thurnham DI, Northrop-Clewes CA (1999). Optimal nutrition: vitamins and the carotenoids. Proc Nutr Soc, 58, 449–57.
Vershinin A (1999). Biological functions of carotenoids—diversity and evolution. Biofactors, 10, 99–104.
Wang XD, Russell RM (1999). Procarcinogenic and anticarcinogenic effect of β-carotene. Nutr Rev, 57, 263–72.
Bischoff-Ferrari HA et al. (2009). Fall prevention with supplemental and active forms of vitamin D: a meta—analysis of randomised controlled trials. BMJ, 339, b3692.
Nutritional aspects of bone; a symposium (1997). Proc Nutr Soc, 56, 903–87.
Wilkinson RJ et al. (2000). Influence of vitamin D deficiency and vitamin D receptor polymorphisms on tuberculosis among Gujarati Asians in west London: a case control study. Lancet, 355, 618–21.
Gabsi S et al. (2001). Effect of vitamin E supplementation in patients with ataxia with vitamin E deficiency. Eur J Neurol, 8, 477–81.
Halliwell B (1996). Antioxidants in human health and disease. Annu Rev Nutr, 16, 33–50.
Moreno-Reyes R et al. (1998). Kashin–Beck osteoarthropathy in rural Tibet in relation to selenium status. N Engl J Med, 339, 1112–20.
Morrisey PA, Sheehy PJA (1999). Optimal nutrition: vitamin E. Proc Nutr Soc, 58, 459–68.
Stevenson VL, Hardie RJ (2001). Acanthocytosis and neurological disorders. J Neurol, 248, 87–94.
Traber MG, Sies H (1996). Vitamin E in humans: demand and delivery. Annu Rev Nutr, 16, 321–47.
Cousins RJ (1994). Metal elements and gene expression. Annu Rev Nutr, 14, 449–69.
Failla ML (1999). Considerations for determining ‘optimal nutrition’ for copper, zinc, manganese and molybdenum. Proc Nutr Soc, 58, 497–505.
Goyer RA (1997). Toxic and essential metal interactions. Annu Rev Nutr, 17, 37–50.
Lukaski HC (1999). Chromium as a supplement. Annu Rev Nutr, 19, 279–302.