3
Vitamins
C. A. Northrop-Clewes and D. I. Thurnham, University of Ulster
3.1 Introduction
Vitamins are classically defined as a group of organic compounds required in very
small amounts for the normal development and functioning of the body. They are
not synthesised by the body, or only in insufficient amounts, and are mainly
obtained through food (Machlin and Huni, 1994).
There are thirteen vitamins: four are fat-soluble, namely vitamins A (retinol),
D (calciferols), E (tocopherols) and K (phylloquinone and menaquinones) and
nine are water-soluble, vitamin C (ascorbate) and the B-complex made up of vita-
mins B
1
(thiamin), B
2
(riboflavin), B
6
(pyridoxal, pyridoxamine and pyridoxine),
B
12
(cobalamin), folic acid, biotin, niacin and pantothenic acid. No single food
contains all of the vitamins and therefore a balanced and varied diet is necessary
for an adequate intake.
3.1.1 Dietary reference values
Prior to 1991 relatively few micro-nutrients were covered in official British
government publications on energy, protein, vitamin and mineral requirements
(Whitehead, 1991). The 1979 definition of the Recommended Dietary Allowance
(RDA) was ‘the average amount of the nutrient which should be provided per
head in a group of people if the needs of practically all members of the group are
to be met.’ However, the RDA values have always been derived differently
depending on whether energy or nutrients were being considered.
In 1987 the Committee on Medical Aspects of Food Policy (COMA) convened
a panel to review the old RDAs of energy, fat, non-starch polysaccharides, sugars,
starches, protein, vitamins and minerals for groups of people in the United
Kingdom (Department of Health and Social Security, 1979). The panel changed
the ‘requirements’ nomenclature, and whereas most sets of RDAs provided only
a single value for the micronutrients, the new Dietary Reference Values (DRVs)
set three levels of values for each age and sex grouping. The aim was to describe
the range of requirements in different individuals more adequately. The estimated
average requirement (EAR) represents the mean requirement of the average indi-
vidual, the reference nutrient intake (RNI) is nominally set at the mean plus two
standard deviations (2 SD), and the lower reference nutrient intake (LRNI) is
nominally the mean minus an estimated 2 SD. The three parameters describe the
spread of requirements.
It was thought that the EAR might be increasingly used in food labelling thus
providing all dietary constituents in food with a common baseline for compari-
son but the RNI is the key value for clinical and health purposes. Table 3.1 is the
summary table of the RNI for six B vitamins, plus vitamins A, C and D. Within
the context of the UK, the reason for considering only 9 out of the 13 vitamins
was that the panel thought only those micronutrients for which some possibility
of deficiency existed needed to be dealt with in such detail. In addition, there was
insufficient information for pantothenic acid, biotin and vitamins E and K to
provide a complete data set of recommendations, hence only single safe intake
recommendations were considered (Whitehead, 1992).
Table 3.2 gives a summary of the principal food sources and major functions
of each vitamin.
3.2 Vitamin A
Vitamin A can be obtained in two forms: pre-formed retinol, usually as retinyl
esters, or as provitamin A carotenoids, such as a- and b-carotene and a- and b-
cryptoxanthin. In the UK about a quarter to a third of dietary vitamin A is obtained
from fruits and vegetables, the majority of this as b-carotene. In many develop-
ing countries up to 100% of dietary intake can be from plant sources and in these
communities, where exposure to infection is usually high, it is most likely to find
vitamin A deficiency disorders (VADD). One of the earliest clinical signs of
vitamin A deficiency (VAD) is night blindness (XN), an impaired ability to see
in dim light. Severe deficiency produces partial or total blindness. The most vul-
nerable groups to VADD are infants and young children and pregnant and lac-
tating women.
3.3 Vitamin A deficiency disorders (VADD)
Vitamin A deficiency disorders may be defined as a level of depletion of total
body stores of retinol and of its active metabolites such that normal physiologic
function is impaired. Dietary intake does not accurately reflect status since intake
may fluctuate considerably in different seasons and the body only stores vitamin
Vitamins 35
36
The nutrition handbook for food processors
Table 3.1 Reference nutrient intakes (RNI) for vitamins
Age
Thiamin Riboflavin Niacin Vitamin B
6
Vitamin B
12
Folate Vitamin C Vitamin A Vitamin D
mg/d mg/d mg/d mg/d# mg/d mg/d mg/d mg/d mg/d
0–6 months 0.2 0.4 3.0 0.2 0.3 50 25 350 8.5
7–9 months 0.2 0.4 4.0 0.3 0.4 50 25 350 7
10–12 months 0.3 0.4 5.0 0.4 0.4 50 25 350 7
1–3 years 0.5 0.6 8.0 0.7 0.5 70 30 400 7
4–6 years 0.7 0.8 11 0.9 0.8 100 30 500 –
7–10 years 0.7 1.0 12 1.0 1.0 150 30 500 –
Males
11–14 years 0.9 1.2 15 1.2 1.2 200 35 600 –
15–18 years 1.1 1.3 18 1.5 1.5 200 40 700 –
19–50 years 1.0 1.3 17 1.4 1.5 200 40 700 –
50 + years 0.9 1.3 16 1.4 1.5 200 40 700 ***
Females
11–14 years 0.7 1.1 12 1.0 1.2 200 35 600 –
15–18 years 0.8 1.1 14 1.2 1.5 200 40 600 –
19–50 years 0.8 1.1 13 1.2 1.5 200 40 600 –
50 + years 0.8 1.1 12 1.2 1.5 200 40 600 ***
Pregnancy/Lactation +0.1* +0.3 ** ** ** +100 +10 +100 10
0–4 months +0.2 +0.5 +2** +0.5 +60 +30 +350 10
4 + months +0.2 +0.5 + +0.5 +60 +30 +350 10
# Based on protein providing 14.7% of EAR for energy.
* For last trimester only.
** No increment.
*** After age 65 the RNI is 10.0mg/d for men and women.
DEPARTMENT OF HEALTH 1991.
V
itamins
37
Table 3.2 Food sources and major functions of principal vitamins
Vitamin Principal food sources Major functions in the body
Vitamin A Animal sources: liver, egg yolk, fish, whole milk, butter, cheese. Helps to keep mucosal membranes healthy, thus increasing
Plant sources (as provitamin A): carrots, yellow and dark green resistance to infections; essential for vision; promotes bones and
leafy vegetables, pumpkin, apricots, melon, red palm oil. tooth development. Vegetable consumption may be protective
against certain cancers.
Vitamin D Fish-liver oils (sardine, herring, salmon, mackerel), eggs, meat, Promotes hardening of bones and teeth, increases the absorption
milk. of calcium.
Vitamin E Vegetable oils (peanut, soya, palm, corn, sunflower etc). Other Protects vitamins A and C and fatty acids; prevents damage to
sources: nuts, seeds, whole grains, leafy green vegetables. cell membranes. Antioxidant.
Vitamin K Green leafy vegetables, soybeans, beef liver, green tea, egg Helps blood to clot. May play a role in bone health.
yolks, potatoes, oats, asparagus, cheese.
Vitamin C Citrus fruits, sweet peppers, parsley, cauliflower, potatoes, Formation of collagen, wound healing; maintaining blood
strawberries, broccoli, mango, Brussels sprouts. vessels, bones, teeth; absorption of iron, calcium, folate;
production of brain hormones, immune factors; antioxidant
Thiamin Dried brewers yeast, animal products, whole grains, nuts, Helps release energy from foods; promotes normal appetite;
(B
1
) pulses, dried legumes. important in function of nervous system.
Folate Main sources: liver, dark green leafy vegetables, beans, wheat Aids in protein metabolism; promotes red blood cell formation;
germ and yeast. Other sources: egg yolk, beet, orange juice, prevents birth defects of spine, brain; lowers homocysteine
whole wheat bread. levels and thus coronary heart disease risk.
Cobalamins Animal products (particularly liver, kidneys, heart, brain) fish, Aids in building of genetic material; aids in development of
(B12) eggs, dairy products. normal red blood cells; maintenance of nervous system.
Vitamin B
6
Chicken, liver of beef, pork, fish (tuna, trout, salmon, herring), Aids in protein metabolism, absorption; aids in red blood cell
peanuts and walnuts, bread, whole-grain cereals. formation; helps body use fats
Biotin Yeast, liver, kidney, egg yolk, soybeans, nuts and cereals. Helps release energy from carbohydrates; aids in fat synthesis.
Pantothenic Yeast, liver, heart, brain, kidney, eggs, milk, vegetables, Involved in energy production; aids in formation of hormones.
acid legumes, whole grain cereals.
Niacin Yeast, liver, poultry, lean meats, nuts and legumes. Less in milk Energy production from foods; aids digestion, promotes normal
and green leafy vegetables. appetite; promotes healthy skin, nerves.
Riboflavin Yeast, liver, milk and milk products, meat, eggs, and green Helps release energy from foods; promotes healthy skin
(B
2
) leafy vegetables. and mucous membranes; possible role in preventing cataracts.
A when the intake exceeds requirements. Tissue concentrations may be assessed
by measuring serum or breast milk retinol, or by using the retinol dose response
(RDR), modified retinol dose response (MRDR), or by dilution with stable iso-
topes. Functional indicators include pupillary dark adaptometry (PDA), conjunc-
tival impression cytology (CIC), and xerophthalmia.
3.4 Bioavailability of provitamin carotenoids
De Pee and West (1996) proposed that control of VADD depends to a large
extent on an adequate supply of vitamin A and the vitamin A supply is determined
by:
Food intake ￥ (pro)-vitamin A content ￥ bioavailability/bioefficacy, where
Bioavailability = fraction of ingested nutrient available for normal physiological
functions and storage (Jackson, 1997)
Bioefficacy = efficiency of absorption and conversion of ingested nutrient to the
active form e.g. b-carotene to retinol (van Lieshout et al, 2001).
3.4.1 Relationship between bioefficacy and vitamin A requirements
The RNI for children up to 5 years of age is 400mg retinol equivalents (RE)/day,
which can easily be met from the diet if animal foods are available e.g. 1 egg
(50 g) contains about 100mg RE, 25 g chicken liver contains 3000mg RE. Plants
also contribute to vitamin A intake e.g. 1 raw carrot (20 g) contains 400mg b-
carotene, a 70 g portion of spinach contains 600mg b-carotene and with a bioef-
ficacy of 100% would supply 400 and 600mg RE respectively. But the
pro-vitamin A carotenoids are absorbed less efficiently than retinol, that is, their
bioefficacy is less than 100%. Therefore the effective supply of vitamin A from
fruits and vegetables is much lower than that from retinol in animal foods (van
Lieshout et al, 2001). If 1 mole b-carotene (Fig. 3.1) yields 2 moles retinol
then, using 100% bioefficacy, 1mmol (0.537mg) b-carotene would be absorbed
and converted totally to 2mmol (0.572mg) retinol, i.e. 0.537/0.572 = 0.94mg b-
carotene is equivalent to 1mg retinol. The results of the ‘Sheffield’ studies carried
out by the Medical Research Council (MRC) during the Second World War pro-
vided important information to establish the relative equivalency of carotenoids
and retinol (Hume and Krebs, 1949). These and other studies suggested that
6mg b-carotene or 12mg of other pro-vitamin A carotenoids in a mixed diet had
the same activity as 1mg retinol (FAO/WHO 1967). Therefore, according to
FAO/WHO the bioefficacy of b-carotene in food is (100% * 0.94)/6 = 16%. But
in the 1990s evidence was accumulating that the bioefficacy of provitamin A
carotenoids in fruit and vegetables was only 20–30% of the FAO/WHO estimates
of 16%. The efficiency with which b-carotene in dark green leafy vegetables
(DGLV) is metabolised to vitamin A was re-examined and various bioconversion
factors have been put forward:
38 The nutrition handbook for food processors
? 4 :1 (Gopalan et al, 1989)
? 6 :1 (Report of a Joint FAO/WHO expert consultation 1988)
? 12 :1 (Report of the Institute of Medicine 2001)
? 21 :1 and 26 :1 (de Pee et al, 1998; Khan et al, 1998; van het Hof et al, 1999)
Assuming that 100 g DGLV contains 3000mg b-carotene and a child of 4 years
needs an RNI of 400mg RE/day then using bioconversion factors of 12 :1,
21 :1 or 26 :1, the child would need 160 g, 280 g or 360 g of DGLV each day to
meet requirements.
3.4.2 Equivalence factor as calculated by IOM
In January 2001, the US National Academy of Sciences/Institute of Medicine
(IOM) announced a new equivalence factor (12 :1) for the conversion of b-
carotene to retinol. The basis for formulating the equivalency factor was to relate
the absorption of b-carotene in a principally mixed vegetable diet to that from oil
in healthy and nutritionally-adequate individuals. The recommendations were
based on a product of relative absorption of b-carotene in mixed vegetable diet
(1 :6) (van het Hof et al, 1999) to the amount of retinol formed (1mg) when 2mg
b-carotene was fed in oil (Sauberlich et al, 1974). In the van het Hof study, the
increase in serum b-carotene concentration after consumption of b-carotene-rich
vegetables was 1/7 or 14% of the increase after consumption of b-carotene in oil.
The Institute of Medicine (IOM) (2001) adjusted the value to 1/6 or 17%, because
of the low fruit content in the diet used. From these two studies, the IOM con-
cluded that the bioefficacy of b-carotene in oil was (100% * 0.94)/2 = 47%
(Sauberlich et al, 1974) and 17% from mixed vegetables (van het Hof et al, 1999).
Thus bioefficacy from vegetables in a mixed diet was 17% * 0.47 = 8%, that
is 12mg b-carotene in food has the same vitamin A activity as 1mg retinol. The
Vitamins 39
Fig. 3.1 Structures of all-trans-retinol and b-carotene.
12 :1 ratio is referred to as the retinol activity equivalence (RAE) (Northrop-
Clewes, 2001a).
In the calculation, the IOM used the mean ratio from only one ‘oil study’.
There were in fact 5 ‘oil’ studies (Booher et al, 1939; Wagner, 1940; Hume and
Krebs, 1949; Sauberlich et al, 1974; Tang et al, 2000) and if the data from all the
studies had been used a bioconversion value of 3.5mg b-carotene to 1mg retinol
would have been calculated giving an RAE of 21 instead of 12 as quoted. The
IOM are at present reconsidering the published data (van Lieshout, 2001). How
we interpret the bioconversion factors for b-carotene proposed by IOM or by
others still needs much more thought before any of them can be put to practical
use. However, even if the RAE is only 12 :1, there are probably not sufficient
vegetables in developing countries to meet that required, therefore, there must be
other, as yet undiscovered factors, that influence bioefficacy. Despite this, intake
of fruit and vegetable-sources of provitamin A should be encouraged, for although
the bioefficacy of b-carotene is apparently so poor, it has to be remembered that
the majority of the world’s children are not VAD.
3.5 Function
Vitamin A, its analogues and metabolites function in vision, cell differentiation,
embryogenesis, the immune response, reproduction and growth.
3.5.1 Vision
In vision, vitamin A is required in two forms for two processes:
1. As 11-cis-retinal in rhodopsin which on exposure to light in the retina iso-
merises to a transoid intermediate, triggering a series of conformational
changes in membrane potential which is transmitted to the brain.
2. As retinoic acid to maintain normal differentiation of the cells of the con-
junctival membranes, cornea and other ocular structures hence preventing
xerophthalmia (Ross, 1999).
3.5.2 Cell differentiation
The role of vitamin A in cell differentiation has been clarified since the identifi-
cation of two sets of nuclear receptors, RXR and RAR, which are activated by
retinoic acid isomers (Olson et al, 2000). Each of the receptors has three distinct
forms a, b and g and six domains involved in transcription of genes. RAR binds
either all-trans or 9-cis retinoic acid, while RXR binds only 9-cis retinoic acid.
Both receptors are dimers, however, the RXR receptors form homodimers with
themselves as well as heterodimers with RAR, the vitamin D receptor, the tri-
iodothyronine receptor and several other nuclear transcription receptors (see also
sections 3.12.3, 3.20.2 and 3.21.3). These interactions usually activate gene
expression but RAR can also be inhibitory.
40 The nutrition handbook for food processors
3.5.3 Embryogenesis
Both retinoic acid and retinol are essential for embryonic development, however,
retinoic acid does not seem to be involved in vertebrate embryogenesis before
gastrulation. After gastrulation specific genes of the Hox family are expressed in
a wave starting about 7.5 days postcoitus in the mouse. The presence of retinoids
together with their binding proteins and receptors in a temporally precise manner
provides strong circumstantial evidence that retinoic acid-activated RARs regu-
late Hox gene expression (Ross, 1999). The Hox family consists of 38 genes
arranged in four chromosomal clusters, which code for transcription factors that
regulate development along the posterior axis. Retinoic acid is also thought to
act in limb development and formation of the heart, eyes and ears. Experimental
VAD has demonstrated that major target tissues of retinoic acid include the heart,
central nervous system and structures derived from it, the circulatory, urogenital
and respiratory systems and the development of skull, skeleton and limbs. Home-
ostasis of retinoic acid is maintained by enzyme systems, which are develop-
mentally regulated by vitamin A. Inadequate vitamin A nutrition during early
pregnancy may account for some paediatric congenital abnormalities (Zile, 2001).
However, the study of this aspect of vitamin A is in its ‘infancy’ and much more
work is still to be done. A high incidence of spontaneous abortions and birth
defects has been observed in the foetuses of women taking therapeutic doses of
13-cis retinoic acid. The drugs Accutane (13-cis retinoic acid) and etretinate, an
aromatic analogue of all-trans retinoic acid have been most implicated in such
effects (Armstrong et al, 1994). The dysmorphogenic effects caused by retinoids
depend on dosage (exposure), the form of the retinoid, its rate of metabolism and
stage of foetal development at the time the retinoid is taken. Retinoids are ter-
atogenic during the period of foetal organogenesis (first trimester) (Ross, 1999).
3.5.4 Immunity
One of the first names for vitamin A was ‘the anti-infective vitamin’, which was
based on the increased number of infections noted in VAD animals and humans
(Ross, 1996). In VAD the humoral response to bacterial, parasitic and viral infec-
tions, cell-mediated immunity, mucosal immunity, natural killer cell activity and
phagocytosis are all impaired. The primary immune response to protein antigens
is markedly reduced but the response to bacterial lipopolysaccharides or the
process of immunological memory essential for secondary response does not
seem to be affected (Ross and Hammerling, 1994).
A major site for vitamin A action in the immune response is the T-helper cell
and studies have shown that the activity of T-helper cells variety 1 (Th1) pre-
cedes that of Th variety 2 (Th2) and that vitamin A supports Th2 development
(Cantorna et al, 1994). Hence, in the presence of VAD the ratio of Th1 :Th2 may
become elevated during an immune response. Retinol, probably in the form of
14-hydroxy-retroretinol (HRR), is thought to be involved in the proliferation of
normal B- and T-cells, an action which can be modulated by some cytokines.
Retinoic acid, the usual active form of vitamin A, appears to be inactive in these
Vitamins 41
systems (Olson et al, 2000). The enzyme responsible for the conversion of retinol
to HRR has not been characterised.
3.6 Health-related roles of b-carotene
3.6.1 b-Carotene as an antioxidant
The ability of carotenoids to act as antioxidants can be measured in vitro, ex vivo,
or in vivo. LDL isolated from an individual who has been supplemented with
carotenoids and then evaluated for its antioxidant activity is an extension of an in
vivo study, i.e. ex vivo. However, when carotenoids are added to plasma and then
the oxidisable value of the LDL is measured it is more like an in vitro model
(Krinsky, 2001). Many studies report using the ex vivo method of measuring the
oxidisability of the LDL particles after feeding increased amounts of carotene-
containing foods. However, when using fruits and vegetables the outcome is vari-
able and difficult to interpret because they also contain vitamin C, polyphenols and
flavonoids, which are also potential antioxidants. One study which gave additional
dietary fruits and vegetables to subjects reported an increase in the resistance of
LDL to oxidation (Hininger et al, 1997) while two other studies found no effect
(Chopra et al, 1996; van het Hof et al, 1999). The differing results obtained may
be due to different time periods on the diets, different degrees of change in the
plasma carotenoids or to different study populations (Krinsky, 2001).
3.6.2 b-Carotene and protection from cancer
The strongest epidemiological evidence suggesting high intake of fruits and veg-
etables might give protection against lung cancer came from prospective studies
in which low plasma b-carotene was associated with a higher incidence of lung
cancer. Carotenoid intake was associated with reduced cancer risk in 8 prospec-
tive studies and 18 out of 20 retrospective studies (Zeigler et al, 1996). Based on
the results of these studies, three major intervention studies investigated the pro-
tective effect of b-carotene in the prevention of lung cancer:
(a) The alpha-tocopherol, beta-carotene (ATBC) Cancer Prevention study, was
a randomised-controlled trial that tested the effects of daily doses of 50 mg (50
IU) vitamin E (all-racemic a-tocopherol acetate), 20 mg of b-carotene, both or
placebo in a population of more than 29 000 male smokers for 5–8 years. No
reduction in lung cancer or major coronary events was observed with any of the
treatments. What was more startling was the unexpected increases in risk of death
from lung cancer and ischemic heart disease with b-carotene supplementation
(ATBC Cancer Prevention Study Group, 1994).
(b) Increases in risk of both lung cancer and cardiovascular disease mortality
were also observed in the beta-Carotene and Retinol Efficacy Trial (CARET),
which tested the effects of combined treatment with 30 mg/d b-carotene and
retinyl palmitate (25 000 IU/d) in 18 000 men and women with a history of ciga-
rette smoking or occupational exposure to asbestos (Hennekens et al, 1996).
42 The nutrition handbook for food processors
(c) The third study was the Physicians Health Study, in which 22 071 US male
physicians were randomised to get 50 mg b-carotene or aspirin (325 mg), or both
or neither every other day for 12 years. There was no evidence of a significant
beneficial or harmful effect on cancer or cardiovascular disease, but the number
of smokers in the study was too small to be certain whether b-carotene was
harmful in the group or not (Hennekens et al, 1996).
One other study should also be mentioned. The Cancer Prevention Study II, a
prospective study on more than one million US adults investigated the effect of
commercially available multivitamins and/or vitamins A, C, and/or E on mortal-
ity during a 7 year follow-up. The use of multivitamins plus A, C and/or E sig-
nificantly reduced the risk of lung cancer in former smokers and in those who
never smoked, but increased the risk in men who smoked and used vitamins A,
C and/or E compared with men who reported no supplement use. Thus the
‘antioxidant’ vitamins A, C and E only appear to benefit male non-smokers. No
association with smoking was seen in women (Watkins et al, 2000).
3.6.3 Reasons for increased cancer risk associated with
b-carotene supplementation
The mechanism for the increased risk associated with b-carotene supplementa-
tion in smokers is unclear. One suggestion is that the subjects of the studies
already had a ‘high risk’ of developing lung cancer and many might have had
undetected tumours at the start. The stage of carcinogenesis that b-carotene might
affect is not known but if mediated by the immune system the effect might be at
the promotional stages preceding the formation of a malignant tumour (Hughes,
2001). The immune system appears to be particularly sensitive to oxidative stress.
Immune cells rely heavily on cell-to-cell communication, particularly via mem-
brane bound receptors, to work effectively. Cell membranes are rich in polyun-
saturated fatty acids and if peroxidised, can lead to a loss of membrane integrity,
altered membrane fluidity and result in alterations in intracellular signalling and
cell function. It has been shown that exposure to reactive oxygen species (ROS)
can lead to a reduction in cell-membrane expression (Hughes, 2001). In addition,
the production of ROS by phagocytic immune cells can damage the cells them-
selves if not adequately protected by antioxidants such as b-carotene, lycopene
and lutein.
One of the major unresolved dilemmas of b-carotene research is the intake
required to optimise immune function and provide other health benefits (Hughes,
2001). Most studies have been done using pharmacological doses of b-carotene
and it is not clear whether different intakes are associated with different outcomes.
It is also possible that supplemental b-carotene might be interfering with intesti-
nal absorption of other possible chemopreventive nutrients e.g. b-carotene can
inhibit absorption of lutein, a-carotene and canthaxanthin, all of which show
good antioxidant properties (Olson, 1999). Another explanation might be that b-
carotene is acting as a pro-oxidant in the presence of high oxygen tension in
the lung.
Vitamins 43
The apparent protection of a diet high in fruit and vegetables is likely to be
the result of a multifactorial effect from a number of components in those foods.
Two recent prospective studies found that subjects entering the studies with
higher plasma b-carotene concentrations from dietary intake had a lower risk of
lung cancer (McDermott, 2000). This finding perhaps suggests more studies using
dietary enrichment with carotenoids rather than pharmacological supplements
should be carried out.
3.7 Safety of vitamin A and b-carotene
3.7.1 Safety of retinol
Symptoms of hypervitaminosis A may occur in the skin, nervous system, mus-
culoskeletal, circulatory systems or in internal organs. Toxicity varies with the
dose, body mass, age, sex, disease conditions, concurrent drugs being taken and
environmental chemical exposure (Blomhoff, 2001). Toxicity is rare from natural
diets, the exception is from high intakes of liver (3–5 mg/100 g) (Northrop-
Clewes, 2001b). Fortified foods are used in industrialised countries and can be
consumed excessively (e.g. children may consume several bowls of breakfast
cereal a day). Supplements, i.e. multiple micro-nutrients containing vitamin A,
are readily available in industrialised countries and the usual content is:
? 1500mg RE non-pregnant, non-lactating women.
? 1000mg RE pregnant/lactating women.
? 750mg RE for children < 5 years.
However, in general, healthy individuals in industrialised countries should not
need supplements, as eating a balanced diet should provide all the nutrients
required. In contrast, VAD is common among women of reproductive age living
in deprived conditions and may be associated with a substantial increase in mater-
nal mortality. The immediate postpartum period represents an opportunity to
provide such women with a large dose of vitamin A, which benefits both mother
and child. Since 1982 WHO/UNICEF/IVACG have recommended supplement-
ing postpartum women and their infants, where VADD are a public health
problem, with:
? Mothers: 200 000 IU < 6 weeks post partum.
? Infants: 25000IU at 6, 10, 14 weeks and 100000IU at 9 months.
However, theoretical calculations and recent data suggest the dose is too small
and so in 2001, new WHO supplement recommendations were proposed:
? Mothers: 200 000 IU at delivery + 200000IU < 6–8 weeks after delivery
? Infants: 50 000 IU at 6, 10, 14 weeks + 100 000 IU 6–11 months + 200 000 IU
every 4–6 months after 12 months.
Acute toxicity
Acute hypervitaminosis A can be defined as any toxicity manifested following a
single very high dose or several very high doses over a few days (Blomhoff,
44 The nutrition handbook for food processors
2001). Symptoms of acute toxicity include: increased cerebrospinal fluid pres-
sure, bulging fontanelle in infants, headache and blurred vision in adolescents
and adults, loss of appetite, nausea, vomiting lassitude and abdominal pain.
An acceptable dose for mothers is 400 000 IU vitamin A (120 mg retinyl
palmitate i.e. 1 IU = 0.3mg retinol) post partum while 50 000 IU is assumed safe
for 0–6 month infants, 100 000 IU for infants > 6 months and 200000IU for those
> 12 months old. However, in infants and children 25 000–50 000 IU occasion-
ally leads to bulging fontanel. In about 6% of infants 300 000 IU can lead to
nausea and vomiting and to diarrhoea in about 16% and although this is transient,
it is unacceptable (IOM, 2001).
Retinyl esters in plasma are indicators of high or recent vitamin A intake i.e.
they transiently increase after a vitamin A-rich meal, 1–2 ￥ RNI and particularly
with supplementation. Plasma retinoic acid will also go up after eating liver or
taking high doses of vitamin A, and in lactating women the retinoic acid may go
into the breast milk although this has not been measured. Breast milk retinyl
palmitate is a useful indicator to monitor total maternal vitamin A intake, that is,
milk retinol concentration will be very similar to plasma retinol. There is no
evidence of an upper concentration of breast milk vitamin A that is harmful to
infants.
Chronic toxicity from retinol
Definitions of upper limits of toxicity (IOM, 2001)
No observed adverse effect level (NOAEL) or the highest dose which has no
adverse effect.
Tolerable upper intake level (UL) is the highest level of intake that is
likely to pose no risk for almost all members of the population, where UL =
NOAEL/uncertainty factor (UF).
Chronic hypervitaminosis A can be defined as any symptom resulting from
continued ingestion of high doses of vitamin A for months or years (Blomhoff,
2001). In adults, there are many reports of chronic toxicity where the intake of
vitamin A exceeds 15 mg/d, characterised by headache, fatigue, anorexia, itchy
skin, liver damage, desquamation of mucous membranes and of skin.
The NOAEL for adults is set 15 mg/d and the UL is 3 mg/d, where the UF =
5 because of severe irreversible effect and inter-individual variability in sensi-
tivity. If the UL was <3 mg, 50% of adults in the US would exceed the UL, yet
few cases of liver toxicity are seen. The mean normal US liver concentration is
100mg/g (range 10–1400mg/g).
In infants and young children toxicity produces bulging fontanelle. The
NOAEL is 6 mg/d and the UL is 0.6 mg/d, where the UF = 10 because of uncer-
tainty and inter-individual variability in sensitivity.
In women of child-bearing age, toxicity can lead to teratogenicity, therefore
the NOAEL is set at 4.5 mg and the UL at 3 mg/d (10 000 IU). The most serious
effects of vitamin A toxicity include foetal resorption, birth defects, abortion
and permanent learning difficulties in the offspring (IOM, 2001) (see section
3.3.3).
Vitamins 45
3.7.2 Safety of b-carotene
Experimental studies with animals have shown that b-carotene is not mutagenic
or teratogenic. In addition, doses of 180 mg/day have been used over many years
to treat patients with erythropoietic protoporphyria, with no evidence of vitamin
A toxicity (Blomhoff, 2001). b-Carotene is considered not to be toxic because
absorption becomes inefficient at high intakes, possibly because conversion of b-
carotene and other provitamin A carotenoids is regulated by the vitamin A status
of the individual. In two studies in which very different large intakes of b-carotene
were given (15 and 40 mg), the mean absorption of b-carotene was <2mg sug-
gesting the human intestine possess only a limited capacity to absorb b-carotene
(van Vliet et al, 1995; O’Neill and Thurnham, 1998).
3.8 Vitamin D
Recognition of the antirichitic effect of meat fat in the 1920s, as well as the pro-
tective effects of sunlight led to the discovery of vitamin D. Vitamin D is the
name given to a group of fat-soluble compounds essential for maintaining the
mineral balance of the body. Vitamin D is also known as calciferol and the anti-
rachitic vitamin and its principal function is to regulate calcium and phosphate
metabolism. It has two main forms: ergocalciferol, vitamin D
2
(plant origin) and
cholecalciferol, vitamin D
3
(animal origin).
Vitamin D is produced from endogenous sources, synthesised in the skin from
7-dehydrocholesterol (7DHC) in a reaction catalysed by the ultra-violet (UV)
light, and exogenous sources from the diet. There are only a few natural food
sources, egg yolk, oily fish, butter and milk (Table 3.2). Margarines and spreads
are fortified with vitamin D. Vitamin D, either natural or added, is stable in foods,
and storage, processing and cooking do not affect its activity. The normal human
diet is, however, a trivial source of vitamin D, since the biggest source results
from exposure to sunlight. However, vitamin D production by the skin is strongly
related to latitude and season, because short UV wavelengths of light are neces-
sary for photoconversion. This means that in the UK and other countries in the
northern latitudes, sunlight during the winter months is ineffective for the pro-
duction of vitamin D because the sun is so low in the sky, the absorption by ozone
too great and UV-B radiation too scattered (Maxwell, 2001).
There are at least 37 metabolites of vitamin D (Norman, 1990) but only three:
25 hydroxyvitamin D (25-OHD), 1,25 dihydroxvitamin D
3
(1,25-OHD) and 24,
25 dihydroxyvitamin D (24,25-OHD) have any important biological activity.
Plasma 25-OHD is an index of availability of vitamin D and the normal range is
20–150 nmol/L (8–60 ng/ml). Values below 25 nmol/L (10 ng/ml) indicate risk of
deficiency and toxicity occurs at levels above 150 nmol/L (60 ng/ml). The average
intake within the UK ranges from 0.5 to 8mg/day, dependent on season, with a
mean around 3mg/day. Table 3.1 shows no DRV for those aged between 4 and
65 years as usual daily activity of able-bodied persons should provide sufficient
exposure to UV light. The elderly and those confined indoors are recommended
46 The nutrition handbook for food processors
to take 10mg/day. Vitamin D is stored in various fat depots throughout the body
and, because of its lipid nature, it is carried by a specific transport protein, a
2
-
globulin, within the circulation.
3.9 Specific nutritional deficiencies
3.9.1 Asian communities in the UK
Osteomalacia in adults and rickets in children occur as a result of vitamin D defi-
ciency or from a disturbance in its metabolism (see Table 3.3). The frequency of
occurrence depends on the distribution of the populations affected. In the nine-
teenth century nutritional rickets were endemic in industrial cities of Britain due
to the poor diet and environmental conditions of children. As a result of effec-
tive public health measures privational rickets virtually disappeared from the UK
by 1940 (Maxwell, 2001). However, in the 1960s low vitamin D status was found
to be common among immigrant Asian children, adolescents and women living
in the UK due to a combination of factors including the type of vegetarian diet,
which was high in phytate from unleavened breads (see section 3.11), low
calcium intake and limited exposure to sunlight. A striking reduction in Asian
rickets occurred in Glasgow when free vitamin D supplements were introduced
for children up to 18 years old (Smith, 2000).
3.9.2 Elderly
The elderly, especially those over 75 years, may have 25-OHD levels less than
12 nmol/L during the winter months because they expose insufficient skin to the
sun during the summer months. Fifteen elderly people, living at 37° latitude who
formerly went outside infrequently, were studied over a 4 week period while
spending 0, 15 and 30 minutes on a veranda exposing the face and legs to
sunlight. At the end of the study period, plasma 25-OHD levels increased by
Vitamins 47
Table 3.3 Main causes of rickets and osteomalacia
Cause Clinical features
Lack of sunlight vitamin D deficiency e.g. in Asian immigrants and the elderly
Malabsorption coeliac disease
postgastrectomy
intestinal bypass surgery
Renal disease: tubular inherited hypophosphataemia
Fanconi syndrome
Renal disease: Osteodystrophy
glomerular Dialysis bone disease
18.5 nmol/L (7.4mg/ml) in the group spending 30 minutes/day and there was a
small but insignificant rise in those who spent 15 minutes outside (Reid et al,
1986). Reid and his co-workers thought this a safe inexpensive method for the
prevention of osteomalacia in frail elderly subjects. Histologically proven osteo-
malacia occurs in 2–5% of the elderly in hospitals (Campbell et al, 1994) and low
plasma 25-OHD in up to 40% of elderly living in homes and hospitals. Dietary
or supplementary vitamin D may be the only effective way of maintaining or
improving status in this group when it is not possible to expose them to sunlight.
3.9.3 Other diseases linked to vitamin D deficiency
After nutritional vitamin D deficiency, coeliac and renal diseases are the most
important causes of osteomalacia and rickets. In coeliac disease there is a patchy
enteropathy of the gut which can potentially lead to fat malabsorption. The effi-
cient absorption of fat is essential for the absorption of fat-soluble vitamins, thus
in patients with inadequate sunlight exposure, vitamin D deficiency can occur.
Patients with kidney diseases are also susceptible to vitamin D deficiency as the
enzyme necessary to convert 25-OHD to the metabolically active vitamin D
metabolite, 1,25-OHD, is located in the kidney.
3.9.4 Vitamin D and type 1 diabetes
A follow-up study in Finland of 10 821 people born in Oulu and Lapland in 1966
showed that those who received recommended supplements of vitamin D during
their first year of life were 80% less likely to develop type 1 diabetes over the
next 30 years. Type 1 diabetes is thought to be an autoimmune disease caused
when immune system cells attack insulin-producing cells in the pancreas. Vitamin
D is known to be an immune system suppressant and the authors believe that
vitamin D might somehow inhibit this autoimmune reaction (Hypp?nen et al,
2001). Finnish children are less exposed to sunlight than those in more southerly
countries, hence most receive vitamin D supplements. It is also suggested that
mothers’ worries over UV exposure and skin cancer may also be contributing to
world-wide increases in type I diabetes. There is evidence that parents are restrict-
ing children’s exposure to sunlight by using more sun-screen than previously and
this might have played a part. Early infections with enterovirus and childhood
obesity were also associated with increased risks of type 1 diabetes (Hypp?nen
et al, 2001). Nevertheless, none of the above explains the molecular role in the
pathogenesis of type 1 diabetes and more work is needed in the area.
3.10 Synthesis and actions of 1,25-OHD
The most biologically active form of calciferol is 1,25-OHD and its synthesis is
tightly controlled (Loveridge, 2000). The kidney produces both dihydroxylated
forms of vitamin D (1,25 and 24,25-OHD); the dominant form is determined by
48 The nutrition handbook for food processors
the circulating level of parathyroid hormone (PTH), plasma calcium concentra-
tions and the current vitamin D status of the body. In response to PTH in the
vitamin D-deficient state, 1,25-OHD production is high (and this directly induces
feedback inhibition of PTH production within the parathyroid gland) and 24,25
OHD is low. In the vitamin D-adequate state, 1a-hydroxylase activity in the
kidney is increased and more 24,25-OHD is produced. Again, 1,25-OHD directly
reduces PTH production in the parathyroid gland (Fig. 3.2). Under most circum-
stances the principal site for 1,25-OHD production is the kidney; however, during
Vitamins 49
Blood
calcium
&
phosphorus
Parathyroid
glands
Kidney
Liver
C
a
l
c
i
f
i
c
a
t
i
o
n
Intestine
Bone
VITAMIN D
25-OH-ase
1a-OH-ase
PTH
PTH
Ca
++
HPO
4
-
Ca
++
HPO
4
-
Pi & other
factors
25-OHD
1
,
2
5
O
H
D
1
,2
5
O
H
D
Fig. 3.2 Metabolism of vitamin D and the biological actions of 1,25 dihydroxyvitamin
D (1,25-OHD) in raising blood calcium from bone resorption and/or the intestinal absorp-
tion. The figure shows the stimulatory role of parathyroid hormone (PTH) on kidney syn-
thesis of 1,25-OHD and the feedback inhibition. (From Holick MF, ‘McCollum Award
Lecture, 1994: vitamin D – new horizons for the 21st century’, Am J Clin Nutr, 1994, 60,
619–30) (Reproduced with permission by the American Journal of Clinical Nutrition.
? Am J Clin Nutr. American Society for Clinical Nutrition.)
pregnancy the placenta also seems to have a role in producing it at the time of
increased calcium requirements by the foetus (Loveridge, 2000).
Vitamin D maintains plasma calcium concentrations by stimulating intestinal
calcium absorption by the small intestine and/or the resorption of calcium from
bone. Calcium transport across intestinal cells is stimulated by 1,25-OHD by
inducing the production of calcium binding protein (CBP) within the villous cells,
and inducing an extremely low concentration of cystolic calcium within the
enterocyte. 1,25-OHD also promotes cell maturation within the intestine (Suda
et al, 1990) and in studies using vitamin D-deficient rats, villous length is only
70% of that of normal rats. Serum calcium is tightly controlled but a number
of studies suggest it peaks in the summer perhaps because of improved vitamin
D status, growth stimulation in children and higher intestinal calcium absorption
(McCance and Widdowson, 1943). More recent studies, using radio-isotopes,
have shown fractional calcium absorption was significantly higher in post-
menopausal women evaluated from August to October than from March to May
(Krall and Dawson-Hughes, 1991). In addition, increased intestinal calcium
retention was associated with a lower rate of bone loss from the radius. The
changes in calcium absorption and excretion could be accounted for by small sea-
sonal increases in 1,25 OHD during the summer months found in some of the
studies (Sherman et al, 1990).
3.11 Bone mineral density and fractures
In the presence of adequate dietary calcium, 1,25-OHD increases bone formation
and growth plate mineralisation by providing sufficient calcium to allow calcifi-
cation to occur. In contrast, prolonged vitamin D deficiency results in a poorly
mineralised skeleton. When calcium is limited the skeleton is sacrificed because
appropriate concentrations of calcium are required for vital nerve and muscle
activity. Seasonal increases in PTH may have an adverse effect on bone loss. It
is known that accelerated bone loss occurs in hyperparathyroidism (Maxwell,
2001) and increased PTH activity is a determinant in vertebral osteoporosis.
However, studies by Krall and Dawson-Hughes (1991) showed that serum con-
centrations of 25-OHD >95 nmol/L prevent a seasonal increase in PTH. This sug-
gests that when vitamin D status is poor, PTH stimulates 1,25-OHD production,
which acts primarily on the bone to release calcium for essential activities. When
vitamin D status is good, sufficient 1,25-OHD is produced to stimulate intestinal
absorption of calcium so the bone is spared.
In the UK and the USA, seasonal changes in hip fractures have been recog-
nised with peak fracture rates occurring during the winter months. The role of
vitamin D deficiency in the pathogenesis of osteoporotic fractures is controver-
sial and the inverse association with hip fracture rates could just be coincidence.
However, seasonal changes in bone mineral density (Krolner, 1983) and the
inverse seasonal changes in PTH, could be related to the accelerated bone loss.
In addition, vitamin D is known to regulate the synthesis of osteocalcin (Reichel
50 The nutrition handbook for food processors
et al, 1989), the matrix protein in bone, thus a causal association between low
25-OHD levels and osteoporosis in postmenopausal women (Villareal et al, 1991)
is possible (see also sections 3.20.2 and 3.21.3).
3.12 Vitamin D and other aspects of health
3.12.1 Behaviour
Specific vitamin D receptors are found in parts of the brain and spinal cord
(Maxwell, 2001). Seasonal changes in 25-OHD and 1,25-OHD could have an
effect on hormonal function, mood and behaviour. For example, seasonal affec-
tive disorders (SAD) appear to have a latitude gradient, with mood changes due
to a reduction in daylight hours and altered circadian secretion of melatonin.
Whether seasonal changes in UV light and vitamin D contribute is unknown.
3.12.2 Colon cancer
Mortality rates from colon cancer are highest in those areas that receive the least
amount of sunlight. A prospective study of 26 000 volunteers investigated the
association between 25-OHD and the risk of colon cancer. In those with 25-OHD
concentrations of 50 nmol/L (20 ng/ml) or more, the risk of colon cancer was
decreased threefold. However, confounding factors such as consumption of milk,
meat or fat in the diet were not considered but these observations and previous
epidemiological and laboratory studies suggest good vitamin D status in con-
junction with calcium nutrition might lower the risk of colon cancer (Garland
et al, 1989).
3.12.3 The immune system
Experimental evidence from animals, both in vitro and in vivo, has shown an
immunological role for 1,25 OHD
3
in both lymphocytes and monocytes (Yang et
al, 1993). Strict lactovegetarians, particularly in immigrant Asians, have an 8.5-
fold increased risk of tuberculosis compared with those who ate meat or fish daily.
Since vitamin D deficiency is more common among vegetarian Asians and it is
known to have effects on immunological function in animals, vitamin D defi-
ciency may be responsible for reduced immunocompetence (Maxwell, 2001).
The mechanism for the immunological role of vitamin D is not known, but
the hormone receptor for 1,25-OHD is now recognised as one of a superfamily,
the so-called ‘Steroid-Thyroid-Retinoid-Superfamily’. It is understood that if
these nuclear receptors and their activating substances are to recognise response
elements within responsive genes, they must act in pairs and a member of the
retinoid family must serve as a partner if the dimer is to function. Thus vitamin
A, usually in the form of retinoic acid, is a regulator for several hormone response
systems including vitamin D (Kliewer et al, 1994) (see section 3.5.2). The
anti-infection properties of vitamin A are widely recognised and interactions
Vitamins 51
between vitamins A and D status may therefore be important in regulating
immune function.
3.13 Safety
Infants are most at risk of developing hypervitaminosis D. There are some reports
of hypercalcaemia in infants given 50mg vitamin D/day and mild hypercalcaemia
at doses of 15 mg orally every 3–5 months (Department of Health, 1991).
3.14 Vitamin E
In 1922, a factor X, an antisterility factor, was found to be a fat-soluble compo-
nent and essential for prevention of foetal death and sterility in rats. Vitamin E,
as it became known, was isolated from wheat germ in 1936 and given its present
name, tocopherol from the Greek words ‘tokos’ and ‘pherein’ which means ‘to
bring forth children’. It is now known that there are several forms of tocopherol
and the term vitamin E is used to denote any mixture of biologically active
tocopherols. Vitamin E activity is currently defined in mg a-tocopherol equiva-
lents (a-TE) where 1 mg a-TE equals the activity of 1 mg RRR-a-tocopherol.
Formerly international units (IU) were used and vitamin E is still occasionally
quoted in this way in clinical trials. Where dl-a-tocopherol acetate is used, 1 IU
equals 0.67 mg a-TE (Duthie, 2000).
There are eight naturally-occurring vitamin E compounds, four tocopherols
and four tocotrienols, all synthesised by plants. The tocopherols are quantitatively
and physiologically the more important. The most active of these compounds is
a-tocopherol, which accounts for 90% of the vitamin E present in human tissues.
Vegetable oils are the major sources of tocopherols. Sunflower and olive oil
contain mainly a-tocopherol while in soya oil, the g form accounts for 60% of
total tocopherol. Other food groups provide substantial amounts of a-tocopherol
including meat, fruit, nuts, cereals and eggs (Table 3.2). There is a widely
available synthetic form of vitamin E, dl-a-tocopherol that consists of eight
stereoisomers in approximately equal amounts. The synthetic form is used in
animal feeds and is available in capsule form as a supplement for humans. Toco-
pherols and tocotrienols are readily oxidised to quinones, dimers and trimers by
light, heat, alkali and divalent metals such as copper and iron so synthetic prepa-
rations are often protected by acetylation and succinylation (Duthie, 2000).
The assessment of vitamin E status is difficult because clinical signs of defi-
ciency are not often seen, except occasionally in premature babies or in persons
with fat malabsorption. This suggests that modern diets, which provide approxi-
mately 10 mg vitamin E/d in the UK are adequate (Gregory et al, 1990). However,
epidemiological evidence that intakes of vitamin E and other antioxidants are
inversely correlated with the risk of some cancers and heart disease have led to
some suggestions that optimal intake should be more than DRVs.
52 The nutrition handbook for food processors
Vitamin E status can be measured as plasma tocopherol concentration but
increased concentration of serum lipids appears to cause tocopherol to leave cell
membranes and go into the circulation hence increasing blood levels. Because of
this, plasma tocopherol is usually expressed in relation to circulating lipids, the
most commonly used ratio being serum tocopherol :cholesterol. Values for
serum a-tocopherol ￡11.6mmol/L or of the a-tocopherol :cholesterol ratio ￡2.2
mmol/mmol indicate a risk of vitamin E inadequacy (Thurnham et al, 1986).
3.15 Biological activity
The biological activity of vitamin E is almost entirely due to its antioxidant prop-
erties. In vivo vitamin E appears to be the major lipid-soluble antioxidant
component in membranes and is particularly effective in preventing lipid perox-
idation, which is a series of chemical reactions involving the oxidative deterio-
ration of polyunsaturated fatty acids (PUFA). Lipid peroxidation may cause the
disruption of cell structure and function and may play an important role in the
aetiology of many diseases e.g. heart disease and cancer. In biological systems
the peroxidative cascade is likely to be terminated by vitamin E:
PUFA:H + R? = PUFA? + RH [3.1]
Non-radical PUFA + Free radical = PUFA Radical + Non-radical product
PUFA? + O
2
= PUFAOO? [3.2]
PUFA Radical + oxygen = peroxyl PUFA Radical
tocopherol-OH + PUFAOO? = tocopherol-O? + PUFAOOH [3.3]
= tocopheroxy radical + lipid hydroperoxide
When vitamin E donates hydrogen it becomes a free radical but is relatively
unreactive and the chain reaction is halted because the unpaired electron on the
oxygen atom becomes delocalised within the aromatic ring structure. The con-
centration of vitamin E in cell membranes is low, about 1 molecule for every
2000 phospholipid molecules, therefore in order for vitamin E to continue to
protect the membranes, it must be reduced to its original structure by vitamin C
or other reducing compounds in the immediate environment. Likewise, lipid
hydroperoxide has to be removed from the membrane for although it is semi-
stable, its structure is altered by oxidation and it is potentially pro-oxidative in
the presence of transition metals. Lipid hydroperoxides can be released from the
phospholipid structure in membranes by phospholipase A2 and then degraded by
selenium-dependent glutathione peroxidase in the cytoplasm or associated with
the cell membrane (Chaudiere and Ferrari-Iliou, 1999).
3.16 Coronary heart disease (CHD)
Epidemiological studies have reported an inverse relationship between the inci-
dence of CHD and vitamin E status using a variety of methods. A descriptive
Vitamins 53
correlation study of 24 developed countries showed that the supply of a-
tocopherol was strongly related to CHD and explained the low rates of heart dis-
ease in some European countries (Bellizzi et al, 1994). For example, Spain, with
low rates of CHD, has estimated intakes of 18–25 mg/day vitamin E, whereas in
the UK, where the number of deaths from CHD is one of the highest, the intake
of vitamin E is only 4.7–11.9 mg/day. Biochemical evidence to support the
epidemiological data suggests that the susceptibility of LDL-cholesterol to
oxidative modification to an atherogenic form is key in the development of
atherosclerosis. Oxidised LDL is taken up by monocytes, which are attracted to
a site of injury on an arterial wall. Monocytes are transformed to macrophages
and oxidised LDL appears to decrease the ability of macrophages to leave the
arterial wall. The enhanced uptake of oxidised LDL may then convert mac-
rophages into foam cells, the precursors of plaque, which block the artery. Studies
in vitro have shown that oxidation of LDL can be inhibited by addition of antiox-
idants to the LDL. If LDL is exposed to a copper-mediated oxidation system,
vitamin E in the outer phospholipid layer of the LDL is depleted first, followed
by the carotenoids, lycopene and b-carotene. LDL oxidation is further delayed if
vitamin C is present in the external medium, probably because vitamin C can
regenerate vitamin E. In addition, studies by Esterbauer et al (1992) suggest that
increasing the vitamin E or ubiquinone content of LDL by dietary supplementa-
tion also inhibits oxidation of LDL to the atherogenic form.
There is also some biochemical evidence which supports the hypothesis that
low dietary vitamin E is correlated with less vitamin E in blood and higher risks
of heart disease (Gey, 1993). However, more recent studies suggest that plasma
a-tocopherol concentrations are very similar in several European countries in
spite of widely differing vitamin E intakes (Howard et al, 1996; Olmedilla et al,
2001). Furthermore, the susceptibility of LDL to oxidation does not differ in
people living in different countries with very different vitamin E intakes (Wright
et al, 2002).
3.16.1 Randomised trials
Although observational studies have provided support for the potential health
benefits of antioxidants, there remains a deficiency of direct experimental evi-
dence from randomised trials. In the ATBC study, mentioned earlier (see section
3.6), there was not only no reduction in lung cancer or major coronary events,
there was in fact an increase in lung cancer (ATBC Cancer Prevention Study
Group, 1994). What was more startling was an unexpected increase in death from
haemorrhagic stroke associated with vitamin E supplementation (50 mg/day).
Some more positive results emerged from a study carried out in China where
130 000 adults from Linxian Province, who did not have cardiovascular disease
at entry, were randomly assigned to receive daily vitamin E (30 mg), b-carotene
and selenium supplements or placebo. During the 5.2 years of follow-up, there
was a 9% decrease in ‘deaths from any cause’ without any significant reduction
in cardiovascular events. However, the dose of vitamin E was smaller than the
ATBC study, other supplements were included, nutritional status was poorer and
54 The nutrition handbook for food processors
cardiovascular risk of this population very much lower than that of the industrial
west. Therefore the beneficial effect cannot be attributed to vitamin E alone (Blot
et al, 1993). Lastly, treatment with vitamin E alone had no apparent benefit on
cardiovascular outcomes in The Heart Outcomes Prevention Evaluation (HOPE)
study. The HOPE study was a double-blind randomised trial with a 2 ￥ 2 facto-
rial design, to evaluate the effect of either 400 IU vitamin E daily from natural
sources or matching placebo, and either ramiprol (an angiotensin-converting-
enzyme inhibitor) or placebo for 4.5 years on 2545 women and 6996 men at high
risk for cardiovascular events (Heart Outcomes Prevention Evaluation Study,
HOPE, 2000).
Secondary prevention trials have been a little more supportive of the idea that
vitamin E provides protection against heart disease. The Cambridge Heart Antiox-
idant Study (CHAOS) in patients with angiographic evidence of coronary
atherosclerosis found high doses of a-tocopherol (400 or 800 IU/d) reduced risks
of myocardial infarction (MI) and all cardiovascular events by 77% and 47%
respectively (Stephens et al, 1996). Furthermore, secondary analysis of the ATBC
study showed individuals with a history of MI at the start of the study had a
reduced risk of subsequent nonfatal MI of 38%; however, risk of fatal coronary
end points was not reduced (ATBC Prevention Study Group, 1994).
In conclusion, the apparent benefits of vitamin E in individuals with existing
coronary disease were not consistent with the proposed role of antioxidants to
prevent initiation or block propagation of lesions (HOPE, 2000). However, Stein-
berg hypothesised that unlike agents that lower cholesterol or blood pressure
which have an immediate benefit, antioxidants may have to be used for more than
5 years to have demonstrable benefits, since the primary mechanism of these
agents may be in the prevention of new lesions (Stephens et al, 1996). Further
work on the possible effects of vitamin E on the clinical aspects of cardiovascu-
lar disease is needed. At present possibly the best advice to give is to recommend
a balanced diet with emphasis on antioxidant-rich fruits, vegetables and whole
grains.
3.17 Other roles of vitamin E
Vitamin E is the most effective chain-breaking lipid-soluble antioxidant in bio-
logical membranes, where it contributes to membrane stability and protects criti-
cal cellular structures against damage from free radicals and reactive products of
lipid oxidation (Burton and Ingold, 1981). There are suggestions however that it
has a variety of other effects, for example on immune function, platelet and vas-
cular functions, prevention of oxidative damage to DNA and DNA repair and
modulation of signal transduction pathways (Morrissey and Sheehy, 1999).
3.17.1 Immuno-enhancement
Immune function in the elderly is often the focus of vitamin-supplementation
studies but, while some workers, e.g. Meydani et al (1997), were able to demon-
Vitamins 55
strate improvements in both cellular and humoral responses from vitamin E
supplements, others who used less vitamin E (67 mg aTE/d) were unable to do
so (de Waart et al, 1997). Meydani and colleagues suggested that a consumption
of at least 147 mg a-TE/d (ie 5–10 times a normal dietary intake) was needed to
benefit the immune response. Dose may play a critical role in the effects of
vitamin E on immune function and high doses may enhance the immune system
of the aged by suppression of prostaglandin E
2
(PGE
2
) production and/or decreas-
ing free radical formation. An increase in oxidant stress associated with an
inadequacy of vitamin E will stimulate nuclear factor kB (NFkB) formation, stim-
ulating inflammation and the formation of PGE
2
(Grimble, 1997). At low con-
centrations PGE
2
is believed to be necessary for certain aspects of cellular
immunity. However, at higher concentrations, it suppresses several indices of cel-
lular and humoral immunity such as antibody formation, delayed-type hypersen-
sitivity (DTH) skin tests, lymphocyte proliferation and cytokine production
(Meydani and Beharka, 2001). Tocopherols decrease the release of arachidonic
acid from membrane phospholipids, resulting in a decreased production of PGE
2
,
hence mega-doses of vitamin E may stimulate immune function reducing the risk
of free radical formation and reducing the substrate for PGE
2
formation, but such
doses can have adverse effects. Mega-dose amounts of vitamin E have been
reported to antagonise the pro-coagulant effects of vitamin K and this can
have serious consequences in elderly people on warfarin therapy (Corrigan and
Marcus, 1974).
3.17.2 Vascular effects of vitamin E
In addition to the protection provided by vitamin E against LDL oxidation, several
studies have consistently reported that platelet aggregation is reduced by vitamin
E supplements but amounts used to demonstrate these effects are generally in
excess of 250 mg a-TE/d (Morrissey and Sheehy, 1999). However, a reduction
in reactive oxygen species (ROS) that is promoted by vitamin E can protect
against vascular cell dysfunction, preventing adhesion molecule expression and
recruitment of monocytes and the production and release of nitric oxide (Baeuerle
and Henkel, 1994; Goldring et al, 1995). a-Tocopherol has also been shown
specifically to inhibit the proliferation of smooth muscle cells through modula-
tion of protein kinase C and not a mechanism related to its antioxidant proper-
ties (Azzi et al, 2001).
3.17.3 Oxidative damage to DNA and DNA repair
Despite the use of high doses of vitamin E, large changes in the vitamin content
of blood and liver and extended periods of study in animals and humans (smokers
and non-smokers), vitamin E does not appear to have affected repair products of
oxidative DNA damage, sister-chromatid exchanges in peripheral lymphocytes
or DNA adducts in lymphocytes (Morrissey and Sheehy, 1999).
56 The nutrition handbook for food processors
3.18 Safety issues
Although the intake from dietary sources is usually not greater than 20 mg/d, few
adverse effects have been reported even from doses up to 3200 mg/d (Bendich
and Machlin, 1988).
3.19 Vitamin K
The parent structure of the vitamin K group is 2-methyl-1,4-napthoquinone, also
known as menadione. Menadione is not a natural constituent of foods but does
possess biological activity in vertebrates (Shearer, 2000). Constituents of the
naturally occurring vitamin K group all possess the napthoquinone ring structure
but differ in the structure of the side chain at the 3 position. There are two main
groups according to whether plants or bacteria produce them. In plants, the major
form of vitamin K is phylloquinone (vitamin K
1
), which has the same phytyl side
chain as chlorophyll. Bacteria synthesise menaquinones (vitamin K
2
) with side
chains based on a number of repeating prenyl units (MKn). The primary func-
tions of vitamin K are in blood coagulation and in bone metabolism.
The major dietary sources of vitamin K are shown in Table 3.2; however, green
vegetables contain the highest concentrations and are the largest contributors
to dietary intakes (Booth et al, 1996). The relative phylloquinone content of
vegetables reflects its association with photosynthetic tissues, with highest values
being found in dark green leafy vegetables, e.g. spinach and cabbage (300–
600mg/100 g). Fermented cheeses contain two major menaquinones MK-8 and
MK-9 at concentrations of 50–100 and 10–20mg/100g respectively. Moulds do
not normally synthesise menaquinones so MK-8 and 9 are thought to be derived
from the starter fermentation bacteria.
Many bacteria present in the human intestine synthesise menaquinones, the
major forms being MK-10 and -11 produced by Bacteroides, MK-8 by Enter-
obacteria, MK-7 by Veillonella and MK-6 by Eubacterium lentum. Most are
found in the distal colon where the menaquinone content is about 20mg/100 g dry
weight, but there is no direct evidence that this pool is bioavailable. However,
the widespread presence of very long-chain forms in the liver, e.g. MK10–13,
does require explanation because these forms are not detected in commonly eaten
foods but are typical of those produced by Bacteroides (Conly, 1992). There is,
therefore, some debate as to the contribution of intestinal-derived K
2
to vitamin
K status (Lipsky, 1994; Suttie, 1995).
3.20 Biological activity
3.20.1 Blood clotting
Vitamin K is required for the biological activity of several coagulation factors
including four procoagulants, factors II (prothrombin), VII, IX and X and two
Vitamins 57
feedback anticoagulants, proteins C and S. A seventh plasma protein (Z) may
have a haemostatic role but its function is currently unknown (Shearer, 2000).
Specifically, vitamin K functions as a cofactor for vitamin K-dependent car-
boxylase, a microsomal enzyme that facilitates the post-translational conversion
of glutamyl (Glu) residues in the protein precursor to g-carboxyglutamic acid
(Gla). The biological relevance of the Gla structure is that it forms a cage struc-
ture to which divalent metal ions such as calcium may be bound (Berkner, 2000).
The synthesis of clotting factors occurs in the hepatic tissue. In its severest
form, vitamin K deficiency results in bleeding syndrome due to lowering of
circulating levels of the procoagulant factors and their replacement by under-
carboxylated species.
3.20.2 Bone
Two vitamin K-dependent bone proteins were identified towards the end of the
twentieth century. In 1975, it was found that bone tissue contains the Gla protein
osteocalcin (BGP), which is one of the most abundant proteins in the body
(Hauschka et al, 1975). In 1985 the matrix Gla protein, MGP, was discovered
in bone, dentine and cartilage (Price, 1988). Osteocalcin is a water-soluble 49-
residue protein and accounts for up to 80% of the total g-carboxyglutamyl content
of mature bone (Olson, 1999). Its discovery changed the thinking about the role
of vitamin K. Although the exact role of osteocalcin in bone metabolism is still
not understood, the available data suggest a regulatory function of osteocalcin in
bone mineral maturation (Weber, 2001). Osteocalcin is synthesised by osteoblasts
and since its concentration in blood reflects osteoblast activity, measurement of
total osteocalcin in blood has become accepted as a marker of bone turnover
(Khosla and Kleerekoper, 1999). In addition, the extent to which osteocalcin is
carboxylated is believed to be a more sensitive measure of vitamin K status than
the conventional tests involving blood coagulation. High serum concentrations of
undercarboxylated osteocalcin (ucOC) are indicators of low vitamin K status and
vice versa (Weber, 2001).
Work during the 1980s using cultured osteosarcoma cells showed that osteo-
calcin and MGP are regulated by 1,25-OHD (Olson, 1999). However, more recent
studies have demonstrated that retinoic acid receptors and vitamin D hormone
receptors may form heterodimers that bind to the osteocalcin promoter in the cells
allowing retinoic acid and 1,25-OHD to work synergistically in the cultured cells,
i.e. osteocalcin and MGP may mediate some of the actions of vitamin D on bone.
It has been shown that a deficiency of osteocalcin does not appear to affect repair
of bone structure but in bone disease, plasma osteocalcin is elevated (Price, 1988).
The author concluded that osteocalcin may stimulate bone remodelling and
calcium mobilisation while MGP may be associated with the inhibition of growth-
plate mineralisation.
There is evidence that vitamin K positively influences calcium balance, a key
mineral in bone metabolism. Humans given a diet rich in vitamin K showed an
increase in calcium retention within the body (Sakamoto et al, 1999). There is
58 The nutrition handbook for food processors
some evidence to suggest that proteins located in the kidney are responsive to
vitamin K and that g-carboxy-glutamic acid may be involved in calcium reten-
tion and contribute to the effect.
3.21 Vitamin K status and health
3.21.1 Adults
Vitamin K deficiency in adults leading to clinical bleeding is almost unheard of,
except as a consequence of hepato-intestinal disorders which disturb the ab-
sorption or utilisation of the vitamin. Use of warfarin or other anti-coagulant
drugs, i.e. vitamin K antagonists, in the management of thromboembolic disease,
reduces circulating concentrations of vitamin K-dependent clotting factors. The
anticoagulants inhibit the biosynthesis of prothrombin and other vitamin K-
dependent factors in the liver and others factors in extrahepatic tissues, leading
to clotting factor deficiencies in the body (Olson, 1999). Studies investigating
the effect of anticoagulants on bone health have shown mixed results but the
participants were severely ill with chronic vascular disease which would have
affected physical activity, a very important factor in bone health and may be the
reason why vitamin K status did not appear to be important (Weber, 2001).
3.21.2 Infants
Newborn infants are a special case in vitamin K nutrition for several reasons:
lipids are not easily transferred across the placenta, the neonatal liver is imma-
ture with respect to prothrombin synthesis, breast milk is low in vitamin K and
the infant gut is sterile at birth. As a result of this unique combination of factors
infants can develop a condition known as haemorrhagic disease of the newborn
(HDN). The term HDN was first used in 1894 by Townsend who described it as
a self-limiting bleeding disorder of newborns of unknown origin (Shearer, 2000).
Until the late 1960s HDN was thought to be a problem of the first week of life;
however, two other forms have now been identified: late HDN, a more serious
condition, that occurs between weeks 3 and 6 of life and early HDN occurring
in the first 24 hours, probably caused by antagonistic drugs taken during pre-
gnancy. Late HDN is rare, about 4–70 cases per 100 000 births but world-wide
is a significant cause of infant morbidity and mortality. Late HDN has a high
incidence of intercranial haemorrhage resulting in death or severe and permanent
brain damage in 50% of cases. One risk factor for both the late and classical forms
of HDN is exclusive breast-feeding, which may be aggravated by the low con-
centrations of vitamin K in breast milk 1–10mg/L (cf. formula milk ~50mg/L).
A second risk factor might be the ‘precarious’ hepatic stores of vitamin K found
in newborns possibly due to poor placental transfer (Shearer, 2000). As a result
of this phenomenon the COMA panel recommended all babies should be given
prophylactic vitamin K at birth (Whitehead, 1991). Recent interest in late HDN
has come from a reported increase in the syndrome since the early 1980s. In many
Vitamins 59
developed countries the increased incidence followed a decline in vitamin K pro-
phylaxis since its introduction in the 1940s and an increase in exclusive breast-
feeding. Prophylaxis was often given as an intramuscular dose at birth but since
1992 concerns about an epidemiological association between the intramuscular
route and later childhood cancer has seen a shift towards the oral route of ad-
ministration (Shearer, 2000). Work published by Autret-Leca and Jonville-Bera
(2001) suggest oral administration of a single dose of vitamin K protects against
classical and early vitamin K deficiency bleeding but that intramuscular propha-
lylaxis is needed for late HDN. Although the risk of solid tumours associated with
vitamin K administration is unlikely there remains a low potential risk of lym-
phoblastic leukaemia in childhood. Autret-Leca and Jonville-Bera suggest
formula-fed neonates without risk of haemorrhage should receive 2 mg oral dose
of vitamin K followed by a second 2 mg dose between days 2 and 7. Infants exclu-
sively or nearly exclusively breast-fed should receive weekly oral administration
of 2 mg (25mg/day) vitamin K, after the first two doses of the vitamin, until com-
pletion of breast-feeding. High-risk neonates (premature, neonatal disease,
hepatic disease, maternal drugs inhibiting vitamin K activity) should receive the
first dose intramuscularly or by a slow intravenous route. Subsequent doses
should be administrated according to the clotting factor profile specific to each
infant.
3.21.3 Bone health
Despite gaps in knowledge it appears that sub-optimal vitamin K status may play
a role in osteoporosis. Patients with osteoporotic fractures tend to have very low
plasma levels of vitamin K. Much of this evidence is circumstantial based on
associations of measures of vitamin K status with fractures or biomarkers of
bone metabolism and some of the evidence is conflicting (Binkley and Suttie,
1995; Shearer, 1995; Vermeer et al, 1995). French workers have shown an age-
dependent impairment of g-carboxylation of osteocalcin and strong associations
of ucOC with hip fracture risk (Szulc et al, 1993) and bone mineral density
(BMD) (Szulc et al, 1994). The work also showed a potential influence of vitamin
D on g-carboxylation, which may indicate interactions between vitamins D and
K, an area requiring further investigation. It has also been shown that ‘matrix Gla
protein’ which is an important regulator of bone strength and growth is deter-
mined by a vitamin A response gene (Gudas et al, 1991). To what extent vita-
mins A and K interact at the gene level is not known.
3.21.4 Intervention studies
A number of adult intervention studies have been carried out, particularly on
postmenopausal women, which have shown vitamin K to be effective in reduc-
ing ucOC in serum. Takahashi et al (2001) used 89 osteoporotic patients with ver-
tebral fractures, 24 patients with hip fractures, 43 pre- and 48 post-menopausal
60 The nutrition handbook for food processors
Japanese women. They gave either a daily dose of 45 mg vitamin K
2
alone or
vitamin K
2
plus1mg 1-a hydroxyvitamin D
3
or vitamin D alone. After four weeks
of treatment with vitamin K alone or vitamin K plus D, ucOC was significantly
decreased, but was not changed in those who received vitamin D alone. There
was a disproportion of ucOC/intact OC in postmenopausal women and those with
hip or vertebral fractures, vitamin K and vitamins K plus D markedly decreased
the ratio of ucOC/intact OC to approximately 80%, but vitamin D did not decrease
the ratio. This work confirmed previous studies (Plantalech et al, 1990; Douglas
et al, 1995; Schaafsma et al, 2000).
The first intervention study to look at the influence of vitamin K on bone
strength was published by Akjba et al (1991) who recruited 17 dialysis patients
losing bone mass due to renal insufficiency. They supplemented the patients with
45 mg vitamin K
2
for 1 year and measured bone mass at different points on the
skeleton. They found loss of bone was reduced in the vitamin K group. The find-
ings were confirmed in subsequent studies by Orimo et al (1992) who carried out
a placebo-controlled trial studying 546 patients with osteoporosis to whom they
gave either 45 mg vitamin K
2
or 1mg 1-a-hydroxy vitamin D
3
for 48 weeks. Arm
BMD increased by 2.1% in the vitamin K group but decreased by 2.4% in the
vitamin D group (P < 0.001); no difference in vertebral BMD was found. A more
recent study reported that 45 mg vitamin K
2
or placebo given for 2 years to 241
osteoporotic women increased BMD and significantly reduced occurrence of new
fractures (14 in the vitamin K vs. 35 in the placebo group) (Shiraki et al, 2000).
Finally, preliminary data from a placebo-controlled, randomised, clinical trial on
244 post-menopausal women aged 60–85 years, after a 2 year intervention period,
showed a significant increase in the BMD of the distal radius in a group receiv-
ing 200mg vitamin K
1
plus 10mg vitamin D and 1 g calcium (Bolton-Smith et al,
2001).
3.22 Safety
There have been no adverse effects associated with the ingestion of natural
sources of vitamin K. One clinical intervention study used up to 90 mg of vitamin
K
2
daily over 2 years without any relevant adverse side-effects (Orimo et al,
1998). The COMA report states that ‘natural vitamin K vitamins seem remark-
ably free from toxic effects when taken orally even in milligram quantities’
(Department of Health, 1991). Guidelines issued by the Institute of Medicine
state, ‘A search of the literature revealed no evidence of toxicity associated with
the intake of either phylloquinone (vitamin K
1
) or menaquinone (vitamin K
2
) form
of vitamin K’ (IOM, 2001). The only studies reporting an adverse effect of
vitamin K were retrospective analyses for factors associated with childhood
cancer that suggested an increased risk in those infants receiving vitamin K at
birth (Golding et al, 1990, 1992). Subsequent studies conducted in both Europe
and the USA, however, failed to confirm the reports (IOM, 2001).
Vitamins 61
3.23 Vitamin C
Humans, other primates and guinea pigs depend on external sources of vitamin
C for their requirements; most other animals synthesise ascorbic acid within the
body. The vitamin C content in body tissues varies widely with highest concen-
trations in the pituitary, adrenals, leukocytes, eye lens and brain and the lowest
levels in plasma and saliva (Thurnham, 2000). The ascorbic acid (AA) body pool
in adults has been determined using isotope labelled AA as a tracer. Pharmaco-
kinetic data was obtained from healthy men given doses of 1-
14
C labelled AA
with steady-state intakes of 30 to 180 mg/d AA. The data showed the body half-
life of AA was inversely related to intake up to a maximum of 20 mg/kg body
weight, corresponding to a plasma AA concentration of 57mmol/L, and an AA
intake of 100 mg/d (Kallner et al, 1981). In normal adults about 2.7% of the
exchangeable body pool of ascorbate is degraded each day, which is independent
of body pool size (Baker et al, 1971; Kallner et al, 1979).
3.24 Absorption and deficiency
Vitamin C intakes are usually similar in men and women and absorption of the
water-soluble vitamin causes few problems, however plasma and leukocyte AA
concentrations tend to be higher in women than men (Thurnham, 1994). AA is
absorbed in the intestine via an energy/sodium dependent carrier-mediated trans-
port mechanism (Stevenson, 1974). AA is converted into dehydroacorbic acid
(DHAA) which is transported across the cell membrane more quickly than AA
and, once in the epithelial cells, DHAA is reduced back to AA. At high intakes
the process is saturated. Up to 180 mg there is an average absorption of 70%, but
absorption decreases to 50% with intakes up to 1.5 g and further decreases to 16%
with intakes of 12 g (Kubler and Gehler, 1970).
When the body pool falls below 300 mg vitamin C there is evidence of
impaired function (Hodges et al, 1969). Plasma vitamin C levels are sensitive to
recent dietary intake with values <11 mmol/L indicating biochemical depletion
(Sauberlich, 1975). The early symptoms of vitamin C deficiency are fatigue,
lassitude, loss of appetite, drowsiness, insomnia, feeling run-down, low resistance
to infection and petechiae (minor capillary bleeding). Those most at risk of
vitamin C deficiency are cigarette smokers, alcohol users, institutionalised elderly
and people on certain drugs. Deprivation of vitamin C for long periods leads to
scurvy, characterised by weakening of collagenous structures, resulting in wide-
spread bleeding. Scurvy in infants results in bone malformations. The earliest
clinical signs of deficiency are bleeding gums and loosening of teeth. Today,
scurvy is relatively rare. An LNRI of 10 mg/d for adults has been shown to prevent
scurvy in the UK and elsewhere but this level has little margin of safety (Bartley
et al, 1953).
62 The nutrition handbook for food processors
3.25 Biochemical functions
Vitamin C can act both as an antioxidant and a pro-oxidant. AA can readily donate
electrons to quench a variety of reactive free radicals and oxidant species and
is easily returned to its reduced form by electron donors such as glutathione,
flavonoids, tocopherol and NADPH (Zeigler et al, 1996; Xu and Wells, 1996).
Vitamin C can scavenge hydroxyl, peroxyl and superoxide radicals as well as
reactive peroxide, singlet oxygen and hypochlorite species (Bendich et al, 1986;
Sies and Stahl, 1995). Vitamin C is believed to be of fundamental importance as
an antioxidant in tissues. The evidence suggests that it protects against plasma
lipid and low density lipoprotein peroxidation by scavenging peroxyl radicals
from the aqueous phase before they can initiate lipid peroxidation, which it does
by regenerating oxidised vitamin E to the active reduced form (Thurnham, 1994).
In vitro vitamin C is rapidly lost when plasma is exposed to peroxyl radicals,
cigarette smoke or activated neutrophils and its disappearance is accompanied
by the onset of lipid peroxidation (Halliwell, 1994). In the eye, high levels of AA
provide antioxidant protection against photolytically-generated free radicals in
the lens, cornea, vitreous humour and retina. High concentrations of AA, 8–10
times higher than plasma levels, are found in seminal fluid to protect sperm
proteins. Oxidative stress has been associated with sperm agglutination and
decreased male fertility (Jacob, 1999).
Vitamin C can also be violently pro-oxidant, as in vitro, it has been shown to
release iron from ferritin and stimulate lipid peroxidation (Halliwell, 1994). The
reducing properties of vitamin C are responsible for the conversion of Fe
3+
to Fe
2+
which is extremely important in the absorption of iron, but within the tissues
could be potentially harmful because Fe
2+
is a potent free-radical catalyst able to
produce hydroxyl radicals (Stadtman, 1991). Stadtman and colleagues have
argued that much ongoing protein damage involves metal ion-dependent OH
?
generation, hence a small rise in OH
?
generation over a lifetime could increase
the incidence of age-related cancer (Tanner, 1976). In the healthy human body,
most transition metal ions are safely sequestered and are not available to catal-
yse free radical reactions, but metals are always in transit within and between
cells, hence it is possible that interactions of metal ions with ascorbate could
contribute to oxidative damage (Halliwell, 1994). However, it is probable that
the antioxidant properties of ascorbate predominate in healthy people most of the
time.
3.26 Disease–nutrient interactions
Disease and stress lower both plasma and leukocyte ascorbate concentrations
(Thurnham, 1994, 1997). It has been recognised for many years that smokers
have lower plasma ascorbate concentrations than non-smokers, even when dietary
intake is taken into account. The effect is similar to that seen during surgical stress
or infection but the stress of smoking is more easily studied (Thurnham, 2000).
Vitamins 63
It has been argued that smokers have an increased turnover of vitamin C, so in
order to maintain their body pool and circulating levels at similar levels to those
of non-smokers, intake would need to be higher, 80 mg/d (Kallner et al, 1981;
Smith and Hodges, 1987). However, an alternative explanation might be that
vitamin C can act as a pro-oxidant in plasma, hence the body may be lowering
concentrations to minimise the potential pro-oxidant damage caused by smoking
and other stresses (Thurnham, 1994). The mechanism which reduces plasma
vitamin C is also linked to the processes activated by the onset of disease.
One of the earliest features of the body’s response to trauma is a release of
neutrophils from the bone marrow (Sipe, 1985). Such neutrophils are able to
actively accumulate vitamin C (Moser and Weber, 1984) and rapid falls in plasma
and leucocyte vitamin C following trauma have been reported (Vallance et al,
1978).
The need to lower circulating vitamin C concentrations in the presence of
trauma may be linked to an increased risk of reaction between vitamin C and iron
(Thurnham, 1994). During infection, the capacity to bind iron in plasma does not
diminish and in fact several acute phase proteins with the capacity to bind iron
increase (eg ferritin and lactoferrin). Nevertheless there is increased likelihood
that iron and other transition metals are released in the immediate locality sur-
rounding damaged tissues (Chevion et al, 1993), and therefore vitamin C con-
centrations may be reduced to minimise the risk of aggravating the damage still
more. Reactions between iron and vitamin C are known to occur in situations
where tissue integrity is reduced. So amounts of dietary vitamin C that would
normally be tolerated easily can cause acute haemolysis and coma in persons with
conditions where their red cells are more susceptible to oxidative stress such
as glucose-6-phosphate dehydrogenase deficiency or nocturnal haematuria
(Thurnham, 1994).
There are also other groups within industrialised countries where there may
be risks associated with elevated intakes of vitamin C. In North European com-
munities, genetic haemochromatosis has a gene frequency of 1 in 20, such that
approximately 1 in every 300 individuals are at risk of iron overload. Although
they appear apparently healthy, giving vitamin C without an iron-chelating agent
to such people can potentially produce serious clinical effects (Halliwell, 1994).
As indicated above, iron is usually bound to transport, storage or tissue proteins
and the body is therefore protected from its damaging reactions. If localised or
more general breakdown of tissue integrity should occur during infection, inflam-
mation, strenuous exercise or other traumas resulting in an acute phase response,
then metal ions are potentially released into the circulation. In addition as we
get older, we get sicker and in humans with advanced atherosclerotic lesions,
catalytic metal ions capable of free radical reactions can be measured. In fact, the
contents of such a lesion will stimulate OH
?
formation in the presence of perox-
ide and ascorbate in vitro. Should older people take large doses of antioxidant
vitamins? The Finnish study suggests that people who have been smokers for
many years and may well be on their way to developing cancer and/or cardio-
vascular disease, are harmed by high dose b-carotene treatment (ATBC Cancer
64 The nutrition handbook for food processors
Prevention Study Group, 1994). Furthermore, there are controversial suggestions
that high body copper and/or iron stores are associated with increased risk of
cancer and cardiovascular disease. This could mean that in the event of injury or
trauma, more iron and copper would be available to catalyse free radical reac-
tions (Halliwell, 1994). As stated earlier, a decline in plasma ascorbate at the onset
of oxidative stress is probably beneficial, for although vitamin C is helping to
scavenge radicals and recycle vitamin E, its reduction in plasma may prevent any
problems if metal ions are released. Furthermore, it must be remembered that
oxidised vitamin C can be regenerated within erythrocytes and other tissues
(Chaudiere and Ferrari-Iliou, 1999), and if the radical scavenging properties of
vitamin C are to be maintained, regeneration of reduced vitamin C has to be
increased if plasma concentrations are lower. Hence, giving high doses of vitamin
C to sick people may not be a good idea.
Fruits and vegetables are associated with a decreased risk of cardiovascular
disease and many types of cancer and neurodegenerative disease, but the role of
vitamin C in this effect is uncertain (Halliwell, 2001). Supplementation trials with
vitamin C using biomarkers of oxidative damage to DNA bases to measure levels
of oxidative DNA damage in vivo showed little evidence of a beneficial effect,
except where vitamin C intakes were low. In addition, no conclusive evidence
of a protective effect of vitamin C in studies on strand breaks, micronuclei or
chromosomal aberrations were found (Halliwell, 2001). There is some evidence
that diet-derived vitamin C may decrease gastric cancer in some populations
but whether this is due to its antioxidant or other properties is uncertain.
3.27 Immune function
Phagocytes and lymphocytes can concentrate vitamin C up to 100 times higher
than the plasma and this may indicate a physiological role for AA in these immune
cells (Thurnham, 2000). The vitamin also affects many immune modulators like
blood histamine, serum complement, prostacyclin, prostaglandins and B- and T-
cell cyclic nucleotides (Jariwalla and Harakeh, 1996; Siegal, 1993). Results from
studies on the effects of supplementation on the immune functions have been
inconsistent. Many studies show the vitamin has beneficial effects while others
show no effect. For example, a double blind study giving 1 or 4 g AA daily to 24
free-living women produced increases in serum AA concentrations but no dif-
ference in leukocyte AA concentrations or function (Ludvigsson et al, 1979). AA
inactivates or inhibits a wide range of viruses in vitro including human immuno-
deficiency virus (HIV), yet no clinical efficacy has been demonstrated (Jariwalla
and Harakeh, 1996). A review of 21 controlled human trials of megadose ascor-
bate intake in those exposed to the common cold showed no consistent effect on
reducing the incidence of colds although the duration of episodes and the sever-
ity of symptoms were reduced by an average of 23% (Jariwalla and Harakeh,
1996). The vitamin may have acted by reducing inflammation associated with
reactive oxidants produced by phagocytic leukocytes or by antihistaminic action.
Vitamins 65
It has been suggested that concentrations of plasma AA of 40–50mmol/L, from
an intake of 50–60 mg/day, are optimal for protection against heart disease (Gey,
1993). However, it is clear that plasma vitamin C is influenced by many factors
other than diet, therefore it is difficult to suggest that any particular plasma con-
centration is optimal for all situations (Thurnham, 2000).
3.28 Toxicity
Claims that intakes of vitamin C above those recommended can protect or cure
various diseases are debatable. Mega-doses of vitamin C (10 g/d or more) were
said to protect against the common cold and could be used in the treatment of
advanced cancer. Neither has been substantiated and in fact, four cancer patients
died of haemorrhagic tumour necrosis soon after vitamin C treatment was started
(Halliwell, 1994). Possible risks associated with high intakes include diarrhoea
(at intakes of ≥4 g/d) and increased production of oxalate leading to kidney stones
in a small number of individuals with a high propensity for synthesising oxalate
(Balcke et al, 1984). ‘Systemic conditioning’ where a sudden cessation of high
intakes may precipitate scurvy due to high turnover were not confirmed follow-
ing studies in guinea pigs and humans.
3.29 Vitamin B
1
(thiamin)
The thiamin molecule consists of a pyrimidine ring with an amine group joined
to a sulphur-containing thiazole ring. In the tissues thiamin occurs most com-
monly as thiamin diphosphate (TDP), a coenzyme important in carbohydrate
metabolism and as thiamin triphosphate (TTP) which is located in nervous tissues
(Thurnham, 2000). Thiamin is also important in fat and alcohol metabolism to
be discussed later (section 3.31). Diets high in carbohydrate require more thiamin
than diets high in fat so symptoms of the deficiency disease beriberi occur where
there is dietary deficiency of thiamin and where energy from the diet is provided
mainly from carbohydrate sources.
Beriberi occurred in Japan, China and South-east Asia in epidemic proportions
during the nineteenth and early twentieth centuries and dated from the introduc-
tion of steam-powered, rice-milling machines which made polished rice more
widely available. At the end of the nineteenth century Takaki, Director General
of the Medical Department of the Japanese navy, found that by supplementing
rice with fish, vegetables, meat and barley, beriberi was almost eradicated
(Sinclair, 1982). However, ordinary people in the poor social conditions prevail-
ing at that time were not able to supplement their diets and the problem of beriberi
became a difficult one for the colonial powers of the day as the death toll rose.
The Dutch government set up an expert committee and a man named Eijkman
was appointed to tackle the problem. Within six years Eijkman had shown
beriberi to be a nutritional problem (Eijkman, 1897). Eijkman fed both stale and
66 The nutrition handbook for food processors
freshly-cooked polished rice to chickens and produced a paralytic condition
closely resembling beriberi (Luyken, 1990). Subsequently, Funk (1911) isolated
the anti-beriberi factor from the rice polishings and first used the word ‘vitamine’
to describe the vital amine essential for life.
Thiamin is present in all natural foods (Table 3.2). However, in many parts of
the developing world thiamin is obtained either from unrefined cereal grains or
starchy roots and tubers. The milling process of cereals removes most of the
thiamin and polished rice is particularly low in thiamin (80mg/100 kg). Rice
which is parboiled before milling retains a lot of its thiamin (190mg/100kg)
because water-soluble vitamins diffuse inward during the process (Padua and
Juliano, 1974). Thiamin is water-soluble and is stable in slightly acidic water up
to boiling point. Thiamin is unstable in alkaline solution and is, therefore, lost
during the distillation process to produce alcohol.
In the British diet 45% of thiamin is obtained from fortified white flour prod-
ucts (fortified at 2.4 mg thiamin/kg white flour). Human milk is low in thiamin.
The mean range reported by The Committee on Medical Aspects of Food Policy
(CMAFP) for analyses from five centres in Great Britain was 0.59mmol/L
(0.49–0.79) which is equivalent to 0.23mg/4.2 MJ (1000 kcal) (CMAFP, 1977).
Thiamin concentration in mothers’ milk is not increased by giving supplements,
but the concentration does increase naturally over the first 6 weeks of lactation
from 0.41 to 0.65mmol/L (Thurnham, 2000).
3.30 Functions and requirements
Thiamin has two distinct functions:
1. TDP is the co-enzyme for pyruvate dehydrogenase (EC 1.2.4.1) and trans-
ketolase (EC 2.2.1.1.) in carbohydrate metabolism, a-ketoglutarate dehy-
drogenase (EC1.2.4.2.) in the citric acid cycle and branched chain keto-acid
dehydrogenase (EC 1.2.4.4.) in the metabolism of branched chain amino
acids.
2. TTP acts in nerves (and maybe muscles) to activate a chloride ion channel
(Bender, 1999). In addition, TTP may play a fundamental role in the control
of conductance of axonal membranes as well as in other neurological
processes.
Estimations of thiamin requirements have been based on a variety of biochemi-
cal methods. The finding that urinary thiamin output falls below 15mg/d in people
with beriberi provided the reference point against which other methods such as
thiamin loading, glucose loading and transketolase (TKL) activation are all com-
pared (Department of Health and Social Security, 1979). Early studies in men
suggested that thiamin intakes of 0.4 mg/d were near to the minimum at low
energy intakes (Williams, 1961). Epidemiological evidence suggested beriberi
occurred when the intake of thiamin was 0.2 mg/1000 kcal or less (Williams
et al, 1943). Later studies found that at a thiamin intake of 0.16 mg/1000 kcal both
Vitamins 67
urinary and TKL measurements were abnormal and thiamin intakes of
0.3 mg/1000 kcal were needed to get these values into the normal range (Sauber-
lich et al, 1979). From these and other studies the COMA panel accepted that
thiamin requirements were linked to energy metabolism, and therefore to energy
intake, and hence set the RNI at 0.4 mg/4.2 MJ for men (Table 3.1) (Department
of Health and Social Security, 1979).
Women are less frequently affected by beriberi than men even when eating
the same food (Platt, 1958; Tang, 1989), but there is no consistent data that
the needs of women are different. Bamji (1970) suggested that 0.21–0.26 mg
thiamin/1000 kcal would normalise measurements of TKL and thiamin excretion
in young women. Others reported urinary thiamin excretion only reached normal
values at intakes of 0.51 mg/1000 kcal. The COMA panel set the same RNI for
women as for men.
3.30.1 Thiaminase enzymes
The thermolabile anti-thiamin factors (ATFs) include thiaminase I (EC 2.5.1.2.)
and II (EC 3.5.99.2). Thiaminase I is found in the viscera of freshwater fish, in
shellfish, ferns, a limited number of seafish, plants and several micro-organisms.
Destruction of thiamin occurs either by base-exchange between thiazole and other
bases or hydrolytic cleavage of the methylene bridge between the pyrimidine
and thiazole moieties. Thiaminase II is found in several micro-organisms e.g.
Clostridium thiaminolyticus, Candida aneurolytic. It hydrolyses thiamin to 2-
methyl-4-amono-5-hydroxymethyl pyrimidine and 4-methyl-5-(2-hydroxyethyl)
thiazole. Cooking food destroys these heat-labile enzymes, but food which is
not generally cooked i.e. is eaten raw or fermented, may lose thiamin during
its preparation or in the gastrointestinal tract. Thermostable ATFs have been
shown to be in ferns, tea, betel nuts, some vegetables and some animal tissues
(Tanphaichitr, 1999).
3.31 Clinical thiamin deficiency
Cardiac failure, muscle weakness, peripheral and central neuropathy and gas-
trointestinal malfunction have been seen in animals and humans on diets defi-
cient in thiamin. The circulatory effects of thiamin deficiency are no doubt linked
to attempts by the body to increase metabolism of energy-forming substances.
Other effects may well be due to impaired nerve transmission and/or the energy
deficiency; the precise biochemical reasons have not been established.
3.31.1 Beriberi
Children and adults may present with dry (paralytic or nervous) or wet (cardiac),
or cerebral (Wernicke-Korsakoff syndrome) forms of beriberi.
68 The nutrition handbook for food processors
a. Wet beriberi
In addition to peripheral neuropathy, common signs in wet beriberi include:
oedema, tachycardia, wide pulse pressure, cardiomegaly and congestive heart
failure. Patients with wet beriberi respond dramatically to intramuscular doses of
25 mg for 7–14 days, followed by oral doses of 10 mg three times a day until the
patient fully recovers (Tanphaichitr, 1999).
b. Dry beriberi
A peripheral neuropathy, dry beriberi is characterised by a symmetric impairment
of sensory, motor and reflex functions affecting the distal segments of the limbs
more severely than the proximal ones (Thurnham, 2000). Dry beriberi can be
more resistant to treatment. Disappearance of impaired sensation occurs between
7 and 120 days, and recovery of motor weakness within 60 days of the start of
treatment (Tanphaichtir, 1999).
c. Wernicke-Korsakoff syndrome
In Wernicke’s disease, failure of energy metabolism predominantly affects
neurons and their functions in selected areas of the central nervous system. Bio-
chemical lesions that affect TKL and nucleic acid metabolism may cause glial
changes. Membranous structures are visibly altered and secondary demyelination
follows (Tanphaichitr, 1999). Prolonged alcohol consumption is associated with
a variety of neuropsychiatric conditions, including the dense amnesic disorder
known as Korsakoff’s syndrome. Korsakoff’s syndrome is frequently diagnosed
in alcoholics after an episode of acute thiamin deficiency. The accepted view
within the medical literature is that the etiology of this disorder lies in thiamin
deficiency or Wernicke’s encephalopathy. However, examination of the published
reports of pure thiamin deficiency unaccompanied by chronic and excessive con-
sumption of alcohol shows that, in this group of patients, the rate of progression
to Korsakoff’s syndrome is low. This result suggests that the memory impair-
ments associated with alcohol-related brain damage cannot be attributed to
thiamin deficiency alone. The etiology of alcohol-related cognitive impairments
such as Korsakoff’s syndrome is still poorly understood, but several lines of evi-
dence suggest multiple causal factors interact to produce deficits in performance
(Homewood and Bond, 1999).
d. Early, less well-recognised thiamin deficiency
What are less widely recognised are the lesser degrees of thiamine deficiency that
can be caused by situations of hypermetabolism, for example, by burns, trauma,
surgery and acute febrile illness. These situations are most likely to be seen in
patients who are given additional carbohydrate loading via glucose-containing
intravenous fluids and in those who already had marginal thiamin status to begin
with. This includes alcohol abusers, the homeless and others on inadequate diets.
Part of the reason for this lack of recognition is the speed with which bio-
chemical evidence of thiamin deficiency can occur on such diets (Brin, 1964) and
also because there is no clearly recognised clinical syndrome corresponding to
Vitamins 69
marginal (biochemical) thiamin status. Another reason is that clinicians simply
do not think of thiamin status often enough. It is relatively easy to test for thiamin
status using activity levels of the thiamin-dependent enzyme erythrocyte TKL;
however, the test is not commonly available in clinical laboratories.
3.32 Toxicity
Chronic intakes in excess of 50 mg/kg or more than 3 g/day are toxic to adults
with a wide variety of clinical signs including headache, irritability, insomnia,
rapid pulse, weakness, contact dermatitis, pruritus (Ibner et al, 1982).
3.33 Folate
The folate molecule is a double aromatic ring of a pteridine attached to para-
aminobenzoate and glutamate. Folic acid (pteroyl glutamic acid) is the parent
molecule of a large number of derivatives known as folates. Folic acid is physi-
ologically inactive until it has been enzymatically reduced to dihydrofolate when
it can enter the body’s folate pool (Fig. 3.3). The main naturally occurring forms
are tetrahydrofolate (THF), 5-methylTHF and 10-formylTHF. The characteristic
sign of folate deficiency is megablastosis, with abnormal, multi-lobed neutrophil
nuclei and giant platelets in peripheral blood. Other rapidly regenerating tissues
like intestinal mucosa might also be affected by folate deficiency (Department of
Health, 1991).
In 1872 Biemer described a severe, rapidly progressive and sometimes fatal
anaemia of pregnancy (McNulty, 1997). However, this condition was not linked
to a dietary deficiency until 1931 when it was established that cases of macro-
cytic anaemia in poor, pregnant women in India responded to treatment with
crude liver or yeast extract (Wills, 1931). The new nutritional factor was called
‘vitamin M’, ‘vitamin Bc’ and ‘factor U’, but isolating and successful chemical
synthesis of folic acid, as it became known, was not achieved until the 1940s.
Subsequently, neural tube defects (NTD) and anencephaly were linked to poor
folate status during pregnancy (Smithells et al, 1976) and, following a randomised
double-blind trial, folate supplements were found to lower significantly the re-
currence of NTD (MRC Vitamin Study Research Group, 1991) (section 3.34.1).
Currently, the greatest interest in folate metabolism is centred around its role in
lowering plasma homocysteine concentrations and links to cardiovascular disease
(section 3.35). Stockstad (1990) describes the history of folic acid in a book which
reviews the key advances in the biochemistry and physiology of folates.
A diet that is rich in other B vitamins and vitamin C is usually rich in folate
(Whitehead, 1991). Rich food sources of folate include liver, yeast extract and
green leafy vegetables (Table 3.2). However, liver is not eaten by a sufficiently
large proportion of the population to make a significant contribution to intake,
but beer (90mg/L folate) may account for 10% of dietary folate intake in British
70 The nutrition handbook for food processors
adults (McNulty, 1997)! There are concerns about the bioavailability of dietary
folate (Gregory, 1995). Food folates are present mainly as polyglutamate deriva-
tives which have to be converted to a monoglutamyl form by folate conjugase to
be absorbed by the jejunum. Changes in the pH of the lumen contents, or the
presence of folate conjugase inhibitors, folate binders or other specific food com-
ponents can all adversely affect the rate of hydrolysis and uptake of the vitamin
(McNulty, 1997). These factors account for the wide range of folate bioavail-
ability of the naturally occurring folate polyglutamates in all foods.
Stable isotope studies have found 50% relative bioavailability of polyglutamyl
compared to monoglutamyl forms of folate (Gregory et al, 1991). Hence the rela-
tive bioavailability of folates from a mixed diet is difficult to predict and is con-
siderably less than when the vitamin is eaten as a supplement or in a fortified food.
Folates are involved in a number of single carbon transfer reactions, especially
in the synthesis of purines, pyrimidines, glycine and methionine. THF can have
Vitamins 71
PROTEIN
FOLATE
CYCLE
METHIONINE
CYCLE
TRANSULPHURATION
PATHWAY
Methionine
Homocysteine
S-adenosyl
methionine
S-adenosyl
homocysteine
Methyl acceptors
e.g. lipid, protein,
DNA etc
Methylated products
2
3
Betaine
Dimethylglycine
Choline
Methyl-tetrahydrofolate
Methylene
tetrahyrodrofolate
Tetrahydrofolate
Serine
Glycine
5 4
Vitamin B
6
dependent
Vitamin B
6
dependent
ATP PPi + Pi
1
6
Riboflavin
dependent
FOLATE
Dihydrofolate
7
8
8
9
Fig. 3.3 Interrelationships between methionine and folate metabolic cycles.
Key:
1. Methionine adenosyl transferase EC 2.5.1.6
2. S-adenosylmethionine-dependent methyltransferase EC 2.1.1
3. S-adenosylhomocysteine hydrolase EC 3.3.1.1
4. Betaine:homocysteine methyltransferase EC 2.1.1.5
5. 5-Methyltetrahydrofolate:homocysteine methyl transferase (vitamin B
12
dependent) EC 2.1.1.13
7. 5,10-Methyltetrahydrofolate reductase (vitamin B
2
dependent) EC 1.1.99.15
7. Serine hydroxymethyl transferase (vitamin B
6
dependent) EC 2.1.2.1.
8. Tetrahydrofolate dehydrogenase 1.5.1.3.
9. Thymidylate synthase EC 2.1.1.45
one-carbon units added, for example formyl, methyl or methylene and these are
attached to either the N5 or N10 nitrogen or both (Scott, 2000). The principal
source of the one-carbon units is serine. The one-carbon units are donated once
to change uracil to a thymine-type base during the biosynthesis of pyrimidine,
and twice during the biosynthesis of purines. Pyrimidine biosynthesis generates
dihydrofolate from 5,10 THF, which is then reduced back to THF. In the two reac-
tions of purine biosynthesis 5,10 formyl THF is the donor of carbon and THF
is generated by both reactions. In all three reactions the cofactor THF is then
available to attach another one-carbon unit and repeat the process. Only a small
amount of the cofactor is required in cells because of the continuous regenera-
tion back to the THF form. These cycles could be called the DNA and/or RNA
biosynthetic cycles since they provide the de novo synthesis of the bases used to
make these structures (Scott, 2000).
In addition to the role described above, folates also participate in the methy-
lation cycle (Fig. 3.3). Enzymes called methyl transferases exist in all mammalian
cells and transfer methyl groups to a wide range of acceptors. In all cases the
source of the methyl groups is the methyl group of methionine, which is passed
on after it has been activated with ATP to form S-adenosylmethionine (SAM).
Donations of the methyl groups from SAM produce S-adenosylhomocysteine
(SAH) which is rapidly hydrolysed to homocysteine. Homocysteine is remethy-
lated either from choline or by the folate cofactor 5-methylTHF. The methylation
cycle is essential in intermediary metabolism requiring a continuous supply of
methyl groups and for which a major source is the folate cofactor.
3.34 Requirements
Biochemical evidence of folate status can be obtained in a variety of ways. The
lower limit of normal serum folate is accepted as 6.8 nmol/L (3mg/L). Tissue
levels are better indicated by the concentration of folate in red cells, which is
buffered against short-term changes (Department of Health, 1991) and concen-
trations below 0.23mmol/L (100mg/L) are considered to be severely deficient.
Values between 0.23 and 0.34mmol/L (100–150mg/L) indicate marginal stores.
Liver values greater than 6.8 nmol/g (3mg/g) indicate adequate reserves (Depart-
ment of Health, 1991). The RNI for folate for different age groups are summarised
in Table 3.1. However, in recent years new evidence suggests that the RNI should
possibly be increased in some groups.
3.34.1 Folate requirements in women
Conventional views on folate deficiency and its role in the aetiology of NTD sug-
gested that the sole adverse outcome was the arrest of cell division because of
inadequate folate-dependent DNA biosynthesis (section 3.39). Recently this was
revised because, although those women whose babies’ NTDs such as spina bifida
(Fig. 3.4) had low folate status, the blood concentrations were often within the
72 The nutrition handbook for food processors
accepted normal range. In addition an increased risk of cardiovascular disease
and stroke has been associated with low folate status (see section 3.41) (Scott,
2000).
NTDs are major malformations in which there is a failure of the developing
neural tube to close properly during the fourth week of embryonic life. Incom-
plete closure of the spinal cord results in spina bifida while incomplete closure
of the cranium results in anencephaly (McNulty, 1997). The latter condition
means the babies will either die in utero or shortly after birth.
The MRC trial provided the first unambiguous confirmation of the effective-
ness of periconceptional folic acid in the prevention of NTDs (MRC Vitamin
Vitamins 73
Fig. 3.4 New-born infant with neural tube defect.
Study Research Group, 1991). The study was a randomised double-blind trial
conducted in 33 centres in 7 countries over 8 years and showed that folic acid
had a 72% protective effect in preventing the recurrence of NTD in nearly 1200
women who had a previously-affected pregnancy. A subsequent study by Czeizel
and Dudas (1992) confirmed the MRC study and added that folic acid also pre-
vented first occurrence NTD (Czeizel and Dudas, 1992).
A possible mechanism for folate and/or folic acid protection against
NTDs was put forward by Whitehead et al (1995) to explain why folate was
effective against NTDs, even though folate status was not noticeably poor. They
suggested that genetic polymorphisms existed within a population where the me-
tabolism of folate was disturbed and higher concentrations were needed for full
functionality. In particular, they identified a variant form of the gene for 5,10-
methyleneTHF reductase (E.C. 1.7.99.5) present in approximately 10% of the UK
population (Whitehead et al, 1995). Possession of this variant gene may increase
the risk of NTDs, etc. even though dietary folate might be marginally adequate.
3.34.2 New recommendations
In 1992, The UK Department of Health revised their recommendations for folic
acid and proposed 5 mg folic acid/day in tablet form to stop the recurrence of NTD
and 400mg/day for the prevention of NTD, to be started before conception and to
be continued until the twelfth week of pregnancy (Department of Health, 1992).
Those women who have had a previously affected pregnancy have a 10-fold
increased risk of having another NTD baby; however, 95% of cases are first time
occurrences and these women are causing the most concern (Department of Health,
1992). Targeting the latter group is difficult because of the number of unplanned
pregnancies (about 50%) and the malformations of NTD occurring so early in
pregnancy. To deal with the problem the Department of Health proposed three
possible ways of preventing first occurrence NTD: (1) increased intake of foods
naturally rich in folate, (2) folic acid supplementation, (3) folic acid fortification.
Supplementation is effective if taken but a study of 411 pregnancies in London
in 1994 indicated only 3% of women had taken folic acid before conception
(Clark and Fisk, 1994). Increasing food folate would require women to eat three
times their current intake, however, even when this is achieved experimentally it
does not appear to optimise folate status (McNulty, 1997). Although food forti-
fication is the most promising there are worries that too high a folate intake might
mask a vitamin B
12
deficiency in the elderly. Extra dietary folic acid might reduce
the erythrocyte effects of vitamin B
12
deficiency (i.e. megablastosis) and delay
the diagnosis. An undiagnosed vitamin B
12
deficiency can progress to a neu-
ropathy following the remission of the anaemia (Cuskelly et al, 1996).
3.34.3 Fortification
In 1998 folic acid fortification of grain foods in the USA was made mandatory.
The Food and Drug Administration opted for a low level of fortification of
74 The nutrition handbook for food processors
1400mg/kg product because of some concerns over safety issues (United States
Department of Health and Human Services, 1996). It was expected that this level
of fortification would result in a mean additional intake of 100mg/day in the
US population which would be low enough to carry no risk of masking vitamin
B
12
deficiency, but others argued it would be ineffective in preventing NTD
(McNulty, 2001). Recent data published indicates that the incidence of NTD in
the US has fallen by almost 20% following the introduction of the fortification
policy (Honein et al, 2001).
COMA recommended mandatory food fortification to the UK government in
2000 (Department of Health, 2000). The main conclusion of the report was ‘uni-
versal folic acid fortification at 2400mg/kg in food products as consumed would
have a significant effect in preventing NTD-affected conceptions and births
without resulting in unacceptably high intakes in any group of the population’.
This level of fortification has been estimated to increase mean folic acid intakes
by 200mg/day which is predicted to reduce NTD pregnancies by 41%.
In the COMA report, estimates of exposure of different sections of the
poulation were made by modelling dietary intake data from 4 National Diet and
Nutrition Surveys for each age group at 5 possible levels of fortification of flour:
1400mg/kg, 2000mg/kg, 2400mg/kg, 2800mg/kg and 4800mg/kg. At each level
of fortification the number of NTD-affected births per year prevented, as well
as the percentage of people over 50 years who would be exposed to a folic acid
intake greater than 1 mg/day (upper tolerable limit), was calculated. If white flour
were fortified with 2400mg/kg, the results predict all subjects would have total
folate intake above the LRNI and mean intakes would increase to 343 and 365
from 153 and 165mg/day for girls and boys aged 11–12 years respectively, 10%
of whom currently do not meet the LRNI (Moynihan et al, 2001).
The report is currently undergoing consultation by the four UK Health Depart-
ments and the Food Standards Agency (McNulty, 2001).
3.35 Folate, homocysteine and cardiovascular disease (CVD)
Cardiovascular disease remains one of the main causes of mortality in the western
world and approximately two-thirds of cases are attributable to traditional envi-
ronmental and genetic factors. However, in the last decade it has emerged that a
moderate rise in the amino acid homocysteine in plasma constitutes a risk factor
for atherosclerotic vascular disease in the coronary and peripheral blood vessels
(Ward, 2001). The B vitamins folate, B
12
and B
6
all play a key role in homocys-
teine metabolism and deficiencies of any one of the three B vitamins can result
in homocysteinaemia. However, folate appears to be the most important and has
been shown to lower plasma homocysteine concentrations at doses of 0.2–
10 mg/day in both healthy and hyper-homocysteinaemic subjects (Ward, 2001)
(see Fig. 3.3).
There is considerable variation throughout Europe in the rate of CVD mor-
tality. In southern Europe more fruit and vegetables are eaten and this appears to
Vitamins 75
be associated with lower incidence of CVD. The consumption of fruit and veg-
etables has traditionally been associated with increased antioxidant intake (see
sections 3.6, 3.15, 3.25) but green vegetables are also one of the main sources of
folate, contributing more than 30% of total dietary folate intake.
A recent meta-analysis of 12 randomised controlled trials, which included
1114 subjects, demonstrated that folic acid in the range of 0.5–5 mg/day lowers
plasma homocysteine by approximately 25% with 0.5 mg/day as effective as
5 mg/day (Clark and Homocysteine Lowering Trials Collaboration 1998).
Furthermore, the greatest lowering was in those with the highest baseline homo-
cysteine concentrations (Ward, 2001). Doses as high as 0.5 mg/day can only
be achieved using supplementation. It was of interest, therefore, to investigate
whether dietary modification or fortification with lower levels of folic acid could
also be effective. Results indicated that homocysteine concentrations were sig-
nificantly lowered in response to doses of 100 and 200mg/day but no additional
lowering was found with 400mg/day (Ward, 2001). When the fall in homocys-
teine was expressed in tertiles, only the top two tertiles showed lowering. Homo-
cysteine and folate (serum or red cell) showed no response with any of the doses
in the bottom tertile. It was concluded that 200mg/day folic acid was the optimal
homocysteine-lowering dose: interestingly the same dose had previously been
predicted to be optimal in reducing the risk of NTD (Daly et al, 1997).
3.36 Causes of decreased folate status
Dietary folate deficiency has been previously associated with poor socio-economic
groups but now is thought to exist in 5–10% of the population of most communi-
ties. Intestinal absorption is impaired in those with coeliac disease or tropical sprue
which if left untreated can lead to folate deficiency (Scott, 2000) (see also section
3.33). Pregnancy is associated with increased folate catabolism, particularly in the
second and third trimesters, when it exceeds intake. Women who enter pregnancy
with adequate stores or receive prophylaxis during pregnancy will avoid defi-
ciency. Haemolytic anaemia, a condition with increased cell division, can also lead
to folate deficiency (Scott, 2000). Anticonvulsant drug therapy is associated with
folate deficiency but the mechanism is not known (Scott, 2000). It was suggested
that the drugs cause folate malabsorption or excretion of folate through hepatic
enzyme induction but this theory has now been discarded. Chronic alcoholics
usually have folate deficiency. The aetiology is unclear. Suggestions are that
alcohol causes decreased intestinal absorption, increased renal excretion and is
directly toxic to bone marrow and other cells (Scott, 2000).
3.37 Safety/toxicity
Claims have been made that very high folate intakes may result in mood changes
but this work has received little or no subsequent support (Hunter et al, 1970).
The most important aspect of folate toxicity has occurred as a result of efforts to
76 The nutrition handbook for food processors
raise folate status, not by dietary means, but by using supplements and fortifica-
tion, to protect against NTDs and/or cardiovascular disease. Supplements con-
taining 400mg/day, an adequate amount to give protection, are probably safe.
However, because only a minority of those at risk, particularly in the poorer socio-
economic groups, can be persuaded to take the supplements, fortification is being
considered as an alternative method (Scott et al, 1994). Fortification is associated
with some difficulties, because in order to deliver the target dose to those who
need it, others who already have a high intake may get up to 10 times that amount.
In addition, high amounts of folate will mask vitamin B
12
deficiency thus allow-
ing the associated neuropathy to develop. High amounts of folate have also been
implicated in destabilising epilepsy, which is being controlled with anticonvul-
sants and accelerating the growth of certain tumours (Scott, 2000). In these
effects, toxicity may be dependent on the sustained appearance of folate, instead
of 5-methylTHF normally presented to cells. The dose of folate at which any of
the above adverse reactions can occur is not known but in calculating safe levels
for fortification, 1 mg/d was taken as the upper tolerable intake level to prevent
masking of vitamin B
12
deficiency in the elderly (McNulty, 2001). The recent
work by Ward et al (1997) to determine the minimum dose to lower homocys-
teine concentrations suggests a supplement of 200mg/day to be effective. Hence
the proposed level of fortification (2.4 mg/kg flour) should provide the extra
200mg folate/day for both the homocysteine-lowering effects and for reducing
risk of NTDs (see section 3.35) and should not exceed safe levels of intake.
3.38 Cobalamin (vitamin B
12
)
Compounds with vitamin B
12
activity consist of a corrinoid ring surrounding an
atom of cobalt, the only known function of cobalt in the human body (Depart-
ment of Health, 1991). Vitamin B
12
is involved in recycling folate coenzymes in
methionine synthesis (see section 3.33) and degradation of valine via methyl-
malonyl CoA. Interaction with the folate group of coenzymes is responsible for
the condition, megaloblastic anaemia, in which vitamin B
12
deficiency results in
the same syndrome as folate deficiency (see section 3.34) as it is needed to regen-
erate THF (Fig. 3.3).
Only microorganisms synthesise vitamin B
12
and the vitamin gets into the food
chain from the bacteria present in the digestive system of herbivores. Herbivores
are then eaten by animals higher in the food chain. For humans, food sources of
vitamin B
12
include almost all animal products, certain algae and bacteria.
Vitamin B
12
is not present in vegetables or fruits. The dietary intake of vitamin
B
12
is about 5mg/day (Weir and Scott, 1998). The current RDA for B
12
in most
countries is between 1 and 20mg/day; the British RNI are given in Table 3.1.
Since food sources of vitamin B
12
are limited to those of animal origin, vegetar-
ians and especially vegans are at risk of becoming deficient and should take an
oral supplement. Studies on these groups have shown evidence of biochemical
deficiency such as raised concentrations of homocysteine and methylmalonic acid
Vitamins 77
but no clinical evidence of anaemia or neuropathy. The exception is breastfed
infants of vegans, who have developed neuropathy.
3.39 Deficiency
Deficiency of vitamin B
12
produces two diseases in humans, megoblastic anaemia
and a specific neuropathy or subacute combined degeneration of the spinal cord.
These complications are seen mainly in pernicious anaemia, first described by
James Coombe in 1824 (Wickramasinghe, 1995). The disease is usually fatal in
1–3 years and was first understood by George Minot and William Murphy (1926)
who demonstrated that feeding a daily diet of lightly cooked beef liver induced
a remission of the anaemia. Subsequently, the beef was shown to have an extrin-
sic factor (vitamin B
12
) which required an intrinsic factor (IF) for its normal
absorption. IF was produced by normal stomachs but not by those with perni-
cious anaemia. IF was shown to complex with vitamin B
12
for uptake and trans-
port by a specific receptor on the ileal enterocytes in the terminal ileum of humans
(Weir and Scott, 1995).
The body has no mechanism to control the effects of vitamin B
12
deficiency.
It may be associated with clinical complications such as atheroma, causing coro-
nary thrombosis, stroke, and peripheral vascular disease, neural tube defects or
hepatic drain on the stores by steatosis.
The most important causes of B
12
deficiency are the various forms of intesti-
nal malabsorption of which the autoimmune disease, pernicious anaemia, is the
most common. Autoantibodies are produced against the parietal cells of the
stomach so the cells can no longer produce IF or hydrochloric acid (HCl). Patients
with hypochlorhydria, such as the elderly and postgastrectomy patients, may
exhibit malabsorption of dietary cobalamin and a lack of IF prevents absorption
of vitamin B
12
.
Reduced secretion of pancreatic enzymes and bicarbonate leads to impaired
digestion of haptocorrins (Hc) (also called R binders, TC I and III or cobalaphilin)
and elevation of intestinal pH. Haptocorrins are glycoproteins which bind to
vitamin B
12
in the terminal ileal cells and transport it in the plasma to the cells
of the body. Raised pH and low pancreatic enzymes result in cobalamin malab-
sorption because the transfer of cobalamin from dietary haptocorrins binders to
IF is impaired.
Grasbeck-Immerslund syndrome (Weir and Scott, 1999) is a rare congenital
disorder where the ileal receptor is either missing or malfunctioning.
Bacterial overgrowth (Weir and Scott, 1999) of the small intestine by colonic
bacteria at concentrations greater than 10
8
organisms/L can result in B
12
defi-
ciency caused by competitive uptake of vitamin B
12
by the micro-organisms.
Patients with AIDS are known to develop plasma vitamin B
12
deficiency (Weir
and Scott, 1999); however, the pathogenic significance of this still needs to be
determined. The condition is thought to be due to failure of the IF-B
12
complex
uptake by the ileal intrinsic factor enterocyte cell wall receptor.
78 The nutrition handbook for food processors
The condition, transcobalamin II (TC II) deficiency (Weir and Scott, 1999),
usually presents within the first or second month of life. TC II transports co-
balamin from the ileum to the liver where it can be stored or to the tissues where
it can be used. Deficiency results in a potentially lethal effect. Early symptoms
are vomiting, weakness, failure to thrive and megaloblastic anaemia. The TC
defect can be due to TC II being absent or it may be immunologically normal but
fail to bind cobalamin, or it may bind cobalamin but fail to be taken up by the
cell wall receptor.
Several drugs, such as colchicine, neomycin, p-aminosalicylate and alcohol
cause vitamin B
12
malabsorption (Halsted and McIntyre, 1972). In addition,
commonly used antacid drugs, such as cimetidine, ranitidine and omeprazole,
decrease gastric acid essential for the absorption of vitamin B
12
from food. The
anaesthetic nitrous oxide is known to inactivate methionine synthesis by oxidis-
ing vitamin B
12
to an inactive form. Prolonged exposure to nitrous oxide has been
shown to cause neuropathy and subacute combined degeneration of the spinal
cord in humans (Layzer et al, 1978).
3.40 Assessment and other issues
The determination of serum vitamin B
12
concentrations has been the method of
choice to assess status for many years. Acceptable concentrations are 147 pmol/L
(200mg/L) and a risk of deficiency is indicated by concentrations <74 pmol/L
(100mg/L). Most circulating vitamin B
12
is carried on TC I, which is biologically
inactive, but representative of the status of the individual. Conventially TC I
was measured by a microbiological assay which has been the ‘gold standard’.
Recently a radiometric assay based on competitive binding between endogenous
vitamin B
12
and an exogenous radioactive form of vitamin B
12
for a binder has
been used. It has been suggested that measuring holo TC II might be a better way
of assessing cobalamin deficiency. However, a recently introduced indirect mea-
surement of intra-cellular cobalamin deficiency may be a more functional measure
of B
12
status (Green, 1995). It is the measurement of the substrates of two
cobalamin-dependent enzymes, methylmalonic acid (MMA) and homocysteine.
3.40.1 Plasma and urinary methylmalonic acid
Normal variations in dietary intake of MMA do not affect plasma levels. MMA
is excreted in the urine and is closely correlated with plasma concentrations.
Plasma MMA levels rise in renal failure and in cobalamin deficiency but not
in folate deficiency. Plasma concentrations rise from normal values of 0.1–
0.4mmol/L to 50–100mmol/L in vitamin B
12
deficiency. MMA in urine or plasma
is a sensitive measure of absolute and/or functional vitamin B
12
deficiency, and
is especially useful in the diagnosis of sub-clinical vitamin B
12
deficiency in the
elderly.
Vitamins 79
3.40.2 Homocysteine
Plasma homocysteine comes predominantly from dietary methionine, although
concentrations are sensitive to dietary vitamin B
12
, folate and pyridoxine, the
vitamin co-factors associated with its metabolism (Fig. 3.3). Normal homo-
cysteine concentrations are higher in men than in premenopausal women, but
increase with age especially after age 60.
Plasma homocysteine concentrations rise in deficiency states of folate, vitamin
B
12
and pyridoxine, in persons with inborn errors of the enzymes of homocys-
teine metabolism and in those with defects associated with the synthesis of
vitamin B
12
coenzymes required for normal function of methionine synthetase.
However, plasma MMA concentrations seem to be more specific and sensitive
indicators of vitamin B
12
status than homocysteine concentrations and are sig-
nificantly better than plasma vitamin B
12
. It is not unknown for normal individ-
uals with normal plasma vitamin B
12
concentrations to have had elevated levels
of MMA that fell after a single injection of vitamin B
12
. In vitamin B
12
deficiency,
elevated levels of both MMA and homocysteine seem to occur before a fall in
plasma vitamin B
12
(Green, 1995).
3.41 Safety/toxicity
Vitamin B
12
has extremely low toxicity. It has been found to be toxic in animals
only at g/kg levels and no toxic effects have been seen in humans (Department
of Health, 1991).
3.42 Biotin
Biotin is a bi-cyclic compound consisting of an imidazole ring fused to a tetrahy-
drothiophene ring with a valeric acid side-chain. Biotin acts as a co-factor for
several carboxylases used in fatty acid synthesis and metabolism and gluconeo-
genesis and branched-chain amino acid metabolism (Halsted and McIntyre,
1972).
Biotin is widely distributed in food sources (Table 3.2) but the bioavailability
of biotin is governed by binding substances in food. For example, biotin is tightly
bound by the imidazole ring to avidin in egg white which is released on cooking
and in an unavailably-bound form in wheat (Halsted, 2000). Biotin is also syn-
thesised by intestinal flora and in vivo studies suggest there is significant absorp-
tion of biotin from the proximal and transverse colon which may be from
intestinal bacteria. The extent to which biotin is available is not known. Biotin
and its metabolites are excreted at a rate of 6–50mg/day in urine and faecal excre-
tion of biotin is 3–6 times the intake (Department of Health, 1991).
Signs of biotin deficiency have been recorded in in-patients receiving total
pareneral nutrition for prolonged periods. Biotin supplements (60–200mg)
appeared to resolve the deficiency. The COMA panel did not set DRVs but the
80 The nutrition handbook for food processors
average intake by British men is 39mg/day (range 15–70mg) and by women
26mg/day (10–58mg), hence the average requirement is thought to lie within
this range (Department of Health, 1991), but if microbial biotin is utilised by
humans, requirements may be much higher.
Three of the four biotin-dependent carboxylases are mitochondrial: propionyl-
CoA carboxylase, methylcrotonyl-CoA carboxylase and pyruvate carboxylase;
the fourth, acetyl-CoA carboxylase, is found in the mitochondria and the cytosol.
Pyruvate carboxylase is a key enzyme in gluconeogenesis, so impairment as a
result of biotin deficiency may lead to fasting hypoglycaemia. Work with chicks
showed a fatal hypoglycaemia called ‘fatty liver-kidney syndrome’ was due to
impaired gluconeogenesis caused by deficient activity of pyruvate carboxylase.
Several workers have suggested that biotin deficiency may cause sudden infant
death syndrome (SIDS) by an analogous pathogenic mechanism (Mock, 1999).
The hypothesis was supported by demonstrating that hepatic biotin concentra-
tions were significantly lower in infants who died from SIDS than those dying
from other causes. Further work is needed to confirm this finding.
There are no reports of biotin toxicity in amounts up to 10 mg/day.
3.43 Pantothenic acid
Pantothenic acid is a dimethyl derivative of butyric acid linked to alanine. The
vitamin is linked through phosphate to form 4￠-phosphopantetheine and coen-
zyme A (CoA), the primary active form. As a constituent of CoA and its esters,
the vitamin is essential for numerous reactions involved in lipid and carbohydrate
metabolism including fatty acid synthesis and degradation, steroid hormone syn-
thesis and gluconeogenesis (Halsted, 2000).
Rich dietary sources of pantothenic acid are listed in Table 3.2. Especially high
levels of pantothenate are found in royal bee jelly (511mg/g) and in ovaries of
tuna and cod (2.3 mg/g). Human breast milk pantothenate content increases five-
fold within 4 days of the birth of the baby from 2.2 to 11.2mmol/L, similar to
that found in cow’s milk.
No biochemical method has been accepted for determining pantothenate con-
centrations in humans. Measurements have been made on blood levels and urine
excretion but it has not been possible to interpret the results in terms of dietary
needs. The COMA panel derived no DRVs for pantothenate and as there are no
signs of pantothenic acid deficiency in the UK, intakes of between 3 and 7 mg/day
were deemed adequate even during pregnancy and lactation (Department of
Health, 1991).
3.44 Deficiency
3.44.1 Nutritional melalgia
Deficiency in humans is rare and has only been detected in conditions of severe
malnutrition. In World War II prisoners of war in the Philippines, Japan and
Vitamins 81
Burma experienced numbness of their toes and painful burning sensations in their
feet (nutritional melalgia). They were treated with yeast extract as a source of all
B vitamins.
3.44.2 Hair colour
Early studies showed a loss of fur colour in black and brown rats fed a pantothenic
acid-deficient diet; as a consequence pantothenate became known as the anti-grey
hair factor. Even though there is no evidence to support the idea that loss of
hair colour with age is related to pantothenic acid deficiency, it is still added to
shampoos (Bender, 1999).
3.44.3 Alzheimer’s disease
In Japan, homopantothenate (pantoyl g-aminobutyrate), the next highest homo-
logue of pantothenic acid, is used to enhance cognitive function, especially in
Alzheimer’s disease. It acts via g-aminobutyrate receptors to increase acetyl
choline release and cholinergic function in the cerebral cortex and hippocampus
(Bender, 1999). A rare side-effect of the treatment is hepatic encephalopathy and
the excretion of a variety of dicarboxylates, both effects being reversed by pan-
tothenate, suggesting homopantothenate may cause pantothenate deficiency. If
this is verified dicarboxylic aciduria may provide a marker of pantothenate status.
However, the alternative theory might be that the adverse effects are due to
toxicity of homopantothenate which is antagonised by pantothenate.
3.45 Toxicity
There are no reports of toxicity with intakes up to 10 g/day.
3.46 Niacin
Niacin is a generic term for nicotinic acid and nicotinamide, both of which are
substrates for the synthesis of nicotinamide nucleotide coenzymes, NAD and the
phosphorylated derivative of NADP (Powers, 1999). The major metabolic role of
NAD(P) is as a coenzyme in oxidation and reduction reactions. Niacin is there-
fore of crucial importance in intermediary metabolism and requirement is related
to energy expenditure (Department of Health, 1991). Deficiency of niacin results
in pellagra, which is fatal if untreated.
Quantitation of dietary niacin must take into account the dual source of the
vitamin, the vitamin nicotinic acid and the amino acid tryptophan. Niacin in food
is quantified as niacin equivalents (NE). Dietary sources are listed in Table 3.2
but meat is a rich source of both tryptophan and niacin.
High intake of tryptophan results in greater efficiency of conversion to niacin.
82 The nutrition handbook for food processors
Oestrogens reduce the rate of tryptophan metabolism, so where pellagra is
common, twice as many women as men are affected. However, before puberty
and after menopause there are no sex differences. It is generally believed that
1 NE is equivalent to 60 mg of tryptophan or 1 mg dietary niacin.
The mean observed requirement for niacin to prevent or cure pellagra, or to
normalise the urinary excretion of N-methyl nicotinamide (NMN) and its onward
metabolite methyl pyridone carboxamide (MPCX), in experimental subjects
maintained on niacin-deficient diets and in energy balance, is 5.5 mg/4.2 MJ
(1000 kcal). A coefficient of variation of 10% gives an RNI of 6.6 mg/4.2 MJ (i.e.
plus 2 SD) and an LNRI of 4.4 mg/4.2 MJ. A summary of niacin requirements is
given in Table 3.1.
Niacin deficiency causes pellagra and occurs in parts of India and the African
continent. Pellagra is frequently associated with the ‘hungry season’, strong sun-
shine and with diets of mainly maize and millet. The clinical features of pellagra
are dermatitis, diarrhoea and dementia. Pellagra is occasionally found in mal-
nourished alcoholic patients and it has been found associated with the intake of
mycotoxins from the mould Fusarium. Pellagra occurs in Hartnup’s disease, an
autosomal recessive disorder with impaired absorption of several amino acids
including tryptophan (Thurnham, 2000).
Classic pellagra responds dramatically to nicotinamide or niacin given at a
dose of 100–300 mg/day in three doses. In addition to niacin, it is usual to give
riboflavin and pyridoxine and a diet high in calories and protein. Mental changes
disappear within 24–48 hours but dermal lesions may take 3–4 weeks. A dose of
40–200 mg niacin/day can be used to treat the symptoms of Hartnup’s disease.
Doses of 1–2 g/day of niacin are used to manage patients with hypertriglyceri-
daemia and/or hypercholesterolaemia. However, nicotinic acid, but not nicoti-
namide, in excess of 200 mg causes vasodilation of cutaneous blood vessels.
Higher doses cause vasodilation of other blood vessels and a transient drop in
blood pressure (Thurnham, 2000).
3.47 Vitamin B
6
(pyridoxine)
The term vitamin B
6
includes three pyridines: pyridoxine, pyridoxal and pyri-
doxamine and their 5￠-phosphorylated derivatives, which are metabolically inter-
convertible. The liver is the main tissue responsible for converting pyridoxine
and pyridoxamine phosphates into pyridoxal 5￠-phosphate (PLP). PLP is the main
coenzyme form of the vitamin and the co-factor for a large number of enzymes
catalysing reactions of amino acids (Department of Health, 1991). PLP is also
the main extra-cellular form of the vitamin, being transported in plasma bound
to albumin. PLP, which is not enzyme-bound, is oxidised to pyridoxic acid and
excreted in urine (Powers, 1999).
The vitamin is widely distributed in foods (Table 3.2), although much of the
vitamin B
6
in some vegetables may be present as glycosides, which are unavail-
able. The intestinal bacteria are also able to synthesise relatively large amounts
Vitamins 83
of pyridoxine (Department of Health, 1991). Intestinal absorption takes place
mainly in the jejunum by non-saturable, passive diffusion of the non-
phosphorylated forms of the vitamin. Post-absorptive phosphorylation of all three
forms is catalysed by pyridoxal kinase and the phosphorylation constitutes a form
of cellular trapping in intestinal cells and other tissues (liver, muscles and brain)
as the charge on the phosphate hinders efflux through the cell membrane.
Pyridoxine exhibits greater stability than other forms of the vitamin. As the
hydrochloride salt, it is used in dietary supplements and food fortification be-
cause of its stability, comparative ease of manufacture and low cost (Gregory,
2001).
The total body pool of vitamin B
6
is about 15mmol/kg body weight. Isotope
tracer studies have indicated a turnover of about 0.13%/day hence the estimated
minimum requirement for replacement is 0.02mmol (5mg)/kg body weight or
350mg/day for a 70 kg adult. Vitamin B
6
is extensively required in protein metab-
olism hence depletion studies have shown that deficiency develops more quickly
on high protein intakes (80–160 g/day) and repletion is faster at lower intakes of
protein (30–50 g/day). Therefore RNIs are based on protein intakes and estimated
at 15–16mg/g protein (Department of Health, 1991). At average intakes of about
100 g protein/day, this gives an RNI of 1.5–1.6 mg vitamin B
6
(Table 3.1). Average
intakes in Britain are between 20 and 30mg/g protein (Thurnham, 2000).
3.48 Deficiency
Clinical deficiencies in vitamin B
6
are rare. However, severe deficiency of vitamin
B
6
was reported in 1954 when infants were fed formula, which had been severely
heated during manufacture. The heating caused the formation of pyridoxyllysine,
which has anti-vitamin metabolic activity. The affected infants suffered seizures,
which responded to B
6
supplements (Coursin, 1954).
Other groups vulnerable to vitamin B
6
deficiency and deficiency of other
water-soluble vitamins, are those suffering from malabsorption, coeliac disease,
chronic alcoholics and those undergoing dialysis for renal failure.
Clinical signs of deficiency are inflammation of the tongue, lesions of the lips
and corners of the mouth; however, these symptoms are also seen in riboflavin
deficiency. Peripheral neuropathy is found in thiamin as well as in pyridoxine
deficiency. Finally, microcytic hypochromic (sideroblastic) anaemia associated
with pyridoxine deficiency is caused by impaired synthesis of haem (Thurnham,
2000).
3.48.1 Vitamin B
6
and plasma homocysteine
Evidence suggests that elevated homocysteine concentrations are a risk factor for
cardiovascular disease. As several steps in the metabolism of homocysteine are
vitamin B
6
-dependent, several workers have investigated the effects of vitamin
84 The nutrition handbook for food processors
B
6
nutrition on plasma homocysteine concentrations and its metabolites.
However, fasting homocysteine concentrations are only weakly related to vitamin
B
6
status. It appears that reduced efficiency of the vitamin B
6
dependent trans-
sulphuration reactions are adequately countered by cellular remethylation capac-
ity, and the steady state of plasma homocysteine is unaffected. That is, plasma
homocysteine is much more strongly influenced by folate and vitamin B
12
status
than vitamin B
6
status (Gregory, 2001).
3.49 Safety/toxicity
Sensory neuropathy developed in 7 patients taking 2–7 g/day pyridoxine and
although there was residual damage in some patients, withdrawal of these
extremely high doses resulted in considerable recovery of sensory nerve function
(Schaumburg et al, 1983). Dalton and Dalton (1987) reported peripheral sensory
neuropathy in 60% of 172 women taking 50–500 mg B
6
/day for between 6 and
60 months, and even in women taking only 50 mg B
6
/day, 40% reported symp-
toms of peripheral sensory neuropathy. Within 6 months of stopping the supple-
ments all the women recovered.
3.50 Riboflavin
The concept of ‘accessory growth factors’ in biological fluids such as milk which
enabled rats to grow on a purified carbohydrate diet led to the characterisation
of a flavin component in 1932 (Bates, 1987). It was given a number of names,
usually indicating the food of extraction e.g. lactoflavin (milk), ovoflavin (egg);
later it was called vitamin G in the USA and finally vitamin B
2
or riboflavin
(Thurnham, 2000). Riboflavin is an isoalloxazine ring linked to a ribityl side-
chain. Modifications of the ribityl side-chain include an ester phosphate linkage
to form flavin mononucleotide (FMN), and the latter can then be linked with
adenine monophosphate to form flavin adenine dinucleotide (FAD). FMN and
FAD are the two coenzyme forms of riboflavin in tissues (Thurnham, 2000).
Principal dietary sources are dairy products, meat and in some countries beer,
and a small amount from green vegetables (see Table 3.2). Cereals are often a
poor source of riboflavin, but because of the amount consumed, they often make
a significant contribution to meeting requirements in developing countries. For
example, in The Gambia the staple food is rice made with a sauce of stewed
groundnuts and both are poor sources of riboflavin (Bates, 1987). In developed
countries cereals are often fortified with riboflavin and eaten with milk; in this
way they make a significant contribution to the dietary intake (Thurnham, 2000).
Riboflavin is heat-stable, so losses through cooking are only about 10%, but it is
easily destroyed by sunlight in such products as milk.
Vitamins 85
Riboflavin coenzymes are required in the Krebs cycle, hence requirements
for riboflavin are often linked to energy requirements. One particularly important
enzyme which requires riboflavin is glutathione reductase (EC 1.6.4.2). This
enzyme is needed to maintain glutathione in the reduced state (GSH). The enzyme
occurs in all aerobic tissues and GSH is an important constituent to maintain the
cellular redox potential and cellular integrity. Riboflavin coenzymes are also
required in the metabolism of other nutrients. Riboflavin is required to convert
pyridoxine phosphate to pyridoxal phosphate, therefore a deficiency in riboflavin
may impair the conversion of tryptophan to niacin (section 3.46). Anaemia is
sometimes associated with riboflavin deficiency but this may be partly related to
impaired conversion of folic acid to 5N-methyl-tetrahydrofolic acid (see Fig. 3.3).
There are few epidemiological studies which can be used to determine
minimum requirements, because classical signs of deficiency are not completely
specific and respond only moderately to riboflavin supplements (Department of
Health, 1991). Requirements are also dependent on the status of other nutrients
and precipitating factors (Thurnham et al, 1971). In 1990, a survey of British
adults showed the median intake of riboflavin for men was 2.03 mg/day and for
women it was 1.56 mg/day. Erythrocyte glutathione reductase activation coeffi-
cient (EGRAC) values below 1.3 represent adequate tissue saturation and satis-
factory long-term riboflavin status. EGRAC values of 1.3 or above were found
in ~2% of British adults on normal diets. Mean dietary intakes in the latter persons
corresponded to intakes of 1.3 mg/day for men and 1.1 mg/day for women. The
COMA panel decided to base their RNIs on those intakes (Table 3.1, Department
of Health, 1991).
The widespread consumption of dairy products and use of fortified foods
in industrialised countries provides a high dietary intake for most people, hence
the very small proportion of poor EGRAC values in the UK National Survey
(Gregory et al, 1990). Furthermore, there were only ~15% with raised EGRAC
results in the Pre-School Child Survey (Gregory et al, 1995). These results con-
trast markedly with values found in developing countries where availability of
dairy foods and animal food in general is restricted. Status is at best marginal and
often influenced by seasonal availability of food.
Clinical signs of deficiency are often associated with damage to the epithe-
lium but are non-specific. For example, cracks in the corners of the mouth
(angular stomatitis) are frequently linked to riboflavin deficiency but can also
occur in other water-soluble, vitamin-deficient states e.g. niacin, pyridoxine and
folate. Cracked lips (cheilosis), swollen red beefy tongue (glossitis) and scrotal
dermatitis are also associated with the deficiency. In experimental studies, growth
inhibition is a typical sign of severe deficiency. In developing countries, poor
growth is characteristic in many infants and children and riboflavin deficiency
undoubtedly contributes to the problem.
The low solubility of riboflavin prevents its absorption from the gastrointesti-
nal tract in amounts sufficient to produce toxic effects. When 120 mg/day were
used for 10 months to treat congenital methaemoglobinaemia, no adverse effects
were reported (Department of Health, 1991).
86 The nutrition handbook for food processors
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96 The nutrition handbook for food processors
| 课件名称： | 食品加工营养手册（英文版） |
| 课件分类： | 食品 |
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